Molecular Mechanisms of Smooth Muscle Contraction (Molecular Biology Intelligence Unit)

COMPANY R.G. LANDES M OLECULAR BIOLOGY I N T E L L I G E N C E U N I T 5 Kazuhiro Kohama • Yasuharu Sasaki KOHAMA • ...

Author: Kazuhiro | Ed. Kohama

85 downloads 1265 Views 2MB Size Report

This content was uploaded by our users and we assume good faith they have the permission to share this book. If you own the copyright to this book and it is wrongfully on our website, we offer a simple DMCA procedure to remove your content from our site. Start by pressing the button below!
Report copyright / DMCA form

DOWNLOAD PDF

COMPANY

R.G. LANDES

M OLECULAR BIOLOGY I N T E L L I G E N C E U N I T

5

Kazuhiro Kohama • Yasuharu Sasaki KOHAMA • SASAKI MBIU

Molecular Mechanisms of Smooth Muscle Contraction

5

Molecular Mechanisms of Smooth Muscle Contraction R.G. LANDES C O M P A N Y

MOLECULAR BIOLOGY INTELLIGENCE UNIT 5

Molecular Mechanisms of Smooth Muscle Contraction Kazuhiro Kohama, M.D., Ph.D. Department of Pharmacology Gunma University School of Medicine Maebashi, Gunma, Japan

Yasuhara Sasaki, Ph.D. Frontier 21 Project Institute for Life Science Research Asahi Chemical Industry, Co., Ltd. Samejima, Fuji, Shizuoka, Japan

R.G. LANDES COMPANY AUSTIN, TEXAS U.S.A.

MOLECULAR BIOLOGY INTELLIGENCE UNIT Molecular Mechanisms of Smooth Muscle Contraction R.G. LANDES COMPANY Austin, Texas, U.S.A. Copyright ©1999 R.G. Landes Company All rights reserved. No part of this book may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the publisher. Printed in the U.S.A. Please address all inquiries to the Publishers: R.G. Landes Company, 810 South Church Street, Georgetown, Texas, U.S.A. 78626 Phone: 512/ 863 7762; FAX: 512/ 863 0081

ISBN: 1-57059-566-6

While the authors, editors and publisher believe that drug selection and dosage and the specifications and usage of equipment and devices, as set forth in this book, are in accord with current recommendations and practice at the time of publication, they make no warranty, expressed or implied, with respect to material described in this book. In view of the ongoing research, equipment development, changes in governmental regulations and the rapid accumulation of information relating to the biomedical sciences, the reader is urged to carefully review and evaluate the information provided herein.

Library of Congress Cataloging-in-Publication Data

Molecular mechanisms of smooth muscle contraction / edited by Kazuhiro Kohama, Yasuharu Sasaki. p. cm. -- (Medical intelligence unit) ISBN 1-57059-566-6(alk. paper) 1. Smooth muscle—Molecular aspects. 2. Myosin. 3. Actin. 4. Muscle contraction. I. Kohama, Kazuhiro. II. Sasaki, Yasuharu. III. Series. [DNLM: 1. Muscle, Smooth—physiology. 2. Muscle Contraction—physiology. 3. Myosin. 4. Actins. 5. Myosin—Light-Chain Kinase. 6. Signal Transduction. WE 500 M7185 1999 QP321.5.M767 1999 612.7'41—dc21 99-40980 CIP

CONTENTS 1. Smooth Muscle Myosin as Revealed by Baculovirus-Sf9 Cell Expression System ............................................. 1 Hirofumi Onishi and Shin-ichiro Kojima Genetic Systems for Mutagenesis ........................................................... 2 Outlines of Smooth Muscle Myosin ....................................................... 2 Mutations of Active Site .......................................................................... 6 Mutations of the Actin Binding Face ...................................................... 8 Swinging of Lever Arm .......................................................................... 10 Conclusion ............................................................................................. 11 2. The Molecular Anatomy of Myosin Light Chain Kinase ...................... 15 Hiroko Kishi, Li-Hong Ye, Kohichi Hayakawa, Yinhuan Xue, Yuan Lin, Akio Nakamura, Tsuyoshi Okagaki and Kazuhiro Kohama Characterization of MLCK as an Actin-Binding Protein .................... 16 Regulation of Actin-Myosin Interaction by the Actin-Binding of MLCK.......................................................... 18 Effects of Other Smooth Muscle Actin-Binding Proteins on Actin- Myosin Interaction ........................................................... 21 Physiological Involvement of the Inhibitory Effect of MLCK in Regulating the Actin-Myosin Interaction of Smooth Muscle Cells .................................................................... 22 Myosin-Binding Properties of MLCK .................................................. 23 Conclusion ............................................................................................. 25 3. Structure and Regulatory Mechanisms of Myosin Phosphatase .......... 31 Kazuhito Ichikawa, Masaaki Ito, Takeshi Nakano and David J. Harshorne Structural Components of Smooth Muscle Myosin Phosphatase ...................................................................................... 32 Regulatory Mechanisms of MP Activity ............................................... 38 Possible Function of MP ....................................................................... 41 Conclusion ............................................................................................. 42 4. Regulation of Smooth Muscle Myosin Phosphatase and Contraction by Rho and Rho Kinase .............................................. 47 Yoh Takuwa Downregulation of Myosin Phosphatase and Consequent Sensitization of MLC20 Phosphorylation ........................................ 49 Involvement of the Low Molecular Weight G Protein Rho in the Downregulation of Myosin Phosphatase and in Ca-Sensitization ..................................................................... 50 Rho Kinase Mediates Rho Dependent Downregulation of Myosin Phosphatase ..................................................................... 50 Conclusion ............................................................................................. 53

5. Caldesmon Phosphorylation and Smooth Muscle Contraction .......... 59 Vladimir P. Shirinsky, Alexander V. Vorotnikov and Nikolai B. Gusev Caldesmon Gene, mRNA and Protein ................................................. 59 Domain Structure and Activities .......................................................... 60 Caldesmon’s Place and Role in Smooth Muscle .................................. 62 Caldesmon Phosphorylation In Vitro .................................................. 67 Phosphorylation of Caldesmon in Smooth Muscle Tissues ................ 70 The Complex Case of In Vivo Caldesmon Kinase ............................... 73 Future Prospects .................................................................................... 74 6. Thick and Thin Filament Regulation of Smooth Muscle Contraction in Health and Disease ........................................................ 81 Kathleen G. Morgan, William E. Butler and InKyeom Kim Regulation of Smooth Muscle Contraction in Health ......................... 81 Regulation of Smooth Muscle Contraction in Disease ....................... 85 Conclusion ............................................................................................. 92 7. Diphosphorylation of Myosin Light Chain and Spastic Contraction of Smooth Muscle ............................................................. 97 M. Seto and Y. Sasaki Quantification Method of MLC20 Phosphorylation ........................... 98 Diphosphorylation of MLC20 in Intact Smooth Muscle .................. 100 Mechanism for the Formation of MLC20 Diphosphorylation in Smooth Muscle .......................................... 101 Diphosphorylation of MLC20 in Spastic Smooth Muscle ................ 103 Conclusion ........................................................................................... 104 8. Intracellular Mechanisms for Coronary Artery Spasm .................................................................. 107 Hiroaki Shimokawa Endothelial Dysfunction vs. Smooth Muscle Hypercontraction ............................................................................ 107 Animal Models of Coronary Artery Spasm ........................................ 108 Mechanisms of Vascular Smooth Muscle Contraction ..................... 110 Coronary Artery Spasm and Vascular Smooth Muscle ..................... 110 Enhanced Myosin Light Phosphorylations and Coronary Spasm ....................................................................... 111 Future Directions of Research on the Pathogenesis of Coronary Artery Spasm .............................................................. 113

9. Antispastic Therapeutic, HA1077 (Eril ) ............................................. 119 Y. Sasaki, M. Shibuya and H. Hidaka Pharmacological Properties ................................................................ 119 Biochemical Property .......................................................................... 120 Attenuation of Spastic Contraction .................................................... 124 Clinical Trial: Prospective Placebo-Controlled Double-Blind Study on a Delayed Cerebral Vasospasm After SAH ..................................................................... 126 10. Molecular Mechanisms of Smooth Muscle Phenotypic Modulation ........................................................................ 133 Ryozo Nagai and Masahiko Kurabayashi Technical Comments .......................................................................... 134 Isolation of Genomic Clones and Sequence Analysis ........................ 137 Reporter Gene Analysis of the 5'-Flanking Region of the SMemb Gene ......................................................................... 137 Nuclear Factor Binding to the Sequence Between -105 and -91 ...................................................................... 138 Isolation of Rabbit BTEB2 cDNA ....................................................... 138 Transactivation of SMemb Promoter by BTEB2 ............................... 139 Expression of the BTEB2 mRNA in Adult Tissues and Developing Aorta ..................................................................... 139 BTEB2 Expression in Neointimal SMCs ............................................ 139 Conclusion ........................................................................................... 139 11. Migration and Proliferation of Smooth Muscle Cells for Arterial Intimal Growth .................................................................. 143 Yoji Yoshida, M. Mitsumata, J. Jiang and Q. Shu Phenotypes of Vascular Smooth Muscle Cells ................................... 145 Growth Factors and Biologically Active Substances Expressed by SMC Particularly Related to Migration ................... 147 Migration of Medial SMC to the Intima ............................................ 150 Proliferation of SMC and Extracellular Matrix in the Vessel Wall ............................................................................ 152 Effects of Inhibitors Against Angiotensin-Converting Enzyme (ACE) on Neointimal Growth After Balloon Injury ................................................................................................ 155 Matrix Metalloproteinases of the Vessel Wall .................................... 155 Elastase of the Vessel Wall ................................................................... 157 Human and Animal Arteries Have Preferential Areas for Atherosclerosis ........................................................................... 157 Index....................................................................................................... 163

EDITORS Kazuhiro Kohama, M.D., Ph.D. Department of Pharmacology Gunma University School of Medicine Maebashi, Gunma, Japan Chapter 2 Yasuharu Sasaki, Ph.D. Frontier 21 Project Institute for Life Science Research Asahi Chemical Industry, Co., Ltd. Samejima, Fuji, Shizuoka, Japan Chapters 7, 9

CONTRIBUTORS William E. Butler, M.D. Neurosurgery Department Massachusetts General Hospital Boston, Massachusetts, USA Chapter 6

Hiroyoshi Hidaka, M.D., Ph.D. Department of Pharmacology Nagoya University School of Medicine 65 Tsurumai, Nagoya, Japan Chapter 9

Nikolai B. Gusev, Ph.D. Dr. Sci., Professor Department of Biochemistry School of Biology Moscow State University Moscow, Russia Chapter 5

Kazuhito Ichikawa, M.D., Ph.D. First Department of Internal Medicine Mie University School of Medicine Tsu, Mie, Japan Chapter 3

David J. Hartshorne, Ph.D. Muscle Biology Group University of Arizona Tucson, Arizona, USA Chapter 3 K. Hayakawa, Ph.D. Chief Investigator Division of Discover Research, Kissei Pharmaceutical Co., Ltd. Hotaka, Nagano, Japan Chapter 2

Masaaki Ito, M.D., Ph.D. First Department of Internal Medicine Mie University School of Medicine Tsu, Mie, Japan Chapter 3 J. Jiang, M.D., Ph.D. Department of Pathology Yamanashi Medical University Nakakoma, Yamanashi, Japan Chapter 11 InKyeom Kim, M.D., Ph.D. Boston Biomedical Research Institute Boston, Massachusetts, USA Chapter 6

H. Kishi, M.D. Graduate Student for Ph.D. Department of Pharmacology Gunma University School of Medicine Maebashi, Gunma, Japan Chapter 2 Shin-ichiro Kojima, Ph. D. Japan Science and Technology Cooperation Domestic Research Fellow Department of Structural Analysis National Cardiovascular Center Research Institute Fujishiro-dai, Suita, Osaka, Japan Chapter 1 Masahiko Kurabayashi M.D. Associate Professor The Second Department of Internal Medicine Gunma University, School of Medicine Showa, Maebashi, Gunma, Japan Chapter 10 Y. Lin, Ph.D. Associate Professor Department of Pharmacology Dalian Medical University Dalian, China Chapter 2 M. Mitsumata, M.D.,Ph.D. Department of Pathology Yamanashi Medical University Nakahoma, Yamanashi, Japan Chapter 11 Kathleen G. Morgan, Ph.D. Cardiovascular Division Beth Israel Deaconess Medical Center and Harvard Medical School Boston Biomedical Research Institute Boston, Massachusetts, U.S.A. Chapter 6

Ryozo Nagai, M.D. Professor Second Department of Internal Medicine Gunma University School of Medicine Showa, Maebashi, Gunma, Japan Chapter 10 A. Nakamura, Ph.D. Research Associate Department of Pharmacology Gunma University, School of Medicine Maebashi, Gunma, Japan Chapter 2 Takeshi Nakano, M.D., Ph.D. First Department of Internal Medicine Mie University School of Medicine Tsu, Mie, Japan Chapter 3 T. Okagaki, Ph.D. Lecturer, Department of Pharmacology Gunma University, School of Medicine Maebashi, Gunma, Japan Chapter 2 Hirofumi Onishi, Ph. D. Section Chief Department of Structural Analysis National Cardiovascular Center Research Institute Fujishiro-dai, Suita, Osaka, Japan Chapter 1 M. Seto, Ph.D. Frontier 21 Project Institute for Life Science Research Asahi Chemical Industry, Co., Ltd. Mifuku, Ohito, Tagata, Shizuoka, Japan Chapter 7 Masato Shibuya, M.D., Ph.D. Chukyo Hospital Sanjou, Nagoya, Japan Chapter 9

Hiroaki Shimokawa, M.D., Ph.D. The Research Institute of Angiocardiology and Cardiovascular Clinic Kyushu University School of Medicine Maidashi, Higashi-ku, Fukuoka, Japan Chapter 8 Vladimir P. Shirinsky, Ph.D. Head of Laboratory of Cell Motility Cardiology Research Center Moscow, Russia Chapter 5 Q. Shu, M.D., Ph.D. Department of Pathology Yamanashi Medical University Nakakoma, Yamanashi, Japan Chapter 11 Yoh Takuwa, M. D. Professor, Department of Physiology Kanazawa University School of Medicine Takara-machi, Kanazawa, Ishikawa, Japan Chapter 4 Alexander V. Vorotnikov, Ph.D. Senior Investigator, Laboratory of Cell Motility Cardiology Research Center Moscow, Russia Chapter 5 Y. Xue, Ph.D. Postdoctoral Fellow Department of Neurobiology and Behavior Gunma University School of Medicine Maebashi, Gunma, Japan Chapter 2

L.-H. Ye, M.D., Ph.D. Postdoctoral Fellow Department of Pharmacology Gunma University School of Medicine Maebashi, Gunma, Japan Chapter 2 Yoji Yoshida, M.D., Ph.D. Department of Pathology Yamanashi Medical University Nakakoma, Yamanashi, Japan Chapter 11

PREFACE

S

mooth muscle is the main component of smooth muscle organs such as blood vessels, digestive tract, uterus etc. Blood vessels perfuse various nonsmooth muscle organs, and so blood vessel disorder directly affects the functions of these organs. Therefore, smooth muscle disorder is widely related to the diseases of almost every organ in the body. Understanding of smooth muscle function under physiological and pathological conditions is essential for the advance of medical science. In the past, electrophysiology has contributed greatly to the understanding of the functions of smooth muscle membrane. Protein chemistry has revealed many unique properties of smooth muscle myosin, which is a molecular motor that contracts smooth muscle. Technical progress in biology in this decade has solved some of the pathways of intracellular linkage between membrane and myosin. Knowledge of this intracellular signal transduction will be particularly important for medical science, because diseases of smooth muscle organs are associated with several types of disorder of the signal transduction, but not with myosin itself. In the field of drug discovery, the vast amount of information about smooth muscle membrane has provided methods for drug screening and development. Indeed, the membrane is the site of action of most smooth muscle acting drugs. Recently, some types of drugs which enter the cytoplasm through the plasma membrane smooth muscle cells can also be developed. This possibility has been realized through the progress in the understanding of the intracellular signal transduction. Furthermore, the understanding of the mode of action of intracellularly acting drug has revealed a novel signal transduction mechanisms for normal or disordered contraction of smooth muscle. In this book, we first explain how smooth muscle contracts through the interaction of myosin with actin, within the context of recent developments in molecular biology. The key enzyme that phosphorylates myosin to convert it from the inactive form to the active form is myosin light chain kinase. The structure and function of this enzyme are described together with the recent findings of its actin- and myosin-binding properties. The counterpart of the kinase is myosin phosphatase. Recently, a few messengers that modify the phosphatase have been reported; these will also be described in this book, with special reference to the regulatory mode involving the phosphorylation and dephosphorylation of myosin. This myosin-linked regulation is not the sole regulatory mode of smooth muscle contraction. Actin-binding proteins such as caldesmon and calponin also take part in the regulation. The increased tone of smooth muscle is the most common disorder of smooth muscle organs. One of the most possible candidates of the abnormality can be detected as an excess in the phosphorylated state of myosin and/or actin-binding proteins. This book describes a new drug that neutralizes abnormal contraction of smooth muscle through the inhibition of the excess phosphorylation. The other disorder of smooth muscle is arterial injury, where smooth muscle cells lose their contractility and thus the ability to proliferate and

migrate. The phenotypic modulation is explained based on pathological as well as molecular studies. Contributors to this book include the speakers at a symposium on the pathology and pharmacology of vascular smooth muscle which was held at the annual meeting of the Japanese Pharmacological Society at Chiba. We have also invited a few specialists to cover the vast area of healthy and diseased smooth muscle. This book provides the up-to-date knowledge that we expect will stimulate further studies on smooth muscle in the medical sciences. Kazuhiro Kohama Yasuharu Sasaki May 1998

CHAPTER 1

Smooth Muscle Myosin as Revealed by Baculovirus-Sf9 Cell Expression System Hirofumi Onishi and Shin-ichiro Kojima

V

ascular smooth muscle myosin plays an important role in the contraction of blood vessels or the maintenance of blood pressure. This motor protein belongs to the myosin II family. Like other members of the family, myosin slides along actin filaments, in coupling with the hydrolysis of ATP, and is thus responsible for force generation in muscle contraction. In vascular smooth muscle cells, signals from vasoactive substances are transmitted via a Ca2+/calmodulin complex to myosin light chain kinase, which can phosphorylate the regulatory light chains of smooth muscle myosin. This phosphorylation is of primary importance for triggering smooth muscle contraction.1,2 Because smooth muscle myosin plays important roles not only in muscle contraction, but also in its regulation, the knowledge of myosin is necessary for studying of physiological and pharmaceutical problems of blood vessels. One of the most fundamental questions is how the myosins II, including smooth muscle myosin, convert the chemical energy of ATP hydrolysis to the sliding movement of actin filaments. In spite of the long history of biochemical and biophysical studies, this primary important question is not yet fully solved. Recently, there have been two lines of breakthrough on this question. One is derived from X-ray cryatallographic studies. Threedimensional structures of actin3,4 and myosin5,6 have been solved at high resolution. This information provides a structural framework to interpret how the myosin head changes its conformation during translocation of actin filaments. Another line is the application of molecular genetics and the development of expression systems for myosin. These techniques have provided excellent tools for manipulating myosin residues and producing various myosins with characteristics different from those of the wild type. By combining results of mutation analyses with knowledge of crystal structure, it has become possible to estimate the role of several important residues for myosin functions. We have recently developed an expression system for wild-type or mutant chicken smooth muscle heavy meromyosin (carboxyl-terminus truncated myosin fragment) using baculovirus and cultured insect cells.7 In this review, we briefly summarize a baculoviral expression system for myosin mutants, and also introduce myosin structures unveiled by X-ray crystallography. We then review our mutation studies on the active site and actinbinding site of smooth muscle myosin, as well as related studies.

Molecular Mechanisms of Smooth Muscle Contraction, edited by Kazuhiro Kohama and Yasuharu Sasaki. ©1999 R.G. Landes Company.

2


Genetic Systems for Mutagenesis Human hypertrophic cardiomyopathy was found to be caused by a single amino acid mutation in the beta-cardiac myosin heavy chain.8 These diseases were characterized by left ventricular hypertrophy and variable phenotypic expression. More than 30 such mutations have been identified from families bearing hypertrophic cardiomyopathy. The locations of the mutations gave important information for functional areas of the myosin molecule. For example, mutations were clustered around the nucleotide binding pocket, the actinbinding interface, the region that connects two reactive cysteines (SH1 and SH2) , and the interface of the heavy chain and the essential light chain.9 However, since serious defects in beta-cardiac myosin are fatal for patients with this disease, the viable mutations tend to have relatively mild defects in myosin function. Spudich and his colleagues have developed a homologous expression system using the silm mold Dictyostelium discoidium.10,11 This organism has only one myosin heavy chain gene, which is dispensable for propagation in plate culture. Therefore, it is possible to introduce the mutated gene to the myosin heavy chain “null” cell and to obtain transformed cells for subsequent analysis. In this system, wild-type and mutant Dictyostelium myosin heavy chains were complexed with endogenous essential and regulatory light chains to form chimeric proteins. Since large amounts of the proteins can be purified, detailed biochemical studies of mutant myosins are possible. Furthermore, the phenotypes of the transformed cells tell us the nature of the mutant myosin at the cell level. This homologous expression system is especially powerful for analyzing both in vivo and in vitro behavior of the mutant myosins. However, this system is limited so far to expression of Dictyostelium myosin. Vertebrate muscle myosin or myosin fragments have been expressed by other systems. Recently, several groups, including ours,7 have succeeded in expression of wild-type or mutant vertebrate muscle heavy meromyosin (HMM; a carboxyl terminal-truncated myosin fragment) in cultured fall armyworm (insect) cells using baculovirus as an expression vector (heterologous expression).12-14 By transfection of recombinant viruses, the HMM heavy chain and the two light chains from vertebrate muscle are coexpressed in insect cells to form the functional HMM (see Fig. 1.1). Usually, Autographa californica baculovirus is used as a vector for the expression of foreign proteins in insect cells. This virus produces occluded viral particles. At the very late stage of infection (48-72 hrs postinfection), occluded particles are accumulated in the nucleus of the infected cells. A polyhedrin with molecular mass 29 kDa is the major matrix protein of the occluded particles. This protein is primarily synthesized during the occlusion phase; its gene is nonessential for infection or replication of the virus. Both the high level expression and the nonessential nature of the polyhedrin gene allow the promoter of this gene to be used for expression of foreign proteins. Simultanous expression of both the HMM heavy chain and the two light chains is required for formation of functional HMM. So, we employ the polyhedrin promoter to express all three genes. To increase the efficiency of co-introduction of all three genes, we constructed a dual-expression system in which one baculoviral vector is responsible for expression of both regulatory and essential light chains (see Fig. 1.1). Recombinant HMM is expressed by coinfecting two different viruses, one for the heavy chain and the other for both light chains. We have obtained about 0.3 mg purified HMM from 0.4 g of viral infected cells.7

Outlines of Smooth Muscle Myosin Smooth muscle myosin, like other types of myosin II, contains two globular heads attached to a long rod. This molecule consists of two heavy chains (200 kDa) and two pairs of the essential (17 kDa) and the regulatory (20 kDa) light chains. Each globular head is

Smooth Muscle Myosin as Revealed by Baculovirus–Sf9 Cell Expression System

3

Fig. 1.1. A schematic diagram outlining the procedure for expression of mutant smooth muscle heavy meromyosin (HMM). The heavy chain gene of wild type chicken smooth muscle HMM was mutagenized with a variety of oligonucleotides. Mutagenesiswas p erformed by the method of Kunkel et al15 The cDNA encoding the mutant HMM heavy chain was inserted into the transfer vector pAcC4, which contains the polyhedrin promoter and viral flanking sequences. Both cDNAs encoding wild type regulatory and essential light chains were inserted into a single transfer vector as described in Onishi et al7 The transfer vector, encoding either the HMM heavy chain or the two light chains, was cotransfected with linearized wild type Autographa californica baculovirus DNA into Sf9 cells by cationic liposome using the manufacture’s protocol (Invitrogen Co.). The heavy chain gene and the two light chain genes were transferred into the baculoviral DNA by homologous recombination. Recombinant viruses were purified by the visual screening of plaques and then amplified to large amounts by the method of Summers and Smith.16 Sf9 cells were coinfected with the resultant viruses to express functional recombinant HMM.


4

formed by about 850 amino acid residues of the heavy chain at the amino-terminal side and a pair of the light chains, and the remaining portions of the two heavy chains form the rod (~1500 Å in length). Each myosin head can hydrolyze ATP and interact with actin filaments. This interaction results in the sliding movement of actin filaments. On the other hand, the rod portion is required for thick filament formation. The head and the rod portions of smooth muscle myosin can be separated by mild proteolysis (papain17 or Staphylococcus V8 protease18). The head fragment—subfragment 1 (S1)—is soluble and retains functions of the original myosin molecule. Because of the simplicity of analysis, S1 has been used to perform kinetic studies of the actomyosin ATPase reaction. The mechanism of the smooth muscle actoS1 ATPase reaction19 can be fitted to a simple model proposed by Lymn and Taylor20 and Bagshaw and Trentham.21,22 M + ATP M•ATP M•ATP* M•ADP•Pi** M•ADP M + ADP

AM + ATP AM•ATP AM•ATP* AM•ADP•Pi** AM•ADP AM + ADP In the absence of ATP, S1 and actin bind tightly to form a rigid complex (AM). The ATP binding is a two step process. ATP initially forms a collision complex with actoS1 (AM•ATP). The structure of S1 and its interaction with actin are not changed by formation of the collision complex. This step is followed by isomerization into the second ATP-bound state, with enhanced tryptophan fluorescence (M•ATP*). The affinity of S1 for actin is greatly decreased by forming the second state. The formation of this state results in dissociation of the rigid complex. Bound ATP is then rapidly hydrolyzed to ADP and Pi, and forms a metastable ternary complex between S1, ADP, and Pi. The tryptophan fluorescence of the protein is further enhanced by this hydrolysis (M•ADP•Pi**). The rate of product release from the complex is very slow in the absence of actin, and rebinding of actin to the complex greatly stimulates this step. The rate of product release in the presence of saturated actin is much lower for smooth muscle S1 (3-4 s-1) than for skeletal muscle S1 (80-90 s-1). This difference is thought to be related to the slow shortening of smooth muscle contraction. The regulation of smooth muscle actomyosin ATPase occurs via phosphorylation of the regulatory light chains. But, in smooth muscle S1, which is a single-headed proteolytic fragment, the ATPase activity is not affected by phosphorylation. This regulation requires the two-headed myosin structure. Heavy meromyosin (HMM), a two-headed myosin fragment obtained from smooth muscle myosin by α-chymotryptic digestion, is a proper model for studying the regulation mechanism.23,24 This fragment lacks about two-thirds (1000 Å in length) of the rod portion, so that it loses the ability to form thick filaments. The ATPase activity of this soluble fragment is regulated by the phosphorylation of its light chain. Two groups have studied the kinetics of the smooth muscle actoHMM ATPase reaction. We have revealed that (thio)phosphorylation is accompanied by a 10-fold increase in the maximal actin-activated ATPase activity (Vmax) and a 10-fold change in the affinity constant for actin (Ka),25 whereas Sellers et al have found about 25-fold increase in Vmax, but only a 4-fold change in Ka upon phosphorylation of the HMM.26 Both groups have concluded that the regulated step is the product release. Since we were also interested in the regulation of smooth muscle, the HMM form was employed in our expression system. The crystal structure of smooth muscle S1 has not yet been obtained. However, the three-dimensional structures of chicken skeletal S1 with no ligand5 and the truncated Dictyostelium S1 (S1Dc) complexed with various nucleotide analogs27 have been determined by X-ray crystallography. As there is clear correspondence of amino acid residues among


5

Fig. 1.2. A space-filling molecular model of chicken skeletal S1. The 25, 50, and 20 kDa segments of the heavy chain are colored in green, red (or orange), and blue, respectively. The essential and regulatory light chains are shown in yellow and purple, respectively. A horizontal cleft divides the central 50 kDa segment of the heavy chain into upper (red) and lower (orange) domains. The nucleotide binding pocket is formed by the N-terminal 25 kDa segment on one side and the central 50 kDa segment on the other side. Coordinates for the crystal structure are obtained from the Brookhaven Protein Data Bank (code: 1MYS)5 and displayed by Rasmol version 2.6.

smooth muscle, skeletal muscle, and Dictyostelium myosins from alignment of their sequences, we can use the structure of chicken skeletal S1 or Dictyostelium S1Dc to discuss our mutation studies. Here, we will briefly describe on the structural features of the myosin head using these structures. As shown in Figure 1.2, the overall appearance of chicken skeletal muscle S1 is highly asymmetric. This molecule consists of both globular and flat portions. The globular portion contains a nucleotide- and an actin-binding site, whereas the flat portion consists of the carboxyl terminal portion of the S1 heavy chain wrapped by both essential and regulatory light chains. From the functional roles of these portions, the globular and the flat portions are called the catalytic and the lever-arm domains, respectively. The catalytic domain is further divided into three subdomains which were predicted by proteolytic digestion studies of S1. The model in Figure 1.2 shows these proteolytic segments with different colors: green (25 kDa amino-terminal), red (50 kDa central), and blue (20 kDa carboxyl- terminal). Prior to the study of the crystal structure by Rayment et al,5 a variety of biochemical techniques had been applied to identify the nucleotide-binding site of myosin. Sequence analyses of various nucleotide-binding proteins28 revealed that these proteins shared a homologous sequence of GXXGXGKT. In myosin, this sequence was located in the 25 kDa amino-terminal segment. Yount and his colleagues have also identified several

6


nucleotide-binding residues using a photoaffinity labeling technique. Both Trp130 in rabbit skeletal myosin29 and Arg131 in Dictyostelium myosin30 were photolabeled by azideATP analogs. Glu185 in chicken gizzard myosin was also photolabeled by reaction with UDP31 or ADP.32 MgADP and vanadate formed a long-lived complex with myosin. Vanadate exhibited an photochemical reactivity that results in modification of either Ser181 or Ser243 of rabbit skeletal myosin.33 The nucleotide-binding residues identified by early biochemical studies could be superimposed on the crystal structure of Rayment et al5 By this strategy, it was found that a narrow cleft of the nucleotide binding pocket was located between the 25 kDa and the 50 kDa segments. In addition to this pocket, there is a long narrow cleft (the 50 kDa cleft) that subdivides the 50 kDa domain into upper and lower segments. The actin-binding face is located on the side of the myosin head opposite from the nucleotidebinding cleft. The 50 kDa cleft opens out towards the actin-binding face and also connects with the nucleotide-binding pocket at its apex. Comparison of the structures of Dictyostelium S1Dc complexed with MgADP-beryllium fluoride and MgADP-aluminum fluoride has provided insights into structural changes of the myosin head which may be related to an ATP hydrolysis transition. Fisher et al27 have proposed that the MgADP-beryllium fluoride complex is an analog of the prehydrolysis state, whereas the MgADP-aluminum fluoride complex is analogous to the transition state for ATP hydrolysis. The structure of the MgADP-beryllium fluoride complex was substantially indistinguishable from that of the chicken skeletal muscle S1 with no nucleotide. The structure of the MgADP-aluminum fluoride complex, however, showed some structural changes, one of which is the partial closure of the 50 kDa cleft. A segment from Ser448 to Tyr496 (of Dictyostelium myosin) is one of the most highly conserved sections of the myosin sequence among all types of myosin. The X-ray crystallographic studies described above suggest a possible role for this segment. This segment consists of a β-sheet from Ser448 toSer456, a random coil from Gly457 to Ser465, and a long α-helix from Phe466 to Tyr496 (see Fig. 1.3). The β-sheet portion belongs to the upper domain of the 50 kDa segment, whereas the α-helix portion is a component of its lower domain. The upper and the lower domains are connected via the random coil portion. From the analysis of the structural changes in the 50 kDa cleft of Dictyostelium S1Dc complexed with MgADP-aluminum fluoride, Fisher et al have revealed that the partial closure of the 50 kDa cleft results from a rotation of the lower domain by about 5˚. This rotation is accomplished by main-chain conformational changes of the highly conserved Ile455 and Gly457 (see Fig. 1.3). They have also found that there are several new interdomain contacts in the 50 kDa cleft of the MgADP-aluminum fluoride complex. Based on these findings, they have proposed the idea that the new interdomain contacts stabilize the “rotated” state of the ADP•Pi-bound head.

Mutations of Active Site In this section, we focus on our mutational studies of the active site of smooth muscle HMM. We have studied functions of the rotation in Gly468 (Gly457 in Dictyostelium) and the salt bridge Glu470-Arg247 (Glu459-Arg238 in Dictoystelium). If the “rotated” structure of the MgADP-aluminum fluoride complex is really an analog of the transition state for ATP hydrolysis, it is expected that hindering the rotation would impair the transition from M•ATP to M•ADP•Pi. We impede the “rotation” by replacing Gly468 with Ala,35 because this replacement is known to limit backbone angle change of the Gly residue.36 An actin filament decorated with G468A HMM showed an arrowhead appearance indistinguishable from that of wild-type HMM. Addition of ATP dissociated the actoHMM complex, indicating that this mutant protein could interact with ATP to change its conformation into a weaklybound state for actin. However, this mutant was clearly deficient in ATP hydrolysis. The


7

Fig. 1.3. An expanded view around the Ile-Ser-Gly loop in the truncated head of Dictyostelium myosin complexed with MgADP•VO4. The numbers in parentheses below correspond to amino acid positions in the chicken gizzard myosin sequence. Backbone atoms of both sequences 226-269 (235-278) and 447-454 (458-465) of the heavy chain are colored red and those of the sequence 458-510 (469-521) are colored orange. Residues Ile455 (466)- Ser456 (467)-Gly457 (468) are colored green. Residues 238 (247), 457 (468), 459 (470), and 501 (512) are shown by sticks and balls in blue for 238 and 459, green for 457, and purple for 501. MgADP•VO4 is shown by sticks and balls in yellow. Coordinates are obtained from the Brookhaven Protein Data Bank (code: 1MND).34

steady-state ATPase activity of this mutant was nearly equal to zero. This mutant also exhibited no initial phosphate burst. Thus, it is clear that the formation of M•ADP•Pi is blocked by the mutation. In addition to blocking of hydrolysis, we found that this mutant has no tryptophan fluorescence enhancement upon adding ATP. This suggests that the mutation causes HMM to lose its ability to isomerize into the state M•ATP*. Our finding supports an idea of Fisher et al27 that the rotation of Gly468 occurs during ATPase reaction. Interestingly, it also suggests that hindering the rotation might affect the conformation of M•ATP itself to block the isomerization to M•ATP*. It is well established that hydrolysis of ATP results in release of a proton. However, very little is known about residues that participate in the proton acceptance phase of ATP hydrolysis. The structure of the γ-phosphate pocket in both MgADP-beryllium fluoride and MgADP-aluminum fluoride complexes, revealed by Fisher et al, showed that no potentially catalytic residue exists within 5 Å of the point of hydrolytic attack.25 However, from computer modeling, we predict that Glu470 is just outside of this environment and could be reached if a small structural change occurs in the γ-phosphate pocket. We tested whether function is affected if this residue is mutated to Ala. The steady-state ATPase activity of this mutant was much lower than that of the wild-type HMM, and no initial phosphate burst was observed. It is thus suggested that this mutant is deficient in ATP

8


hydrolysis. However, this protein produced the fluorescence enhancement expected from binding unsplit ATP. This result could be interpreted by assuming that the mutation blocks the step from M•ATP* to M•ADP•Pi**. This is just the behavior expected if Glu470 is a catalytic base in the hydrolytic process. However, an alternative explanation proposed by Fisher et al27 is possible: The salt bridge between Glu470 and Arg247, one of several new interdomain contacts in the 50 kDa cleft, stabilizes the transition for ATP hydrolysis and mutation of Glu470 to Ala impairs the salt bridge formation. This may inhibit the structural changes needed for hydrolysis to take place. To examine whether the two explanations, the “proton acceptor” idea or the “salt bridge” idea, is true, we have created a series of smooth muscle mutant HMMs at both Glu470 and Arg247.37 When Glu470 or Arg247 was singly mutated to Arg or Glu, respectively, no steadystate ATPase activity and initial phosphate burst were exhibited. On the other hand, when Glu470 and Arg247 were doubly substituted with Arg and Glu, both ATPase activity and phosphate burst were comparable to those of wildtype HMM. The fluorescence was also enhanced to the level expected from forming M•ADP•Pi**. All our data indicate that the ATP hydrolysis reaction proceeds normally in the doubly mutated HMM as in wildtype HMM, even though Glu470, a potential “proton acceptor”, is removed. These results can be explained simply by assuming that the presence of Glu and Arg at positions 470 and 247 is essential for the ATPase reaction, whichever the direction is. Thus, as proposed by Fisher et al27, the salt bridge formation is likely to be important for ATP hydrolysis, probably via stabilization of the transition state. In contrast to the restored intrinsic activity, this double mutant had a very low value of actin-activated ATPase activity compared to wildtype HMM. Some conformational change required for actin activation is likely disrupted by switching Glu to Arg and Arg to Glu. Dependency on actin concentration revealed that the double mutation resulted in a great decrease in the maximal actin-activated ATPase activity, but no significant change in the affinity for actin. Therefore, in the acto-mutant HMM system, the release step of ADP or Pi from AM•ADP•Pi must be abnormally slow. Our findings support the idea that both unhindered rotation and the salt-bridge formation are essential for ATP hydrolysis. Using the Dictyostelium system, it has been shown that the E459V38 and the R247A39 myosins block ATP hydrolysis. Our findings are along the same line, but, more importantly, this is the first direct evidence that the interdomain interaction between the upper and lower domain of the 50 kDa segment is actually neccesary for ATP hydrolysis reaction.

Mutations of the Actin Binding Face In this section, we focus on our mutation studies of the actin-binding face of smooth muscle HMM. Knowledge on the actin-binding face is very important for understanding functions of this motor protein. This face must form a strong interaction with actin during force generation. After a cycle of ATP hydrolysis is completed, it must be dissociated from actin by rebinding ATP to the active site. In addition, the binding of this face to actin must transmit its signal to the active site. This signal results in the acceleration of the product release steps. Several lines of evidence have demonstrated that the junction region between the 50 and 20 kDa segments is involved in actin binding. Monet et al have found that when myosin binds to actin, this region is protected from proteolytic attack.40 Studies using an aminocarboxyl group crosslinker have also shown that this Lys-rich, positively charged region can crosslink to the negatively charged amino-terminus of actin.41,42 This junction itself is not seen in the crystal structure of S1. Nevertheless, by superimposing crystal structures of actin and S1 to the three-dimensional image of the actin-S1 complex reconstituted from


9

Fig. 1.4. An expanded view around the actin-binding face in the model of chicken skeletal muscle S1. Residues Gly627 to Phe646 and Lys572 to Lys574 are not seen in the crystal structure because of their flexibility. The two Lys-rich loops are involved in the ionic interaction between actin and myosin. On the other hand, the exposed hydrophobic triplet (Met541-Phe542-Pro543) probably participates in the stereospecific interaction. Coordinates are obtained from the Brookhaven Protein Data Bank (code: 1MYS).5

electron micrographs, it has been suggested that the junction (the Lys-rich flexible loop shown in Fig. 1.4) is close enough to interact with six negatively charged residues (Asp1, Glu2, Glu3, Glu4, Asp24, and Asp25) on actin. Crosslinking study has also shown another region in the 50 kDa segment crosslinked to actin.40 Superimposing study has indicated that this region (another Lys-rich loop shown in Fig. 1.4) makes contact with residues Tyr91 to Glu100 of the second actin molecule of an actin filament. Identification of residues participating in ionic interactions has been easier, because the amino-carboxyl group crosslinker is useful for detecting such an interaction. Compared with ionic interaction, evidence of the presence of the hydrophobic interaction between actin and myosin has been more indirect. Botts et al have observed the enviromental change of Cys540 in skeletal muscle myosin upon ATP binding.43 As a hydrophobic triplet of myosin residues (541-543 in the skeletal myosin sequence) is just adjacent to Cys540, they have speculated that this hydrophobic triplet might participate in the hydrophobic interaction with actin.44 Superimposing studies of the crystal structures of actin and myosin have more clearly demonstrated that the hydrophobic triplet (see Fig. 1.4) could contact with residues Ile341 to Gln354 and Ala144 to Thr148 of actin. To test the significance of the hydrophobic triplet (Trp546-Phe547-Pro548 in smooth muscle sequence), we have analyzed a mutant smooth muscle HMM in which Trp546 and Phe547 were mutated to hydrophylic residues Ser and His, respectively.45 This mutant had normal ATPase activity in the absence of actin, but a very limited actin-activated ATPase activity. The actin filaments decorated with this mutant did not show the regular arrowhead

10

Molecular Mechanisms of Smooth Muscle Contraction Fig. 1.5. A revised swinging model. In this model, the barbed end of an actin filament is toward the bottom. Therefore, the actin filament moves upward. The lever-arm domain of S1 rotates along the direction shown by the arrow to generate movement of the actin filament. The line in the lever-arm domain represents a long α-helical portion of the S1 heavy chain. The pivot of the rotation is located near the base of the lever arm. The molecular model is based on the actoS1 complex of Rayment et al.6

pattern observed with wild-type HMM. The impairment of Trp546 and Phe547 decreases the affinity for actin and destroys the stereospecificity in the strong binding state. We thus proposed that these residues form a hydrophobic site for actin binding. Giese and Spudich, using Dictyostelium, purified and characterized a myosin mutant P536R (corresponding to Pro548 in the chicken gizzard sequence).46 Pro536 is at the last position of the hydrophobic triplet. The P536R mutant has normal activity in the absence of actin, but a limited actin-activated activity. This result also supports the idea that the hydrophobic triplet is important for binding to actin. Rayment et al have speculated that this triplet is involved in a stereospecific interaction of myosin with actin.6 This hydrophobic interaction may play an important role to maintain the strong and stereospecific binding to actin during force generation.

Swinging of Lever Arm Accumulating evidence supports an idea that the carboxyl-terminal domain of the S1 heavy chain acts as a lever arm. The three-dimensional structure of chicken skeletal S1 has revealed that this domain forms a long α-helix wrapped with the essential and the regulatory light chains. Comparison of the structures of Dictyostelium S1Dc complexed with various ATP analogs suggests that the orientation in the carboxyl-terminal region of the motor domain (beyond Thr688 in Dictyostelium sequence) changes coupled with the transition from M•ATP to M•ADP•Pi.27,34 Based on these structural changes, Rayment and his colleagues have proposed a new swinging model (see Fig. 1.5). In contrast to the classical model, in which swing is produced by movement of the interface of the myosin head to actin, their revised model assumes that the cue is a small structural change in the region close to the nucleotide binding site. A series of conformational changes are transmitted via this region, which contains two reactive cysteines, to the lever-arm domain. The resulting lever-arm swing could produce a mechanical stroke. Therefore, the reactive cysteine region, in addition to the 50 kDa cleft we have studied, must be important for the sliding movement.


11

This revised swinging model has been supported by other studies. Three-dimensional reconstruction from cryoelectron micrographs of actin filaments decorated with smooth muscle S1 in the presence of ADP have shown about 23˚ swing of the lever arm domain with respect to the actin filament axis.47 The observed lever-arm movement is not thought to be related to the power stroke, because the ADP release step does not contribute to the power stroke directly. Nevertheless, this result shows that the lever-arm can make a swinging movement, of which the pivot point is within or near the reactive cysteine region. To further test the revised model, a series of mutant Dictyostelium myosins that have different lever-arm lengths were characterized.48 The length change was introduced by deleting or inserting light chain binding site(s). The sliding velocities of these myosins were linearly related to the number of light chain binding sites. This result is consistent with the idea that the swinging motion of the lever-arm domain occurs around the reactive cysteine region. Kinose et al mutated Gly699 (a conservative residue in the reactive cysteine region) of the rat skeletal muscle myosin gene to Ala.49 They developed a new system to express the mutant protein in a mouse myogenic cell line, and found that the mutation markedly reduces the actin-sliding velocity although its actin-activated ATPase activity is comparable to wild-type. This result suggests that this Gly residue may play a role in a swinging pivot of the lever arm.

Conclusion Both X-ray crystallography and mutagenetic techniques are very powerful tools to understand myosin function at the atomic level. Mutagenetic studies have just been started to analyze roles of various residues in the motor domain of smooth muscle myosin. We have identified several important residues in the nucleotide binding site and the actin binding face. Although the crystal structure of smooth muscle S1 itself has not be reported, information on skeletal S1 and Dictyostelium S1Dc help us to interpret our data. Combining two lines of study, it became possible to understand how each residue functions during a power production cycle. We believe future studies along these lines will yield greater knowledge about the energy transduction mechanism of smooth muscle myosin, and its regulation mechanism.

Acknowledgments This work was supported by a Research Grant for Cardiovascular Diseases from the Ministry of Health and Welfare of Japan and a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan. S. K. is a Japan Science and Technology Cooperation Domestic Research Fellow.

Note added in proof: While this article was in print, we learned that the crystal structures of a vertebrate smooth muscle myosin motor domain complexed with nucleotide analogs (MgADP.BeFx and MgADP.A1F4-) were determined [Dominguez R, Freyzon Y, Trybus, KM et al. Crystal structure of a vertebrate smooth muscle myosin motor domain and its complex with the essential light chain: Visualization of the pre-power stroke state. Cell 1998; 94:559-571]. The structures of vertebrate smooth muscle and Dictyostelium myosin motor domains with tthe nucleotide analogs were similar.

12


References 1. Adelstein RS, Eisenberg E. Regulation and kinetics of the actin-myosin-ATP interaction. Ann Rev Biochem. 1980; 49:921-956. 2. Hartshorne DJ, Siemankowski RF. Regulation of smooth muscle actomyosin. Ann Rev Plysiol 1981; 43:519-530. 3. Kabsch W, Mannherz HG, Suck D et al. Atomic structure of the actin:DNase I complex. Nature 1990; 347:37-44. 4. Holmes KC, Popp D, Gebhard W, et al. Atomic model of the actin filament. Nature 1990; 347:44-49. 5. Rayment I, Rypniewski WR, Schmidt-Base K et al. Three-dimensional structure of myosin subfragment-1: A molecular motor. Science 1993; 261:50-58. 6. Rayment I, Holden HM, Whittaker M et al Structure of the actin-myosin complex and its implications for muscle contraction. Science 1993; 261:58-65. 7. Onishi H, Maéda K, Maéda Y et al. Functional chicken gizzard heavy meromyosin expression in and purification from baculovirus-infected insect cells. Proc Natl Acad Sci USA 1995; 92:704-708. 8. Geisterfer Lowrance AAT, Kass S, Tanigawa G et al. A molecular basis for familial hypertrophic cardiomyopathy: A beta cardiac myosin heavy chain gene missense mutation. Cell 1990; 62:999-1006. 9. Rayment I, Holden HM, Sellers JR et al. Structural interpretation of the mutations in the beta-cardiac myosin that have been implicated in familial hypertrophic cardiomyopathy. Proc Natl Acad Sci USA 1995; 92:3864-3868. 10. Manstein DJ, Ruppel KM, Spudich JA. Expression and characterization of a functional myosin head fragment in Dictyostelium discoidium. Science 1989; 246:656-658. 11. Ruppel KM, Egelhoff TT, Spudich JA. Purification of a functional recombinant myosin fragment from Dictyostelium discoidium. Ann New York Acad Sci. 1990; 582:147-155. 12. Pato MD, Preston YA, Sellers JR et al. Expression of a truncated form of chicken brain myosin that binds to actin in an ATP-dependent manner using the baculovirus expression system. Biophys J 1993; 64:A144 (Abstr.) 13. Sweeney HL, Straceski AJ, Leinwand LA et al. Heterologous expression of a cardiomyopathic myosin that is defective in its actin interaction. J Biol Chem 1994; 269:1603-1605. 14. Trybus KM. Regulation of expressed truncated smooth muscle myosins. Role of the essential light chain and tail length. J Biol Chem 1994; 269:20819-20822. 15. Kunkel TA, Roberts JD, Zakour RA. Rapid and efficient site-specific mutagenesiswithout phenotypic selection. Methods Enzymol 1987; 154:367-382. 16. Summers MD, Smith GE. A manual of methods for baculovirus vectors and insect cell culture procedures. Bull 1555, Texas Agric Exp Stn, College Station, TX, 1987. 17. Kendrick-Jones J. The subunit structure of gizzard myosin. Philos Trans R Soc London Ser B 1973; 265:183-189. 18. Ikebe M, Hartshorne DJ. Proteolysis of smooth muscle myosin by Staphylococcus aureus protease: Preparation of heavy meromyosin and subfragment 1 with intact 20000-dalton light chains. Biochemistry 1985; 24:2380-2387. 19. Rosenfeld SS, Taylor EW. The ATPase mechanism of skeletal and smooth muscle actosubfragment 1. J Biol Chem. 1984; 259:11908-11919. 20. Lymn RW, Taylor EW. Transient state phosphate production in the hydrolysis of nucleoside triphosphates by myosin. Biochemistry 1970; 9:2975-2983. 21. Bagshaw CR, Trentham DR. The characterization of myosin-product complexes and of product-release steps during the magnesium ion-dependent adenosine triphosphatase reaction. Biochem J 1974; 141:331-349 22. Bagshaw CR, Eccleston, JF, Eckstein F et al. The magnesium ion dependent adenosine triphosphatase of myosin. Two-step processes of adenosine triphosphate association and adenosine diphosphate dissociation. Biochem J 1974; 141:351-364. 23. Onishi H, Watanabe S. Chicken gizzard heavy meromyosin that retains the two light-chain components, including a phosphorylatable one. J Biochem 1979; 85:457-472.


13

24. Seidel JC. Fragmentation of gizzard myosin by alpha-chymotrypsin and papain, the effects on ATPase activity, and the interaction with actin. J Biol Chem 1980; 255:4355-4361. 25. Ikebe M, Tonomura Y, Onishi H et al. Elementary steps in the F-actin activated Mg2+-ATPase reaction of gizzard H-meromyosin: Effects of phosphorylation of the lightchain subunit. J Biochem 1981; 90:61-77. 26. Sellers JR, Eisenberg E, Adelstein, RS. The binding of smooth muscle heavy meromyosin to actin in the presence of ATP. Effect of phosphorylation. J Biol Chem 1982; 257: 13880-13883. 27. Fisher AJ, Smith CA, Thoden JB et al. X-ray structures of the myosin motor domain of Dictyostelium discoidium complexed with MgADP•BeFx and MgADP•AlF4-. Biochemistry 1995; 34:8960-8972. 28. Walker JE, Saraste M, Runswick MJ et al. Distantly related sequences in the alpha- and beta-subunits of ATP synthase, myosin, kinases and other ATP-requiring enzymes and a common nucleotide binding fold. EMBO J 1982; 1:945-951. 29. Okamoto Y, Yount RG. Identification of an active site peptide of skeletal myosin after photoaffinity labeling with N-(4-azido-2-nitrophenyl)-2-aminoethyl diphosphate. Proc Natl Acad Sci USA 1985; 82:1575-1579. 30. Kerwin BA, Yount RG. Photoaffinity labeling of scallop myosin with 2-[(4-azido-2nitrophenyl) amino]ethyl diphosphate. Bioconjugate Chem 1992; 3:328-336. 31. Garabedian TE, Yount RG. Direct photo affinity labeling of gizzard myosin with [3H]uridine diphosphate places Glu-185 of the heavy chain at the active site. J Biol Chem 1990; 265:22547-22553. 32. Garabedian TE, Yount RG. Direct photoaffinity labeling of gizzard myosin with vanadatetrapped ADP. Biochemistry 1991; 30:10126-10132. 33. Cremo CR, Grammer JC, Yount RG. Direct chemical evidence that serine 180 of the glycine-rich loop of myosin binds to ATP. J Biol Chem 1989; 264:6608-6611. 34. Onishi H, Morales MF, Kojima S et al. Functional transitions in myosin: Role of highly conserved Gly and Glu residues in the active site. Biochemistry 1997; 36:3767-3772. 35. Richardson JS, Richardson DC. Principles and patterns of protein conformation. In: Fasmann GD, ed. Prediction of protein structure and the principles of protein conformation. Plenum Press, New York 1989; 1-98. 36. Onishi H, Kojima S, Katoh K et al. Functional transitions in myosin: Formation of a critical salt-bridge and transmission of effect to sensitive tryptophan. Proc Natl Acad Sci USA 1998; 95:6653-6658. 37. Ruppel KM, Spudich JA. Structure-function studies of the myosin motor domain: Importance of the 50-kDa cleft. Mol Biol Cell 1996; 7:1123-1136. 38. Shimada T, Sasaki N, Ohkura R et al Alanine scanning mutagenesisof the switch I region in the ATPase site of Dictyostelium discoidium myosin II. Biochemistry 1997; 36:14037-14043. 39. Mornet D, Pantel P, Audemard E et al. The limited tryptic cleavage of chymotryptic S-1: An approach to the characterization of the actin site in myosin heads. Biochem Biophys Res Commun 1979; 89:925-932. 40. Sutoh K. Mapping of actin-binding sites on the heavy chain of myosin subfragment-1. Biochemistry 1983; 22:1579-1585. 41. Yamamoto K. Binding manner of actin to the lysine-rich sequence of myosin subfragment 1 in the presence and absence of ATP. Biochemistry 1989; 28:5573-5577. 42. Botts J, Thomason JF, Morales MF. On the origin and transmission of force in actomyosin subfragment 1. Proc Natl Acad Sci USA 1989; 86:2204-2208. 43. Morales MF. Preface. In: Morales MF, ed. Muscle as a machine. Natl Inst Health, Bethesda, MD 1992; 1-4. 44. Onishi H, Morales MF, Katoh K et al. The putative actin-binding role of hydrophobic residues Trp546 and Phe547 in chicken gizzard heavy meromyosin. Proc Natl Acad Sci USA 1995; 92:11965-11969. 45. Giese KC, Spudich JA. Phenotypically selected mutations in myosin’s actin binding domain demonstrate intermolecular contacts important for motor function. Biochemistry 1997; 36:8465-8473.

14


46. Smith CA, Rayment I. X-ray structure of magnesium (II)•ADP•vanadate complex of the Dictyostelium discoidium myosin motor domain to 1.9 Å resolution. Biochemistry 1996; 35:5404-5417. 47. Whittaker M, Wilson-Kubalek EM, Smith JE et al. A 35-Å movement of smooth muscle myosin on ADP release. Nature 1995; 378:748-751. 48. Uyeda TQP, Abramson PD, Spudich JA. The neck region of the myosin motor domain acts as a lever arm to generate movement. Proc Natl Acad Sci USA 1996; 93:4459-4464. 49. Kinose F, Wang SX, Kidambi US et al. Glycine 699 is pivotal for the motor activity of skeletal muscle myosin. J Cell Biol. 1996; 134:895-909.

CHAPTER 2

The Molecular Anatomy of Myosin Light Chain Kinase Hiroko Kishi, Li-Hong Ye, Kohichi Hayakawa, Yinhuan Xue, Yuan Lin, Akio Nakamura, Tsuyoshi Okagaki, and Kazuhiro Kohama

M

yosin light chain kinase (MLCK) plays a regulatory role in smooth muscle contraction by controlling the actin-myosin interaction. The interaction is regulated by Ca2+,1 with calmodulin (CaM) as its site of action. CaM in the presence of Ca2+ (Ca/CaM) activates MLCK, which then phosphorylates the regulatory light chain of myosin. Phosphorylated myosin is in an active form to interact with actin filaments.2 This mode of regulation is assumed to operate upon the stimulation of smooth muscle and has been extensively studied from a biochemical perspective. However, recent technical progress, such as measurement of intracellular Ca2+, skinning of cell membranes and the development of CaM and MLCK antagonists, have suggested that the actual regulation of smooth muscle contraction appears to be much more complex.3 Uchida and his colleagues induced the contraction of uterine smooth muscle in EGTA-containing, Ca2+-free medium by using oxytocin as an agonist.4 Monitoring intracellular concentration of Ca2+ by the use of fura-2 failed to detect any increase in Ca2+ concentration. Accordingly, there are no signs of phosphorylation during contraction. Karaki and his colleagues also observed that phorbol ester is able to contract aortic smooth muscle in the Ca2+free medium. 5 During the contraction, there was no increase in either Ca2+ concentration or myosin phosphorylation. Therefore, we realized that there are regulators which are able to induce smooth muscle contraction without myosin phosphorylation and that myosin phosphorylation is not obligatory for inducing the contraction.3 The above view is supported by our biochemical experiments using a MLCK antagonist. 6 We prepared crude actomyosin, which contained regulatory protein(s) including MLCK and CaM, from chicken gizzard. Then we measured myosin phosphorylation and superprecipitation of the actomyosin in the presence of various concentrations of an antibiotic produced by Actinomadura.7 Our measurement showed that the regulation of superprecipitation is not strictly related to that of myosin phosphorylation, which leads us to characterize MLCK by solving a paradoxical property of MLCK: That is, it binds to actin filaments, although its kinase activity is related to myosin. This property has been known for some time,8-11 but the effect on the actin-myosin interaction had not yet been analyzed because of complications due to the kinase activity of MLCK. This difficulty has been overcome by the use of recently developed motility assays in vitro12 and by producing actin-binding fragments devoid of kinase activity. 13 These developments enable us to write this review of the structure and function of MLCK, including the novel regulatory modes of MLCK. We do not refer to how MLCK Molecular Mechanisms of Smooth Muscle Contraction, edited by Kazuhiro Kohama and Yasuharu Sasaki. ©1999 R.G. Landes Company.


16

Table 2.1. Actin-linked activities of MLCK and its fragments Actin-binding activitya) Ca/CaMsensitive MLCK (parent) CNBr-fragment (native)d) NTCB-fragment (native) N-fragment (recombinant)d) NC-fragment (recombinant) NN-fragment (recombinant)

+ + + + – +

Actin-b) bundling activity

insensitive + + – + + –

+ + – + – –

Inhibitionc) of actinmyosin interaction + + + + – +

a) Actin-binding activity was measured by the centrifugation assay in the presence and absence of Ca/ CaM (13). b) Mixtures with actin filaments were observed with a phase contrast microscope and analyzed by an electron microscope (14). c) The effect was examined in vitro motility assay on a glass surface coated with phosphorylated smooth muscle myosin (13). d) Unpublished fragments. CNBrfragment and NTCB-fragment are composed of Asp2-Met213 and Met1-Lys114 sequences of chicken gizzard MLCK, respectively. For the topology of the recombinant fragments see Figure 2.2. NB actinbundling activity requires both Ca/Ca-sensitive and -insensitive activities. Ca/CaM-sensitive site is required to inhibit the actin-myosin interaction. This inhibitory effect is detectable only when myosin is in a phosphorylated form (see Fig. 2.9).

phosphorylates smooth muscle myosin, because that mode is well documented in other chapters of this volume.

Characterization of MLCK as an Actin-Binding Protein Studies with Intact MLCK MLCK from smooth muscle of chicken gizzard was mixed with actin filaments at various concentrations, and the mixture was then centrifuged. The amount of MLCK precipitated with actin filaments was related to its concentration.13 A similar relationship was also obtained in the presence of Ca2+ and CaM (Ca/CaM). Analysis on Scatcherd plots shows that the actin binding sites of MLCK include both Ca/CaM-sensitive and -insensitive sites (Table 2.1). Observation of the above mixture with a phase contrast microscope followed by electron microscopy showed that actin filaments assembled into thick bundles.14 Because multiple actin binding sites are required for assembling actin-filaments by crosslinking*, the bundling activity supports the idea of two actin binding sites in MLCK. Accordingly, when actin binding at the Ca/CaM-sensitive site was broken by Ca/CaM, the bundling activity was abolished.

Studies with Native MLCK Fragments The actin-binding core of MLCK was identified to lie in the sequence Asp2-Met213 by cleaving MLCK with Cyanogen bromide (CNBr) at Met residues (see Fig. 2.1 for MLCK topology). *At its higher concentrations, MLCK tends to polymerize.15 However, only the monomeric form was demonstrated under our experimental conditions subjecting MLCK to analytical ultracentrifugation.

The Molecular Anatomy of Myosin Light Chain Kinase

17

Fig. 2.1. Domain structure of MLCK. The numbers refer to amino acid sequence deduced from cDNA coding for MLCK from chicken gizzard smooth muscle.16 The N-terminal binding sites for actin and CaM are cited from ref. 13. The C-terminal myosin binding site is cited from ref. 17. a) Experiments to narrow the sequence responsible for this actin binding have not yet done.

Like parent MLCK, the CNBr fragment is composed of Ca/CaM-sensitive and Ca/CaM-insensitive sites for actin binding and able to assemble actin filaments in a Ca/CaM-sensitive manner. On the other hand, the fragment obtained by cleavage with 2-nitro-5-thiocyanatobenzoic acid (NTCB) at Cys residues is composed of the Met1-Lys114 sequence.18 Actin-binding experiments with the NTCB fragment showed that it contains only the Ca/CaM-sensitive site, because: 1. Its binding activity was totally abolished by Ca/CaM;13 and 2. Because it is unable to assemble actin filaments.

Studies with Recombinant Fragments To confirm two actin binding sites in MLCK as described above, we expressed various fragments of bovine stomach MLCK cDNA in E. coli as recombinant proteins (see footnote* and Fig. 2.2 for the constructs) and examined their actin-binding activities.13 The N-fragment bound actin filaments by both Ca/CaM-sensitive and-insensitive mechanisms. Ca/CaM-sensitive binding was detected in the NN-fragment located in the N-terminal half of the N-fragment, and Ca/CaM-insensitive binding in the NC-fragment located in the C-terminal half of the N-fragment (see Fig. 2.2). Because both of the binding sites are in the N-fragment, this construct assembled actin filaments, but neither the NC-fragment nor the NN-fragment did (Table 2.1). To narrow definition of the Ca/CaM-sensitive site for actin-binding in the NN-fragment,13 we deleted the Met1-Pro41 sequence from the NN-fragment. This NN/41-fragment failed to bind actin filaments (Fig. 2.2). The NN/25-fragment, devoid of the Met1-Gly25 sequence, showed only weak actin binding activity (Fig. 2.2). We interpreted the entire 141 sequence to be responsible for the actin binding of Ca/CaM-sensitive sites.

Analysis of CaM-Binding Site Regulating Actin-Binding

The CaM-binding sequence in the NN-fragment was identified as follows.13 The NN-fragment was allowed to bind to CaM immobilized to the cuvette surface of the IAsys

*Compared with MLCK from chicken gizzard, bovine stomach has an additional sequence of unknown function, i.e., 20 repeats of 12 residues, indicated by an asterisk in Figure 2.2.19 For simplicity, the numbering of amino acid residues in this review is based on the sequence of chicken gizzard16 as shown in Figure 2.1.

18


Fig. 2.2. Schematic diagram of recombinant fragment constructs.13 The constructs are derived from cDNA encoding bovine stomach MLCK.19 Therefore, it contains 12 residue repeats at the position shown by an asterisk. See text for details of actin binding and inhibitory activities.

Cuvette System. Binding was detected by surface plasmon resonance (Fig. 2.2 in Ref. 13). We synthesized Met1-Gly25 and Pro26-Pro41 peptides and allowed them to compete against the CaM-binding of the NN-fragment. Because the 26-41 peptide abolished the binding, we identified the 26-41 sequence as the sequence responsible for CaM-binding (Table 2.2). This identification was also supported by our observation that the NN/25-fragment, but not the NN/41-fragment, binds to CaM as well as did the NN/fragment (Table 2.2).

Two Distinct Sites for CaM-Binding in Parent MLCK In addition to the CaM-binding site (Pro26-Pro41) that regulates actin binding of MLCK, another CaM-binding site (Ala796-Ser815) is known to be involved in regulating kinase activity.16 The 26-41 peptide did not affect the kinase activity that was activated by Ca/CaM, indicating that the peptide does not interact with the 796-815 sequence (Table 2.3). We also synthesized the Ser787-Ser815 peptide and confirmed its inhibitory effect on kinase activity by allowing it to compete against the binding of CaM to the 787-815 sequence. On the other hand, this peptide failed to affect Ca/CaM-sensitive binding of MLCK to actin filaments (Table 2.3). Taken together, the findings show that the amino acid sequence of MLCK that binds to CaM to regulate its kinase activity must be totally different from the one that regulates its actin-binding activity (Fig. 8 in ref. 13).

Regulation of Actin-Myosin Interaction by the Actin-Binding of MLCK Studies with a Nitella-Based Motility Assay To examine how the ATP-dependent interaction between actin and myosin is modified by the actin-binding properties of MLCK, we carried out a Nitella-based motility assay that utilized Nitella’s actin cables, composed of unidirectional actin filaments.20 As shown in Figure 2.3A, MLCK was introduced to the cables to allow MLCK to bind to actin filaments.12 Then, the unbound MLCK, if any, was removed by repeated washing. Finally, we allowed latex beads (about 2 µm in diameter) coated with skeletal muscle myosin to move by


19

Table 2.2. CaM-binding site regulating actin-binding activity of MCLK13 CaM-binding MLCK NN-fragment NN/25-fragment NN/41 fragment Met1-Gly25 peptide Pro26-Pro41 peptide

+ +a) + + – – +b)

CaM-binding activity was monitored by surface plasmon resonance from CaM-coated surface. a) Parent MCLK has additional CaM-binding site that regulates kinase activity (see Fig. 2.1 and Table 2.3). b) The CaM-binding activity was monitored by the competition against CaM-binding of NN-fragment.

interacting with the cables in an ATP-dependent manner. The average velocity of their movement was 0.14 µm/s, as observed under a phase-contrast microscope (Fig. 2.3B). However, the same beads moved along the control cable that had not been treated with MLCK at an average velocity of 1.90 µm/s. We related the reduction in the velocity directly to MLCK bound to actin filaments.12 When designing this experiment, we deliberately used skeletal muscle myosin, whose interaction with actin filaments is minimally modified by phosphorylation.21 Thus, it is not likely that the inhibitory effect is brought about by phosphorylating myosin with MLCK bound to actin cables.13 This suggestion will be confirmed by the use of a MLCK fragment devoid of its kinase domain (see below). We confirmed the inhibitory effect of MLCK with smooth muscle myosin.* Further confirmation was achieved by the most popular method, namely, measuring the actinactivated ATPase activity of skeletal muscle12 and smooth muscle23 myosins, whose activity decreased with an increase in the MLCK concentration.

Studies with a Motility Assay on a Myosin-Coated Glass Surface The inhibitory effect is detectable in an alternative motility assay by the use of a glass surface coated with myosin.12 An aliquot of a solution containing fluorescent actin filaments and ATP was mounted on the surface. The motility of actin filaments was then observed under a fluorescence microscope. The effect of MLCK on this movement was examined by adding specified amounts of MLCK to the above solution. The mean velocity of this movement in the absence of MLCK was 0.5 µm/s. The velocity was reduced as the MLCK concentration increased (Fig. 2.4, open circles). MLCK at 16 nM abolished the movement; half-maximal inhibition was at about 10 nM.**

*Unless otherwise specified, in this chapter myosin refers to smooth muscle myosin that was phosphorylated by chicken gizzard MLCK22 in the presence of Ca/CaM. For the use of unphosphorylated smooth muscle myosin, see the section on myosin binding properties of MLCK, below. **MLCK and its fragments are mostly derived from smooth muscle of the digestive tract, i.e., chicken gizzard and bovine stomach smooth muscle.12,13,24 MLCKs from smooth muscle of bovine aorta27 and from skeletal muscle of chicken breast muscle28 also show the actin-linked inhibitory effect on the actin-myosin interaction, and the inhibition is relieved by Ca/CaM.

20


Table 2.3. Two distinct sites for CaM-binding in parent MCLK13

Pro26-Pro41 peptide Ser787-Ser815 peptide

Regulation of a) actin-binding

Regulation of b) kinase activity

+ –

– +

a) Actin-binding of NN-fragment (see Fig. 2.2, Table 2.1) was monitored in the presence of Ca/CaM and various concentrations of the 26-41 peptide or 787-815 peptide. Only the former is effective. b) Smooth muscle was phosphorylated by MCLK in the presence of Ca/CaM. This reaction was carried out in the presence of various concentrations of the 26-41 peptide or 787-815 peptide. The latter inhibited the reaction.

It must be noted that the concentration of actin filaments in the myosin-coated surface assay was extremely low: 3 nM. Given the dissociation constant of 1.3 µM between MLCK and actin,13 only 0.8% of actin filaments were calculated to be in the MLCK-bound form when MLCK was present at 10 nM. Thus, only partial decoration of the actin filaments by MLCK is sufficient to inhibit their movements. The effect of Ca/CaM is to retard this inhibition (Fig. 2.4, filled circles). When we varied the Ca2+ concentration, micromolar amounts of Ca2+ relieved the inhibition. Relief of inhibition is also observed by increasing the concentration of actin filaments used for the assay, as shown in Fig. 2.5; this is attributable to the reduction in the percentage of MLCK-bound forms per total actin filaments, and explains the actin-linked nature of the inhibition.*

Studies with Actin-Binding Fragments of MLCK The relief by Ca/CaM of the motility inhibition imposed by MLCK suggests that the site responsible for the inhibition is the Ca/CaM-sensitive site for actin binding. To demonstrate this suggestion, we examined the effect of an actin-binding fragment of MLCK containing only Ca/CaM-sensitive site, i.e., native NTCB-fragment and recombinant NN-fragment. They inhibited movement, and the inhibition was relieved by Ca/CaM.13 However, the NC-fragment, which contains the Ca/CaM-insensitive site, failed to modify movement. The truncated forms of the NN/41-fragment devoid of the 1-41 sequence, and the NN/25-fragment devoid of the 1-25 sequence, also failed to modify movement (Fig. 2.2). Taken together, these results indicate that the inhibitory effect of MLCK is exerted through the Ca/CaM-sensitive site for actin binding, i.e., the 1-41 sequence of MLCK. The Ca/CaM-insensitive site has no regulatory role. This conclusion was confirmed by measuring the actin-activated ATPase activity of smooth muscle myosin; the NN-fragment inhibited the activity, but not the NN/41- or the NN/25-fragment (Fig. 3 in ref. 13). *There is a discrepancy between the concentration of MLCK required for inhibiting the movement of actin filaments on a myosin-coated surface, and the actin-activated ATPase activity of myosin (compare Fig. 1 with Fig. 2 in ref. 27). The former is much lower than the latter. The discrepancy is explained in terms of the concentration of actin filaments used in the respective assays. The concentration of actin filaments is much lower in the motility assay (3 nM) than in the ATPase measurement (2 µM) (see ref. 27). The lower concentration of actin filaments requires less MLCK.


21

Fig. 2.3. Actin-linked property of MLCK and its effect on the actin-myosin interaction.12 (A) Actin cables in internodal cells of Nitellopsis obutusa were exposed by intracellular perfusion. MLCK was then introduced into the cells and allowed to bind to the cables . The free unbound MLCK, if any, was removed by subsequent perfusion. As a control, the buffer containing MLCK was introduced and treated in the same way. Latex beads coated with skeletal myosin were suspended in a solution containing MgATP and EGTA and injected into control and MLCK-treated cells. (B) The injected cells were examined by a phase contrast microscope connected to a videoCaMera, and the velocities of the movement of the beads were measured. Arrows refer to mean velocity.

For in vitro motility assays and actin-activated ATPase measurement, in this section and those immediately preceding and following, we use exclusively the phosphorylated form of myosin. With unphosphorylated myosin, actin binding fragments have no effect at all. What is suggested by the difference in such results will be described in the section on myosin binding properties of MLCK.

Effects of Other Smooth Muscle Actin-Binding Proteins on Actin-Myosin Interaction Caldesmon (CaD), calponin (CaP) and tropomyosin (TM) are other regulatory proteins of smooth muscle that bind to actin filaments (Table 2.4). The effects of these proteins on the actin-myosin interaction were monitored by myosin-coated surface assay.* CaD inhibits the interaction (Fig. 2.6), and the inhibition is relieved by Ca/CaM.29,30 This mode of action is similar to that of MLCK. On a structural basis, the Met1-Pro41 sequence of MLCK, mentioned above, contains a sequence highly homologous to one within the actin-binding sequence of CaD (Fig. 2.7, asterisks). When MLCK was allowed to bind to actin filaments in the presence of various concentrations of CaD , the binding of MLCK was affected (Fig. 2.7). This competition between MLCK and CaD is in accordance with their homology. Resembling CaD, CaP binds to actin filaments and inhibits the actin-myosin interaction (Fig. 2.6). This inhibition is abolished by Ca/CaM.31-34 CaP competes with MLCK *A slight stimulatory effect on the actin-myosin interaction was detectable at the lower concentrations of MLCK, CaD and CaP (Figs. 2.4 and 2.6). This effect will be discussed in the section on myosin binding properties of MLCK.

22


Fig. 2.4. Inhibitory effect of MLCK as examined with in vitro motility assay on a myosin-coated glass surface.12,23,24 (A) Fluorescent actin filaments (thick lines) in MgATP were mounted on a coverslip coated with myosin (double headed symbols) and allowed to move.25,26 (B) The movement of actin filaments was observed under a fluorescence microscope in the presence of CaM, MLCK, and Ca2+ (filled circles) or EGTA (open circles). The velocities of their movement were plotted against MLCK concentrations.12

for binding to actin filaments (Fig. 2.7). The Met1-Pro41 sequence of MLCK also contains homology to the actin-binding sequence of CaP (Fig. 2.7). Unlike CaD and CaP, the effect of TM is quite different from that of MLCK; TM does not inhibit movement, but works so as to stimulate movement. The stimulatory effect of TM, however, is not potent enough to overcome the inhibitory effect of CaD or MLCK. Because Ca/CaM is not known to interact with TM, we do not comment on it further.

Physiological Involvement of the Inhibitory Effect of MLCK in Regulating the Actin-Myosin Interaction of Smooth Muscle Cells The physiological role of MLCK in regulating smooth muscle contraction was addressed by the following three experiments. However, the arguments are indirect, obviously requiring another approach (see last section) to establish this role.

Inhibitory Strengths of MLCK and CaD The inhibitory effect of MLCK is brought about by its action as a cytoskeletal protein, not as an enzyme. The inhibition produced by MLCK has features in common with those of CaD (see above), a typical cytoskeletal protein of smooth muscle. Therefore, we compared the inhibitory strength of MLCK with that of CaD.35 As shown in Figure 2.6, the concentration of CaD that abolishes movement was 88 times that of MLCK on a molar basis. We obtained myofibrils of chicken gizzard as cytoskeleton of smooth muscle, and subjected them to SDS PAGE (Table 2.5). Densitometry indicated that the amount of CaD was only 7.1 times that of MLCK on a molar basis (mean value of four experiments). This amount is 12.4 times smaller than that producing the same inhibitory effect, suggesting physiological relevance in terms of the concentrations. A similar argument is possible for CaP. The inhibitory strength of CaP in myofibrils is much smaller than that of CaD (Figure 2.6), which also provides an indication for the physiological relevance of MLCK.


23 Fig. 2.5. Increase in actin filaments relieves the inhibition imposed by MLCK. 27 Myosin-coated surface assay was performed in the presence (open circles) and absence (filled circles) of MLCK. The concentration of fluorescent actin filaments was fixed at 3 nM, to which unlabeled actin filaments were added.

Effect of Calmodulin Inhibitor Calmodulin inhibitors relax smooth muscle that has been contracted by various methods.38-40 Movement of actin filaments on a myosin-coated surface in the presence of MLCK and Ca/CaM was abolished by the calmodulin inhibitor trifluoperazine (Fig. 2.8A). The concentration giving half-maximal inhibition was in the micromolar range, reflecting the concentration at which trifluoperazine relaxes smooth muscle. This also presents evidence for the physiological relevance of the actin-linked effect of MLCK. It must be noted that MLCK may not be the sole site of action for Ca/CaM. CaD and CaP have similar actin-linked inhibitory effects on the actin-myosin interaction as described above, and Ca/CaM relieves this inhibition.29,30 However, in the preceding section, we discussed evidence that the actin-linked inhibition of contraction is totally attributable to MLCK. Therefore, the action of Ca/CaM will be on MLCK, providing a rationale for relating the effect of trifluoperazine to MLCK in smooth muscle.

Effect of Ca2+

Reasonable concentrations are also evident for Ca2+, as shown in Figure 2.8B. We allowed actin filaments to move on a myosin-coated surface in the presence of MLCK, CaM and various concentrations of Ca2+ .36 The Ca2+ concentration that allowed half-maximal velocity of movement was in the micromolar range, and was similar to that required to activate smooth muscle contraction.41 The similarity supports the physiological relevance of the inhibitory effects of MLCK.

Myosin-Binding Properties of MLCK Studies with Recombinant Myosin-Binding Fragments As shown in Figure 2.1, MLCK is composed of an N-terminal actin-binding domain, a central kinase domain and a C-terminal telokin domain, which is the same as the gene product of the 816-972 sequence of MLCK.17 The portion between the kinase and telokin domains has been characterized as the regulatory domain for kinase activity.16 Using cDNA of MLCK,19 we expressed the telokin domain, Met816-Glu972, in E coli. The recombinant 816-972 fragment bound to myosin, but exerted no effect on the

24


Table 2.4. Regulatory proteins in smooth muscle Myosin light chain kinase (MLCK) Caldesmon (CaD) Calponin (CaP) Common properties: 1. Calmodulin-binding 2. Actin-binding 3. Myosin-binding MLCK, CaD, and CaP form a family due to their common properties. NB Tropomyosin, although present in smooth muscle, does not belong to this family.

actin-myosin interaction. A construct with Asp777-Gly972, which contains a regulatory domain in its N-terminal, stimulated the interaction slightly, as shown by the filled circles in Figure 2.9B. It must be noted that the myosin used for the assay is smooth muscle myosin in a phosphorylated form. When both fragments were tested using unphosphorylated forms, the 777-972 fragment stimulated the activity by 7-8 fold (open circles in Fig. 2.9B).*

Different Effects of Parent MLCK on the Actin-Myosin Interaction According to the Phosphorylated State of Myosin42 In the above discussion, the effect of parent MLCK on the actin-myosin interaction is inhibitory (Figs. 2.3-2.6), where the smooth muscle myosin used for the assay is exclusively in the phosphorylated form.** As shown by the filled circles in Figure 2.9C, MLCK inhibited the actin-activated ATPase activity of phosphorylated smooth muscle myosin. However, with unphosphorylated myosin, the effect of MLCK was stimulatory (Fig. 2.9C, open circles). Because the effect was assayed in the absence of Ca/CaM, the stimulation is hardly attributable to the kinase activity of MLCK. Taken together with the stimulatory effect of the myosinbinding 777-972 fragment, the stimulation of ATPase activity (Fig 2.9) is attributable to the myosin-binding properties of MLCK.

*The effect of the N-fragment on the actin activated ATPase activity of myosin is also different, depending on whether we use phosphorylated myosin or unphosphorylated myosin.42 (Fig. 2.9A). Unlike the myosin-binding 777-972 fragment, the inhibitory effect of the N-fragment was observed with phosphorylated, but not with unphosphorylated myosin. **We can measure the actin-activated ATPase activity of myosin irrespective of whether or not smooth muscle myosin is phosphorylated. However, the myosin-coated surface assay is only possible using phosphorylated smooth muscle myosin, because unphosphorylated myosin does not support motility of actin filaments. Nonetheless, the surface assay is superior to the ATPase for detecting the effect of MLCK and its fragment in the following respects:43 1. The assay detects mechanical aspects of the actin myosin interaction in real time; 43 2. The assay is not complicated by the kinase activity of MLCK, because the motility is not altered once myosin is phosphorylated (Fig. 4 in ref. 26); 3. We ruled out the actin-bundling activity of MLCK and its fragments by ignoring the motility of assembled actin filaments and observing only isolated actin filaments; 4. Similarly, the assay is not complicated by the assembly/disassembly of myosin, if any.


25

Fig. 2.6. Strength of the inhibitory effect of MLCK compared to those of CaD and CaP (see Table 2.5).35 Myosin-coated surface assays were carried out in the presence of various concentrations of MLCK (open circles), CaD (closed circles), and CaP (triangles). The velocity of movement of actin filaments on the surface was plotted against the molar ratio relative to CaD.

The stimulatory effect of MLCK is notably in accordance with the superprecipitationinducing activity of bovine stomach MLCK detected by Ebashi and his colleagues, because they use unphosphorylated myosin for the assay.45 They attributed the activity to its actinbinding property. However, our data relate the activity to its myosin-binding property. Another difference from their data is that Ca/CaM was not required for the activity.

Stimulatory Effect of CaD and CaP Under the specified conditions, we detected a stimulatory effect of CaD and CaP on the actin-myosin interaction23,46-50 (Fig. 2.6). Although the effect is not very strong, we can relate it to their myosin binding property (Refs. 36, 43 for CaD, ref. 50 for CaP). This stimulation has been assayed with phosphorylated smooth muscle myosin. In light of the similarity of MLCK to CaD and CaP (Table 2.4), their effects must be reexamined using unphosphorylated myosin.

Conclusion When myosin is in the unphosphorylated form, MLCK is bound to myosin at its myosin-binding domain to stimulate the actin-myosin interaction. However, MLCK barely binds to myosin once it is phosphorylated by the kinase domain.10 The effect of MLCK on the interaction turns from stimulation to inhibition. As described above, inhibition is exerted by the actin binding domain. Such a scheme among the three domains has been indicated only by in vitro experiments, although physiological relevance has been discussed

26


Fig. 2.7. Binding of MLCK to actin filaments in the presence of CaD or CaP.36 (Left panel) Effect of CaD. MLCK was mixed with actin filaments in the presence of various concentrations of CaD and subjected to centrifugation. The precipitated amounts of MLCK relative to actin were plotted against the concentration of CaD. (Right panel) Effect of CaP. Similarly to the left panel, MLCK was bound to actin filaments in the presence of various concentrations of CaP. (Bottom panel) Comparison of amino acid sequence among MLCK,16 CaD, 29,30 and CaP33,37 from chicken gizzard. Numbering of amino acids is from the N-terminal.

for the kinase domain,1 the actin-binding domain, and the myosin-binding domain, especially in terms of MLCK concentration in smooth muscle cells*. To obtain in vivo data, we expressed the N-terminal, actin-binding domain of MLCK in CHO cells in a transient manner. Cultures of CHO cells transfected by a plasmid coding this domain often contained polynuclear cells, suggesting that actin-myosin interaction at the contractile ring during cell division was affected by the overexpressed actin-binding domain (Fig. 2.10). The alternative approach is to abolish the expression of MLCK in smooth muscle cells. Recently, we transfected a plasmid containing antisense DNA for MLCK mRNA to SM3, a cell line derived from rabbit aorta,51 and screened for a stable transformant. The lack of expression of the MLCK molecule was examined by both Northern and Western blots. We found that the transformant only minimally developed a ruffling membrane when induced by platelet-derived growth factor.52 Because the ruffling membrane is the site of the actin-myosin interaction to support cell migration, an experiment is now planned to see which of the actin binding domain, kinase domain, or myosin-binding domain will rescue the ruffling when the respective gene fragments are electroporated into the transformed cell. This rescue experiment will enable us to investigate the physiological importance of these three fragments.

*The concentration of MLCK in gizzard smooth muscle cells has been estimated as 4 µM.44 The stimulatory effect of MLCK and its fragments is detectable at µM levels (Fig 2.9), suggesting physiological relevance in terms of concentration. However, physiological studies are required, as described in the final section of this chapter.


27

Table 2.5. Amount of MLCK and CaD present in chicken gizzard myofibrils35 MLCK: CaD (molar ratio) 1.0 : 6.5 1.0 : 6.6 1.0 : 5.7 1.0 : 9.8 1.0 : 7.15 ± 1.57 (mean ± SEM , n=4) MLCK, myosin light chain kinase; CaD, caldesmon Myofibril was prepared from chicken gizzard and subjected to SDS PAGE. The amount of MLCK relative to that of CaD was determined by the densitometry.

References 1. Ebashi S, Iwakura H, Nakajima H et al. New structural proteins from dog heart and chicken gizzard. Biochem Z 1966; 345:201-211. 2. Kamm KE, Stull JT. The function of myosin and myosin light chain kinase phosphorylation in smooth muscle. Annu Rev Pharmacol Toxicol 1985; 25:583-620. 3. Kohama K, Saida K. Ed. Smooth muscle contraction: new regulatory modes. Basel:Karger, 1995:1-159. 4. Oishi K, Takano-Ohmuro H, Minakawa-Matsuo N et al. Oxytocin contracts rat uterine smooth muscle in Ca2+-free medium without any phosphorylation of myosin light chain. Biochem Biophys Res Commun 1991; 176:122-128. 5. Sato K, Hori M, Ozaki H. Myosin phosphorylation-independent contraction induced by phorbol ester in vascular smooth muscle. J Pharmacol Exp Ther 1992; 261:497-505. 6. Kohama K, Hiranuma T, Takano-Ohmuro H et al. Effects of NAO344, a new smooth muscle relaxant, on the actin-myosin-ATP interaction and myosin light chain phosphorylation in vitro. Gen Pharmac 1991; 22:465-474. 7. Sezaki M, Sasaki T, Tanaka T et al. A new antibiotic SF-2370 produced by actin omadura. J Antibiot 1985; 38:1437-1439. 8. Guerriero VJr, Rowley DR, Means AR. Production and characterization of an antibody to myosin light chain kinase and intracellular localization of the enzyme. Cell 1981; 27:447-458. 9. De Lanerolle P, Adelstein RS, Feramisco JR et al. Characterization of antibodies to smooth muscle myosin kinase and their use in localizing myosin kinase in nonmuscle cells. Proc Natl Acad Sci USA 1981; 78:4738-4742. 10. Sellers JR, Pato MD. The binding of smooth muscle myosin light chain kinase and phosphatase to actin and myosin. J Biol Chem 1984; 259:7740-7746. 11. Yamazaki K, Ito K, Sobue K et al. Purification of caldesmon and myosin light chain (MLC) kinase from arterial smooth muscle: Comparisons with gizzard caldesmon and MLC kinase. J Biochem 1987; 101:1-9. 12. Kohama K, Okagaki T, Hayakawa K et al. A novel regulatory effect of myosin light chain kinase from smooth muscle on the ATP-dependent interaction between actin and myosin. Biochem Biophys Res Commun 1992; 184:1204-1211. 13. Ye L-H, Hayakawa K, Kishi H et al. The structure and function of the actin-binding domain of myosin light chain kinase of smooth muscle. J Biol Chem 1997; 272:32182-32189. 14. Hayakawa K, Okagaki T, Higashi-Fujime S et al. Bundling of actin filaments by myosin light chain kinase from smooth muscle. Biochem Biophys Res Commun 1994; 199:786-791. 15. Sobieszek A, Strobl A, Ortner B et al. Ca2+-calmodulin-dependent modification of smoothmuscle myosin light-chain kinase leading to its cooperative activation by calmodulin. Biochem J 1993; 295:405-411.


Velocity (µm/sec)

Velocity (µm/sec)

28

Trifluoperazine (µM)

pCa2+

Fig. 2.8. Effect of trifluoperazine and Ca2+. 36 (A) Myosin-coated surface assay was carried out in the presence of MLCK, Ca2+, CaM and various concentrations of trifluoperazine, a CaM inhibitor. (B) A similar assay was carried out in the presence of MLCK, CaM and various concentrations of Ca2+. pCa2+ = log[Ca2+]M. 16. Olson NJ, Person RB, Needleman DS et al. Regulatory and structural motifs of chicken gizzard myosin light chain kinase. Proc Natl Acad Sci USA 1990; 87:2284-2288. 17. Shirinsky VP, Vorotnikov AV, Birukov KG et al. A kinase-related protein stabilizes unphosphorylated smooth muscle myosin minifilaments in the presence of ATP. J Biol Chem 1993; 268:16578-16583. 18. Kanoh S, Ito M, Niwa E et al. Actin-binding peptide from smooth muscle myosin light chain kinase. Biochemistry 1993; 32:8902-8907. 19. Kobayashi H, Inoue A, Mikawa T et al. Isolation of cDNA for bovine stomach 155 kDa protein exhibiting myosin light chain kinase activity. J Biochem 1992; 112:786-791. 20. Shimmen T, Yano Y. Active sliding movement of latex beads coated with skeletal muscle myosin on Chara actin bundles. Protoplasma 1984; 121:132-137. 21. Morgan M, Perry SV, Ottaway J. Myosin light-chain phosphatase. Biochem J 1976; 157:687-697. 22. Adelstein RS, Klee CB. Purification and characterization of smooth muscle myosin light chain kinases. J Biol Chem 1981; 256:7501-7509. 23. Lin Y, Ishikawa R, Kohama K. Role of myosin in the stimulatory effect of caldesmon on the interaction between actin, myosin and ATP. J Biochem 1993; 114:279-283. 24. Ye L-H, Hayakawa K, Lin Y et al. The regulatory role of myosin light chain kinase as an actin-binding protein. J Biochem 1994; 116:1377-1382. 25. Warrick HM, Spudich JA. Myosin structure and function in cell motility. Annu Rev Cell Biol 1987; 3:379-421. 26. Okagaki T, Higashi-Fujime S, Ishikawa R et al. In vitro movement of actin filaments on gizzard smooth muscle myosin: Requirement of phosphorylation of myosin light chain and effects of tropomyosin and caldesmon. J Biochem 1991; 109:858-866. 27. Sato M, Ye L-H, Kohama K. Myosin light chain kinase from vascular smooth muscle inhibits the ATP-dependent interaction between actin and myosin by binding to actin. J Biochem 1995; 118:1-3. 28. Fujita K, Ye L-H, Sato M et al. Myosin light chain kinase from skeletal muscle regulates an ATP-dependent interaction between actin and myosin by binding to actin. Mol Cell Biochem 1998; 190:85-90.


29

Fig. 2.9. Distinct effects of MLCK and its fragments according to the state of myosin phosphorylation.42 The actin-activated ATPase activity of smooth muscle myosin in both phosphorylated (filled circles) and unphosphorylated (open circles) forms was measured in various concentrations of actin-binding N-fragment (A), myosin binding 777-972 fragment (B) and parent MLCK (C). For the constructs of the N-fragment and 777-972 fragment, see Figures 2.2 and 2.1, respectively. 29. Sobue K, Sellers JR. Caldesmon, a novel regulator protein in smooth muscle and nonmuscle actomyosin system. J Biol Cehm 1991; 266:12115-12118. 30. Marston SB, Redwood CS. The molecular anatomy of caldesmon. Biochem J 1991; 279:1-16. 31. Abe M, Takahashi K, Hiwada K. Effect of calponin on actin-activated myosin ATPase activity. J Biochem 1990; 108:835-838. 32. Winder SJ, Walsh MP. Smooth muscle calponin: Inhibition of actomyosin MgATPase and regulation by phosphorylation. J Biol Chem 1990; 265:10148-10155. 33. Takahashi K, Nadal-Ginard B. Molecular cloning and sequence analysis of smooth muscle calponin. J Biol Chem 1991; 266:13284-13288. 34. Shirinsky VP, Biryukov KG, Hettasch JM et al. Inhibition of the relative movement of actin and myosin by caldesmon and calponin. J Biol Chem 1992; 267:1588615892. 35. Kohama K, Ye L-H, Hayakawa K et al. Myosin light chain kinase: An actin-binding protein that regulates an ATP-dependent interaction with myosin. Trend Pharmacol Sci 1996; 17:284-287. 36. Ye L-H, Hayakawa K, Okagaki T et al. Actin-binding property of myosin light chain kinase and its role in regulating actin-myosin interaction of smooth muscle. In: Nakano, T., Hartshorne, D.J. Ed. Tokyo: Springer-Verlag, 1995:159-173. 37. Mezgueldi M, Fattoum A, Derancourt J et al. Mapping of the functional domains in the amino-terminal region calponin. J Biol Chem 1992; 267:15943-15951. 38. Van Belle H. The effect of drugs on calmodulin and its interaction with phosphodiesterase. Adv Cyclic Nucleotide Protein Phosphorylation Res 1984; 17:557-567. 39. Kreye VAW, Ruegg JC, Hofmann F. Effect of Calcium-antagonist and calmodulin-antagonist drugs on calmodulin dependent contractions of chemically skinned vascular smooth muscle. Naunyn Schimiedebergs Arch Pharmacol 983; 323:85-89. 40. Asano M. Effects of the calmodulin antagonist W-7 on isometric tension development and myosin light chain phosphorylation in bovine tracheal smooth muscle. Jpn J Pharmacol 1990; 52:471-481. 41. Saida K. Ca2+ sensitization of smooth muscle in relation to small GTP-binding protein. In: Kohama K, Saida K, Ed. Smooth muscle contraction: New regulatory modes. Basel: Karger, 1995:103-112. 42. Ye L-H, Kishi H, Nakamura A et al. Domain structure of myosin light chain kinase (MLCK) and the cross-talk among the domains. Jpn J Pharmacol 1997; 73:Supple. I:282P.

30


Fig. 2.10. Expression of N-terminal MLCK in CHO cells. To the pMIKNeo vector, which was kindly donated by Dr. K. Maruyama at Tokyo Medical and Dental University, we inserted the cDNA fragment encoding Met1-Val708 of bovine stomach MLCK, 19 together with its 5'-untranslated region. The obtained construct was transfected to CHO cells transiently. The transfected cells were cultured in the presence of neomycin. Multinuclear cells were identified by phase contrast microscopy (Phase Contrast) and fluorescent microscopy after nuclear staining with diamidinophenylindole (DAPI stain). 43. Okagaki T, Kohama K. Characterization of myosin light chain kinase as an actin-binding protein that regulates the ATP dependent interaction between actin and myosin of smooth muscle. In: Kohama K, Saida K, Ed. Smooth muscle contraction, new regulatory modes. Basel:Karger, 1995:17-35. 44. Erdodi F, Ito M, Hartshorne DJ. Myosin light chain phosphatase. In: Barany M, Eds. Biochemistry of smooth muscle contraction. San Diedo: Academic Press, 1996:131-142. 45. Kuwayama H, Suzuki M, Koga R et al. Preparation of protein components exhibiting myosin light chain kinase activities from bovine aorta: Discrepancies between its enzyme activity and actomyosin activating effect. J Biochem 1988; 104:862-866. 46. Ishikawa R, Okagaki T, Higashi-Fujime S et al. Stimulation of the interaction between actin and myosin by Physarum caldesmon-like protein and smooth muscle caldesmon. J Biol Chem 1991; 266:21784-21790. 47. Ishikawa R, Okagaki T, Kohama K. Regulation by Ca2+- calmodulin of the actin-binding activity of Physarum 210-kDa protein. J Muscle Res Cell Motil 1992; 13:321-328. 48. Haeberle JR, Trybus KM, Hemric ME et al. The effects of smooth muscle caldesmon on actin filament motility. J Biol Chem 1992; 267:23001-23006. 49. Lin Y, Ishikawa R, Okagaki T et al. Stimulation of the ATP-dependent interaction between actin and myosin by a myosin-binding fragment of smooth muscle caldesmon. Cell Motil Cytoskeleton 1994; 29:250-258. 50. Lin Y, Ye L-H, Ishikawa R et al. Stimulatory effect of calponin on myosin ATPase activity. J Biochem 1993; 113:643-645. 51. Sasaki Y, Uchida T, Sasaki Y. A variant derived from rabbit aortic smooth muscle: Phenotype modulation and restoration of smooth muscle characteristics in cell in culture. J Biochem 1989; 106:1009-1018. 52. Kishi H, Mikawa T, Seto M et al. Stable transformants of smooth muscle cell line lacking myosin light chain kinase, and their characterization with respect to actomyosin system. Jpn J Pharmacol 1998:76 Supple. I:303P.

CHAPTER 3

Structure and Regulatory Mechanisms of Myosin Phosphatase Kazuhito Ichikawa, Masaaki Ito, Takeshi Nakano and David Harshorne

I

t is now evident that reversible phosphorylation of the 20 kDa light chain of myosin is an important regulatory mechanism in controlling the contractile activity of smooth muscle.1,2 The phosphorylation state of myosin is determined by two enzymes; the Ca2+-calmodulindependent myosin light chain kinase (MLCK) and myosin phosphatase (MP). The activity of MLCK is regulated by intracellular Ca2+ and the formation of the Ca2+-calmodulin complex. Phosphorylation of myosin by MLCK activates the actin-dependent ATPase activity of myosin and initiates smooth muscle contraction. Relaxation generally follows dephosphorylation of myosin and thus is mediated by MP. At present MLCK has been well characterized. Several cDNAs have been isolated and cloned and some of the functional regions of the molecule have been identified.3 Less is known about MP, and it is only recently that information regarding structure/function relationships of the MP holoenzyme have become available. MP is a serine/threonine phosphatase. Serine/threonine phosphatases are divided into two groups; protein phosphatase 1 (PP1) and 2 (PP2) on the basis of their biochemical properties.4,5 PP2 was further divided into three subgroups, 2A, 2B, and 2C, based on metal dependence.6-8 PP1 enzymes preferentially dephosphorylate the β subunit of phosphorylase kinase, are sensitive to inhibitors 1 and 2, and are inhibited by higher concentrations of okadaic acid (OA). The PP2 enzymes preferentially dephosphorylate the α subunit of phosphorylase kinase, are insensitive to both protein inhibitors, and are inhibited by low concentrations of OA. In addition to these two classes of phosphatases, many other phosphatases which may not be classified according to the above scheme have recently been discovered.9 Previously it was assumed that the activity of MP is constant and that a fixed relationship should exist between the Ca 2+ concentration, myosin phosphorylation, and force. However, such was not observed in many studies carried out with intact or skinned smooth muscle preparations.10 The increased application of fluorescent dyes to measure internal Ca2+ concentration showed that the Ca2+ dependence of force could vary under different conditions. Usually, higher force was achieved following agonist stimulation, compared with K+ depolarization.11-13 Subsequently, it was shown that the changes in force reflected parallel changes in myosin phosphorylation, and thus the balance of MLCK and phosphatase was altered.14,15 Inhibition of phosphatase activity or activation of MLCK could account for this phenomenon. This would increase myosin phosphorylation at a given Ca2+ concentration and lead to increased Ca2+ sensitivity (Ca 2+ sensitization). It has been reported that Molecular Mechanisms of Smooth Muscle Contraction, edited by Kazuhiro Kohama and Yasuharu Sasaki. ©1999 R.G. Landes Company.

32

Molecular Mechanisims of Smooth Muscle Contraction

guanosine-5’-O-(3-thio-triphosphate) (GTPγS) increased the Ca2+ sensitivity of contraction, thus implicating a G protein-linked mechanism.13,16 Kitazawa et al15 demonstrated that this mechanism resulted in inhibition of phosphatase activity. Kubota et al17 showed the same phenomenon in a homogenate of tracheal muscle. Thus, from the above evidence it was apparent that myosin phosphatase could be regulated and that this might be an important component in the contractile properties of smooth muscle, particularly with respect to its Ca2+ dependence. In this chapter the structure/function relationships of MP will be considered and the regulation of MP will be documented further.

Structural Components of Smooth Muscle Myosin Phosphatase Many phosphatases have been prepared from several smooth muscle sources and shown to dephosphorylate either isolated light chains or intact myosin.5 Also, under physiological conditions more than one phosphatase was implicated. In order to limit the number of phosphatases the following restrictions were used, i.e., that the phosphatase binds to myosin or the contractile apparatus and that the phosphatase is effective with intact phosphorylated myosin (P-myosin) as substrate. Several years ago it was found that smooth muscle MP is a type 1 enzyme based on sensitivity to OA.18 Recently, three laboratories obtained similar preparations from chicken gizzard19,20 and pig bladder.21 The MP holoenzyme was composed of 3 subunits: a catalytic subunit of about 38 kDa; a large subunit of about 110 kDa; and a subunit of 20 to 21 kDa (based on the calculated molecular mass). As the 110 kDa subunit of MP can bind to myosin, this subunit has been termed the myosin-binding subunit.20 This subunit also binds to the catalytic subunit and may interact with substrates other than myosin (Kaibuchi K, unpublished data). Thus, we will refer to the large non-catalytic subunit as the myosin phosphatase target subunit (MYPT). The small non-catalytic subunit is referred to as M20.

38 kDa Catalytic Subunit It has been reported that the PP1 catalytic subunit (PP1c) has at least 5 isoforms. Four cDNA clones, i.e., PP1α, γ1, γ2, δ were isolated from rat libraries22 and a fifth, PP1α2, from a human lymphocyte library.23 Sometimes the δ isoform is referred to as the β-isoform.19 These isoforms are thought to be products of three genes, with the isoforms produced by alternative splicing. Except for PP1cα2, the difference in primary structure is mainly in the C-terminal region, and the central 250 amino acids (residues 40 to 299) are very similar.5 The C-terminal differences allow the preparation of selective antibodies for PP1 isoforms. The 38 kDa subunit of MP was shown to be the δ isoform (PP1cδ) by Western blotting using isoform-specific antibodies.20 The PP1cδ from chicken gizzard has been sequenced.20 The deduced amino acid sequence was identical to that of rat PP1cδ.22 The physiological functions of the PP1 isoforms are of great interest, but remain unknown. Zhang et al24 reported the expression and characterization of PP1α, γ1, γ2 and δ and demonstrated that the four isoforms had similar enzymatic properties in terms of substrate specificity and sensitivity to okadaic acid and inhibitor-2. It is thought that the PP1c isoforms might form complexes with distinct regulatory subunits to generate different holoenzymes. However, all of the various recombinant isoforms were reported to be able to associate with both the G (glycogen) subunit and M (myosin) subunit of PP1 in vitro.25 Furthermore, a recent immunofluorescent study showed that the PP1cα isoform colocalized with the 130 kDa phosphatase subunit in smooth muscle cells and fibroblasts.26 Thus the relationship between the PP1c isoforms and different types of regulatory subunits is obscure, and further studies are required to establish these interactions in different cell types. An unusual feature of MP is its activation by Co2+ and Mn2+ ions (Mn2+ ions are less effective). Most of the previous studies with the isolated catalytic subunit show that there is

Structure and Regulatory Mechanisms of Myosin Phosphatase

33

Fig. 3.1. Structure of human MYPT.

an active form that is independent of metal ions, and also metal ion dependent forms of PP1. All of the recombinant forms of PP1c are dependent on Mn2+.27 The Co2+ response is irreversible, temperature dependent and slow.28 These factors may reflect an unusual conformational change in PP1c or chemical modification of residue(s) associated with catalysis. Oxidation of Cys127 has been suggested as one possibility.29 Chu et al27 showed that the activation of PP1c by Co2+ is associated with a stoichiometrical incorporation of Co2+ into the recombinant PP1c. It was also shown that treatment of PP1c with a combination of Fe2+ and Zn2+ (but not the individual metal ions) significantly activated PP1c. These results suggest that at least two metal binding sites exist on the enzyme and that PP1c may be an iron/zinc metalloprotein in vivo. Consistent with this are the crystal structures for rabbit muscle PP1c complexed with microcystin-LR and for the human PP1c in complex with tungstate.29,30 These structural studies show that the catalytic subunit forms a compact ellipsoidal structure and the two metal ions are positioned by a central β-α-β-α scaffold at the active site, from which emanates three surface grooves that are potential binding sites for substrates and inhibitors. The catalytic site is situated at the C-terminal side of the two central β-sheets and it is here that the two metal ions, which bridge the two β-sheets, are coordinated by His, Asp, and Asn residues. The C-terminal part of PP1c seems to play a regulatory role in catalytic activity as demonstrated by proteolysis31 and phosphorylation studies.32,33 Cyclin-dependent protein kinase (cdk) phosphorylates a consensus sequence present in the C-terminus of all PP1c isoforms and inhibits their activity.33 Recently, it was also found that phosphorylation of PP1cα by PKC is involved in the regulation of signal transduction in response to the stimulation of B-cells through a cell surface IgM.34 However, it is not known whether the phosphorylation of PP1c occurs during normal smooth muscle function.

Regulatory Subunits An important question is: How can so few phosphatase catalytic subunits cope with the wealth of kinase reactions? One intriguing possibility is that non-catalytic subunits can

34


provide a specific targeting function by which the substrate and catalytic subunit are colocalized. Such proteins are called targeting subunits,7,35 and these bind to the catalytic subunit and substrate. This may determine subcellular localization, may also determine substrate specificity, and could regulate catalytic activity of the phosphatase. The classical example is the G subunit involved in glycogen metabolism.35 Targeting subunits have also been implicated for smooth muscle MP. The two non-catalytic subunits, i.e., MYPT and M20, may act as targeting subunits to colocalize the substrate, myosin, and PP1c. In support of this, it was shown that addition of MYPT and M20 to PP1c increased phosphatase activity toward phosphorylated 20 kDa light chain of myosin (P-MLC20) and phosphorylated heavy meromyosin (P-HMM), which is a substitute for myosin that is insoluble at low ionic strength.19 Similar findings were reported by Shirazi et al,21 who in addition found that the trimeric phosphatase was more effective in relaxing permeabilized smooth muscle preparations than the isolated PP1c. Tissue Distribution Using a monoclonal antibody to the N-terminal 58 kDa fragment of chicken gizzard MYPT, the distribution of MYPT in chicken tissues was investigated.36 MYPT was found in all smooth muscles analyzed (gizzard, aorta, stomach, small intestine, and oviduct) but also was present in other tissues, including brain, spleen, cardiac muscle, kidney and lung (see below). It was not detected in skeletal muscle and liver. In gizzard, a major band of a 130 kDa and a minor band of 133 kDa were detected. Two bands of 137 and 125 kDa were found in oviduct and small intestine. In the stomach, a 137 kDa protein and an additional 58 kDa protein were observed. The presence of these distinct immunoreactive components suggests the presence of different isoforms of this subunit in various tissues. Northern blots confirmed these data, and, using a labeled cDNA probe, an mRNA of 5.7 kilobases was detected in many tissues, but not in liver or skeletal muscle.20 Our preliminary results show that a similar distribution was observed in human and rat tissues. The distribution of the 20 kDa subunit is not established. Our preliminary results show that this subunit is predominant in smooth muscle. General Structure of MYPT The structure of human MYPT is shown schematically in Figure 3.1. MYPT contains 1030 amino acid residues with a calculated molecular mass of approximately 115 kDa. The N-terminal region of the molecule contains the characteristic sequence motif called the cdc10/SW16 or ankyrin repeat. In human MYPT, this consists of seven repeat sequences between residues 39 to 296. The region 171 to 197 was not considered as an ankyrin repeat since it is less homologous. Each of seven repeats contains 33 residues, and twenty of the 33 positions are conserved. The ankyrin motif is present in modified forms in many proteins and links several integral membrane proteins and the spectrin-based membrane cytoskeleton. The anion exchanger, Na-K-ATPase, a voltage-dependent Na+ channel, a ryanodine receptor and an adhesion molecule have been reported to interact with ankyrin.37 One possibility is that the ankyrin repeats may provide a platform for interaction with myosin and/or substrate(s) other than P-myosin. The central region (Ala653 to Ile711) in human MYPT is conserved among several isoforms, as described in the next section. This is of particular interest since this region includes the sequence around the putative inhibitory site. Phosphorylation of this site (Thr695) by an endogenous kinase in MP holoenzyme preparations (from chicken or turkey gizzard) inhibited phosphatase activity.38 In the human MYPT, this site is Thr696. Recently, it was found that an N-terminal peptide preceding the ankyrin repeats was required for interactions with PP1c.39,40 This is based on evidence that a similar sequence is


35

Fig. 3.2. Relationship of the MYPT isoforms.

found in many PP1c bindinG proteins, and the sequence R/K-V/I-X-F was proposed as a common PP1c binding motif.41 In human MYPT, this motif corresponds to K-V-K-F for residues 35 to 38. It is thought that the 4 residue motif is important in the recognition of the target molecule. Recently, Zhao and Lee42 proposed a modification of the 4 residue sequence and suggested that binding to PP1c required the conserved motif Val-X-Phe or Val-X-Trp, where X was frequently His or Arg. In addition, it was found that this sequence was commonly preceded by 2-5 basic residues and followed by an acidic residue. The extreme C-terminal region of the molecule contains the leucine zipper motifs. These are characterized by a leucine residue at every 7th position and are relatively common among DNA bindinG proteins. The C-terminal part of MYPT is homologous to the chicken gizzard M20, with the similarity being particularly striking in the C-terminal two-thirds of the M20 subunit, which terminates in the leucine zipper motifs. The leucine zippers are thought to form dimers, and it is possible that these structures in the M20 and MYPT subunits are either involved in mediating interactions between the two subunits or in binding to other partners which also contain this motif. Other features of the human MYPT molecule include an acidic cluster (326-372); two ionic clusters (719-755 and 814-848); and a Ser/Thr-rich sequence (770-793). Rotary shadowing electron microscopy recently revealed an elongated structure for MYPT, with three globular domains connected by flexible strands (using a full length MYPT expressed by the baculovirus system).43 Isoform Diversity of MYPT Several MYPT isoforms have recently been isolated by several groups. These include human MYPT,44 rat isoforms (M110 (rat1, 2, 3)45,46 and a partial N-terminal fragment from kidney47), and chicken gizzard (termed M133 and M13020). Furthermore, a gene was

36


identified in C. elegans that encoded a protein similar to MYPT, termed MEL-11.48 The structural relationships of the various isoforms are indicated in Figure 3.2. With the exception of MEL-11, these isoforms are quite similar and show >80% sequence identity. The human MYPT and rat3 isoforms are almost the same. However, some differences exist to distinguish the distinct isoforms. The differences in structure, and thus the main factors in generation of different isoforms, are due to central inserts and the presence or absence of the C-terminal leucine zipper motifs. Three isoforms, i.e., human MYPT, rat3 and chicken M133, have central insert regions which are not similar to each other. In rat aorta, rat1 and 3 differ by an insert in the central part of molecule, residues 553-608.46 In chicken gizzard M133 and M130, the insert region spans residues 512-552.20 The C-terminal leucine zipper motif is present in human and rat isoforms. Rat1 and 2 differ by the presence of the leucine zipper motif. The rat uterus isoforms (rat2) and chicken isoforms (M133/130) do not contain these repeats.20,46 It is suggested that the various MYPT isoforms are generated by splicing of the same primary RNA transcript, and thus it is speculated that another isoform(s) may exist. Some of the regions are more conserved than others; these include the conserved 4 residue repeat (R/K-V/I-X-F) at the N-terminal edge of the ankyrin repeats, the N-terminal ankyrin repeats, and a conserved central region including the putative inhibitory phosphorylation site, as described above. The MYPT of C. elegans, i.e., MEL-11, is less related to the vertebrate MYPT family members (total sequence identity is only 35%), but similar structural elements, i.e., the ankyrin repeats and the central region, are relatively conserved. For the latter, the short sequence peptide around Thr696 in human MYPT, i.e., 693RRSTQGVT701L, is identical in all other isoforms. In addition, MEL-11 contains leucine zipper motifs at its C-terminal end. It has been determined44 that the MYPT family,discussed above, originates from a gene located on chromosome 12 (12q15-q21.2). A second MYPT gene has also been localized to chromosome 1 (1q32.1).49 This second gene product is found in heart and brain. It is similar to the smooth muscle MYPT isoform (61% identical), although the N-terminal regions of the two classes of isoforms (i.e., preceding the ankrin repeats) are distinct. 20 kDa Regulatory Subunit The M20 subunit of MP has been cloned from chicken gizzard45 and comprises 186 amino acids with a leucine zipper domain at its C-terminus. The whole molecule is similar to the C-terminal part of the MYPT isoform. A second cDNA for M20 has been recently reported43 which contains a 53 bp difference insert and codes for a truncated version of the subunit (18.5 compared to 21.3 kDa). The smaller isoform would not contain the leucine zipper motif. The function of M20 has not been determined. The kinetic parameters of the earlier preparations of MP (a complex of an N-terminal fragment of MYPT and PP1c: 58 plus 38 kDa) and the trimeric MP holoenzyme with P-myosin and P-MLC20 were similar.28,50 It was also found that selective removal of M20 from the MP holoenzyme did not affect MP activity.40 Thus, it is assumed that M20 is not required for activation of phosphatase activity. Since M20 was recently shown to bind to myosin,46 it may serve an alternative function, such as targeting MP to actomyosin or other proteins, and thus determining cellular localization.

Interaction between Subunits To analyze the physiological function of MP and to understand its mechanism of action and regulation, the interactions among the three subunits, i.e., MYPT, M20 and PP1cδ, need to be documented. On the basis of the various findings described above, a tentative plan of the MP holoenzyme can be proposed as shown by the model in Figure 3.3. PP1cδ was found to bind to the N-terminal part of MYPT.39,50 One location for a PP1δ binding site


37

Fig. 3.3. A model of the structure of the myosin phosphatase holoenzyme and its interactions with substrate and other molecules

was shown to be the N-terminal segment preceding the ankyrin repeats, namely residues 1 to 38 of human MYPT.39 The N-terminal mutants lacking this sequence did not bind to PP1c. As discussed above, the 4 residue motif (35KVK38F in human MYPT) was proposed as a requirement for binding to PP1c. However, Hirano et al39 found using the two-hybrid system that the binding of PP1cδ to the N-terminal 1-38 sequence was weaker than for the longer N-terminal sequence including ankyrin repeats. They suggested, therefore, that at least two PP1c binding sites were present, a relatively strong site in the N-terminal 1-38 sequence and a second weaker site, possibly in the C-terminal part of the ankyrin repeats. Activation of phosphatase activity (towards P-myosin) also indicates the binding of PP1c to MYPT and substrate (as defined by the target subunit concept). N-terminal fragments of MYPT, such as N-terminal 75 kDa fragment47, the chicken MYPT sequences 1-633, 1-67450 and 1-374,39 increased phosphatase activity. Johnson et al reported that the N-terminal 1-38 peptide also activated PP1c, but high concentrations of peptide are needed for activation.40 Hirano et al showed that the same peptide had no effect on activation.39 These results suggest that the 1-38 peptide is not sufficient for full activation of MP activity and thus an additional site is required, probably in the ankyrin repeats. The M20 subunit interacted with the C-terminal half of MYPT (residues 514-963 of gizzard MYPT (M130)39 and the C-terminal 72 residues of rat M110 (residues 933-1004).45 This site corresponds to residues 934-1030 of human MYPT. These reports further indicate that the N-terminal part of M20 interacts with MYPT and that the leucine zipper motifs of M20 are not required. Also, it has been shown that M20 does not bind to PP1c.39

Interaction of MP with Substrate and Other Molecules From earlier studies it was suspected that MP bound to myosin, since phosphatase was a persistent contaminant of myosin preparations. Subsequently, it was demonstrated that MP bound to myosin and is not dissociated under physiological ionic conditions.28,51 Several procedures have added information on the interaction of the MP holoenzyme with myosin. Using a cosedimentation assay with myosin, it was found that most of the MP holoenzyme bound to myosin.20 In addition, the binding of PP1c was relatively weak.

38


Substrate affinity columns and competitive assays of phosphatase activity (with thiophosphorylated substrates) suggested the following:50 In the presence of ATP, and therefore under conditions approaching physiological, MP bound only with the phosphorylated substrate, while in the absence of ATP, MP bound to the dephosphorylated protein; also, it was suggested that the dominant recognition site for binding of MP to myosin was the phosphorylated light chain,21,50 although binding to other sites cannot be eliminated; results from affinity columns clearly indicated an independent binding site on MYPT for P-myosin, also.50 Hirano et al reported that the substrate binding site in MYPT was in the ankyrin repeats, probably the C-terminal repeats 5 to 8, using an overlay assay with thiophosphorylated biotinylated MLC20.39 However, cosedimentation assays with myosin showed that proteolyzed MP (58 plus 38 kDa complex) also bound to myosin; its affinity appeared weaker compared to the intact holoenzyme. It is known that the 58 kDa fragment represents the N-terminal part of MYPT.20,28 An additional site (not present in the 58 kDa fragment) may therefore be implicated. Consistent with this idea, Johnson et al found that C-terminal mutants of MYPT (714-933) cosedimented with myosin46 (see Fig. 3.3). These authors also found that M20 bound to myosin dimers, and many of the binding sites were located in the myosin rod domain. If MP is attached to myosin and does not relocate during the contractile cycle, then the stoichiometry of phosphatase relative to myosin heads becomes an important issue. The concentration of myosin heads, about 50 µM,1 is considered higher than the concentration of MP, 1-2 µM.19 Thus, MP may be highly mobile within the contractile apparatus and move rapidly from one myosin molecule to another. The pertinent constants relating to the binding of MP to myosin have not been calculated. The discrimination of MP binding to P-myosin represents a simple regulatory mechanism in which the phosphatase would be recruited only by the phosphorylated cross bridges. Once the myosin is dephosphorylated, the MP would become available for further reactions. This mechanism would eliminate the problem of sequestration of phosphatase by dephosphorylated myosin. Also, it is suggested that the presence of multiple binding sites on myosin for the M20 subunit (and perhaps for the MYPT subunit as well) allows MP to slide rapidly from one myosin molecule to another. However, there are many points to be resolved about this scheme. It has been recently reported that the MYPT molecule can interact with other proteins and lipids. These include RhoA,52 acidic phospholipids53 and arachidonic acid54 (Fig. 3.3). Kimura et al52 reported that activated RhoA (RhoAV14) (substitution of glycine by valine) interacted with the C-terminal part of rat MYPT (699-976) using the yeast two-hybrid screening. Our recent data further define the binding site, namely to residues 930-1030 of human MYPT. But RhoA itself does not modulate the activity of MP. Ito et al showed the interaction of MP with various membrane components, i.e., the acidic phospholipids, phosphatydylserine, phosphatidylinositol and phosphatidic acid.53 Neutral phospholipids did not bind. The binding of acidic phospholipids inhibited phosphatase activity. The interaction appeared to be electrostatic. The relevant phospholipid binding site is within the C-terminal sequence, Ser668-Lys1030 of the human isoform.53 It was further shown that MYPT and M20 were phosphorylated by PKA and that phosphorylation of the MP holoenzyme decreased phospholipid binding with a recovery of phosphatase activity. These results support the idea that MP may interact with membranes and that phosphorylation by PKA could modify this interaction. Also, it is known that arachidonic acid interacts with the C-terminal part of rat MYPT.54 (see below)

Regulatory Mechanisms of MP Activity

The possible pathways from agonist stimulation to Ca2+ sensitization of smooth muscle are shown in Figure 3.4, and include monomeric G protein, arachidonic acid and protein


39

Fig. 3.4. Possible pathway to Ca2+ sensitization.

kinase (PKC) cascades. These pathways are not mutually exclusive and may be interactive (cross-talk) and may contain redundant components. A considerable body of evidence has suggested that the agonist-induced Ca2+ sensitization mechanism is associated with inhibition of smooth muscle MP. The pathways generated by agonist binding at the membrane and proceeding to the contractile apparatus are complex, but appear to converge at the level of the myosin phosphatase. This realization has prompted considerable research in this area. One hypothesis about the mechanism of MP inhibition is that it occurs via phosphorylation of the MYPT subunit. This is based on the evidence of Trinkle-Mulcahy et al,55 who found that treatment of α-toxin-permeabilized portal vein with ATPγS resulted in increased Ca2+ sensitivity of force output and reduced phosphatase activity. This was achieved under conditions where MLC20 thiophosphorylation by MLCK was minimum. During the Ca2+ sensitization, several high molecular weight proteins were thiophosphorylated; among these was the large regulatory subunit of MP. Thus, it was suggested that phosphorylation of the MYPT subunit of MP may be a regulatory mechanism for phosphatase activity. Subsequently, it was suggested that the phosphorylation-induced inhibition of MP could be caused by at least two kinases. One is the endogenous kinase of the MP preparation, which phosphorylated the holoenzyme, primarily on the MYPT subunit and resulted in inhibition of phosphatase activity.38 The major site of phosphorylation was Thr654 and Thr695 of the M130 and M133 isoforms, respectively. It appeared that phosphorylation did not dissociate the holoenzyme. The associated kinase was activated by arachidonic acid and oleic acid. This protein kinase was not identified. The other possibility is the Rho-associated kinase

40


(Rho-kinase) which also phosphorylated the MYPT subunit and inactivated the MP holoenzyme in vitro.52 Several isoforms of Rho-associated kinase were isolated and called either ROKα,56 Rho-kinase57 or p160 ROCK.58 These kinases show significant homology in the N-terminus to myotonic dystrophy kinase.56-58 One signaling pathway which leads to Ca2+-sensitization involves the monomeric G protein RhoA.59-63 As shown recently, the Rho-kinase is a target protein for RhoA.56-58 The phosphorylation site(s) of MYPT by Rho-kinase appears to be different and located to the C-terminal side of the endogenous kinase site.38,52 Since the N-terminal part of MYPT binds to PP1c, it is difficult to account for inhibition of activity following phosphorylation in the C-terminal segment of the molecule. One possibility is that the molecule folds so that the phosphorylated residue (e.g., Thr654) occupies the active site. This situation would be analogous to inhibition by phosphorylated Inhibitor-1.64 Another interesting finding was that Rho-kinase stoichiometrically phosphorylates myosin light chain at the same site that is phosphorylated by MLCK and thereby activates myosin ATPase.65 It is possible, therefore, that activation of Rho-kinase influences Ca2+ sensitization via a two stage process. One is the phosphorylation of MYPT and resulting inhibition of MP activity, and the other is a direct phosphorylation of myosin. Obviously, the two processes would amplify the change in Ca2+ sensitivity, compared to simple inhibition of MP activity. Kureishi et al66 demonstrated that constitutively active Rho-kinase, the recombinant catalytic domain of Rho-kinase, can induce contraction in Triton-X-100permeabilized smooth muscle of rabbit portal vein. Both in the absence and presence of Ca2+, contractions were accompanied by proportional increases in monophosphorylated myosin light chain. A recent report also indicated the involvement of Rho-kinase in smooth muscle function. A pyridine derivative, Y-27632, was found to be a Rho-kinase inhibitor, and when applied to smooth muscle strips inhibited the GTPγS and phenylephrine-induced Ca2+ sensitization.67 A second pathway is thought to involve arachidonic acid. It is known that arachidonic acid increases Ca2+ sensitivity in smooth muscle and inhibits phosphatase activity.14 Agonists such as phenylephrine act on phospholipase A2, which in turn releases arachidonic acid from the membrane phospholipids. It is suggested that arachidonic acid interacts with the C-terminal part of MYPT and dissociates the catalytic subunit from the MP holoenzyme, resulting in a decrease of phosphatase activity.14 Dissociation of the catalytic subunit from the targeting subunit is analogous to the mechanism originally proposed to regulate the glycogen-associated form of protein phosphatase 1.7 It is also possible that the activation of atypical PKC isoforms, known to be stimulated by arachidonic acid and other unsaturated fatty acids, leads to inhibition of MP activity, possibly via the direct phosphorylation of MYPT or through a more indirect mechanism. The third pathway is via protein kinase C (PKC). Because excitatory agonists which activate PKC, such as diacylglycerol (DG) and phorbol esters, caused Ca2+ sensitization of force, it was suggested that PKCs play a role in the Ca2+ sensitivity of tension and MLC20 phosphorylation in smooth muscle.68,69 It was also shown that this Ca2+ sensitization could involve the inhibition of MP activity. The major phosphorylation site during Ca 2+ sensitization by phorbol ester is Ser19 (i.e., the MLCK site). Using β-escin skinned single cells of vascular muscle, the addition of PKM (the active fragment of PKC) increased force and this was suggested to reflect an inhibition of MP activity.70 However, Gong et al71 reported that downregulation in skinned fibers with GTPγS did not affect the phorbol ester-induced Ca2+ sensitization. Further, it was found that inhibitors of the conventional PKCs (cPKC; α, β and γ) and the novel PKCs (nPKC; δ, ε, η and θ) completely abolished phorbol ester-induced Ca2+ sensitization, but not that induced by phenylephrine.72 These results support the idea that the pathways to Ca2+ sensitization by the two classes of agonists


41

(phorbol ester and G-protein activators) are separate, and that cPKC and nPKC play only a minor role in the G-protein-mediated Ca2+ sensitization. But, the above evidence does not exclude the possibility that atypical PKCs (aPKC; ξ, λ and ι), which are not downregulated by phorbol esters, may be involved in the G-protein mediated mechanism of Ca2+ sensitization. In this context, it should be noted that a novel phosphorylation-dependent inhibitory protein of MP has been purified and characterized from porcine aorta smooth muscle.73,74 This inhibitory protein, molecular mass of 20 kDa, is termed CPI17. It was found that CPI17 phosphorylated by PKC or an unidentified kinase inhibited phosphatase activity. The regulation of phosphatase activity via CPI17 might provide one mechanism for PKC-mediated Ca2+ sensitization in smooth muscle. An interesting and potentially important finding is that MP may also be activated. Recently, it was shown that cGMP causes a Ca2+-desensitization in smooth muscle and activation of MP was proposed as the underlying mechanism.75,76 Preliminary results show that protein kinase G phosphorylates the MYPT subunit of MP in vitro, but no change of phosphatase activity is observed (Nakamura M, Ichikawa K, Ito M, unpublished data). Thus, for activation of MP, another indirect mechanism may be involved. Because cGMP has also been shown to activate CaMP-dependent kinase (PKA),77 the possibility that cGMP stimulates both PKG and PKA signaling pathways remains.

Possible Function of MP As described above, it is evident that one of the functions of myosin phosphatase is to regulate force production in smooth muscle and that MP is the central molecule responsible for the Ca2+ sensitization mechanism. However, as discussed above, Western blotting studies indicate that MYPT is expressed not only in smooth muscle but also in a wide range of tissues, including brain and cardiac muscle. Since MYPT can function as a target subunit of PP1 with smooth muscle myosin, it is reasonable to expect that at least part of its function is associated with the dephosphorylation of myosin in tissues other than smooth muscle. In heart, MP could play a role in the modification of cardiac muscle contraction.78,79 In many nonmuscle cells myosin II phosphorylation plays an important role in motile processes, e.g., shape changes in platelets. In brain, myosin may be linked to filopodial.extension in neurons.80 Thus, it is possible that MP modulates these phenomena. Immunofluorescent studies using REF52 and nonconfluent MDCK cells revealed that MYPT was detected in the cytosol and also associated with stress fibers. These findings are consistent with binding to the substrate, P-myosin.26,81 Another example is the hypodermal cell shape changes that occur during elongation of the Caenorhabditis elegans embryo.48 Two genes are involved in this event. The first gene, called let-502, encodes a protein with high similarity to Rho kinase and the human myotonic dystrophy kinase. The second gene, mel-11, which was identified by mutations that act as extragenic suppressors of LET-502, encodes a protein similar to the M130 regulatory subunit of MP, as described in the previous section. LET-502 phosphorylates myosin and MEL-11, resulting in the activation of the myosinbased hypodermal contractile system that drives embryonic elongation in a small GTPase Rho dependent manner. However, several reports also indicate that MP might have alternative binding sites. Kimura et al reported that the MYPT subunit, in complex with PP1cδ, could be isolated from a bovine brain membrane fraction using a Rho-affinity column.52 Inagaki et al demonstrated that MYPT was concentrated at the cell membrane, probably the cell-cell adhesion site in confluent MDCK cells.81 Since myosin is not expected to be concentrated at these membrane locations, it is reasonable to assume that other substrates exist for MP. It was also shown that MYPT was colocalized with β-catenin, a component protein of the adherens junction. At least part of the interaction of MYPT with cell membranes may be


42

rationalized by its interaction with phospholipids. However, since the membrane association often occurs during certain stages of cell function, e.g., at confluence, it is also reasonable to suggest that additional substrates, presumably membrane proteins, are generated by phosphorylation and act as additional ligands. Obviously one of the key requirements in this theory is to identify the putative alternative substrates. Murata et al showed that M130 is also localized in the nucleus.26 PP1c interacts with at least two nuclear proteins, an inhibitory NIPP182 and the Saccharomyces cerevisiae protein sds22+,83 which is essential for exit from mitosis. It is not known if the nuclear MYPT is complexed with PP1c or if other molecules are involved.

Conclusion Significant progress has been made in research on myosin phosphatase. The structure of MP has been characterized in some detail and various regulatory mechanisms have been proposed. Obviously, MP is a key molecule in the regulation of smooth muscle contractile activity. Elucidation of the regulation of MP activity and the signal transduction pathways involved will provide clues to understand the pathogenesis of contractile and proliferative abnormalities of smooth muscle, such as asthma, high blood pressure, and coronary artery disease. Furthermore, this may lead to the development of improved methods and new drugs for the treatment of smooth muscle diseases. Another important aspect of MP appears to be the cross-talk involving the small GTPase Rho signaling pathway. Rho may be involved not only in the regulation of smooth muscle activity, but also other cellular events such as cell adhesion, motility, cytokinesis and cell growth. Thus, it is possible that MP may play a more diverse role and could be involved in many physiological processes.

Acknowledgment This work was supported in part by grants-in-aid for Scientific Research, for Cancer Research (to M.I and T.N.) and for International Scientific Research (to M.I., T.N. and D.J.H.) from the Ministry of Education, Science and Culture, Japan, by grants from Kanae Foundation of Research for New Medicine, Japan Heart Foundation (to K.I.) and by grants HL 23615 and HL 20984 from National Institutes of Health (to D.J.H.)

References 1. Hartshorne DJ. Biochemistry of the contractile process in smooth muscle. In: Johnson LR, eds. Physiology of the Gastrointestinal Tract. New York: Raven Press, 1987:423-482. 2. Somlyo AP, Somlyo AV. Signal transduction and regulation in smooth muscle. Nature 1994; 372:231-236. 3. Gallagher PJ, Herring BP, Stull JT. Myosin light chain kinase. J Mus Res Cell Motil 1997; 18:1-16. 4. Ingebritsen TS, Cohen P. The protein phosphatase involved in cellular regulation 1. Classification and substrate specificities. Eur J Biochem 1983; 132:255-261. 5. Erdodi F, Ito M, Hartshorne DJ. Myosin light chain phosphatase. In: Barany M, eds. Biochemistry of Smooth Muscle Contraction. San Diego: Academic Press, 1996:131-142. 6. Mumby MC, Walter G. Protein serine/threonine phosphatase: Structure, regulation, and functions in cell growth. Physiol Rev 1993; 73:663-673. 7. Cohen P. The structure and regulation of protein phosphatases. Annu Rev Biochem 1989; 58:453-508. 8. Shenolikar S. Protein serine/threonine phosphatases—New avenues for cell regulation. 1994; 10:55-86. 9. Cohen PTW. Novel protein serine/threonine phosphatases: Variety is the spice of life. TIBS 1997; 22:245-251.


43

10. Gerthoffer WT. Regulation of the contractile element of airway smooth muscle. Am J Physiol 1991; 261:L15-L28. 11. Morgan JP, Morgan KG. Stimulus-specific patterns of intracellular calcium levels in smooth muscle of ferret portal vein. J Physol 1984; 351:155-167. 12. Rembold CM, Murphy RA. Myoplasmic Ca 2+ determines myosin phosphorylation in agonist-stimulated swine arterial smooth muscle. Circ Res 1988; 63:593-603. 13. Kitazawa T, Kobayashi S, Horiuchi K et al. Receptor-coupled, permeabilized smooth muscle: Role of the phosphatidylinositol cascade, G-proteins, and modulation of the contractile response to Ca2+. J Biol Chem 1989; 264:5339-5342. 14. Gong, MC, Fuglsang A, Alessi D et al. Arachidonic acid inhibits myosin light chain phosphatase and sensitizes smooth muscle to calcium. J Biol Chem 1992; 267:21492-21498. 15. Kitazawa T, Masuo M, Somlyo AP. G protein-mediated inhibition of myosin light chain phosphatase in vascular smooth muscle. Proc Natl Acad Sci USA 1991; 88:9307-9310. 16. Fujiwara T, Itoh T, Kubota Y et al. Effects of guanosine nucleotides on skinned smooth muscle tissue of the rabbit mesenteric artery. J Physiol 1989; 408:535-547. 17. Kubota Y, Nomura M, Kamm KE et al. GTPγS-dependent regulation of smooth muscle contractile elements. Am J Physiol 1992; 262:C405-C410. 18. Ishihara H, Martin BL, Brautigan DL et al. Calyculin A and okadaic acid: Inhibitors of protein phosphatase activity. Biochem Biophys Res Commun 1989; 159:871-877. 19. Alessi D, Macdougall LK, Sola MM et al. The control of protein phosphatase-1 by targeting subunits: The major myosin phosphatase in avian smooth muscle is a novel form of protein phosphatase-1. Eur J Biochem 1992; 210:1023-1035. 20. Shimizu H, Ito M, Miyahara M et al. Characterization of the myosin-binding subunit of smooth muscle myosin phosphatase. J Biol Chem 1994; 269:30407-30411. 21. Shirazi A, Iizuka K, Fadden P et al. Purification and characterization of the mammalian myosin light chain phosphatase holoenzyme. The differential effects of the holoenzyme and its subunits on smooth muscle. J Biol Chem 1994; 269:31598-31606. 22. Sasaki K, Shima H, Kitagawa Y et al. Identification of members of the protein phosphatase1α in rat hepatocellular carcinoma. Jpn J Cancer Res 1990; 81:1271-1280. 23. Durfee T, Becherer K, Chen PL et al. The retinoblastoma protein associates with the protein phosphatase type 1 catalytic subunit. Genes and Devel 1993; 7:555-569. 24. Zhang Z, Bai G, Shima M et al. Expression and characterization of rat protein phosphatase-1α, -1γ1, -1γ2, and -1δ. Arch Biochem Biophys 1993; 303:402-406. 25. Alessi DR, Street AJ, Cohen P et al. Inhibitor-2 functions like a chaperone to fold three expressed isoforms of mammalian protein phosphatase-1 into a conformation with the specificity and regulatory properties of the native enzyme. Eur J Biochem 1993; 213:1055-1066. 26. Murata K, Hirano K, Villa-Maruzzi et al. Differential localization of myosin and myosin phosphatase subunits in smooth muscle cells and migrating fibroblasts. Mol Biol Cell 1997; 8:663-673. 27. Chu Y, Lee EYC, Schlender KK. Activation of protein phosphatase 1. Formation of a metalloenzyme. J Biol Chem 1996; 271:2574-2577. 28. Okubo S, Erdodi F et al. Characterization of a myosin-bound phosphatase from smooth muscle. Adv Prot Phosphatase 1993; 7:295-314. 29. Goldberg J, Huang H-B, Kwon YG et al. Three-dimensional structure of the catalytic subunit of protein serine/threonine phosphatase-1. Nature 1995; 376:745-753. 30. Egloff M-P, Cohen PTW, Reinemer P et al. Crystal structure of the catalytic subunit of human protein phosphatase 1 and its complex with tungstate. J Mol Biol 1995; 254:942-959. 31. Martin BL, Shriner CL,Brautigan DL et al. Modulation of type-1 protein phosphatase by synthetic peptides corresponding to the carboxyl terminus. FEBS Lett 1991; 285:6-10. 32. Johanson JW, Ingebritsen TS. Phosphorylation and inactivation of protein phosphatase-1 by pp60V-SRC. Proc Natl Acad Sci USA 1986; 83:207-211. 33. Dohadwala M, Da Cruz E, Hall FL et al. Phosphorylation and inactivation of protein phosphatase-1 by cyclin-dependent kinases. Proc Natl Acad Sci USA 1994; 91:6408-6412.

44


34. Takizawa N, Mizuno Y, Komatsu M et al. Alterations in type-1 serine/threonine protein phosphatase PP1α in response to B-cell receptor stimulation. J Biochem 1997; 122:730-737. 35. Hubbard MJ, Cohen P. On target with a new mechanism for the regulation of protein phosphorylation. Trends Biochem Sci 1993; 18:172-177. 36. Okubo S, Ito M et al. A regulatory subunit of smooth muscle myosin bound phosphatase. Biochem Biophys Res Commun 1994; 200:429-434. 37. Michaelly P, Bennett V. The ANK repeat: A ubiquitous motif involved in macromolecular recognition. Trends Cell Biol 1992; 2:127-129. 38. Ichikawa K, Ito M, Hartshorne DJ. Phosphorylation of the large subunit of myosin phosphatase and inhibition of phosphatase activity. J Biol Chem 1996; 271:4733-4740. 39. Hirano K, Phan BC, Hartshorne DJ. Interaction of the subunits of smooth muscle myosin phosphatase. J Biol Chem 1997; 272:3683-3688. 40. Johnson DF, Moorhead G, Caudwell FB et al. Identification of protein-phosphatase1-binding domains on the gycogen and myofibrillar targeting subunits. Eur J Biochem 1996; 239:317-325. 41. Egloff, M-P, Johnson DF, Moorhead G et al. Structural basis for the recognition of regulatory subunits by the catalytic subunit of protein phosphatase 1. EMBO J 1997; 16:1876-1887. 42. Zhao S, Lee EYC. A protein phosphatase-1-binding motif identified by the panning of a random peptide display library. J Biol Chem 1997; 272:28368-28372. 43. Zhang Y, Mabuchi K, Tao T et al. Expression in insect cells and characterization of the 110 kDa anchoring subunit of myosin light chain phosphatase. Biochim Biophys Acta 1997; 1343:51-58 44. Takahashi N, Ito M, Tanaka J et al. Localization of the gene coding for myosin phosphatase, target subunit 1 to human chromosome 12q15-q21. Genomics 1997; 44:150-152. 45. Chen YH, Chen MX, Alessi DR et al. Molecular cloning of cDNA encoding the 110 kDa and 21 kDa regulatory subunits of smooth muscle protein phosphatase 1 M. FEBS Lett 1994; 356:51-55. 46. Johnson DF, Cohen P, Chen MX et al. Identification of the regions on the M110 subunit of protein phosphatase 1M that interact with the M21 subunit and with myosin. Eur J Biochem 1997; 244:931-939. 47. Haystead CMM, Gailly P, Somlyo AP et al. Molecular cloning and functional expression of a recombinant 72.5 kDa fragment of the 110 kDa regulatory subunit of smooth muscle protein phosphatase 1 M. FEBS Lett 1995; 377:123-127. 48. Wissman A, Ingles J, MacGhee JD et al. Caenorhabditis elegans LET-502 is related to Rho-binding kinases and human myotonic dystrophy kinase and interacts genetically with a homolog of the regulatory subunits of smooth muscle myosin phosphatase to affect cell shape. Genes Devel 1997; 11:409-422. 49. Fujioka M, Takahashi N et al. A new isoform of human myosin phosphatase targeting/ regulatory subunit (MYPT2): cDNA cloning, tissue expression and chromosomal mapping. Genomics 1998; in press. 50. Ichikawa K, Hirano K, Ito M et al. Interactions and properties of the smooth muscle myosin phosphatase. Biochemistry 1996; 35:6313-6320. 51. Mitsui T, Inagaki M et al. Purification and characterization of smooth muscle-associated phosphatase from chicken gizzard. J Biol Chem 1992; 267:16727-16735. 52. Kimura K, Ito M, Amano M et al. Regulation of myosin phosphatase by Rho and Rho-associated kinase (Rho-kinase). Science 1996; 273:245-248. 53. Ito M, Feng J, Tsujino S et al. Interaction of smooth muscle myosin phosphatase with phospholipids. Biochemistry 1997; 36:7607-7614. 54. Gailly P, Wu X, Haystead TA et al. Regions of the 110-kDa regulatory subunit M110 required for regulation of myosin-light-chain-phosphatase activity in smooth muscle. Eur J Biochem 1996; 239:326-332. 55. Trinkle-Mulcahy L, Ichikawa K, Hartshorne DJ et al. Thiophosphorylation of the 130-kDa subunit is associated with a decreased activity of myosin light chain phosphatase in α-toxin permeabilized smooth muscle. J Biol Chem 1995; 270:18191-18194.


45

56. Leung T, Manser E, Tau L et al. A novel serine/threonine kinase binding the Ras-related RhoA GTPase which translocates the kinase to peripheral membranes. J Biol Chem 1995; 270:29051-29054. 57. Matsui T, Amano M, Yamamoto T et al. Rho-associated kinase, a novel serine/threonine kinase, as a putative target for small GTP binding protein Rho. EMBO J 1996; 15:2208-2216. 58. Ishizaki T, Maekawa M et al. The small GTP-binding protein Rho binds to and activates a 160 kDa Ser/Thr protein kinase homologous to myotonic dystrophy kinase. EMBO J 1996; 15:1885-1893. 59. Hirata K, Kikuchi A, Fujisawa K et al. Involvement of Rho p21 in the GTP-enhanced calcium ion sensitivity of smooth muscle contraction. J Biol Chem 1992; 267:8719-8722. 60. Gong MC, Iizuka K, Nixon G et al. Role of guanine nucleotide-binding proteins— ras-family or trimeric proteins or both—in Ca2+ sensitization of smooth muscle. Proc Natl Acad Sci USA 1996; 93:1340-1345. 61. Fujita A, Takeuchi T, Nakajima H et al. Involvement of heterotrimeric GTP-binding protein and Rho protein, but not protein kinase C, in agonist-induced Ca2+ sensitization of skinned muscle of guinea pig vas deferens. J Pharmacol Exp Ther 1995; 274:555-561. 62. Noda M, Yasuda-Fukazawa C, Moriishi K et al. Involvement of Rho in ΓΤΠγS-induced enhancement of phosphorylation of 20 kDa myosin light chain in vascular smooth muscle cells: Inhibition of phosphatase activity. FEBS Lett 1995; 367:246-250. 63. Otto B, Steusloff A, Just I et al. Role of Rho proteins in carbachol-induced contractions in intact and permeabilized guinea-pig intestinal smooth muscle. J Physol 1996; 496:317-329. 64. Endo S, Zhou X, Connor J et al. Multiple structural elements define the specificity of recombinant human inhibitor-1 as a protein phosphatase inhibitor. Biochemistry 1996; 35:5220-5228. 65. Amano M, Ito M, Kimura K et al. Phosphorylation and activation of myosin by Rhoassociated kinase (Rho-kinase). J Biol Chem 1996; 271:20246-20249. 66. Kureishi Y, Kobayashi S, Amano M et al. Rho-associated kinase directly induces smooth muscle contraction through myosin light chain phosphorylation. J Biol Chem 1997; 272:12257-12260. 67. Uehata M, Ishizaki T, Satoh H et al. Calcium sensitization of smooth muscle mediated by a Rho-associated protein kinase in hypertension. Science 1997; 389:990-994. 68. Masuo M, Reardon S, Ikebe M et al. A novel mechanism for the sensitizing effect of protein kinase C on vascular smooth muscle: Inhibition of myosin light chain phosphatase. J Gen Physiol 1994; 104:265-286. 69. Itoh H, Shimomura A, Okubo S et al. Inhibition of myosin light chain phosphatase during Ca2+-independent vasocontraction. Am J Phyiol 1993; 34:C1319-C1324. 70. Ikebe M, Brozovich FV. Protein kinase C increases force and slows relaxation in smooth muscle: Evidence for regulation of the myosin light chain phosphatase. Biochem Biophys Res Commun 1996; 255:370-376. 71. Gong MC, Fujihara H, Walker LA et al. Down-regulation of G-protein-mediated Ca2+ sensitization in smooth muscle. Mol Biol Cell 1997; 8:279-286. 72. Gailly P, Gong MC, Somlyo AV et al. Possible role of atypical protein kinase C activated by arachidonic acid in Ca 2+ sensitization of rabbit smooth muscle. J Physiol 1997; 500:95-109. 73. Eto M, Ohmori T, Suzuki M et al. A novel protein phosphatase-1 inhibitory protein potentiated by protein kinase C. Isolation from porcine aorta media and characterization. J Biochem 1995; 118:1104-1107. 74. Eto M, Senba S, Morita F et al. Molecular cloning of a novel phosphorylation-dependent inhibitory protein of protein phosphatase-1 (CPI17) in smooth muscle: Its specific localization in smooth muscle. FEBS Lett 1997; 410:356-360. 75. Lee MR, LI L, Kitazawa T. Cyclic GMP causes desensitization in vascular smooth muscle by activating the myosin light chain phosphatase. J Biol Chem 1997; 272:5063-5068. 76. Wu X, Somlyo AV, Somlyo AP. Cyclic GMP-dependent stimulation reverses G-proteincoupled inhibition of smooth muscle myosin light chain phosphatase. Biochem Biophys Res Commun 1996; 220:658-663.

46


77. Shabb JB, Ng L, Corbin JD. One amino acid change produces a high affinity cGMPbinding site in CaMP-dependent protein kinase. J Biol Chem 1990; 265:16031-16034. 78. Morano I, Hofmann F, Zimmer M et al. The influence of P-light chain phosphorylation by myosin light chain kinase on the calcium sensitivity of chemically skinned heart fibers. FEBS Lett 1985; 189:221-224. 79. Sweeney HL, Stull JT. Phosphorylation of myosin in permeabilized mammalian cardiac and skeletal muscle cells. Am J Physiol 1986; 250; C657-C660. 80. Tritus MA. Motor protein; myosin V—the multipurpose transport motor. Curr Biol 1997; 7:R301-R304. 81. Inagaki N, Nishizawa M, Ito M et al. Myosin binding subunit of smooth muscle myosin phosphatase at the cell-cell adhesion sites in MDCK cells. Biochem Biophys Res Commun 1997; 230:552-556. 82. Van Eynde A, Beullen M, Stalmans W et al. Full activation of a nuclear species of protein phosphatase-1 by phosphorylation with protein kinase A and casein kinase-2. Biochem J 1994; 297:447-449. 83. Dinischiotu A, Beullen M, Stalmans W et al. Identification of sds 22 as an inhibitory subunit of protein phosphatase-1 in rat liver nuclei. FEBS Lett 1997; 402:141-144.

CHAPTER 4

Regulation of Smooth Muscle Myosin Phosphatase and Contraction by Rho and Rho Kinase Yoh Takuwa

I

t has been established in smooth muscle as well as in a variety of non-smooth muscle cells that binding of excitatory agonists to specific cell surface receptors, most of which belong to a seven membrane-spanning type of G protein-coupled receptors, causes the gating of Ca2+ channels on the plasma membrane and the activation of phospholipase C1, 2 (Fig. 4.1). Phospholipase C catalyzes the hydrolysis of phosphatidylinositol-4, 5-bisphosphate (PIP2) to give rise to inositol-1,4,5-trisphosphate (IP3) and 1,2-diacylglycerol (DAG). IP3 acts as a second messenger to cause Ca2+ release from an intracellular store, which, together with stimulated Ca2+ influx across the plasma membrane, brings about an increase in the [Ca2+]i. On the other hand, an increase in the DAG content was shown to lead to activation of protein kinase C.3 Protein kinase C activates a contractile mechanism independently of or synergistically with an increase in the [Ca2+]i.1,2 A well-defined molecular target of the message of the [Ca2+]i increase is the calmodulin-dependent enzyme, myosin light chain kinase (MLCK).4-6 It is generally accepted that the resulting phosphorylation of the 20 kDa myosin light chain (MLC20) leads to an interaction of actin with myosin, which, in turn, underlies the initiation of a contractile response. The supporting evidence for the central role of MLC20 phosphorylation by MLCK in smooth muscle contraction is as follows. First, phosphorylation of myosin by MLCK stimulates actin-activated myosin MgATPase activity in vitro.7 Second, in in vitro motility assay using isolated myosin and actin, phosphorylation of myosin by MLCK stimulates the movement of myosin along actin cables.8 Third, many smooth muscle constrictors cause an increase in the level of MLC20 phosphorylation with a rise in the [Ca 2+ ] i . 9 Fourth, either calmodulin inhibitors or MLCK inhibitors induce inhibition of contraction with a decrease in the level of MLC20 phosphorylation.10,11 Fifth, in permeabilized smooth muscle thio-phosphorylation of MLC20, which is resistant to dephosphorylation by phosphatase, causes a Ca2+-independent contraction.12 Sixth, the introduction of Ca2+-calmodulin-independent active catalytic fragment of MLCK into smooth muscle causes a Ca2+-independent contraction.13 Seventh, the introduction of the MLCK-specific inhibitor peptides into smooth muscle cells inhibits contraction.14 Thus, MLC20 phosphorylation by MLCK appears to be the major mechanism for smooth muscle contraction. However, an increase in [Ca2+]i is not likely the sole regulator of the extent of MLC20 phosphorylation and contraction: the relationships between the [Ca2+]i and Molecular Mechanisms of Smooth Muscle Contraction, edited by Kazuhiro Kohama and Yasuharu Sasaki. ©1999 R.G. Landes Company.

48


Fig. 4.1. Excitatory receptor agonist increases the level of MLC20 phosphorylation by both increasing the [Ca2+]i and downregulating myosin phosphatase activity. Agonists bind to a heterotrimeric G protein (G)-coupled receptors (GPCR) and cause activation of phospholipase C (PLC) and the gating of Ca2+ channels on the plasma membrane. PLC catalyzes hydrolysis of of phosphatidylinositol-4, 5-bisphosphate (PIP2) to generate inositol-1, 4, 5-trisphosphate (IP3) and 1, 2-diacylglycerol (DAG). IP3 is a second messenger to cause release of Ca2+ from intracellular Ca2+ stores, and DAG causes translocation of protein kinase C (PKC) to the plasma membrane, where PKC become activated. An increase in the intracellular free Ca2+ concentration ([Ca2+]i) activates myosin light chain kinase (MLCK) in a calmodulin (CaM)–dependent manner. MLCK phosphorylates the 20 kDa myosin light chain (MLC20) and trigger the actin-myosin interaction to initiate contraction. Agonists attenuates myosin phosphatase activity toward MLC20 to enhance MLCK-mediated MLC20 phosphorylation and contraction. Activation of PKC causes phosphorylation of caldesmon, desmin and other proteins, which likely contributes to contraction. It is likely that PKC is also involved in agonist–induced inhibition of MLC20 phosphatase (see the text).

tension and between the [Ca2+]i and the phosphorylation level of MLC20 in contracted smooth muscle are not always straightforward. Morgan and Morgan15 first noted that when they compared the magnitude of contraction induced by a receptor agonist vs. KCl depolarization, they found that receptor activation by an agonist consistently induces a larger contraction than KCl depolarization despite similar increases in [Ca2+]i. Rembold and Murphy16 correlated an increase in the [Ca2+]i with increases in either the extent of MLC20 phosphorylation or tension in agonist—or

Regulation of Smooth Muscle Myosin Phosphatase and Contraction by Rho and Rho Kinase 49

KCl-stimulated smooth muscle, and found that an agonist produces larger increases in the extent of MLC20 phosphorylation and the tension at a given level of the [Ca2+]i than does KCl depolarization. Subsequent studies17-19 from several other laboratories confirmed this observation. The findings indicate that agonists somehow enhance the Ca2+ sensitivity of MLC20 phosphorylation and contraction. Thus, these observations suggest that receptor activation by excitatory agonists modulates the Ca2+ sensitivity of smooth muscle contraction. It has recently been demonstrated in staphylococcal α-toxin-, β-escin-, or saponinpermeabilized smooth muscle that an agonist causes potentiation of contraction in a GTP-dependent manner at a constant level of [Ca 2+]i, and that this GTP-dependent sensitization of contraction is accompanied by an increase in the phosphorylation level of MLC20.20-24 This action of an agonist is mimicked by the nonhydrolyzable GTP analog guanosine 5'-(3-O-thio)trisphosphate (GTPγS), whereas it is inhibited by the nonhydrolyzable GDP analog guanosine 5'-O-(β-thio)diphosphate (GDPβS). These findings suggest that an agonist sensitizes Ca2+-dependent MLC20 phosphorylation by a mechanism that involves a G protein. The purposes of this chapter are to describe the G protein-regulated mechanisms of Ca2+ sensitization of MLC20 phosphorylation and contraction.

Downregulation of Myosin Phosphatase and Consequent Sensitization of MLC20 Phosphorylation

The Ca2+ sensitization of MLC20 phosphorylation in an agonist—or GTPγS-stimulated permeabilized smooth muscle could be explained by either of the two possibilities: enhancement of MLCK activity or inhibition of protein phosphatase activity for MLC20. Kitazawa et al25 demonstrated that the addition of GTPγS or the α-adrenergic agonist phenylephrine decreased the rate of dephosphorylation of phosphorylated MLC20 in permeabilized vascular smooth muscle, suggesting that these stimulations inhibited protein phosphatase activity toward MLC20. They also showed that GTPγS did not enhance thio-phosphorylation of MLC20. This latter observation indicates that GTPγS does not enhance MLCK activity, because thio-phosphorylated MLC20 is a very poor substrate for phosphatase. In accordance with this report, Kubota et al 26 showed that GTPγS caused inhibition of the protein phosphatase activity toward both MLC20 and heavy meromyosin, but not stimulation of the MLCK activity, in homogenate of tracheal smooth muscle. It is also known that when the phosphatase activity in permeabilized smooth muscle is inhibited by the addition of phosphatase inhibitors such as calyculin A and microcystin LR, Ca2+induced contraction is enhanced. These results suggest that excitatory agonists and GTPγS alter the Ca2+ sensitivity of MLC20 phosphorylation through inhibition of phosphatase activity toward myosin, but not through enhancement of MLCK activity, by a G proteindependent mechanism. We tried to determine whether the Ca2+-sensitization by GTPγS stimulation is indeed accompanied by a decrease in the activity of myosin phosphatase. Recent studies revealed that myosin phosphatase consists of three subunits of 130, 38, and 21 kDa.27-29 These studies demonstrated that the 130-kDa protein is a myosin binding, regulating subunit (MYPT) and enhances the phosphatase activity toward myosin, and that the 38-kDa subunit is the catalytic subunit, protein phosphatase type 1(PP1) δ isoform. The 21 kDa protein (M21 subunit) is also a regulatory subunit, but its function is not well known so far. We raised polyclonal antibodies against each subunit of the myosin phosphatase, and immunoprecipitated myosin phosphatase from permeabilized vascular smooth muscle cells (SMCs) stimulated with GTPγS, by using anti-130 kDa MYPT antibody.30 When the anti-130 kDa MYPT immunoprecipitate was analyzed by each of the antibodies, i.e., anti-130 kDa MYPT, anti-38 kDa PP1δ and anti-21 kDa M21 subunit, bands immunoreactive with respective antibodies were detected. The anti-MYPT immunoprecipitate showed phosphatase activity

50


toward MLC20. Moreover, we found that the anti-MYPT immunoprecipitate from GTPγSstimulated SMCs displayed a decrease in the phosphatase activity toward MLC20, as compared to non-stimulated SMCs. These observations directly demonstrate that the G protein activation by GTPγS stimulation is accompanied by a decrease in the activity of myosin phosphatase in permeabilized SMCs.

Involvement of the Low Molecular Weight G Protein Rho in the Downregulation of Myosin Phosphatase and in Ca-Sensitization

Hirata et al31 first reported that the low-molecular-weight G protein Rho is involved in GTPγS enhancement of Ca2+-induced contraction in permeabilized vascular smooth muscle. They observed that GTPγS increased the Ca2+ sensitivity of contraction and that the GTPγSinduced Ca2+ sensitization of contraction was abolished by each of the staphylococcal exoenzyme epidermal cell differentiation inhibitor (EDIN) and the Clostrium botulinum exotoxin C3, both of which specifically ADP-ribosylates and inactivates Rho. Furthermore, they showed that the introduction of an active GTPγS-bound form of Rho, but not an inactive GDP-bound form of Rho, enhanced Ca2+-induced contraction in permeabilized vascular smooth muscle either pretreated or non-pretreated with EDIN. Fujita et al,32 Itagaki et al,33 Kokubu et al34 and Otto et al35 independently demonstrated that C3 toxin inhibited receptor agonist-induced contraction as well as GTPγS-induced one in permeabilized smooth muscle, indicating that Rho is involved in receptor agonist-induced contraction as well. Gong et al36 examined the involvement of Rho in receptor agonist-induced contraction in more detail. They found that the addition of EDIN strongly inhibited contraction by GTPγS or endothelin-1, but not by phenylephrine, in permeabilized mesenteric artery. Thus, endothelin-1 uses the Rho-dependent mechanism, whereas phenylephrine appears to activate a Rho-independent mechanism, to cause Ca2+ sensitization. However, these studies did not examine the role for Rho in Ca2+-sensitization of MLC20 phosphorylation. To explore how Rho is involved in the G protein-mediated Ca2+ sensitization of contraction, we examined the effect of C3 toxin on GTPγS-induced enhancement of MLC20 phosphorylation.37-39 We first demonstrated that the pretreatment of permeabilized vascular smooth muscle cells with C3 completely inhibited GTPγS enhancement of Ca2+-induced MLC20 phosphorylation, indicating the involvement of Rho in this phenomenon. We then examined whether C3 pretreatment reversed the inhibitory effect of GTPγS on dephosphorylation of MLC20. C3 pretreatment completely abolished the inhibition by GTPγS of MLC20 dephosphorylation. We further examined whether C3 toxin affected the activity of myosin phosphatase immunoprecipitated from permeabilized SMCs. The pretreatment of SMCs with C3 toxin abolished GTPγS-induced decrease in the myosin phosphatase activity.30 Thus, GTPγS inhibits the myosin phosphatase activity and induces enhancement of MLC20 phosphorylation in a Rho-dependent manner. These observations suggest that Rho is a critical G protein to mediate Ca2+ sensitization in agonist—or GTPγSstimulated smooth muscle. However, a downstream effector of Rho for regulating myosin phosphatase was not known.

Rho Kinase Mediates Rho Dependent Downregulation of Myosin Phosphatase It is a question of particular interest how active Rho can bring about the inhibition of myosin phosphatase. In liver and skeletal muscle, PP1, with a subunit structure similar to that of smooth muscle myosin phosphate, is associated with glycogen particles and regulates the activities of glycogen particle-associated enzymes involved in glycogen metabolism by dephosphorylating them.40,41 It is known that in addition to its roles in determining the location of the catalytic subunit and in enhancing phosphatase activity, the regulatory subunit


of glycogen-associated phosphatase confers a mechanism for the regulation of phosphatase activity by phosphorylation and dephosphorylation. For example, cyclic AMP-dependent protein kinase phosphorylates the regulatory subunit of glycogen particle-associated phosphatase and thereby triggers dissociation of the catalytic subunit from glycogen particles, leading to inhibition of dephosphorylation of glycogen particle-associated enzymes. 41 Recently Trinkle-Mulcahy et al42 demonstrated that incubation of permeabilized vascular smooth muscle with ATPγS under a certain condition induced an increase in the thio-phosphorylation level of the 130-kDa MYPT of myosin phosphatase with concomitant inhibition of dephosphorylation of MLC20. This observation suggests that phosphorylation and dephosphorylation of the 130 kDa MYPT of myosin phosphatase is one of the mechanisms for regulating the myosin phosphatase activity in smooth muscle. Ichikawa et al 43 reported that phosphorylation of the 130 kDa MYPT by a protein kinase that was associated with myosin phosphatase partially purified from chicken gizzards resulted in inhibition of phosphatase activity. Phosphorylation of the 130 kDa MYPT did not dissociate the holoenzyme. They identified the major site of phosphorylation to be threonine 654 of the 130 kDa MYPT. The associated kinase responsible for MYPT phosphorylation was inhibited by the protein kinase C inhibitor chelerythrine, and activated by arachidonic acid and oleic acid. However, it is not known whether the associated protein kinase was a protein kinase C or another kinase. We examined whether the activation of Rho with GTPγS could induce an increase in the extent of MYPT phosphorylation in permeabilized SMCs. The addition of GTPγS caused a 3-fold increase in the extent of MYPT phosphorylation in a C3-sensitive manner, with a concomitant reduction in the myosin phosphatase activity of the immunoprecipitates, which is also a C3-sensitive process.30 These observations strongly suggest that Rho regulates the activity of myosin phosphatase through phosphorylation of MYPT in SMCs. (Fig. 4.2) Recently, several groups identified two of downstream target molecules for Rho. They are serine-threonine protein kinases, protein kinase N (PKN)44,45and Rho kinase.46,47 Activated Rho directly interacts with PKN and Rho kinase, and stimulates their kinase activities. PKN, which was initially isolated from a human hippocampus cDNA library, is an approximately 120-kDa protein with a kinase domain homologous to protein kinase C and an unique N-terminal leucine-zipper-like sequences.48 Unlike protein kinase C, PKN does not possess consensus Ca2+- or phorbol ester-binding motifs which are present in the protein kinase C family. However, the substrates for protein kinase C such as the pseudosubstrate peptides of protein kinase C are also good substrates for PKN. PKN phosphorylates neurofilament subunits and can affect neurofilament function in neuronal cell types.49 Rho kinase, also called ROKα47,50 or ROCKII,51 and its isoform ROKβ50/ROCKI52 are members of a kinase family including the myotonic dystrophy kinase, and was purified on the basis of its affinity for the GTP-bound form of RhoA. Rho kinase is an approximately 160 kDa protein with an N-terminal kinase domain which is highly related to that of myotonic dystrophy kinase, a central region of coiled-coil structure and a C-terminal cysteine rich domain and pleckstrin homology (PH) domain. The expression of Rho kinase resulted in the formation of stress fibers and focal adhesions, implicating Rho kinase in the regulation of actin cytoskeleton. The expression of a mutant lacking kinase activity or the C-terminal PH domain resulted in the disassembly of stress fibers and focal adhesions, suggesting that they acted in a dominant-negative fashion. Kimura et al53 recently demonstrated that Rho kinase, but not PKN, phosphorylates the 130 kDa MYPT of purified myosin phosphatase in vitro. Importantly, Rho kinasecatalyzed phosphorylation of MYPT decreases phosphatase activity. They also showed that the inducible expression of a constitutively active Rho mutant in NIH3T3 fibroblasts caused an increase in the extent of MLC20 phosphorylation with a concomitant increase in the

52


Fig. 4.2. Regulation of myosin phosphatase activity by Rho and Rho kinase. Extracellular receptor agonists trigger the activation of Rho presumably through a hetertrimeric G protein, G12/13. An active GTP-loaded Rho activates Rho kinase, which phosphorylates the 130 kDa MYPT of myosin phosphatase and reduces its phosphatase activity leading to enhancement of MLC20 phosphorylation. Direct phosphorylation of MLC20 by Rho kinase (indicated by the dashed line) was also reported.

extent of MYPT phosphorylation. These observations raise an interesting possibility that Rho kinase may be a mediator for Rho-dependent downregulation of myosin phosphatase activity in vivo in SMCs. We examined this by using a novel Rho kinase inhibitor HA1077 (1-2-(isoquinolinesulfonyl)-homopiperazine) that we have found. This compound was originally found as an agent with a vasodilator activity,54, 55 and is currently employed as a therapeutic agent for cerebral vasospasm after subarachnoid hemorrhage. HA1077 inhibits contraction of isolated intact vascular smooth muscle with a decrease in MLC20 phosphorylation.56 We found that HA1077 abolished GTPγS-induced Ca2+ sensitization of MLC20 phosphorylation in permeabilized SMCs, but not MLC20 phosphorylation induced by Ca2+ alone.30 We found the HA1077 is a strong inhibitor of Rho kinase, with a Ki value of 0.35 µM, which is about 100 times lower and that for MLCK. HA1077 inhibits lysophosphatidic acid-induced stress fiber formation in Swiss 3T3 fibroblasts,30 which is a well-established Rho kinase-mediated process.47,57 The addition of HA1077 abolished both the decrease in myosin phosphatase activity and the increase in the MYPT phosphorylation induced by


GTPγS. We also observed that the addition of a Rho kinase inhibitor protein, the pleckstrin homology region of Rho kinase, also abolished GTPγS-induced sensitization of MLC20 phosphorylation and phosphorylation of the 130 kDa MYPT.30 These observations provide evidence that Rho kinase mediates GTPγS-induced, Rho-dependent downregulation of myosin phosphatase activity most likely through phosphorylation of MYPT in vivo in smooth muscle. The in vitro phosphorylation site of MYPT by Rho kinase is yet unknown. We have not identified the phosphorylation site of MYPT in GTPγS-stimulated SMCs. Identification of phosphorylation sites of MYPT both in vivo and in vitro should reveal whether or not Rho kinase directly phosphorylates MYPT to regulate the phosphatase activity. We have not directly tested the effect of HA1077 on myosin phosphatase activity in receptor agonist-contracted smooth muscle. However, HA1077 at a reasonably low concentration potently inhibits agonist-induced smooth muscle contraction with a decrease in MLC20 phosphorylation.56 Therefore, Rho kinase-mediated sensitization mechanism of MLC20 phosphorylation and contraction is most likely operative in receptor agonist-stimulated smooth muscle. In vascular smooth muscle tissues, Rho kinase/ROKα/ROCKII, and its isoform ROKβ/ROCKI are both expressed.58 Precise distribution of each isoform in various smooth muscle tissues is not known. As the two isoforms are highly homologous, it is very likely that both isoforms are involved in the Rho-dependent regulation of myosin phosphatase activity(Fig. 4.3). It was recently reported that the addition of the constitutively active catalytic fragment of Rho kinase to permeabilized vascular smooth muscle preparations caused a Ca2+, calmodulin-independent phosphorylation of MLC20 and contraction.59 It was also previously demonstrated that Rho kinase phosphorylates purified myosin in vitro at the site of Ser19 of MLC20 (the phosphorylation site by myosin light chain kinase) and increases actinactivated myosin ATPase activity.60 These observations may suggest that direct phosphorylation of MLC20 by Rho kinase could at least in part contribute to Rho kinase-mediated increase in MLC20 phosphorylation under certain experimental conditions. However, as mentioned above, we37 and others35 observed that in permeabilized SMCs and vascular strips, GTPγS did not increase Ca2+-induced thio-phosphorylation of MLC20, indicating that the myosin kinase activity in GTPγS-stimulated smooth muscle was not enhanced. Furthermore, we observed that the Rho kinase inhibitor HA1077 reversed inhibition of MLC20 dephosphorylation, but did not lower thio-phosphorylation level of MLC20 in SMCs stimulated with GTPγS.30 These results strongly suggest that in GTPγS-stimulated SMCs, the observed Ca2+ sensitization of MLC20 phosphorylation is mediated largely through downregulation of myosin phosphatase activity. It still remains to be clarified whether the Rho kinase-mediated mechanism is the sole mechanism for the Rho-dependent downregulation of myosin phosphatase activity. Kimura et al53 reported that the 130 kDa MYPT could interact with a GTPγS-bound active form of RhoA in vitro. They also observed that the overexpression of RhoA protein in COS-7 cells caused a decrease of the 130 kDa MYPT in the cytosolic fraction and its reciprocal increase in the particulate fraction. Thus, Rho appears to promote the association of the 130 kDa MYPT to membranes, most likely by forming a complex with MYPT. However, the binding of a GTPγS-bound form of RhoA to purified myosin phosphatase did not alter its phosphatase activity. Therefore, Rho may be involved in regulating the cellular localization of myosin phosphatase rather than regulating its activity.

Conclusion and Perspective The recent results described above indicate that the small molecular weight G protein of the Ras superfamily, Rho, plays a critical role in the regulation of myosin phosphatase activity. Rho is active in its GTP-bound form, whereas Rho is inactive in its GDP-bound

54


Fig. 4.3. Structure of Rho kinase/ROKα/ROCKII and its isoform ROKβ/ROCKI. Rho kinase contains a N-terminal kinase domain, a centrally located coiled-coil domain, which contains the Rho-binding domain near its C-terminus, and a C-terminal PH (pleckstrin homology) domain which is split by a cysteine-rich domain. The amino acid identity in each domain between Rho kinase/ROKα/ROCKII and its isoform ROKβ/ROCKI is shown as a percent.50

form. Excitatory receptor agonists are thought to trigger conversion of Rho from an inactive form to an a active form by stimulating GDP/GTP exchange. This is brought about by the activation of guanine nucleotide exchange factor(s) (GEF) for Rho.61,62 There are several known GEFs for Rho. However, it is unknown which is dominantly expressed in smooth muscle and activated in response to agonist stimulation. The GTP/GDP cycle of Rho is also modulated by changes in the activities of a GDP-dissociation inhibitor (GDI) and a GTPaseactivatinG protein (GAP). It is also unknown whether or not the activities of these proteins are changed upon agonist stimulation. It is also an interesting question what signaling pathway mediates changes in the activities in Rho GEFs and/or GDI and GAP in agoniststimulated SMCs. Recent studies demonstrated that the heterotrimeric G proteins of the G12/G13 class trigger activation of Rho-dependent signaling pathways in non-muscle cells.63 It was recently demonstrated that a tyrosine kinase inhibitor reduced GTPγS-induced enhancement of MLC20 phosphorylation, suggesting the possibility that a tyrosine kinase may mediate the activation of Rho.64 It was also reported that in permeabilized SMCs, phorbol ester, a protein kinase C activator, sensitized Ca2+-induced MLC20 phosphorylation through inhibition of dephosphorylation of MLC20, suggesting that the activation of protein kinase C leads to inhibition of myosin phosphatase activity.65,66 It is unknown whether protein kinase C could act upstream of Rho or be located in a pathway independent of Rho. Very recently, it was shown that ADP-ribosylation and inactivation of endogenous Rho by chimeric toxin (DC3B) in vascular smooth muscle failed to inhibit phorbol ester-induced Ca2+ sensitization of contraction, indicating that protein kinase C-mediated sensitization is not dependent upon Rho. Further studies are required to clarify mechanisms of protein kinase C-mediated sensitization. This chapter described the functional role for Rho kinase in the regulation of myosin phosphatase activity in smooth muscle. Rho kinase is also expressed widely in nonsmooth muscle cells, in which Rho kinase is involved in formations of stress fiber and focal adhesions. 57,62,68 A recent study showed that phosphorylation of MLC20 is a critical prerequisite for stress fiber formation.69 The expression of an active mutant of Rho in


nonmuscle cells leads to stress fiber formation.70 A Rho kinase inhibitor such as HA1077 efficiently inhibits Rho-induced stress fiber formation,71 indicating that Rho kinase mediates Rho-induced stress fiber formation. Generally, the content of MLCK in non-muscle cells is very low, in contrast to its high level expression in smooth muscle. As mentioned above, Rho kinase can phosphorylate myosin at Ser19 of MLC20 (the phosphorylation site of myosin by MLCK).60 It is an interesting possibility that in non-muscle cells, Rho kinase could act as a myosin kinase, in addition to its role as a regulator of myosin phosphatase. It is also becoming evident that the 130 kDa MYPT and the catalytic subunit (PP1δ) are present in complexes with cytoskeletal structures. Rho kinase may affect functions of the cytoskeleton by regulating phosphatase activity through phosphorylation of MYPT. Thus, Rho kinase appears to participate in the regulation of cell contraction and morphological changes through multiple mechanisms.

Acknowledgments I am very grateful to my coworkers Hiromitsu Nagumo, Yasuharu Sasaki, Masakuni Noda and Noriko Takuwa. This work was supported by grants from the Ministry of Education, Science and Culture of Japan and the Yamanouchi Foundation for Research on Metabolic Disease, funds for Cardiovascular Research from Tsumura Co., and funds from Asahi Chemical Industry Co.

References 1. Rasmussen H, Takuwa Y et al. Protein kinase C in the regulation of smooth muscle contraction. FASEB J 1995; 1:177-185. 2. Rasmussen H, Haller H et al. Messenger Ca2+, protein kinase C, and smooth muscle contraction. In: Sperelakis N, Wood JD, ed. Frontiers in smooth muscle research. New York: Wiley-Liss, 1990; P89-106. 3. Nishizuka Y. Intracellular signalling by hydrolysis of phospholipids and activation of protein kinase C. Science 1992; 258:607-614. 4. Kamm KE, Stull JT. The function of myosin and myosin light chain kinase phosphorylation in smooth muscle. Annu Rev Pharmacol Toxicol 1985; 25:593-620. 5. Walsh MP. Calmodulin and the regulation of smooth muscle contraction. Mol Cell Biochem 1994; 135:21-41. 6. Somlyo AP, Somlyo AV. Signal transduction and regulation in smooth muscle. Nature 1994; 372:231-236. 7. Sellers JR, Pato MD et al. Reversible phosphorylation of smooth muscle myosin, heavy meromyosin and platelet myosin. J Biol Chem 1981; 256:13137-13142. 8. Sellers JR, Spudich JA et al. Light chain phosphorylation regulates the movement of smooth muscle myosin on actin filaments. J Cell Biol 1985; 101:1897-1902. 9. Takuwa Y, Kelley G et al. Protein phosphorylation changes in bovine carotid artery smooth muscle during contraction and relaxation. Mol Cell Endocrinol 1988; 60:71-86. 10. Kanamori M, Naka M et al. Effects on N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide and other calmodulin antagonists (calmodulin interacting agents) on calciuminduced contraction of rabbit aortic strips. J Pharmacol Exp Ther 1981; 217:494-499. 11. Silver PJ, Stull JT. Effects of the calmodulin antagonist, fluphenazine, on phosphorylation of myosin and phosphorylase in intact smooth muscle. Mol Pharmacol 1983; 23:665-670. 12. Hoar PE, Kerrick WGL et al. Chicken gizzard: relation between calcium-activated phosphorylation and contraction. Science 1979; 204:503-506. 13. Walsh MP, Bridenbaugh R et al. Phosphorylation-dependent activated tension in skinned gizzard muscle fibers in the absence of Ca2+. J Biol Chem 1982; 257:5987-5990. 14. Itoh T, Ikebe M et al. Effects of modulators of myosin light-chain kinase activity in single smooth muscle cells. Nature (London). 1989; 338:164-167. 15. Morgan JP, Morgan KG. Stimulus-specific patterns of intracellular calcium levels in smooth muscle of ferret portal vein. J Physiol (London) 1984; 351:155-167.

56


16. Rembold CM, Murphy RA. Myoplasmic [Ca2+]i determines myosin phosphorylation in agonist-stimulated swine arterial smooth muscle. Cir Res 1988; 63:593-603. 17. Small JV, Furst DO et al. Localization of filamin in smooth muscle. J Cell Biol 1986; 102:210-220. 18. Suematsu E, Resnick M et al. Change of Ca2+ requirement for myosin phosphorylation by prostaglandin F2α. Am J Physiol 1991; 261:C253-C258. 19. Tang DC, Stull JT et al. Regulation of the Ca2+ dependent of smooth muscle contraction. J Biol Chem 1992; 267:11839-11845. 20. Nishimura J, Kolber M et al. Norepinephrine and GTPrS increase myofilament Ca2+ sensitivity in a-toxin permeabilized arterial smooth muscle. Biol Bio Res Commu 1988; 157:677-683. 21. Fujiwara T, Itoh T et al. Effects of guanosine nucleotides on skinned smooth muscle tissues of the rabbit mesenteric artery. J Physiol (Lond) 1989; 408:535-547. 22. Kitazawa T, Kobayashi S et al. Receptor-coupled, permeabilized smooth muscle. J Biol Chem 1989; 264:5339-5342. 23. Kitazawa T, Gaylinn BD et al. G-protein-mediated Ca2+ sensitization of smooth muscle contraction through myosin light chain phosphorylation. J Biol Chem 1991; 26:1708-1715. 24. Moreland S, Nishimura J et al. Transient myosin phosphorylation at constant Ca2+ during agonist activation of permeabilized arteries. Am J Physiol 1992; 263:C540-C544. 25. Kitazawa T, Masuo M et al. G protein-mediated inhibition of myosin light-chain phosphatase in vascular smooth muscle. Proc Natl Acad USA 1991; 88:9307-9310. 26. Kubota Y, Nomura M et al. GTPrS-dependent regulation of smooth muscle contractile elements. Am J Physiol 1992; 262:C405-C410. 27. Alessi D, Macdougall LK et al. The control of protein phosphatase-1 by targeting subunits. The major myosin phosphatase in avian smooth muscle is a novel form of protein phosphatase-1. Eur J Biochem 1992; 210:1023-1035. 28. Shimizu H, ito M et al Characterization of the myosin-binding subunit of smooth muscle myosin phosphatase. J Biol Chem 1994; 269:30407-30411. 29. Chen YH, Chen MX et al. Molecular cloning of ‘cDNA encoding the 110 kDa and 21 kDa regulatory subunits of smooth muscle protein phosphatase 1M. FEBS Lett 1994; 356:51-55. 30. Nagumo H, Takuwa N et al. Suppression by a novel Rho kinase inhibitor HA1077 of Rho-mediated myosin phosphatase inhibition and resultant sensitization of myosin phosphorylation in vascular smooth muscle cells. Submitted. 31. Hirata K, Kikuchi A, Sasaki S, Kuroda S, Kaibuchi K, Matsuura Y, Ski H, Saida K, Takai T. Involvement of rho p21 in the GTPγS-enhanced calcium ion sensitivity of smooth muscle contraction. J Biol Chem 1992; 267:8719-8722. 32. Fujita A, Takeuchi T et al. Involvement of heterotrimeric GTP-binding protein and rho protein, but not protein kinase C, in agonist-induced Ca2+ sensitization of skinned muscle of guinea pig vas deferens. J Pharmacol Exp Ther 1995; 274:555-561. 33. Itagaki M, Komori S et al. Possible involvement of a small G-protein sensitive to exoenzyme C3 of clostridium botulinum in the regulation of myobilament Ca 2+ sensitivity in β-escin skinned smooth muscle of quinea pig ileum. Jpn J Pharmacol 1995; 67:1-7. 34. Kokubu N, Satoh M et al. Involvement of botulinum C3-sensitive GTP-binding protein sin α1-adrenoceptor subtypes mediating Ca2+-sensitization. Eur J Pharmacol 1995; 290:19-27 35. Otto B, Steuslogg A et al. Role of Rho proteins in carbachol-induced contractions in intact and permeabilized quinea-pig intestinal smooth muscle. J Physiol 1996; 496:317-329. 36. Gong MC, Iizuka N et al. Role of quanine nucleotide-binding proteins-ras-family or trimeric proteins or both-in Ca2+ sensitization of smooth muscle. Proc Natl Acad Sci USA 1996; 93:1340-1345. 37. Noda M, Fukazawa C, Moriishi K et al. Involvement of rho in GTPγS-induced enhancement of phosphorylation of 20kDa myosin light chain in vascular smooth muscle cells: inhibition of phosphatase activity. FEBS Lett 1995; 367:246-250.

Regulation of Smooth Muscle Myosin Phosphatase and Contraction by Rho and Rho Kinase 57 38. Takuwa Y, Noda M et al. Regulation of Ca2+-dependent pholsphoaryltaion of 20-kDa myosin light chain by the small molecular weight G protein Rho p21 in vascular smooth muscle cells. In: Nakano T, Hartshorne DJ, ed. Regulation of the contractile cycle in smooth muscle. Tokyo: Springer, 1995. 39. Takuwa Y. Regulation of vascular smooth muscle contraction. The roles of Ca2+, protein kinase C and myosin light chain phosphatase. Jpn Heart J 1996; 37:793-813. 40. Hubbard MJ, Cohen P. On target with a new mechanism for the regulation of protein phosphorylation. Trend Biochem Sci 1993; 18:172177. 41. Hubbard MJ, Cohen P. Regulation of protein phosphatase-1G from rabbit skeletal muscle. 1. Phosphorylation by CaMP-dependent protein kinases at site. 2. Releases catalytic subunit from the glycogen-bound holoenzyme. Eur J Biochem 1989; 186:701-709. 42. Trinkel-Mulcahy L, Ichikawa K, Hartshorne DJ et al. Thiophosphorylation of the 130-kDa subunit is associated with a decreased activity of myosin light chain phosphatase i α-toxinpermeabilized smooth muscle. J Biol Chem 1995; 270:18191-18194. 43. Ichikawa K, Ito M et al. Phosphorylation of the large subunit of myosin phosphatase and inhibition of phosphatase activity. J Biol Chem 1996; 271:4733-4740. 44. Watanabe G, Saito Y et al. Protein kinase N (PKN) and PKN-related protein Rhophilin as targets of small GTPase Rho. Science 1996; 271:645-648. 45. Amano M, Mukai H et al. Identification of a putative target for Rho as the serine-threonine kinase protein kinase N. Science 1996; 271:648-650. 46. Matsui T, Amano M et al. Rho-associated kinase, a novel serine/threonine kinase, as a putative target for the small GTp binding protein Rho. EMBO J 1996; 15:2208-2216. 47. Keung T, Manser E et al. A novel serine/threonine kinase binding the ras-related RhoA GTPase which translocates the kinase to peripheral membranes. J Biol Chem 1995; 256:29051-29054. 48. Mukai H, Ono Y. A novel protein kinase with leucine zipper like sequences—its catalytic domain is highly homologous to that of protein kinase C. Biochem Biophys Res Commun 1994; 199:897-904. 49. Mukai H, Tochimori M et al. PKN associates and phosphorylates the head-rod domain of nuerofilament protein. J Biol Chem 1996; 271:9816-9822. 50. Leung T, Chrn XQ et al. The p160 RhoA-binding kinase ROKα is a member of a kinase family and is involved in the reorganization of the cytoskelton. Mol Cell Biol 1996; 16:5313-5327. 51. Nakagawa O, Fujisawa K et al. ROCK-I and ROCK-II two isoforms of Rh o-associated coiled-coil forming protein serine/threonine kinase in mice. FEBS Lett 1996; 392:189-196. 52. Ishizaki T, Maekawa M et al. The small GTP-binding protein Rho binds to and activates a 160 kDa Ser/Thr protein kinase homologous to myotonia dystrophy kinase. EMBO J 1996; 15:1885-1893. 53. Kimura K, Ito M et al. Regulation of myosin phosphatase by Rho and Rho-associated kinase (Rho-kinase). Science 1996; 273:245-248. 54. Takayasu M, Suzuki Y et al. The effects of HA compound calcium antagonists on delayed cerebral vasospasm in dogs. J Neurosurg 1986; 65:80-85. 55. Asano T, Suzuki T et al. vasodilator actions of HA1077 in vitro an din vivo putatively mediated by the inhibition of protein kinase. Br J Pharmacol 1989; 98:1091-1100. 56. Seto M, Sasaki Y et al. Effects of HA1077, a protein kinase inhibitor, on myosin phosphorylation and tension in smooth muscle. Eur J Pharmacol 1991; 195:267-272. 57. Amano M, Chihara K et al. Formation of actin stress fibers and focal adhesions enhanced by Rho-kinase. Science 1997; 275:1308-1311. 58. Nagumo H, Takuwa Y. Unpublished observations. 59. Kureishi Y, Kobayashi S et al. Rho-associated kinase directly induces smooth muscle contraction through myosin light chain phosphorylation. J Biol Chem 1997; 272:12257-12260. 60. Amano M, Ito M et al. Phosphorylation and activation of myosin by Rho-associated kinase (Rho-kinase). J Biol Chem 1996; 271:20246-20249. 61. Boguski MS, McCormick F. Proteins regulating ras and its relatives. Nature 1993; 336:643-654.

58


62. Aelst LV, D’Souza-Schorey C. Rho GTPases and signaling networks. Genes & Dev 1997; 11:2295-2322. 63. Fromm C, Coso O et al. The small GTP-binding protein RHo links G protein-coupled receptors and Gα12 to the serum response element and to cellular transformation. Proc Natl Acad Sci USA 1997; 94:10098-10103. 64. Ridley A, Hall A. Signal transduction pathways regulating rho-mediated stress formation: Requirement for a tyrosine kinase. EMBO J 1994; 13:2600-2610. 65. Fujiwara T, Itoh T, Kubota Y, Kuriyama H. Actions of a phorbol ester on factors regulating contraction in rabbit mesenteric artery. Circ Res 1988; 63:893-902. 66. Masuo M, Reardon S, Ikebe M, Kitazawa T. A novel mechanism for the Ca2+-sensitizing effect of protein kinase C on vascular smooth muscle: Inhibition of myosin light chain phosphatase. J Gen Physiol 1994; 104:265-286. 67. Fujihara H, Walker LA et al. Inhibition of RhoA translocation and calcium sensitization by in vivo ADP-ribosylation with the chimeric toxin DC3B. 68. Lim L, Manser E et al. Regulation of phosphorylation pathways by p21 GTPases. The p21 Ras-related Rho subfamily and its role in phosphorylation signalling pathways. Eur J Biochem 1996; 242:171-185. 69. Chrzanowska-wodnicka M, Burridge K. Rho-stimulated contractility drives the formation of stress fibers and focal adhesions. J Cell Biol 1996; 133:1403-1415. 70. Ridley AJ, Hall A. The small GTP-binding protein rho regulates the assembly of focal adhesions and actin stress fiber in response to growth factor. Cell 1992; 70:389-399. 71. Sasaki Y, Sasaki Y et al. Disorganization by calcium antagonists of actin microfilament in aortic smooth muscle cells. Am J Physiol 1987; 253:C71-C78.

CHAPTER 5

Caldesmon Phosphorylation and Smooth Muscle Contraction Vladimir P. Shirinsky, Alexander V. Vorotnikov, Nikolai B. Gusev

H

istorically a division between cross-striated and smooth muscles was drawn on the basis of contraction regulation. Although both muscle types use a universal sliding filament mechanism of shortening and employ Ca2+ as a trigger, different molecular targets are involved in the two types of muscle. In sarcomeric muscle an actin filament-bound troponin complex receives the Ca2+ signal and, together with tropomyosin, converts thin filaments to a contraction-competent state. In smooth muscle, Ca2+ has been shown to bind calmodulin and activate a myosin light chain kinase whose function is to turn the thick filament “ON” and ready for contraction. Once clear, this distinction has been blurred since the discovery of the thin filamentbased smooth muscle protein caldesmon. Biochemical evidence strongly suggests that caldesmon fulfills troponin-like functions in smooth muscle and is regulated by Ca2+calmodulin and phosphorylation. It may also be involved in smooth muscle latching. However, the physiological significance of caldesmon as a modulator of smooth muscle contraction and the role of its phosphorylation still remains a matter of controversy. In this chapter we will summarize the main features of caldesmon, the current state of caldesmon phosphorylation research and the relation of both to smooth muscle contraction. Caldesmon (CaD) was discovered by Kakiuchi and colleagues in 1981.1 Soon after this it was recognized as the major smooth muscle protein. According to various estimations, its concentration in smooth muscle cells is approximately 16-33 µM.2 Initially, this protein was found to interact with F-actin, the main constituent of muscle thin filaments and with calmodulin, a ubiquitious Ca2+-bindinG protein involved in regulation of a variety of cellular processes.1 The ability of CaD to inhibit the actin-activated Mg2+-ATPase (ATP hydrolysis) activity of myosin, and reduction of this inhibition by Ca2+-calmodulin (CaM),1,3-5 readily put CaD in the focus of smooth muscle contraction regulation research. The biochemical elucidation of CaD was greatly facilitated in 1984 when Bretscher introduced a heat treatment step in the purification protocol for this protein.6 The following years brought about the notion that CaD is also capable of interacting with tropomyosin (TM) and with smooth muscle myosin (SMM).7-9 Many features of CaD may be regulated by phosphorylation. For better understanding of the molecular basis of this regulation we will first summarize the modern views on CaD structure and function.

Caldesmon Gene, mRNA and Protein Full-length CaD cDNA was originally cloned by two groups using chicken-based cDNA libraries.10,11 Sequencing results were in close agreement except for the lack of a 15 amino Molecular Mechanisms of Smooth Muscle Contraction, edited by Kazuhiro Kohama and Yasuharu Sasaki. ©1999 R.G. Landes Company.

60


acid stretch in the middle part of CaD in the sequence of Bryan et al.10 The deduced CaD amino acid sequence contained 756 (771 in the version of Hayashi et al11) amino acids. The computed mass of the protein was around 87-89 kDa, although its mobility in SDS-PAGE suggested a longer polypeptide of 120-150 kDa. The anomalous mobility is due to the high glutamic and aspartic acid content (26%) which modifies the binding of SDS to CaD. The CaD gene was found to consist of at least 14 exons. In the human genome it is represented by a single copy located on chromosome 7 (7q33-q34).11 CaD isoforms of smaller molecular mass (70-80 kDa as judged by SDS-PAGE, computed mass about 60 kDa) were detected primarily in nonmuscle tissues and cells.12,13 Sequence comparison revealed that both the 87-89 kDa and 60 kDa CaD variants, also identified as hCaD and l-CaD respectively, differ mainly by the presence of a central domain which includes a multiply repeated motif of 15 amino acids based on the sequence Glu3-(Lys/Arg)2-Ala2-Glu2(Lys/Arg)-X-(Lys/Arg)-Ala-Lys-Ala, where X stays for Glu, Gln or Ala. The size difference between the CaD sequences mentioned above is due to the presence of an additional such repeat in the sequence reported by Hayashi et al.11 This amino acid stretch forms a substantially long a-helix with repeated alternating charge disposition. This part of the CaD molecule is about 40 nm long and appears rodlike in electron micrographs.14 The repetitive sequence restricted to h-CaD is encoded by the 3'-terminal part of exon 3 (3b) in the CaD genomic sequence. The 5'-part of this exon (3a) is common for h-CaD and l-CaD. l-CaDs are thus generated by a tissue specific alternative splicing of exons 3b and 4 out of h-CaD pre-mRNA.15 Other differences exist between h-CaD and l-CaD as far as mRNA structure and N-terminal and C-terminal sequences of the protein molecules.14,15 Of course, there are interspecies sequence variations between caldesmons. Nevertheless, human and chicken CaD primary structure reveals about 70% identity, whereas in the most critical parts of the molecule it approaches 89%.16,17 We will limit further description to h-CaD, since this isoform is specific to differentiated smooth muscle. However, since all CaDs share extensive structural similarities except in the central repeat region of the molecule, we feel it is appropriate to present the evidence collected while studying l-CaD when it is consonant with the main topic of this chapter. We will be mostly using the abbreviation CaD for h-CaD and the sequence nomenclature of Bryan et al,10 since historically it was widely adopted in publications in the smooth muscle field.

Domain Structure and Activities On the basis of the available structural and functional information, Marston and collegues suggested a four domain model of CaD (Fig. 5.1) and assigned the known CaD features to particular domains.2,14,18 Domain 1 has an extended structure and may bind to the S2 subfragment of SMM.8,9,19 It could also bind TM.20 Domain 2 contains the highly repetitive intervening charged motifs (see above) and represents the binding site for TM. TM is a rod-shaped coiled-coil protein which lies in the groove of the actin filament and displays a similar charge pattern on the surface. Therefore, CaD-TM interaction in this region is mostly electrostatic. Domain 3 has an extensive homology with the Ca2+-insensitive TM binding site of skeletal muscle troponin T.10,21 However, it binds weakly, if at all, to smooth muscle TM in vitro.22-25 The same weak, if any, binding affinity of domain 3 has been reported with regard to actin.26 Yamakita et al27 demonstrated the existence of an additional myosin-binding site in CaD, most likely positioned in domains 3/4.25 Recent evidence casts a doubt that it may be functional at physiologic ionic strength and when CaD is bound to thin filament.19

Caldesmon Phosphorylation and Smooth Muscle Contraction

61

Fig. 5.1. Domain model of caldesmon. Domains are marked by circled numbers. Their approximate boundaries are shown by the numbers corresponding to caldesmon amino acid residues.10 An expanded scheme of domain 4 is represented below. Activities associated with domains are depicted by the patterns (see legend at bottom of figure). Arrows indicate positions of the major phosphorylation sites in chicken gizzard CaD. Phosphorylated residues are indicated. For kinase abbreviations, see text.

Domain 4 hosts the most important activities of CaD, including the binding sites for actin, TM and CaM. This domain has little regular secondary structure and instead is stably folded in an extended though flexible fashion.14,18,28,29 Experiments with C-terminal fragments of CaD prepared by limited proteolysis, peptide synthesis and recombinant techniques pinpointed several scattered sequence stretches involved in binding with the abovementioned proteins (for a review see ref. 2). It is, however, important to realize that in the threedimensional organization of CaD domain 4 these parts of the sequence may be positioned close to each other and constitute a single contact area. Likewise, the affinities of limited CaD fragments towards correspondinG proteins have only an approximate value, since they may be different within the context of the whole domain 4. Three sites of interaction with CaM have been found in domain 4 and designated as A, B, and B’.14,24 They all contain tryptophan residues which respond by alteration of intrinsic fluorescence to the binding of Ca2+-CaM.26 CaM-binding sites B and B’ overlap an extended region involved in TM-dependent actin binding and ATPase inhibition. Another actin binding and TM-dependent inhibitory sequence is located towards the N-terminus and overlaps TM-binding region. Again, in the proposed folding scheme these parts of the CaD sequence are thought to be close enough to constitute a cooperative unit.18 Other linear models of CaD structure are also available.31-34 They mostly differ in details of the assignment of binding sites in domain 4. Thus, along its overall 80 nm length14 CaD displays multiple binding sites which allow it to interact with the major protein constituents of the thick and thin filaments and also be regulated in a Ca2+-dependent manner. Nevertheless, the way that CaD fits in the smooth muscle contractile system remains largely hypothetical.

62


Caldesmon’s Place and Role in Smooth Muscle CaD is localized in the contractile domain of smooth muscle, together with actin and myosin.35,36 It can be isolated from smooth muscle, tightly bound to the thin filaments.37 The molecular arrangement of CaD on actin filaments still constitutes a point of controversy. Although CaD may directly compete with SMM and inhibit myosin ATPase through a stoichiometric binding to actin,28 this type of inhibition is unlikely to happen in smooth muscle cells because of the lack of the necessary amount of CaD. A more physiological method for CaD action seems to be through partnership with TM. Addition of TM to an assay increases CaD binding to actin and attenuates ATPase and SMM in vitro motility.2,38 In the presence of TM, 1 molecule of CaD per 7-14 actin monomers is required to achieve inhibition. Therefore, CaD apparently confers a troponin-like regulation to smooth muscle thin filaments controlling the ability of TM to “lock” the extended blocks of actin monomers in an “OFF” state.2 According to the model, CaD lies continuously along TM in the actin filament groove spanning three TM molecules.14 Unlike TM, CaD can apparently detach from the thin filament at its N-terminal domain. For a more detailed survey of CaD-TM-actin interaction, the reader is directed to recent reviews on CaD2,14 and periodics. It is worth noting that CaD and TM gene expression are closely correlated in smooth muscle cells,39 suggesting that the cell maintains a tight control over the coordinate levels of these proteins. The interaction of CaD with myosin is not a certain issue either. Conflicting electron microscopic data depicts CaD as being mostly longitudinally bound to actin, or peeling off from the filament backbone and crosslinking actin and myosin filaments.40,41 CaD definitely projects out of the thick filaments assembled from the myosin rod, a headless SMM subfragment.42 Independent assessment of CaD domain 1 mobility demonstrates it to be mostly away from the actin filament.43 Therefore, it seems plausible that at least in some instances CaD may participate in the thin and thick filament crosslinking in smooth muscle. CaD-mediated actomyosin crosslinking was suggested to play a role in the latching of smooth muscle in addition to or instead of the dephosphorylated crossbridges.2,19 It is, however, not entirely clear whether CaD bonds are strong enough to maintain the load. An evidence that cultured vascular smooth muscle cells increase h-CaD content when subjected to a long term stretch44 indirectly suggests that CaD may be involved in counteraction to a mechanical deformation. Another possible manifestation of CaD myosin-binding ability is an integration and stabilization of the contractile filaments.45,46 Our recent studies suggest that CaD together with the kinase-related protein telokin may, indeed, be an indispensable component in the control of SMM filament assembly (Vorotnikov AV, Shirinsky VP, manuscript in preparation). The role of a C-terminal myosin binding site in CaD is much less clear, although its contribution in CaD-SMM binding is noticeable in terms of interaction affinity.47 Besides, this site seems to be regulated by Ca2+-CaM,48 whereas the purified N-terminal myosin-binding site (domain 1) is not. The CaD features described above can be integrated in a hypothetical scheme, the essence of which has been adopted from Marston and Huber2 and modified by us (Fig. 5.2). CaD controls the position of TM in the actin filament groove ensuring the filament “OFF” state, e.g., the state when only a weak (actin-S1-ADP-Pi) interaction of myosin heads with actin is possible (Fig. 5.2, top) This state does not provide for force generation since the transition to the strong binding state (actin-S1-ADP) and accompanying conformational change is inhibited. Besides, in this state CaD may physically link actin and myosin filaments. When smooth muscle contraction is initiated by a Ca2+ increase, CaD binds Ca2+CaM at the C-terminus; this association bring about several consequences (Fig. 5.2, bottom):


63

Fig. 5.2. Possible role of caldesmon in smooth muscle contraction. Caldesmon and tropomyosin control the availability of the strong myosin-binding sites on actin (shown as deep cavities along the actin filament). Movement of CaD-TM across a filament makes these sites either open (empty) or closed (brick pattern-filled) for interaction with myosin heads. Shallow pits indicate the weak myosin-binding sites. CaD N-terminus is on the left. All shapes, distances and relative positions are approximate. See text for detailed description.

1. The interaction of CaD with actin is changed in such a way that TM assumes an alternative position on the actin filament which somehow makes both strong and weak myosin binding sites available for myosin heads. Since myosin gets concurrently phosphorylated by a Ca 2+ CaM-dependent enzyme, myosin light chain kinase (MLCK), force-generating cycles may now be initiated. 2. A myosin-CaD link through the N-terminal binding site may have two functions. First, it serves to bring myosin and actin filaments closer to each other, which might facilitate their interaction and ATPase activation. Second, it increases the internal load that might be relieved by a Ca2+dependent or independent phosphorylation (see below).

Kinasea

CaM PK II

Endogenous (CAM PK II)

Endogenous (CaM PK II)

Endogenous (CaM PK II)

CaM PK II CK II PKC

CK II

CK 1I

CK II

CK II

CK II

CaD source

Turkey gizzard

Turkery gizzard

Chicken gizzard

Chicken gizzard

Duck gizzard

Chicken gizzard

Chicken gizzard

Sheep aorta

Duck gizzard

Duck gizzard 0.6

1.0

1.0

ND

1.0

2.0-4.0 1.0 3.0-3.5

2.0

2.0

3.0

5.65

molPi/ molCaD

Ser-73

Ser-73

ND

Ser-73

Ser-73, Thr-83

N- and C-termini N-terminus C-terminus

N-terminus

ND

N-terminus

Ser-73, Ser-26, Ser-726, Ser-587

Location of main phosphoresidues

Table 5.1. Caldesmon phosphorylation in vitro

Reduced SMM and TM binding

ND

ND

Reduced SMM binding

Abolished SMM binding

ND ND ND

Reduced SMM binding

Reversed inhibition of SMM ATPasec

Reduced SMM Binding

NDb

Functional consequences

Equal phosphorylation in solution and in thin filaments

Using recombinant N-terminal CaD fragment

Equal phosphorylation in solution and in thin filaments

Comments

20

59

58

57

56

55

54

48

53

Ref.

64 Molecular Mechanisms of Smooth Muscle Contraction

Kinasea

PKC

PKC

PKC

PKC

PKC endogenous proteolytic fragment

p44 MAPK

p42 MAPK p44 MAPK

CaD source

Turkey gizzard

Porcine stomach

Chicken gizzard

Duck gizzard

Sheep aorta

Chicken gizzard

Porcine stomach >1.0

2.0

2.9

2.0

3.5-3.8

ND

1.9

molPi/ molCaD

ND

Thr-696 and/or Ser-702 or Thr-470 and/or Ser-582 Less: Ser-667, Thr-673

Ser-600 and/or Ser-702 127

C-terminus

35 kDa C-terminal fragment of SMM ATPase

Ser-587, Ser-600

Ser-587, Ser-726


Table 5.1. Caldesmon phosphorylation in vitro, cont.

ND

Attenuated F-actin binding but not Tm and CaM

Reversed inhibition of skletal HMM ATPase, reduced F-actin/TM binding and bindinginhibition coupling

ND

Reduced F-actin, CaMe binding; reversed inhibition

ND

Reversed inhibition of skeletal HMMd ATPase


64

63

62

61

Ref.

Phosphopeptide maps were identical for both kinases

66

Phosphorylation is blocked 65 by CaM, attenuated by F-actin and TM; both Ser and Thr are phophorylated

Corresponding to sequence of Bryan et al. 10

No influence on CaM binding

Corresponding to sequence of Bryan et al. 10

60

Comments

Caldesmon Phosphorylation and Smooth Muscle Contraction 65

4.0 1.2 Ser-667, Thr-673. Less: Thr-696, Ser-702, Ser-582

ND

Ser-702, Ser-?

ND


Reduced F-actin binding

ND

ND

Reduced inhibition of F-actin sliding velocity


68

67

Ref.

Phosphorylation is blocked 69 by F-actin, CaM

Ser-? belongs to sequence VTS*PTKV not present in chicken CaD

In in vitro motility assay

Comments

aFor kinase abbreviation see section 5.5; bND, not detertmned; cHere and below actin-activated Mg2+-ATPase activity; dHeavy meromyosin oCa2+ -CaM

cdc2

cdc2

Chicken gizzard

2.0

p44 MAPK

Porcine stomach

0.7

p44 MAPK

Turkey gizzard

molPi/ molCaD

Kinasea

CaD source

Table 5.1. Caldesmon phosphorylation in vitro, cont.

66 Molecular Mechanisms of Smooth Muscle Contraction


67

Lowering free cytoplasmic Ca2+ concentration leads to the relaxation of smooth muscle, accompanied by dephosphorylation of myosin heads and reinstatement of the CaD-TM inhibitory position on the thin filament following CaM dissociation. Although the proposed action of CaD functionally resembles that of troponin in striated muscle, the observed structural events are totally different. In the absence of Ca2+, CaD shifts TM in the opposite direction to what has been shown for troponin. Thus, instead of sterically blocking strong myosin-binding sites on actin, CaD rolls TM away from them. In the presence of Ca2+, the TM-CaD complex moves back and appears to flank the strong binding sites.49,50 The interpretation of this structural data calls for the assumption that CaD-TM modifies the structure of actin monomers rather than merely hinders an actomyosin interaction. According to the scheme in Figure 5.2, CaM plays the central role in CaD regulation. This had been appreciated in the very first reports devoted to this protein. Later its involvement was questioned because of unphysiologically high concentrations of CaM required to relieve CaD induced SMM ATPase inhibition.14 The search for an alternative Ca2+ binding regulator, however, failed to provide any candidate protein abundant enough in the smooth muscle to substitute for CaM. The recent findings that CaM interacts 10 times more strongly with CaD purified without heat treatment,51 and that other smooth muscle proteins (gelsolin and filamin) improve this interaction,52 return to CaM all credit as the most probable Ca2+ sensitizer of CaD in vivo. In the times of CaM depreciation, the regulation of CaD activity by phosphorylation has been extensively studied. It will be described in the following sections.

Caldesmon Phosphorylation In Vitro Several protein kinases phosphorylate CaD in vitro (Table 5.1 and Fig. 5.1). They demonstrate preferences with regard to location of the phosphorylation site within the CaD molecule. All the sites for protein kinase C (PKC), p34 cdc2 kinase (cdc2) and p42/p44 mitogen activated protein kinases (MAPK) were mapped to the C-terminal part of CaD, mostly in domain 4. Ca2+-CaM-dependent protein kinase II ( CaM PK II) phosphorylates sites both in the C-terminal and N-terminal parts of CaD. Casein kinase II (CK II) was found to be entirely tropic to domain 1. A 39-42 kDa endogenous PKC proteolytic fragment phosphorylated CaD at a somewhat different array of sites than a parent kinase. It is known that proteolysis may alter substrate specificity of PKC.70 Due to the interspecies variations in CaD sequence (which include the absence of a particular Ser or Thr residue and the unfavorable amino acid context around a putative phosphorylation site), the location of phosphorylated residues is not the same in CaD from different sources. Phosphorylation of gizzard CaD53 and pig arterial CaD14 by CaM PKII is an example. Likewise, l-CaD from human platelets is phosphorylated by cAMP dependent protein kinase (PKA)71 while gizzard CaD is not (Vorotnikov AV, unpublished results). Since h-CaD is assumed to include all the primary sequence of l-CaD, it is possible that in h-CaD of human origin PKA phosphorylation may, indeed, take place. Identification of the phosphorylated residues was usually achieved by direct phosphopeptide sequencing. In a number of studies, mutations of the putative phosphorylation sites of CaD were created to verify whether they, in fact, are phosphorylated by a particular kinase.33,72 This approach is informative and preferable when the natural phospho-CaD can not be recovered in analytical quantities. However, it is indirect and may not be as sensitive as the conventional method.33 Initial reports claimed CaD to be a self-kinase,73 but this has not been confirmed. The kinase activity copurifying with CaD was later identified as CAM PK II.74 Besides, examination of CaD sequence shows no consensus motifs characteristic for serine/threonine and tyrosine protein kinases.10

68


In spite of the differences mentioned, it could be concluded that CaD is the substrate for at least 5 serine/threonine protein kinases in vitro and therefore has multiple phosphorylation sites. These sites demonstrate an endwise clustering in the CaD molecule, being mainly concentrated in domain 4 while domain 1 contains few of them. No phosphorylation in the TM binding domain 2 has been reported. Phosphorylation of CaD alters its activity. As shown in Table 5.1, phosphorylation in the C-terminal part of CaD attenuates its interactions with actin-TM, CaM and SMM. The inhibitory action of CaD over myosin ATPase is reversed. The most profound effect on CaD is apparently produced by the cdc2 kinase from mitotic cells, which incorporated up to 7 phosphate residues in the C-terminus and virtually blocked all the functions of domain 4.27 Although both cdc2 and MAPK may phosphorylate the same sites in CaD in vitro, their preferences are different. cdc2 kinase primarely modifies Ser667 and Thr673, whereas MAPK kinase preferentially phosphorylates Ser and Thr residues from the following set: Thr696, Ser702, Thr470, Ser582. Phosphorylation of Ser702 has been confirmed in vivo (see below). Probably due to these differences, the functional consequences of kinase action may not be similar. In contrast to cdc2, MAPK phosphorylation only slightly inhibited F-actin binding and did not affect CaD-CaM interaction.65 It is interesting to note that the reverse is not always true. The presence of Ca2+-CaM completely inhibited CaD phosphorylation by MAPK regardless of whether CaD existed in solution or it was bound to F-actin.65 Thus, Ca2+-CaM binding seems to induce a profound conformational alteration in the CaD molecule which makes the potential phosphorylation sites for MAPK unavailable. Apparently, in the threedimensional structure of CaD, MAPK sites are positioned in between CaM attachment points and their phosphorylation does not prevent CaM binding. Hence, MAPK and Ca2+-CaM may cooperate in relieving CaD inhibition of SMM ATPase. In our opinion, the most important alteration in CaD behavior related to MAPK phosphorylation was noted by Redwood et al.72 They substituted Ser702 in a recombinant CaD fragment with Asp in order to mimic the effects of phosphorylation. The mutant fragment expressed a reduced ability to interact with actin at the low capacity, high affinity TM dependent binding sites (see previous section). In agreement with these results, the mutant CaD fragment had an attenuated potency to inhibit actin-TM-activated myosin ATPase.72 In another study, CaD phosphorylated by MAPK was shown to exert a reduced inhibition on the actin-TM filament sliding velocity in in vitro motility assay.67 The uncoupling of CaD binding to actinTM from ATPase inhibition was also proposed to take place as a result of CaD phosphorylation by a PKC fragment.64 It remains to be established whether other caldesmon kinases produce the same effect. Functionally, it would be reasonable for smooth muscle CaD to regulate ATPase without being totally released from the thin filament, as is shown in Figure 5.2. A very different aim is apparently achieved by cdc2 phosphorylation of l-CaD in nonmuscle cells during mitosis. In this situation, l-CaD has to be temporarily prevented from participating in the motile events and cytoskeleton integration.45 Consequently, a more profound phosphorylation of lCaD is required.27 Phosphorylation at the N-terminus by CK II and CaM PK II most consistently affects Ser73 in chicken CaD or analogous residues in other animals (Fig. 5.1). As a result of phosphorylation, CaD-SMM and CaD-TM interaction are reduced. Apparently, Ser73 is positioned in a critical region of the myosin-binding site.19 Phosphorylation of both Ser26 and Ser73 by CaM PK II almost completely abolishes the binding of SMM to CaD domain 1.48 In vitro, CaM PK II may also phosphorylate the same sites in the C-terminus of CaD as PKC, e.g., Ser587, Ser726 (see Fig. 5.1).53 Although it has not been shown directly, the available evidence suggests that this phosphorylation de-inhibits SMM ATPase.


69

Table 5.2. Caldesmon phosphorylation in smooth muscle Muscle origin

Porcine carotid artery

Same

Same

Canine colonic muscle

Basal level (molPi/mol CaD)

Stimulant/ duration (min)

Level at stimulation molPi/mol CaD)

Tension (% of maximal)

Level at relaxation (molPi/mol CaD)

0.57

0.48

KCIa/1

0.57

27b

0.63 0.63 0.51 0.65 0.53 0.69 0.7 0.45 0.45 0.45

KCI/5 KCI/60 100µM Histc/5 100µM Hist/60 100µM NEd/2 100µM NE/60 8µM PDBue/60 KCI/15 KCI/60 1µM PDBu/60

0.83 0.88 0.59 0.84 0.65 0.87 1.64 0.8 0.96 1.08

MD 73 83 100 13 5 60 51 100 75

0.45 0.45 0.45

10µM Hist/60 1µM ouabain/60 ?µM carbaCHOl/60

69 57 0

0.3 0.3

50nM Ag-II / 10 50nM Ag-II/ 60

0.3 0.3 0.3 0.6

50nM ET-1g/ 10 50nM ET-1/ 60 KCI/60 10 µM Ach/2

0.93 1.08 0.54 Contraction is over at 60 Min 0.4 0.6 Contraction is over at 60 min 0.3 0.5 0.6 1.1

Ref.

81

ND 1.01 .057 0.69 0.55 0.87 0.71 0.6 82 ND No relaxation after PDBu removal ND ND 0.54

54 0

ND 0.6

61 46 88 80

ND ND N ND

83

67

a109-110 mM KCI bpercent of maximum contraction produced by the most potent stimulant81,82 or a particular stimulant.83,67 cHistamine. dNorepinephrine. ePhorbol 12, 13-dibutyrate. fAngiotensin-II. gEndothelin-1. hAcetylcholine.

70


The overview of CaD phosphorylation in vitro allows formulation of several projections into the pertinent to smooth muscle. 1. Domain 4 of CaD, which is involved in the control of actin-TM-activated myosin ATPase, is phosphorylated by at least four kinases (cdc2, PKC, CaM PK II, MAPK) which act to reduce its inhibitory action on ATPase and its binding to actin. Therefore, CaD phosphorylation in domain 4 by at least some kinases should be related to the force output in the smooth muscle. However, this relation may not be as straightforward as, for instance, that between SMM phosphorylation by MLCK and tension development. 2. CaD has a capability to crosslink thin and thick filaments by means of its myosinbinding and actin-TM-binding sites in domains 1 and 4, respectively (Fig. 5.2, top panel). Such nonproductive linkages were proposed to contribute to the latching of smooth muscle and/or to play an integrative role.46 Phosphorylation at these domains may reduce CaD interaction with either filament system and attenuate latching or increase filament disorder. 3. All of the above mentioned protein kinases, except perhaps cdc2 kinase,68 are abundant in differentiated, quiescent smooth muscle. They are involved in different signal transduction pathways and, hence, stay under control of different receptors and second messengers (Ca2+, DAG, tyrosine phosphorylation). Nevertheless, the cross-talk between alternative pathways in living muscle makes them all interrelated in their action on CaD. CaM may cooperate with some of the kinases (MAPK, CK II) in its effect on CaD, since CaD phosphorylation does not affect its binding. Other kinases (PKC?, cdc2) may prevent CaM involvement. Thus, depending on the nature of the stimulus, the set of kinases modifying CaD may be different. Casein kinase II has no known bona fide endogenous regulators, although phosphorylation and/or compartmentalization are suspected. 75 CK II might be constantly active, and the fate of CaD phosphorylation by CK II (as well as by other protein kinases) would depend on the activity of phosphatases. 4. Finally, the wide variety of protein kinases implicated in regulation of CaD functional features in vitro indirectly suggests that this regulation has to occur under many different circumstances. Investigations of CaD phosphorylation in vivo initiated in 1989 proved it to be a complicated problem. Investigations of CaD phosphorylation in vivo initiated in 1989 proved it to be a complicated problem.

Phosphorylation of Caldesmon in Smooth Muscle Tissues Using permeabilized muscle tissue techniques, CaD was demonstrated to influence contractility. Exogenously added CaD and a C-terminal CaD fragment reduced isometric tension in chemically skinned smooth and skeletal muscle fibers and increased the rate of smooth muscle relaxation.76-80 A short CaD peptide, Gly651 to Ser667, which included the sequence of CaM binding site A (see Fig. 5.1) was shown to induce a dose-dependent contraction of the skinned arterial smooth muscle cells at pCa 7.0.32 Apparently, this peptide competed with the corresponding sequence of CaD for actin-TM binding and displaced CaD from its inhibitory position on the thin filament, as would CaM at high Ca2+. In agreement with this suggestion, pretreatment of cells with CaM or elevation of Ca2+ above pCa 6.6 abolished the peptide effects. These experiments provided the first indication that CaD may be responsible for the inhibition of the basal vascular tone and suggested that CaM-based regulation of CaD may, indeed, be possible in the smooth muscle. A number of studies have addressed the issue of CaD phosphorylation in smooth muscle tissue in order to discern the patterns of CaD modification with regard to the type and


71

Fig. 5.3. Two-dimensional phosphopeptide map analysis of caldesmon phosphorylation in gizzard smooth muscle. Purified chicken gizzard CaD (0.5 mg/ml) was phosphorylated by 1 µM activated recombinant p44 GST-MAPK to a stoichiometry of 1.4 mol Pi/mol CaD in buffer containing 10 mM MOPS (pH 7), 50 mM NaCl, 4 mM MgCl2, 0.1 mM EGTA, 0.5 mM o-vanadate, 120 µM [γ-32P]ATP and 1 mM dithiothreitol. Reaction was stopped by the addition of SDSsample buffer. Chicken gizzard muscle strips (4 x 2 x 0.4 mm) incubated in Hank’s solution saturated with carbogen were labelled with [γ-32P] orthophosphate (0.125 mCi/ml) for 6 hours at 37˚C. Strips were stimulated by the addition of 2 µM PDBu and then frozen in liquid nitrogen at specified times. The tissue strips were pulverized while frozen and extracted with standard RIPA buffer at 90˚C for 5 min. CaD was immunoprecipitated using polyclonal caldesmon antibodies21 and separated on a 7% SDS gel. CaD bands were excised and, together with the caldesmon phosphorylated by MAPK in vitro, were further processed for phosphopeptide mapping. Autoradiography was as described in ref. 68 with minor modifications. The identification of the phosphorylated spots was performed by comapping of in vivo phosphorylated samples with those phosphorylated in vitro by purified protein kinases (comaps are not shown). See text for discussion of results.

duration of stimulation and dynamics of force (Table 5.2). Several generalizations can be made upon analysis of these reports: 1. A certain level of CaD phosphorylation is maintained in smooth muscle at rest. It comprises about a half of the maximum level achieved upon tissue stimulation. 2. Both hormonal receptor-mediated stimulation of smooth muscle and depolarization by KCI produce an increase in CaD phosphorylation ranging from the very modest to twice as high as the basal level. CaD phosphorylation is more pronounced following longer exposures to the stimulant. 3. There is no consistent correlation between CaD phosphorylation and the state of smooth muscle contraction. The latter statement requires some comment. There is a great deal of controversy in the literature about the time course of CaD phosphorylation and force output. While Adam et

72


Fig. 5.4. Time course of caldesmon phosphorylation in chicken gizzard in response to PDBu. CaD was immunoprecipitated from chicken gizzard strips prepared as described in the legend to Figure 5.3. The relative [32P] phosphate incorporation was determined by SDS-PAGE, autoradiography and densitometry of CaD bands and normalized by protein loading. Representative results are shown.

al82 observed a delayed decrease in CaD phosphorylation following the washout of KCI, Barany et al81 reported a continuous buildup of CaD phosphorylation after a 60 min KCI stimulation of porcine arterial tissue. Abe et al found no increase of CaD phosphorylation in KCI-induced sustained contraction of porcine coronary artery.84 Gerthoffer and Pohl stated that CaD phosphorylation in canine tracheal smooth muscle followed the time course of myosin phosphorylation, stress and intracellular free Ca2+ concentration.85 On the other hand, evidence has been presented that angiotensin II may elicit only transient contraction but sustained CaD phosphorylation (Table 5.2).83 A consensus has been reached that the stimulation of smooth muscle with phorbol 12,13-dibutyrate (PDBu) produces a substantial and reversible increase in CaD phosphorylation. PDBu is known to elicit a slow developing, strong contraction. Likewise, it takes several hours to wash this agent out of the muscle.81 This, is a possible explanation of why Adam et al were unable to relax PDBu-contracted muscle preparations in their experiments.82 Upon PDBu stimulation, about one mole of phosphate was incorporated per mole of CaD above the background level. One-dimensional phosphopeptide maps of phosphorylated CaD in resting smooth muscle and muscle stimulated with KCI and PDBu were similar, but different from the maps of CaD phosphorylated in vitro by PKC and CaM PK II.82 Direct peptide sequencing demonstrated that CaD is phosphorylated at two sites in canine aortic strips stimulated for 30 min by 1 µM PDBu. These sites were Val-Thr-Ser*-Pro-Thr-Lys-Val and Asn-LysSer*-Pro-Ala-Pro-Lys, with an asterisk indicating a phosphorylated residue.61 Sequence alignment revealed that both sites are present in mammalian CaD, but only the second site corresponded to the chicken CaD sequence encompassing Ser702 (see Fig. 5.1). The


73

presence of a Pro residue near the phosphorylated Ser pointed to MAPK as the responsible kinase, while another proline-directed kinase, cdc2, was ruled out because of its preference for other residues in vitro, e.g., Ser667 and Thr673 (Table 5.1). Besides, cdc2 has not been detected in differentiated smooth muscle,68 whereas MAPK concentration in this tissue is rather high, approximately 3 µM.86

The Complex Case of In Vivo Caldesmon Kinase MAPK has attracted a lot of attention in recent years, since it was shown to be activated in response to naturally occurring smooth muscle contractile stimuli. Both p42 MAPK and p44 MAPK isoenzymes are activated on the challenging of smooth muscle with hormones and pharmacological agents, membrane depolarization and mechanical stress.66,67,87 On a molecular level, MAPK activation requires it to be double phosphorylated at both Thr and Tyr residues by a MAPK kinase. 88 Thus, MAPK involvement includes tyrosine phosphorylation in an already complex scheme of regulation of smooth muscle contraction. MAPK was demonstrated to undergo a distinct redistribution within sarcoplasm following agonist-induced contraction of smooth muscle cells. Its initial accumulation and activation at the plasma membrane requires PKC activity.89 Hence, the elevation of free intracellular Ca2+ concentration by a variety of means, including membrane depolarization and hormonal receptor stimulation, will activate PKC and its downstream targets, such as MAPK. PDBu may directly activate PKC regardless of Ca2+ and promote MAPK activation as well. Once activated, MAPK translocates to the contractile system and this redistribution is dependent upon tyrosine phosphorylation. In contracting cells, MAPK is colocalized with CaD.89 Several laboratories have presented evidence that CaD can be phosphorylated by active MAPK isolated from smooth muscle.66,67,87 Most importantly, MAPK phosphorylates the same sites in CaD as those identified in vivo.68 The assignment of MAPK as an endogenous caldesmon kinase has partially explained the controversial behavior of CaD phosphorylation in smooth muscle. CaD kinase itself has demonstrated a variable relationship to muscle tension. On the one hand, its activity was shown to correlate with the stress development in arterial tissue.87 On the other, it remained high and unchanged during the contraction-relaxation cycles of the phasic gut smooth muscle. 67 Moreover, KCI induced a transient increase in MAPK activity, whereas CaD phosphorylation level and force were sustained.66 Given a complex path for enzyme activation via the Ras/Raf/MAPK kinase cascade, and involvement of Ca2+-dependent and independent PKC pathways, MAPK might not be equally activated and translocated in response to different stimuli. Consequently, this would be reflected in the downstream MAPK targets such as CaD. Accordingly, a potent and direct activator of PKC, such as PDBu, will induce the most prominent MAPK activation and CaD phosphorylation (Table 5.2). Finally, it is worth mentioning that MAPK activity in intact animal arterial vessels frozen immediately after dissection was low, while in the same vessels ready for experiment it was 10-fold higher.66 Many explanations may be suggested for the current variability of experimental results concerning MAPK and CaD phosphorylation in vivo. Only one thing remains certain: MAPK is, indeed, an in vivo CaD kinase. The direct effect of endogenous CaD phosphorylation within skinned smooth muscle fibers was assessed by the addition of exogenous MAPK, and again controversial results were obtained. MAPK either did not alter muscle contraction and its Ca2+ sensitivity in vascular tissue,86 or it potentiated Ca2+-induced contraction in permeabilized airway smooth muscle90. The latter result is in agreement with the activating effect of CaD phosphorylation by MAPK on actin-TM filament sliding velocity in in vitro motility assay67 and with MAPK’s ability to reverse the cooperative inhibition of acto-TM-activated myosin ATPase by CaD in

74


vitro.72 The former suggests that MAPK may affect other CaD functions, not directly related to regulation of contraction, such as cytoskeleton integration.45,46

Future Prospects Experimental evidence does not clearly demonstrate the involvement of CaD phosphorylation by MAPK as a general phenomenon in regulation of smooth muscle tension. However, across the performed studies, the stimulation of smooth muscle consistently increased the level of CaD phosphorylation (Table 5.2). Additional work is required to unveil its functional significance. Perhaps, returning attention to the alternative kinases implicated in in vitro phosphorylation of CaD (Table 5.1) may be promising. Is there any basis for considering that phosphorylation of CaD other than by MAPK exists in vivo? We believe that the evidence presented below provides a positive answer to this question: 1. The discrepancy between a sustained CaD phosphorylation and a transient MAPK activation in arterial muscle depolarized by KCI has been noted by Adam et al, raising the speculation that another proline-directed kinase may be involved.66 2. Using an in-gel activity assay, Gerthoffer et al revealed a substantial CaD phosphorylating activity recovered from the acetylCHOline-stimulated colonic muscle. This activity was unrelated to p42 MAPK and p44 MAPK both by mobility in electrophoresis and the lack of cross-reactivity with anti-MAPK antibodies.67 Our data also indicate the presence of a 50-58 kDa CaD-kinase in smooth muscle homogenates that can be monitored by in-gel assay. This kinase activity is inhibited in vivo by the MAPK inhibitor PD98059 (Vorotnikov AV, Krymsky MA, Chibalina MV, Shirinsky VP, manuscript in preparation). 3. Other investigators have succeeded in isolating tightly CaD-bound and abundant smooth muscle protein kinases which are active towards CaD in vitro.58,74 4. Finally, two-dimensional phosphopeptide mapping of CaD immunoprecipitated from [32 P] labeled tissue directly demonstrates that more than one kinase phosphorylates CaD in smooth muscle (Vorotnikov AV, Krymsky MA, Chibalina MV, Shirinsky VP, manuscript in preparation). In resting unloaded chicken gizzard muscle, CaD is appreciably phosphorylated (Fig. 5.3, bottom left). The phosphopeptide distribution pattern is similar to that of purified CaD phosphorylated in vitro by a recombinant p44 MAPK (Fig. 5.3, top left). Three major spots are resolved (labelled 1-3). Following PDBu addition, the CaD phosphorylation pattern undergoes distinct changes. At 15 min of stimulation the relative labelling of MAPK-related spots 1 and 2 is altered. Also, several additional spots (A-C) appear, which are not present in the MAPK-treated CaD even after extensive phosphate incorporation (Fig. 5.3, top right). Spot 4, which was minor in resting muscle, now increases. Altogether, spots A-C and 4 account for approximately one-half of the total CaD labelling. At 30 min of stimulation, spots A-C and 4 continue to persist, although their relative intensity is changed. The MAPK-induced phosphorylation pattern becomes clearly dominant at this time (Fig. 5.3, bottom right). Preliminary results indicate that spot 4 may be attributable to CK II. The identity of the kinase responsible for spots A-C is not clear. However, according to the phosphopeptide map comparison, it is not PKC or CK II (data not shown). The involvement of additional kinase(s) is also supported by the time course of CaD phosphorylation in gizzard strips stimulated by PDBu (Fig. 5.4). A biphasic rather than a monotonic type of phosphate incorporation suggests a superposition of several kinase activities modifying CaD at different times after the beginning of stimulation. Figures 5.3 and 5.4 represent a paradigm that has probably been overlooked in the previous studies. We confirm the findings of Adam et al that in both unstimulated and long term


75

PDBu-stimulated smooth muscle, phosphorylation of CaD by MAPK is a prevailing event.61,68,82 However, in between these time points there is a significant change in the CaD phosphorylation pattern. Apparently, at least one more kinase phosphorylates CaD at 15 min of stimulation, and its contribution could hardly be neglected. Interestingly, PDBu appears to stimulate this new kinase activity, which is distinct from PKC. Work is in progress to identify a novel in vivo CaD kinase. This is additionally encouraged by a recent report demonstrating that CaD may be an in vivo substrate for a tyrosine kinase.91 It is tempting to wonder how CaD phosphorylation patterns induced by the short term ac tion of various natural agonists compare with each other. The discovery of more CaD kinases may be expected along the way. Further studies are also needed to correlate the non-MAPK dependent CaD phosphorylation and force development in the smooth muscle. The door is not closed. The proof of caldesmon is in experimenting.

Acknowledgments We thank Drs. M.A. Krymsky, M.V. Chibalina and A.V. Lapshin for assistance in obtaining experimental results presented in this review. This work was supported in part by HHMI International Scholar grant 75195-546901 (VPS), Welcome Trust collaboration grants (AVV, NBG), grants from the Russian Fund for Fundamental Research N 95-04-12260 (VPS), N 96-04-49106 (AVV) and RFFI (NBG).

References 1. Sobue K, Muramoto Y, Fujita M, Kakiuchi S. Purification of a calmodulin-binding protein from chicken gizzard that interacts with F-actin. Proc Natl Acad Sci USA 1981; 78:5652-5655. 2. Marston SB, Huber PAJ. Caldesmon. In: Barany M, ed. Biochemistry of smooth muscle contraction. San Diego: Academic Press, 1996:77-90. 3. Ngai PK, Walsh MP. Inhibition of smooth muscle actin-activated myosin Mg2+-ATPase activity by caldesmon. J Biol Chem 1984; 259:13646-13659. 4. Dabrowska R, Goch A, Galazkiewicz B, Osinska H. The influence of caldesmon on ATPase activity of the skeletal muscle actomyosin and bundling of actin filaments. Biochim Biophys Acta 1985; 842:70-75. 5. Marston SB, Lehman W. Caldesmon is a Ca2+-regulatory component of native smoothmuscle thin flaments. Biochem J 1985; 231:517-522. 6. Bretscher A. Smooth muscle caldesmon. Rapid purification and F-actin cross linking properties. J Biol Chem 1984; 259:12873-12880. 7. Graceffa P. Evidence for interaction between smooth muscle tropomyosin and caldesmon. FEBS Lett 1987; 218:139-142. 8. Ikebe M, Reardon S. Binding of caldesmon to smooth muscle myosin. J Biol Chem 1988; 263:3055-3058. 9. Hemric ME, Chalovich JM. Characterization of caldesmon binding to myosin. J Biol Chem 1990; 265:19672-19678. 10. Bryan J, Imai M, Lee R et al Cloning and expression of a smooth muscle caldesmon. J Biol Chem 1989; 264:13873-13879. 11. Hayashi K, Kanda K, Kimizuka F et al. Primary structure and functional expression of h-caldesmon complementary DNA. Biochem Biophys Res Commun 1989; 164:503-511. 12. Dingus J, Hwo S, Bryan J. Identification of monoclonal antibodies and characterization of human platelet caldesmon. J Cell Biol 1986; 102:1748-1757. 13. Ueki N, Sobue K, Kanda K et al. Expression of high and low molecular weight caldesmons during phenotypic modulation of smooth muscle cell. Proc Natl Acad Sci USA 1987; 84:9049-9053.

76


14. Marston SB, Redwood CS. The molecular anatomy of caldesmon. Biochem J 1991; 279:1-16. 15. Haruna M, Hayashi K, Yano H et al. Common structural and expressional properties of vertebrate caldesmon genes. Biochem Biophys Res Commun 1993; 197:145-153. 16. Humphrey MB, Herrera-Sosa H, Gonzales G et al. Cloning of cDNA encoding human caldesmon. Gene 1992; 112:197-204. 17. Hayashi K, Yano H, Hashida T et al. Genomic structure of the human caldesmon gene. Proc Natl Acad Sci USA 1992; 89:12122-12126. 18. Fraser IDC, Copeland O, Bing W et al. The inhibitory complex of smooth muscle caldesmon with actin and tropomyosin involves three interacting segments of the C-terminal domain 4. Biochemistry 1997; 36:5483-5492. 19. Vorotnikov AV, Marston SB, Huber PAJ. Location and functional characterization of myosin contact sites in smooth muscle caldesmon. Biochem J 1997; 328:211-218. 20. Bogatcheva NV, Vorotnikov AV, Birukov KG et al. Phosphorylation by casein kinase II affects the interaction of caldesmon with smooth muscle myosin and tropomyosin. Biochem J 1993; 290:437-442. 21. Shirinsky VP, Biryukov KG, Vorotnikov AV et al. Caldesmon150, caldesmon77 and skeletal muscle troponin T share a common antigenic determinant. FEBS Lett 1989; 251:65-68. 22. Birukov KG, Shirinsky VP, Vorotnikov AV et al. Competitive binding of the troponin T-specific pool of caldesmon antibodies and tropomyosin to skeletal troponin T and smooth muscle caldesmon. FEBS Lett 1990; 262:263-265. 23. Hayashi K, Fujio Y, Kato I et al. Structural and functional relationship between h- and l-caldesmon. J Biol Chem 1991; 266:355-361. 24. Marston SB, Fraser IDC, Huber PAJ et al. Location of two contact sites between human smooth muscle caldesmon and Ca2+-calmodulin. J Biol Chem 1994; 269:8134-8139. 25. Hnath EJ, Wang C-LA, Huber PAJ et al. Affinity and structure of complexes of tropomyosin and caldesmon domains. Biophys J 1996; 71:1920-1933. 26. Leszyk J, Mornet D, Audemard E et al. Amino acid sequence of a 15 kilodalton actinbinding fragment of turkey gizzard caldesmon: similarity with distrophin, tropomyosin, and the tropomyosin-binding region of troponin T. Biochem Biophys Res Commun 1989; 160:210-216. 27. Yamakita Y, Yamashiro S, Matsumura F. Characterization of mitotically phosphorylated caldesmon. J Biol Chem 1992; 267:12022-2029. 28. Levine BA, Moir AJG, Audemard E et al. Structural study of gizzard caldesmon and its interaction with actin. Binding involves residues of actin also recognized by myosin subfragment 1. Eur J Biochem 1990; 193:687-696. 29. Czurylo EA, Venyaminov SY, Dabrowska R. Studies on the secondary structure of caldesmon and its C-terminal fragments. Biochem J 1993; 293:363-368. 30. Shirinsky VP, Bushueva TL, Frolova SI. Caldesmon-calmodulin interaction. Study by the method of protein intrinsic tryptophan fluorescence. Biochem J 1988; 255:203-208. 31. Sobue K, Sellers JR. Caldesmon, a novel regulatory protein in smooth muscle and nonmuscle actomyosin systems. J Biol Chem 1991; 266:12115-12118. 32. Katsuyama H, Wang C-LA, Morgan KG. Regulation of vascular smooth muscle tone by caldesmon. J Biol Chem 1992; 267:14555-14558. 33. Yamashiro S, Yamakita Y, Yoshida K et al. Characterization of the COOH terminus of nonmuscle caldesmon mutants lacking mitosis-specific phosphorylation sites. J Biol Chem 1995; 270:4023-4030. 34. Wang Z, Yang Z-Q, Chacko S. Functional and structural relationship between the calmodulin-binding, actin-binding, and actomyosin-ATPase inhibitory domains on the C-terminus of smooth muscle caldesmon. J Biol Chem 1997; 272:16896-16903. 35. Furst DO, Cross RA, De Mey J et al. Caldesmon is an elongated, flexible molecule localized in the actomyosin domains of smooth muscle. EMBO J 1986; 5:251-257. 36. Mabuchi K, Li Y, Tao T et al. Immunocytochemical localization of caldesmon and calponin in chicken gizzard smooth muscle. J Muscle Res Cell Motil 1996; 17:243-260. 37. Marston SB. Stoichiometry and stability of caldesmon in native thin filaments from sheep aorta smooth muscle. Biochem J 1990; 272:305-310.


77

38. Shirinsky VP, Biryukov KG, Hettasch JM et al. Inhibition of the relative movement of actin and myosin by caldesmon and calponin. J Biol Chem 1992; 267:15886-15892. 39. Kashiwada K, Nishida W, Hayashi K et al. Coordinate expression of a-tropomyosin and caldesmon isoforms in association with phenotypic modulation of smooth muscle cells. J Biol Chem 1997; 272:15396-15404. 40. Mabuchi K, Lin JJ-C, Wang CL-A. Electron microscopic images suggest both ends of caldesmon interact with actin filaments. J Muscle Res Cell Motil 1993; 14:54-64. 41. Katayama E, Ikebe M. Mode of caldesmon binding to smooth muscle thin filament: Possible projection of the amino-terminal of caldesmon from native thin filament. Biophys J 1995; 68:2419-2428. 42. Marston SB, Pinter K, Bennett P. Caldesmon binds to smooth muscle myosin and myosin rod and crosslinks thick filaments to actin filaments. J Muscle Res Cell Motil 1992; 13:206-208. 43. Graceffa P. Arrangement of the COOH-terminal and NH2-terminal domains of caldesmon bound to actin. Biochemistry 1997; 36:3792-3801. 44. Birukov KG, Stepanova OV, Shirinsky VP et al. Stretch affects phenotype and proliferation of vascular smooth muscle cells. Molec Cell Biochem 1995; 144:131-139. 45. Yamashiro S, Matsumura F. Mitosis-specific phosphorylation of caldesmon: Possible molecular mechanism of cell rounding during mitosis. BioEssays 1991; 13:563-568. 46. Hemric ME, Tracy PB, Haeberle JR. Caldesmon enhances the binding of myosin to the cytoskeleton during platelet activation. J Biol Chem 1994; 269:4125-4128. 47. Redwood CS, Marston SB, Bryan J et al. The functional properties of full length and mutant chicken gizzard smooth muscle caldesmon expressed in Escherichia coli. FEBS Lett 1990; 270:53-56. 48. Hemric ME, Lu FWM, Shrager R et al. Reversal of caldesmon binding to myosin with calcium-calmodulin or by phosphorylating caldesmon. J Biol Chem 1993; 268:15305-15311. 49. Vibert P, Craig R, Lehman W. Three-dimensional reconstruction of caldesmon-containing smooth muscle thin filaments. J Cell Biol 1993; 123:313-321. 50. Hodgkinson JL, Marston SB, Craig R et al. Three dimensional image reconstruction of reconstituted smooth muscle thin filaments: Effects of caldesmon. Biophys J 1997; 72:2398-2404. 51. Zhuang S, Mabuchi K, Wang C-LA. Heat treatment could affect the biochemical properties of caldesmon. J Biol Chem 1996; 271:30242-30248. 52. Gusev NB, Pritchard K, Hodgkinson JL et al. Filamin and gelsolin influence Ca2+-sensitivity of smooth muscle thin filaments. J Muscle Res Cell Motil 1994; 15:672-681. 53. Ikebe M, Reardon S. Phosphorylation of smooth muscle caldesmon by calmodulindependent protein kinase II. J Biol Chem 1990; 265:17602-17612. 54. Ngai PK, Walsh MP. The effects of phosphorylation of smooth-muscle caldesmon. Biochem J 1987; 244:417-425. 55. Sutherland C, Walsh MP. Phosphorylation of caldesmon prevents its interaction with smooth muscle myosin. J Biol Chem 1989; 264:578-583. 56. Vorotnikov AV, Shirinsky VP, Gusev NB. Phosphorylation of smooth muscle caldesmon by three protein kinases: Implication for domain mapping. FEBS Lett 1988; 236:321-324. 57. Sutherland C, Renaux BS, McCay DJ et al. Phosphorylation of caldesmon by smooth muscle casein kinase II. J Muscle Res Cell Motil 1994; 15:440-456. 58. Vorotnikov AV, Gusev NB, Hua S et al. Identification of casein kinase II as a major endogenous caldesmon kinase in sheep aorta smooth muscle. FEBS Lett 1993; 334:18-22. 59. Wawrzynow A, Collins J, Bogatcheva NV et al. Identification of the site phosphorylated by casein kinase II in smooth muscle caldesmon. FEBS Lett 1991; 289:213-216. 60. Ikebe M, Hornick T. Determination of the phosphorylation sites of smooth muscle caldesmon by protein kinase C. Arch Biochem Biophys 1991; 288:538-542. 61. Adam LP, Gapinski CJ, Hathaway DR. Phosphorylation sequences in h-caldesmon from phorbol ester-stimulated canine aortas. FEBS Lett 1992; 302:223-226.

78


62. Tanaka T, Ohta H, Kanda K et al. Phosphorylation of high-Mr caldesmon by protein kinase C modulates the regulatory function of this protein on the interaction between actin and myosin. Eur J Biochem 1990; 188:495-500. 63. Vorotnikov AV, Gusev NB. Some properties of duck gizzard caldesmon. Biochem J 1991; 273:161-163. 64. Vorotnikov AV, Gusev NB, Hua S et al. Phosphorylation of aorta caldesmon by endogeneous proteolytic fragments of protein kinase C. J Muscle Res Cell Motil 1994; 15:37-48. 65. Childs TJ, Watson MH, Sangheras JS et al. Phosphorylation of smooth muscle caldesmon by mitogen activated protein (MAP) kinase and expression of MAP kinase in differentiated smooth muscle cells. J Biol Chem 1992; 267:22853-22859. 66. Adam LP, Franklin MT, Raff GJ et al. Activation of mitogen activated protein kinase in porcine carotid arteries. Circ Res 1995; 76:183-190. 67. Gerthoffer WT, Yamboliev IA, Shearer M et al.Activation of MAP kinase and phosphorylation of caldesmon in canine colonic smooth muscle. J Physiol (Lond) 1996; 495:597609. 68. Adam LP, Hathaway DR. Identification of mitogen activated protein kinase phopshorylation sequences in mammalian h-caldesmon. FEBS Lett 1993; 322:56-60. 69. Mak AS, Watson MH, Litwin CME et al. Phosphorylation of caldesmon by cdc2 kinase. J Biol Chem 1991; 266:6678-6681. 70. Nakabayashi H, Sellers JR, Huang KP. Catalytic fragment of protein kinase C exhibits altered substrate specificity toward smooth muscle myosin light chain. FEBS Lett 1991; 294:144-148. 71. Hettasch JM, Sellers JR. Caldesmon phosphorylation in intact human platelets by CaMP dependent protein kinase and protein kinase C. J Biol Chem 1991; 266:11876-11881. 72. Redwood CS, Marston SB, Gusev NB. The functional effects of mutations Thr-673 → Asp and Ser-702 → Asp at the Pro-directed kinase phosphorylation sites in the C-terminus of chicken gizzard caldesmon. FEBS Lett 1993; 327:85-89. 73. Scott-Woo GC, Walsh MP. Autophosphorylation of smooth muscle caldesmon. Biochem J 1988; 252:463-472. 74. Scott-Woo GC, Sutherland C, Walsh MP. Kinase activity associated with caldesmon is Ca2+/ calmodulin-dependent kinase II. Biochem J 1990; 268:367-370. 75. Sanghera JS, Charlton LA, Paddon HB et al. Purification and characterization of echinoderm casein kinase II. Regulation by protein kinase C. Canad Biochem J 1992; 283:829-837. 76. Szpacenko A, Wagner J, Dabrowska et al. Caldesmon-induced inhibition of ATPase activity of actomyosin and contraction of skinned fibers of chicken gizzard smooth muscle. FEBS Lett 1985; 192:9-12. 77. Taggart MJ, Marston SB. The effects of vascular smooth muscle caldesmon on force production by “desensitized” skeletal muscle fibres. FEBS Lett 1988; 242:171-174. 78. Brenner B, Yu LC, Chalovich JM. Parallel inhibition of active force and relaxed fiber stiffness in skeletal muscle by caldesmon: Implications for the pathway to force generation. Proc Natl Acad Sci USA 1991; 88:5739-5743. 79. Pfitzer G, Zeugner C, Troschka M et al. Caldesmon and a 20-kDa actin-binding fragment of caldesmon inhibit tension development in skinned gizzard muscle fiber bundles. Proc Natl Acad Sci USA 1993; 90:5904-5908. 80. Albrecht K, Schneider A, Liebetrau C et al. Exogenous caldesmon promotes relaxation of guinea-pig skinned taenia coli smooth muscles: Inhibition of cooperative reattachment of latch bridges? Pflugers Arch-Eur J Physiol 1997; 434:534-542. 81. Barany M, Polyak E, Barany K. Protein phosphorylation during the contraction-relaxationcontraction cycle of arterial smooth muscle. Arch Biochem Biophys 1992; 294:571-578. 82. Adam LP, Haeberle JR, Hathaway DR. Phosphorylation of caldesmon in arterial smooth muscle. J Biol Chem 1989; 264:7698-7703. 83. Adam LP, Milio L, Brengle B et al. Myosin light chain and caldesmon phosphorylation in arterial muscle stimulated with endothelin-1. J Mol Cell Cardiol 1990; 22:1017-1023.


79

84. Abe Y, Kasuya Y, Kudo M et al. Endothelin-1-induced phosphorylation of the 20-kDa myosin light chain and caldesmon in porcine coronary artery smooth muscle. Japan J Pharmacol 1991; 57:431-435. 85. Gerthoffer WT, Pohl J. Caldesmon and calponin phosphorylation in regulation of smooth muscle contraction. Can J Physiol Pharmacol 1994; 72:1410-1414. 86. Nixon GF, Iizuka K, Haystead CMM et al. Phosphorylation of caldesmon by mitogenactivated protein kinase with no effect on Ca2+ sensitivity in rabbit smooth muscle. J Physiol (Lond) 1995; 487:283-289. 87. Katoch SS, Moreland RS. Agonist and membrane depolarization induced activation of MAP kinase in the swine carotid artery. Am J Physiol 1995; 269:H222-H229. 88. Boulton TG, Yancopoulos GD, Gregory JS et al. An insulin stimulated protein kinase similar to yeast kinases involved in cell cycle control. Science 1990; 249:64-67. 89. Khalil RA, Menice CB, Wang C-LA, Morgan KG. Phosphotyrosine-dependent targeting of mitogen activated protein kinase in differentiated contractile vascular cells. Circ Res 1995; 76:1101-1108. 90. Gerthoffer WT, Yamboliev IA, Pohl J et al. Activation of MAP kinases in airway smooth muscle. AM J Physiol 1997; 272:L244-L252. 91. McManus MJ, Lingle WL, Salisbury JL et al. A transformation-associated complex involving tyrosine kinase signal adapter proteins and caldesmon links v-Erb B signaling to actin stress fiber disassembly. Proc Natl Acad Sci USA 1997; 94:11351-11356.

CHAPTER 6

Thick and Thin Filament Regulation of Smooth Muscle Contraction in Health and Disease Kathleen G. Morgan, William E. Butler, and InKyeom Kim

I

n this review we will first summarize the current thinking on mechanisms of contraction of the normal, “healthy” smooth muscle cell. The importance of regulation of smooth muscle contractility through phosphorylation of the 20 kDa myosin light chains (MLC20), i.e., “thick filament regulation”, has been recognized for the last two decades; however, it has also become clear that the largest elevations in MLC20 phosphorylation are generally seen in the first 30-60s, during tone development, and thereafter, during tone maintenance, phosphorylation drops to much lower levels, pointing to additional mechanisms to support the continued maintenance of tone. Therefore we will also summarize current thought on potential mechanisms of thin filament regulation as well. In the second half of the review, we will discuss two disease states, preterm labor and posthemorrhagic cerebral vasospasm. Both are diseases of increased smooth muscle tone. The molecular nature of the smooth muscle defect is not known with certainty in either case, but we will review the current state of knowledge in these fields. Preterm labor is of interest because it is one of the first smooth muscle diseases to implicate a possible abnormality in thin filament regulation. Conversely, posthemorrhagic cerebral vasospasm has been reported to involve thick filament regulation and is unusual in that MLC20 phosphorylation levels appear to be at high levels not only during tension development, but also during sustained tone maintenance.

Regulation of Smooth Muscle Contraction in Health Thick Filament Regulation Smooth Muscle Myosin Smooth muscle myosin II is a hexamer, composed of two heavy chains and two pairs of light chains of 20 kDa (MLC20) and 17 kDa (MLC17). There are four myosin heavy chain isoforms, SM1a, SM1b, SM2a, and SM2b. SM1 and SM2 are produced by alternative splicing of primary RNA transcripts from a single genomic DNA. The SM1 isoform, but not the SM2 isoform, has an extra C-terminal tail piece. Each isoform is further subclassified into isoforms depending on the absence or presence of an insert in the N-terminal end of the heavy chain. For more details see Horowitz et al.1 Molecular Mechanisms of Smooth Muscle Contraction, edited by Kazuhiro Kohama and Yasuharu Sasaki. ©1999 R.G. Landes Company.

82

Molecular Mechanisms of Smooth Muscle Contraction Fig. 6.1. Schematic drawing of thick and thin filament regulation of smooth muscle contraction.

Contraction of smooth muscle is regulated primarily by phosphorylation of myosin in the thick filament. The level of myosin phosphorylation is controlled by a balance of the activities of two key enzymes, myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP) (Fig. 6.1). MLCK Since MLCK is activated by the Ca2+-calmodulin complex, it provides the link between intracellular Ca2+ increases and myosin phosphorylation. The kinase primarily phosphorylates Ser19 of MLC20. An excess amount of MLCK will catalyze the diphosphorylation of isolated gizzard myosin light chain at Thr18 as well as Ser19. Diphosphorylated myosin has been detected not only in smooth muscle tissues such as carbachol-stimulated and neurally stimulated bovine trachea and PGF2α-stimulated rabbit aorta, but also in actively growing cultured SM-3 smooth muscle cells.2 The physiological role of MLC20 diphosphorylation is, however, still unclear. MLC20 diphosphorylation further increases actin-activated myosin ATPase activity over MLC20 monophosphorylation.3 However, monophosphorylation at Ser19 generates a maximum force in permeabilized smooth muscle. An additional phosphorylation at Thr18 neither augments force development nor increases sliding speed of actin filament in in vitro motility assays.4 Thus, whether diphosphorylation plays any role in the regulation of contractility is uncertain. MLC20 can be phosphorylated by both PKC and cdc2 kinase5 at three sites, Ser1, Ser2, and Thr9, which are different from the sites phosphorylated by MLCK. PKC phosphorylates three sites on MLC20 in isolated 20 kDa light chain as well as HMM subfragment 1. Thr9 on MLC20 is phosphorylated most rapidly, followed by either Ser1 or Ser2 as compared to Ser19 followed by Thr18 in MLC20 phosphorylation by MLCK. In vitro, phosphorylation of HMM by PKC causes a progressive inhibition of the subsequent rate of phosphorylation of Ser19 by MLCK and of actin-activated ATPase activity of HMM, prephosphorylated by MLCK.6,7 In intact bovine tracheal smooth muscle stimulated with a high concentration of carbachol (which is expected to increase [Ca2+]i as well as activate PKC), the myosin light chain is phosphorylated, however, at sites which correspond to MLCK phosphorylation.8

Thick and Thin Filament Regulation of Smooth Muscle Contraction in Health and Disease

83

Therefore, the physiological significance of MLC20 phosphorylation by PKC remains to be elucidated. MLCP MLCP (SMPP-1M) is composed of three subunits, a 37 kDa catalytic subunit, a 130 kDa myosin binding targeting subunit and a 20 kDa regulatory subunit of as yet unknown function. It was initially assumed that phosphatase was not involved in regulation of myosin phosphorylation in smooth muscle cells. However, more recent data have indicated that under certain conditions phosphatase plays an important role in regulation of myosin phosphorylation.9 Application of GTPγs to permeabilized smooth muscle preparations activates small GTPase Rho, which ultimately results in inhibition of phosphatase activity (see below). The resulting inhibition of phosphatase activity increases the “Ca2+ sensitivity of phosphorylation”, the extent of myosin phosphorylation at a given Ca2+ level. The mechanism of Ca2+ sensitization by inhibition of phosphatase activity is not fully established, but some plausible mediators and/or signaling mechanisms are arachidonic acid, cGMP, PKC, and Rho kinase . Arachidonic acid inhibits SMPP-1M by dissociating PP1c from the native holoenzyme and alleviating the action of the M110/M21 regulatory complex on it.10 Although cGMP has been known to lower [Ca2+]i by increased Ca2+ sequestration, increased Ca2+ efflux, decreased Ca2+ influx, and decreased Ca2+ release, it also causes Ca2+ desensitization in vascular smooth muscle by activating MLCP.11 In permeabilized rabbit femoral artery, PKC activators such as phorbol esters, short chain synthetic diacylglycerols and a diacylglycerol kinase inhibitor increased MLC20 phosphorylation (Ser19) and force development at constant [Ca2+]i by inhibition of MLCP.12 Small GTPase Rho, one of the Ras family G-proteins, appears to play a role in the Ca2+ sensitization of smooth muscle via Rho-associated kinase. Constitutively active Rho-kinase provoked a contraction and a proportional increase in levels of monophosphorylation of myosin light chain in permeabilized smooth muscles in a wortmannin (a MLCK inhibitor)-insensitive manner.13

Thin Filament Regulation Smooth muscle thin filaments consist of F-actin and tropomyosin. Unlike skeletal and cardiac muscle, there is no troponin in smooth muscle thin filaments. Tropomyosin does play an auxiliary role in smooth muscle, however, by conferring cooperatively between these molecules along the thin filament.1 Two proteins are known to be associated with the thin filament and have been postulated to regulate smooth muscle contractility: caldesmon and calponin. Caldesmon Caldesmon (CaD) is an elongated protein consisting of a single polypeptide chain extending to 75-80 nm. It binds to F-actin, tropomyosin, myosin, and Ca/CaM. Caldesmon appears to play a role in modulating smooth muscle contractile activity by inhibiting actomyosin ATPase without affecting the level of light chain phosphorylation. In the absence of caldesmon, which was induced by incubating permeabilized smooth muscle in solutions containing high Mg2+ concentrations, the maximal active force per cross-sectional area was unaffected, while the Ca2+ dependence of active force was shifted towards lower Ca2+ concentrations.14 Moreover, the effects of extraction of caldesmon could in part be reversed by incubation in a solution containing purified caldesmon. Similarly, the use of a peptide designed to compete with the high affinity binding site of endogenous CaD for actin in

84


permeabilized single cells resulted in a disinhibition of the interaction of actin and myosin and contraction of the cells.15 Phosphorylation of CaD in vivo reverses inhibitory influences of caldesmon on crossbridge cycling.16 CaD is known to be phosphorylated in vitro by a number of protein kinases such as CaMKII, casein kinase II, PKA, cdc2 kinase, MAP kinase, and PKC. However, all of them are not likely to be involved in CaD phosphorylation in vivo. Among them, MAP kinase appears to be the endogenous CaD kinase.17 Stimulation of intact mammalian vascular smooth muscle results in CaD phosphorylation at two specific prolinedirected serine residues, Ser702 and Ser759. MAP kinase-catalyzed phosphorylation sites on purified chicken gizzard CaD are consistent with one of those sites which is phosphorylated in situ. Thus, the proposed signaling cascade is as follows: Contractile agonists, by binding to their receptor, activate Erk MAP kinase through a protein kinase Cascade, leading to phosphorylation of caldesmon (Fig. 6.1), and finally, disinhibition of the filaments and contraction (see Chapter 5). Calponin Calponin is an actin, tropomyosin and Ca2+/calmodulin binding protein that inhibits in vitro the actomyosin MgATPase. Basic (h1), neutral and acidic variants of calponin have been described to date. Calponin (h1) is exclusive to SMC, although the other isoforms have been found in nonmuscle cells such as neural cells, platelet, cardiomyocyte, endothelial cells and keratinocyte as well as in pig and mouse smooth muscle. Calponin has been proposed to regulate smooth muscle contractility through inhibition of actomyosin MgATPase. To inhibit the actomyosin MgATPase, calponin would need to colocalize with and bind to actomyosin. However, the subcellular localization of calponin has been controversial.1 There have been reported to be two calponin binding sites on actin: one from which calponin is readily displaced by myosin subfragment 1 (S1); and the other which is not affected by S1, but which may also be responsible for inhibition of the ATPase.18 The former may be associated with bundling of actin filaments by calponin, because bundle formation is inhibited by S1ADP over a ratio of calponin to actin of 1:3. This binding of calponin to actin may be at least in part by electrostatic interactions, which is supported by the fact that the polycationic nature of calponin helps to reduce the electrostatic repulsion between the negatively charged actin filaments during the F-actin bundle formation.19 Calponin has been reported to be phosphorylated in vivo by stimulation with agonists such as 1 µM carbachol in bovine tracheal smooth muscle.20 The functional effect of phosphorylation is loss of the ability to inhibit the actomyosin MgATPase in solution and loss of the inhibition of actin sliding in in vitro motility assays. If calponin is phosphorylated, the signaling cascades of calponin phosphorylation could involve PKC or Ca2+/calmodulindependent protein kinase II. However, others have reported that calponin is not phosphorylated at all in vivo.21 Thus, the physiological role of calponin as a contractile regulatory protein in vivo remains controversial. For more details, see Horowitz et al.1 Conversely, it has recently been reported that calponin may have a different function in the smooth muscle cell, that of a signaling molecule linking the targeting of PKC and MAPK to the plasmalemma.22 In the resting state, calponin is associated with MAPK in differentiated ferret vascular smooth muscle. Upon phenylephrine-induced activation of PKC-epsilon, calponin also associates with PKC-epsilon and may act as an adapter protein which facilitates the translocation of MAPK and PKC-epsilon to the membrane. Recently, calponin was reported to inhibit cell proliferation. Cultured smooth muscle cells and fibroblasts usually express negligible calponin. When the smooth muscle calponin


85

gene was introduced to these cells via an adenoviral vector, cell growth and DNA synthesis were inhibited to one-third of control values.23

Regulation of Smooth Muscle Contraction in Disease Preterm Labor The Problem Premature delivery continues to be one of the major causes of perinatal morbidity and mortality in newborns. In the United States, preterm labor affects approximately 1 in 10 births. Preterm birth, even when the infant survives, can result in lifelong disabilities including mental retardation, cerebral palsy, deafness and blindness. Approximately onethird of all premature births are the result of premature labor. Premature labor is defined as repetitive uterine contractions resulting in cervical dilatation prior to 37 completed weeks of gestation. It is a surprising fact that the incidence of premature labor has remained essentially unchanged for the past 40 years.24 In recent years there have been a number of “tocolytic” agents introduced to the market. These agents include β-mimetics, MgS04, indomethacin, Ca2+ channel blockers and, most recently, oxytocin receptor antagonists. All of these agents aim to inhibit uterine smooth muscle contraction. Although some of these tocolytic agents appear to prevent contractions over a period of hours, randomized clinical trials have repeatedly demonstrated that they do not significantly prolong gestation. It is generally agreed that the lack of effective therapeutics reflects a poor fundamental understanding of the basic physiology and cell biology underlying the mechanism of normal and preterm labor. In most cases of preterm labor the cause is unknown. The causes include infection, maternal medical complications and antepartum fetal compromise.24 We will confine our discussion to possible subcellular mechanisms of idiopathic preterm labor. The Role of Oxytocin It is clear that during normal labor there is generally a dramatic increase in the number of oxytocin receptors and the oxytocin blood levels in pregnant women. The subcellular mechanism of oxytocin-induced contraction of myometrial smooth muscle cells is only partially understood. Szal et al25 have shown that in human pregnant myometrium the addition of exogenous oxytocin in vitro leads to an increase in the intracellular ionized calcium levels, which, as discussed above, will presumably lead to an increased myosin light chain phosphorylation level. Although, to the best of our knowledge, this has not been directly measured in human myometrium, it has been shown26 that myosin light chain phosphorylation is increased in rat uterine strips exposed to oxytocin. Interestingly, however, in the same tissue, Oishi et al27 have shown that in calcium-free bathing media oxytocin causes a persistent contraction that is not associated with any increase in light chain phosphorylation. Nohara et al28 have also shown recently that oxytocin can activate MAPK in pregnant rat myometrium. These latter two reports raise the possibility that oxytocin results in changes not only in thick filament regulation, but also in thin filament regulation in myometrial smooth muscle. Other Targets Simmons and Bigbee29 have found that, in some animal models of preterm labor such as lipopolysaccharide-induced labor, increased myometrial contractility and labor can occur in the absence of an increase in the number of oxytocin receptors. Also, it is to be noted that oxytocin levels and binding to oxytocin receptors actually peak a day prior to the

86


onset of labor. This raises the interesting possibility that primary changes in the smooth muscle cell may be responsible for some cases of preterm labor and point to possible novel therapeutic approaches. In this context, it is of interest that the myometrial cell undergoes profound changes in contractile protein and cytoskeletal content during pregnancy. There have also been reported dramatic, reciprocal changes in contractile protein content and the cytoskeleton during postpartum uterine involution.30 The recent application of two-dimensional protein gel electrophoresis31 and differential display of messenger RNA32 to this question offers the possibility of rapidly identifying numerous new potential targets in the near future. Caldesmon A specific actin-binding protein that has been the focus of recent investigations is caldesmon. Although smooth muscle contains no troponin, a major actin-binding protein in striated muscle, the actin filaments of smooth muscle do contain two novel proteins, caldesmon and calponin. Word et al33 have reported that caldesmon levels increase with pregnancy, whereas calponin levels remain unchanged in pregnant human myometrium. These findings were confirmed and extended in a preliminary report by Ricciotti et al34 that caldesmon levels not only increased in human myometrium during pregnancy but also decreased during normal and preterm labor. However, these results have become controversial, given that the myometrial smooth muscle cell appears to undergo changes in many cytoskeletal proteins simultaneously during pregnancy, and even more dramatically during labor and postpartum involution. Thus, the question remains of whether the caldesmon levels, when properly normalized to reflect changes that are important at the subcellular level, will be consistent with these initial reports. It is well known that normalization and protein matching of human samples, often grossly contaminated with blood proteins, can be quite challenging. In spite of the controversy, the concept of caldesmon being a novel therapeutic target in preterm labor in quite attractive and is supported by considerable circumstantial evidence. Haeberle et al35 have reported especially high concentrations of caldesmon in myometrial smooth muscle compared to vascular smooth muscle, to some extent higher than that in other visceral smooth muscles. As discussed above, caldesmon is an inhibitory protein interfering with the interaction of actin and myosin and, given the dramatic myogenic stimulus represented by the growing fetus during pregnancy, the concept that nature provides parallel increases in this inhibitory protein to exactly counterbalance the myogenic stimulus is an attractive concept. Whether this concept holds up to rigorous experimental investigation, however, remains to be seen though it certainly warrants additional study.

Cerebral Vasospasm The Problem The major sources of blood supply meet in an anastomotic network at the base of the brain called the circle of Willis. This anastomotic network is contained within the subarachnoid space. Since the most common sites of cerebral aneurysm formation are within or near the circle of Willis, aneurysmal rupture commonly causes a coagulum of blood to accumulate on the external surfaces of cerebral arteries within or near the circle of Willis. The presence of a blood clot over the course of several days on the extra luminal side of a blood vessel is etiologically connected to the development of vasospasm in that blood vessel.36 Moreover, the development of vasospasm is associated with the presence of blood products within the vessel wall.37 When vasospasm occurs in a cerebral blood vessel, it can


87

Fig. 6.2A. Head computed tomogram of a patient after subarachnoid hemorrhage. The subarachnoid clot (arrow) follows the contours of the subarachnoid space.

potentially produce ischemia in the territory of the brain parenchyma supplied by that vessel (Fig. 6.2A and B). When the ischemia is sufficiently severe, infraction can result. Vasospasm is a delayed phenomenon in relation to aneurysm rupture and subarachnoid hemorrhage. Typically, there is a 4 to 10 day interval between subarachnoid hemorrhage and vasospasm. While calcium channel blockers have been shown to reduce the incidence of cerebral infarction following subarachnoid hemorrhage, once vasospasm is established it is largely refractory to pharmacologic intervention. Instead, established vasospasm responds best to mechanical methods of dilating the vessel such as hypertension, balloon angioplasty, and laser angioplasty. The chain of events between the appearance of blood on the extra luminal side of the blood vessel and the subsequent development of vasospasm is complex. After introducing the experimental models of cerebral vasospasm, this discussion will be divided into two sections. In the first we will review the evidence concerning the mechanisms whereby subarachnoid hemorrhage leads to excitation of contractile cells in the arterial wall. In the second section we will view evidence concerning the pathophysiology in vasospasm of intracellular contractile pathways in arterial smooth muscle cells. Experimental Models of Chronic Cerebral Vasospasm All animal models of chronic vasospasm share the features of placement of blood about an artery, followed days later by narrowing of that artery. The oldest and most prevalent method of monitoring the status of arterial diameter in an animal model of vasospasm has been arteriography. The need to be able to image arterial diameter and discriminate levels of narrowing by arteriography has obligated the use of large animals for experimental expansion models. An initial model was the canine double subarachnoid hemorrhage model of vasospasm.38 In this model, the animal is anesthetized in two sessions about three days apart and blood is injected into the sterna magna. The blood then pools around the basilar artery. In a third session about seven days after the first, the animal is anesthetized, undergoes

88


Fig. 6.2B. Angiogram of the left vertebral artery shows the left vertebral and basilar arteries before (left) and after (right) the onset of vasospasm. The vasospasm was diffuse, but there were focal areas where it was particularly intense (arrow). This patient succumbed to vasospasm despite the use of calcium channel blockers, systemic hypertension, hemodilution, intraarterial papaverine, and balloon angioplasty.

a cerebral arteriogram, and is sacrificed for harvesting of the vasospastic basilar artery. The reliability with which this model produces chronic cerebral vasospasm has resulted in it being a commonly employed model. This model has been modified to include a transclival exposure of the basilar artery, a feature that gives the researcher an opportunity to directly apply pharmacologic agents to a vasospastic basilar artery and directly visualize the effect on outer arterial diameter.39 There are primate models of chronic cerebral vasospasm.40,41 These presumably mimic the human disease better by virtue of the relative phylogenetic proximity of primates and humans (correspondingly, this proximity obliges researchers to use primate models sparingly). Moreover, the relatively large size of the employed primates facilitates arteriographic monitoring of arterial narrowing. Rabbit models of vasospasm are commonly employed because they are of the smallest size that still permits effective arteriography. Rodent models of vasospasm cerebral vasospasm have been less commonly employed because of the small size of those animals. However, it has been observed that the extraluminal application of blood to the rat femoral artery produces narrowing in that artery if the blood is held in place with a perivascular sheath.42 While in vitro methods are used to study aspects of vasospasm, the specimens are obtained from animal vasospasm models. There are currently no satisfactory methods of reproducing with cultured cells or tissues a phenomenon analogous to chronic vasospasm. Excitation of Contractile Cells in Vasospasm Hemoglobin and Calcium The accumulation of clot on the extraliminal surface of a cerebral artery after subarachnoid hemorrhage leads to suspicion concerning the role of the clot in the pathogenesis of vasospasm. Indeed, there is a significant body of evidence indicating an important role for hemoglobin in the evidence on pathogenesis of vasospasm.43 Since such a clot must


89

contain hemoglobin, evidence that hemoglobin causes Ca2+ influx would suggest a mechanism whereby subarachnoid hemorrhage might elevate intracellular Ca2+. Using fluorescence microscopy and the fluorescent indicator fura-2, Takenaka et al44 found that exposure to cerebrospinal fluid obtained from subarachnoid hemorrhage (SAH) patients (which would contain hemoglobin) caused Ca2+ influx in smooth muscle cells. Steele et al45 found electrophysiologic evidence of hemoglobin-induced calcium influx in smooth muscle cells. These studies suggest a mechanism whereby SAH might cause increased intracellular calcium, but do not document increased intracellular calcium. Excitation of PKC-Dependent Pathways There is conflicting evidence over whether subarachnoid hemorrhage provokes increased production of diacylglycerol in the cellular membrane.46,47 If such increased production were to be conclusively established, it would imply a mechanism whereby subarachnoid hemorrhage might lead to activation of PKC-dependent pathways of smooth muscle contraction. Endothelin There is a considerable body of evidence indicating a role for endothelin in excitation of the smooth muscle contractile pathways. Not only is it known that endothelin causes contraction of cerebral arteries,48-52 there is considerable evidence that endothelin activity is elevated in cerebrospinal fluid (CSF) obtained from experimental models of vasospasm.53-56 In this vein, the finding that oxyhemoglobin causes endothelin synthesis in cultured endothelial cells is intriguing.57 Moreover, there is a report of elevated endothelin receptor levels in experimental vasospasm models.58 Evidence concerning the presence of elevated CSF endothelin levels in human subarachnoid hemorrhage patients is conflicting,59 but one study reports a tantalizing correlation between the quantity of subarachnoid clot and the subsequent CSF endothelin concentration.60 The introduction of endothelin receptor antagonists provided further opportunities to examine the contribution of endothelin to vasospasm and to explore opportunities for pharmacologic intervention in the disease. Several studies have demonstrated some efficacy of endothelin blockers in attenuating experimental vasospasm,54-56,58,61-65 including a doubleblinded, randomized trial in a primate model.66 Some of the circumstances in which endothelin antagonists were ineffective against experimental vasospasm may have been due to insufficient levels of the antagonists in the CSF.66,67 These studies aggregately demonstrate an important role for endothelin in the development of experimental vasospasm, and point to opportunities for pharmacologic intevention in the human clinical disease. Endothelial-Dependent Relaxation In addition to excess excitation of contractile pathways, vasospasm may result from diminished activation of relaxation pathways. Endothelium-dependent relaxation is attenuated in vasospasm.68,69 This phenomenon may be due to impaired response of smooth muscle to endothelium-dependent relaxation factor70-72 or to decreased synthesis of nitric oxide.73,74 While intravenously administered nitrates are clinically ineffective against vasospasm, this may be because the systemic hypotensive effects of nitrates act to decrease cerebral perfusion in a way that might nullify any local benefit on vasospastic vessels. Of note, nitric oxide has a short half life, and when administered into the carotid artery in a primate model of vasospasm was found to reverse angiographic vasospasm.75

90


The Importance of Contractile Versus Noncontractile Pathways The scientific literature on vasospasm contains evidence for and against the role of smooth muscle cell contractile pathways in the pathogenesis of vasospasm. The evidence against a primary role of contractile pathways in the pathophysiology of vasospasm consists mainly of observations of abnormal biophysical properties of vascular smooth muscle obtained from animal models of subarachnoid hemorrhage (SAH). These observations include decreased contactility, increased stiffness, increased tunica media collagen, and arterial well inflammation.70,76-80 Most significantly, this view is suggested by the relatively pharmacologically unresponsive clinical behavior of vasospasm in humans, a situation that is clinically frustrating and injurious. The relative pharmacologic unresponsiveness of chronic vasospasm in humans has led to the development and clinical acceptance of direct balloon angioplasty of vasospastic arteries.81,82 These observations favor the concept that the phenomenon arises from changes in the static structural properties of the walls of vasospastic vessels. The evidence favoring the importance of contractile pathways in vasospasm consists of observations of the continued presence of cytoskeletal and extracellular matrix proteins (although not necessarily at normal levels and ratios), and of the presence of increased spontaneous myogenic tone in vasospastic vessels.70,80,83-85 Chiefly, however, support for a role played by contractile proteins stems from a large and complex body of evidence from biochemical and pharmacologic investigations of contraction mechanisms in smooth muscle cells during vasospasm. However, there may not be an incompatibility between pathogenic pathways that lead to changes in passive properties of the vessel wall and pathways that lead to activation of cellular contractile elements. Several investigators, for example, have found both increased spontaneous myogenic and passive wall tension.70,80,83-85 These investigators have found that application of the vasodilatory cocktails to vasospastic vessels does not result in the same degree of dilation as when the same cocktails are applied to control vessels. Thus, properly understood, the issue may be one of resolving quantitatively the relative contributions of each. Evidence Pertaining to the Thick Filament Regulatory Pathways As described above, these pathways involve Ca2+-calmodulin-dependent activation of myosin by the phosphorylation of MLC20 by MLCK and dephosphorylation by MLC20 phosphatase. Thus, the lines of evidence to be reviewed concern intracellular calcium levels, the role of calmodulin, the activity of MLCK, the percentage of myosin light chain phosphorylation, and the activity of MLC20 phosphatases. Studies reporting direct measurements of intracellular calcium in vasospastic vessels have yielded conflicting results. Using vessel segments loaded with fura-2, Yamada failed to demonstrate increased resting intracellular calcium (but did demonstrate increased slope of the force-calcium curve).86 Electron microscopy studies in basilar arteries from a canine model of subarachnoid hemorrhage demonstrated decreased cellular calcium.87 These data conflict with measurements of intracellular calcium in vasospastic arteries made with the luminescent calcium indicator aequorin.85 Moreover, the latter study found decreased slope of the force-calcium curve and decreased maximal force generation to maximal potassium stimulation. There is pharmacologic evidence indicating a role for calmodulin in the pathogenesis of cerebral vasospasm that has been provided by studies of the influence on vasospasm by calmodulin antagonists belonging to the phenothiazine family. The phenothiazines employed, chlorpromazine and trifluoperazine, are ligands for calmodulin and inhibitors of its biological activity.88,89 In particular, trifluoperazine has been shown to slightly reduce


91

vasospasm when administered systematically.90 However, the degree to which these agents reduced vasospasm was not sufficient to imply its effective clinical use. The effectiveness of this drug may have been attenuated by the inability to achieve satisfactory CSF concentrations by intravenous administration (due to the presence of a blood-brain barrier for these agents). By contrast, the direct topical application of chlorpromazine to an in vitro vasospastic canine basilar artery was more effective against vasospasm.91 This pharmacologic evidence of a contributory role for calmodulin is difficult to reconcile with the observation of decreased calmodulin levels in vasospastic arteries.87 On the other hand, in a rabbit model of vasospasm the calmodulin inhibitors W-7 and R 24571 were not seen to dilate the vasospastic basilar artery upon topical application.46 Further evidence for an abnormality of thick filament regulatory mechanisms is provided by the observation that the MLCK inhibitor ML-9 reverses vasospasm.92 Using the canine double hemorrhage model modified to include transclival exposure of the basilar artery, Kokubu et al92 observed arterial dilation upon topical application of ML-9. There is controversy concerning the level of MLC20 phosphorylation in vasospasm. Using a 2-D gel assay (with isoelectric focusing in the first dimension and sodium dodecyl polyacrylamide gel in the second dimension), different investigators have found either increased or unchanged percent myosin light chain phosphorylation.85,93 A third investigator employed glycerol-polyacrylamide gel electrophoresis to find increased percent myosin light chain phosphorylation.94 Another line of evidence supporting a contribution by increased MLC20 phosphorylation is the observation of a decrease in the levels of phosphatase (PP1 and PP2A) activity in vasospastic basilar artery from a rabbit model of vasospasm.95 A major source of variation in the reported results may be the critical nature of the method used to prevent changes in phosphorylation levels after removal of the tissue from the animal. The likelihood of a contribution by the calcium dependent pathway of smooth muscle contraction to the clinical entity of vasospasm motivated the clinical use of L-type calcium channel blockers. On balance, nimodipine and L-type calcium channel blockers confer a mild protective value against vasospasm as evidenced by a mega-analysis of revised clinical trials of nimodipine.96 However, this protective effect is mild, and there is disagreement as to whether the protective effect results from attenuation of arterial narrowing or from some other effect. In the aggregate, the presence of evidence of calcium influx by smooth muscle cells upon exposure to hemoglobin, increased intracellular calcium in vasospastic models, the inhibition of vasospasm by calmodulin antagonists and MLCK antagonists, and the disputed evidence of increased myosin light chain phosphorylation favor a contribution by the calcium-dependent pathway of contraction in vasospasm. The Role of Non-Classical Pathways of Smooth Muscle Contraction Protein kinase C (PKC) plays a central role in putative classical pathways of smooth muscle contraction. There are several lines of evidence that point toward a role of PKC in the pathogenesis of vasospasm. Phorbol esters, which are known to activate PKC, cause contraction of cerebral blood vessels.97 In the rabbit model of vasospasm, the topical application of H-7 and staurosporine were found to cause reversal of arterial narrowing.46 Moreover, that same study reported increased levels of diacylglycerol, a cofactor in the activation of PKC. The activation of PKC is correlated with an increased ratio of membrane to cytosol PKC enzymatic activity. Such increased ratios have been found in vasospastic tissue.97,98 A separate study found increased turnover of phosphatidylcholine and ethanolamine in vasospastic tissue.

92


As discussed above, there exist actin binding proteins that exert an inhibitory effect on smooth muscle contraction. Two studies have found decreased levels of calponin in vasospasm,99,100 and one study has found a decreased level of caldesmon.99 These studies are tantalizing evidence of a role for actin binding proteins in the pathogenesis of vasospasm, but should be confirmed before such a role is accepted.

Conclusion An analysis of the state of knowledge of the pathogenesis of vasospasm is hampered by the presence of conflicting data as reported in the literature. Nonetheless, the preponderance of the published evidence points toward increased excitation of contractile pathways by hemoglobin and endothelin, as well as decreased endothelium-dependent relaxation. Furthermore, it favors a contributory role in vasospasm for both the thick filament pathway as well as possibly thin filament pathways of contraction. The relative contributions by and the interrelationships between these pathways remain to be elucidated. The state of knowledge on mechanisms of normal and preterm labor is even more sketchy—in some part due to a lesser availability of appropriate animal models. The determination of the true molecular etiologies of these and other functional pathologies of the smooth muscle cell is of interest to both basic and clinical scientists, since they are likely to point both to new mechanisms of signal transduction and to new and important therapeutics.

References 1. Horowitz A, Menice CB, Laporte R et al. Mechanisms of smooth muscle contraction. Physiol Rev 1996; 76:967-1003. 2. Seto M, Sakurada K, Kamm KE et al. Myosin light chain dephosphorylation is enhanced by growth promotion of cultured smooth muscle cells. Pflugers Arch 1996; 432:7-13. 3. Ikebe M, Hartshorne DJ, Elzinga M. Identification, phosphorylation and dephosphorylation of a second site for myosin light chain kinase on the 20,000-dalton light chain of smooth muscle myosin. J Biol Chem 1986; 261:36-39. 4. Umemoto S, Bengur AR, Sellers JR. Effect of multiple phosphorylation of smooth muscle and cytoplasmic myosins on movement in an in-vitro motility assay. J Biol Chem 1989; 264:1431-1436. 5. Satterwhite LL, Lohka MJ, Wilson KL et al. Phosphorylation of myosin-II regulatory light chain by cyclin-p34cdc2: A mechanism for the timing of cytokinesis. J Cell Biol 1992; 118:595-605. 6. Ikebe M, Hartshorne DJ, Elzinga M. Phosphorylation of the 20,000-dalton light chain of smooth muscle myosin by the calcium-activated, phospholipid-dependent protein kinase. Phosphorylation sites and effects of phosphorylation. J Biol Chem 1987; 262(20):9569-73. 7. Nishikawa M, Sellers JR, Adelstein RS et al. Protein kinase C modulates in vitro phosphorylation of the smooth muscle heavy meromyosin by myosin light chain kinase. J Biol Chem 1983; 259:8808-8814. 8. Colburn JC, Michnoff CH, Hsu LC et al. Sites phosphorylated in myosin light chain in contracting smooth muscle. J Biol Chem 1988; 263 (35):19166-19173. 9. Somlyo AP, Somlyo AV. Signal transduction and regulation in smooth muscle. Nature 1994; 372:231-6. 10. Gailly P, Wu X, Haystead TAJ et al. Regions of the 110-kDa regulatory subunit M110 required for regulation of myosin-light-chain-phosphatase activity in smooth muscle. Eur J Biochem 1996; 239:326-332. 11. Wu X, Somlyo AV, Somlyo AP. Cyclic GMP-dependent stimulation reverses G-proteincoupled inhibition of smooth muscle myosin light chain phosphatase. Bioch Biophy Res Comm 1996; 220:658-663.


93

12. Masuo M, Reardon S, Ikebe M et al. A novel mechanism for the Ca(2+)-sensitizing effect of protein kinase C on vascular smooth muscle: Inhibition of myosin light chain phosphatase. J Gen Physiol 1994; 104(2):265-86. 13. Kureishi Y, Kobayashi S, Amano M et al. Rho-associated kinase directly induces smooth muscle contraction through myosin light chain phosphorylation. J Biol Chem 1997; 272:12257-12260. 14. Malmqvist U, Arner A, Makuch R et al. The effects of caldesmon extraction on mechanical properties of skinned smooth muscle fiber preparations. Pflugers Arch 1996; 432:241-247. 15. Katsuyama H, Wang CL, Morgan KG. Regulation of vascular smooth muscle tone by caldesmon. J Biol Chem 1992; 267(21):14555-8. 16. Sutherland C, Walsh MP. Phosphorylation of caldesmon prevents its interaction with smooth muscle myosin. J Biol Chem 1989; 264(1):578-83. 17. Adam LP, Franklin MT, Raff GJ et al. Activation of mitogen activated protein kinase in porcine carotid arteries. Circ Res 1995; 76:183-190. 18. Kolakowski J, Karkucinska A, Dabrowska R. Calponin inhibits actin-activated MgATPase of myosin subfragment 1 (S1) without displacing S1 from its binding site on actin. Eur J Biochem 1997; 243:624-629. 19. Tang JX, Szymanski PT, Janmey PA et al. Electrostatic effects of smooth muscle calponin on actin assembly. Eur J Biochem 1997; 247:432-40. 20. Pohl J, winder SJ, Allen BG et al. Phosphorylation of calponin in airway smooth muscle. Am J Physiol 1997; 272:L115-123. 21. Bárány M, Bárány K. Calponin phosphorylation does not accompany contraction of various smooth muscles. Bioch Biophys Acta 1993; 1179:229-233. 22. Menice CB, Hulvershorn J, Adam LP et al. Calponin and mitogen activated protein kinase signaling in differentiated vascular smooth muscle. J Biol Chem 1997; 272:25157-25161. 23. Jiang Z, Grange RW, Walsh MP et al. Adenovirus-mediated transfer of the smooth muscle cell calponin gene inhibits proliferation of smooth muscle cells and fibroblasts. FEBS Lett 1997; 413(3):441-445. 24. American College of Obstetricians and Gynecologists. Preterm labor. Committee on Technical Bulletins 206. Washington, DC: ACOG, 1995. 25. Szal SE, Repke JT, Seely EW et al [Ca2+]i signaling in pregnant human myometrium. Am J Physiol 1994; 267(30):E77-E87. 26. Csabina S, Barany M, Barany K. Stretch-induced myosin light chain phosphorylation in rat uterus. Arch Biochem Biophys 1986; 249:374-81. 27. Oishi K, Takano-Ohmuro H, Minakawa-Matsuo N et al. Oxytocin contracts rat uterine smooth muscle in Ca2(+)-free medium without any phosphorylation of myosin light chain. Biochem Biophys Res Commun 1991; 176(1):122-8. 28. Nohara T, Asai T, Nakai M et al. Cytochrome f encoded by the chloroplast genome is imported into thylakoids via the SecA-dependent pathway [published erratum appears in Biochem Biophys Res Commun 1996 14;227(2):644]. Biochem Biophys Res Commun 1996; 224(2):474-8. 29. Simmons CF, Bigbee DS. Alternative molecular mechanisms of preterm labor: Differential effects of lipopolysaccharide and Onapristone on oxytocin receptor expressions in the pregnant mouse. Pediatric Res 1997; 4PT2:p52A. 30. Dessouky DA. Myometrical changes in postpartum uterine involution. Am J Obstet Gynecol 1971; 110:318-29. 31. Phillippe M, Harrison HH. Gestational modulation of myometrial proteins in the timedpregnant Sprague-Dawley rat. Life Sci 1992; 50(16):1189-1200. 32. Chien EK, Tokuyama Y, Rouard M, Bell GI. Identification of gestationally regulated genes in rat myometrium by use of messenger ribonucleic acid differential display. Am J Obstet Gynecol 1997; 177(3):645-652. 33. Word RA, Stull JT, Casey ML et al. Contractile elements and myosin light chain phosphorylation in myometrial tissue from nonpregnant and pregnant women. J Clin Invest 1993; 92:29-37.

94


34. Ricciotti HA, Alvarez J, Morgan K et al. Labor is associated with decreased levels of caldesmon in human myometrium. J Soc Gynecol Invest 1996; 3(2)supplement:337A. 35. Haeberle JR, Hathaway DR, Smith CL. Caldesmon content of mammalian smooth muscles. J Muscle Res and Cell Motility 1992; 13:81-89. 36. Fisher CM, Kistler JP, Davis JM. Relation of cerebral vasospasm to subarachnoid hemorrhage visualized by computerized tomographic scanning. Neurosurgery 1980; 6:1-9. 37. Liszczak TM, Varsos VG, Black PM et al. Cerebral arterial constriction after experimental subarachnoid hemorrhage is associated with blood components within the arterial wall. J Neursurg 1983; 58(1):18-26. 38. Varsos VG, Liszczak TM, Han DH et al. Delayed cerebral vasospasm is not reversible by aminophylline, nifedipine, or papaverine in a “two-hemorrhage” canine model. J Neurosurg 1983; 58(1):11-17. 39. Nakamo M, Tani E, Fukumori T et al. Effects of chlorpromazine on experimental delayed cerebral vasospasm. J Neurosurg 1984; 61(5):857-63. 40. Frazee JG. A primate model of chronic cerebral vasospasm. Stroke 1982; 13(5):612-4. 41. Findlay JM, Weir BK, Kanamaru K et al. Arterial wall changes in cerebral vasospasm. Neurosurgery 1989; 25(5):736-45; discussion 745-6. 42. Okada T. Harada T, Bark DH et al. A rat femoral artery model for vasospasm. Neurosurgery 1990; 27(3):349-56. 43. MacDonald Rl, Weir BKA. A review of hemoglobin and the pathogenesis of cerebral vasospasm. Stroke 1991; 22:971-82. 44. Takenaka K, Yamada H, Sakai N et al. Induction of cytosolic free calcium elevation in rat vascular smooth-muscle cells by cerebrospinal fluid from patients after subarachnoid hemorrhage. J Neurosurg 1991; 75(3):452-7. 45. Steele JA, Stockbridge N, Maljkovic G et al. Free radicals mediate actions of oxyhemoglobin on cerebrovascular smooth muscle cells. Circ Res 1991; 68(2):416-23. 46. Matsui T, Takuwa Y, Johshita H et al. Possible role of protein kinase C-dependent smooth muscle contraction in the pathogenesis of chronic cerebral vasospasm. J Cereb Blood Flow Metab 1991; 11:(1):143-9. 47. Yokota M, Peterson JW, Kaoutzanis MC et al. Protein kinase C and diacylglycerol content in basilar arteries during experimental cerebral vasospasm in the dog. J Neurosurg 1995; 82:(5):834-40. 48. Asano T, Ikegaki I, Suzuki Y et al. Endothelin and the production of cerebral vasospasm in dogs. Biochem Biophys Res Commun 1989; 159(3):1345-51. 49. Kobayashi H, Hayashi M, Kobayashi S et al. Cerebral vasospasm and vasoconstriction caused by endothelin. Neurosurgery 1991; 28(5):673-8; discussion 678-9. 50. Ide K, Yamakawa K, Nakagomi T et al. The role of endothelin in the pathogenesis of vasospasm following subarachnoid hemorrhage. Neurol Res 1989; 11(2):101-4. 51. Alafaci C, Jansen I, Arbab MA et al. Enhanced vasoconstrictor effect of endothelin in cerebral arteries from rats with subarachnoid hemorrhage. Acta Physiol Scand 1990; 138(3):317-9. 52. Papadopoulos SM, Gilbert LL, Webb RC et al. Characterization of contractile responses to endothelin in human cerebral arteries: implications for cerebral vasospasm. Neurosurgery 1990; 26(5):810-5. 53. Suzuki H, Sato S, Suzuki Y et al. Increased endothelin concentration in CSF from patients with subarachnoid hemorrhage. Acta Neurol Scand 1990; 81(6):553-4. 54. Hirose H, Ide K, Sasaki T et al. The role of endothelin and nitric oxide in modulation of normal and spastic cerebral vascular tone in the dog. Eur J Pharmacol 1995; 277(1):77-87. 55. Roux S, Loffler BM, Gray GA et al. The role of endothelin in experimental cerebral vasospasm. Neurosurgery 1995; 37(1):78-85; discussion 85-6. 56. Zimmermann M, Seifert V, Loffler BM et al. Prevention of cerebral vasospasm after experimental subarachnoid hemorrhage by RO 47-0203, a newly developed orally active endothelin receptor antagonist. Neurosurgery 1996; 38(1):115-20. 57. Ohlstein EH, Storer BL. Oxyhemoglobin stimulation of endothelin production in cultured endothelial cells. J Neurosurg 1992; 77(2):274-8.


95

58. Itoh S, Sasaki T, Asai A et al. Prevention of delayed vasospasm by an endothelin ETA receptor antagonist, BQ-123: Change of ETA receptor mRNA expression in a canine subarachnoid hemorrhage model. J Neurosurg 1994; 81(5):759-64. 59. Gaetani P, Rodriguez y Baena R et al. Endothelin and aneurysmal subarachnoid hemorrhage: A study of subarachnoid cisternal cerebrospinal fluid. J Neurol Neurosurg Psychiatry 1994; 57(1):66-72. 60. Seifert V, Loffler BM, Zimmermann M et al. Endothelin concentrations in patients with aneurysmal subarachnoid hemorrhage. Correlation with cerebral vasospasm, delayed ischemic neurological deficits, and volume of hematoma. J Neurosurg 1995; 82(1):55-62. 61. Willette RN, Zhang H, Mitchell MP et al. Nonpeptide endothelin antagonist. Cerebrovascular characterization and effects on delayed cerebral vasospasm. Stroke 1994; 25(12):2450-2455; discussion 2456. 62. Shigeno T, Clozel M, Sakai S et al. The effect of bosentan, a new potent endothelin receptor antagonist, on the pathogenesis of cerebral vasospasm. Neurosurgery 1995; 37(1):87-90; discussion 90-1. 63. Itoh S, Sasaki T, Ide K et al. A novel endothelin ETA receptor antagonist, BQ-485, and its preventive effect on experimental cerebral vasospasm in dogs. Biochem Biophys Res Commun 1993; 195(2):969-75. 64. Zuccarello M, Lewis AI, Rapoport RM. Endothelin ETA and ETB receptors in subarachnoid hemorrhage-induced cerebral vasospasm. Eur J Pharmacol 1994; 259(1):R1-2. 65. Nirei H, Hamada K, Shoubo M et al. An endothelin ETA receptor antagonist, FR139317, ameliorates cerebral vasospasm in dogs. Life Sci 1993; 52(23):1869-74. 66. Hino A, Weir BK, MacDonald RL et al. Prospective, randomized, double-blind trial of BQ-123 and bosentan for prevention of vasospasm following subarachnoid hemorrhage in monkeys. J Neurosurg 1995; 83(3):503-9. 67. Clozel M, Watanabe H. BQ-123, a peptidic endothelin ETA receptor antagonist, prevents the early cerebral vasospasm following subarachnoid hemorrhage after intracisternal but not intravenous injection. Life Sci 1993; 52(9):825-34. 68. Kanamaru K, Waga S, Kojima T et al. Endothelium-dependent relaxation of canine basilar arteries. Part 2: Inhibition by hemoglobin and cerebrospinal fluid from patients with aneurysmal subarachnoid hemorrhage. Stroke 1987; 18:938-43. 69. Nakagomi T, Kassell NF, Sasaki T et al. Impairment of endothelium-dependent vasodilation induced by acetylcholine and adenosine triphosphate following experimental subarachnoid hemorrhage. Stroke 1987; 18:482-9. 70. Kim P, Lorenze RR, Sundt TM et al. Release of endothelium-derived relaxing factor after subarachnoid hemorrhage. J Neursurg 1989; 70:108-14. 71. Kim P, Schini VB, Sundt TM et al. Reduced production of cGMP underlies the loss of endothelium-dependent relaxation in the canine basilar artery after subarachnoid hemorrhage. CircRes 1992; 70:248-56. 72. Kajita Y, Suzuki Y, Oyama H et al. Combined effect of L-arginine and superoxide dismutase on the spastic basilar artery after subarachnoid hemorrhage in dogs. J Neurosurg 1994; 80(3):476-83. 73. Pluta RM, Thompson BG, Dawson TM et al. Loss of nitric oxide synthase immunoreactivity in cerebral vasospasm. J Neurosurg 1996; 84(4):648-54 . 74. Hino A, Tokuyama Y, Weir B et al. Changes in endothelial nitric oxide synthase mRNA during vasospasm after subarachnoid hemorrhage in monkeys. Neurosurgery 1996; 39(3):562-7; discussion 567-8. 75. Afshar JK, Pluta RM, Boock RJ et al. Effect of intracarotid nitric oxide on primate cerebral vasospasm after subarachnoid hemorrhage. J Neurosurg 1995; 83(1):118-22. 76. Bevan JA, Bevan RD, Frazee JG. Functional arterial changes in chronic cerebrovasospasm in monkeys: An in vitro assessment of the contribution to arterial narrowing. Stroke 1987; 18(2):472-81. 77. Nagasawa S, Handa H, Naruo Y et al. Experimental cerebral vasospasm arterial wall mechanics and connective tissue composition. Stroke 1982; 13(5):595-600.

96


78. Bevan JA, Bevan RD, Frazee JG. Experimental chronic cerebrovascular spasm in the monkey: An assessment of the functional changes in the cerebral arteries and their protection by diltiazem. Am J Cardiol 1985; 56(16):15H-20H. 79. Duckles SP, Bevan RD, Bevan JA. An in vitro study of prolonged vasospasm of a rabbit cerebral artery. Stroke 1976; 7(2):174-8. 80. Vorkapic P, Bevan RD, Bevan JA. Longitudinal time course of reversible and irreversible components of chronic cerebrovasospasm of the rabbit basilar artery. J Neurosurg 1991; 74(6):951-5. 81. Findlay JM. Current management of aneurysmal subarachnoid hemorrhage guidelines from the Canadian Neurosurgical Society. Can J Neurol Sci 1997; 24:2:161-70. 82. Weir B, MacDonald L. Cerebral vasospasm. Clin Neurosurg 1993; 40:40-55. 83. Matsui T, Kaizu H, Itoh S et al. The role of active smooth-muscle contraction in the occurrence of chronic vasospasm in the canine two-hemorrhage model. J Neurosurg 1994; 80(2):276-82. 84. Findlay JM, Weir BK, Kanamaru K et al. Arterial wall changes in cerebral vasospasm. Neurosurgery 1989; 25(5):736-45. 85. Butler WE, Peterson JW, Zervas NT et al. Intracellular calcium, myosin light chain phosphorylation and contractile force in experimental cerebral vasospasm. Neurosurgery 1996; 38(4):781-7. 86. Yamada T, Tanaka Y, Fujimoto K et al. Relationship between cytosolic Ca2+ level and contractile tension in canine basilar artery of chronic vasospasm. Neurosurgery 1994; 34(3):496-503. 87. Sakaki S, Ohue S, Kohno K et al. Impairment of vascular reactivity and changes in intracellular calcium and calmodulin levels of smooth muscle cells in canine basilar arteries after subarachnoid hemorrhage. Neurosurgery 1989; 25(5):753-61. 88. Levin RM Weiss B. Mechanism by which psychotropic drugs inhibit adenosine 3',5'-monophosphate phosphodiesterase in brain. Mol Pharmacol 1976; 12:581-9. 89. Levin RM, Weiss B. Binding of trifluoperazine to the calcium-dependent activator of cyclic nucleotide phosphodiesterase. Mol Pharmacol 1977; 13:690-7. 90. Peterson JW, Candia G, Spanos AJ et al. The calmodulin antagonist trifluoperazine provides mild prophylactic protection against cerebral vasospasm after subarachnoid hemorrhage, but no therapeutic value. Neurosurgery 1989; 25(6):917-22. 91. Nakano M, Tani E, Fukumori T et al. Effects of chlorpromazine on experimental delayed cerebral vasospasm. J Neurosurg 1984; 61:(5):857-63. 92. Kokubu K, Tani E, Nakano M et al. Effects of ML-9 on experimental delayed cerebral vasospasm. J Neurosurg 1989; 71(6):916-22. 93. Takuwa Y, Matsui T, Abe Y et al. Alterations in protein kinase C activity and membrane lipid metabolism in cerebral vasospasm after subarachoid hemorrhage. J Cereb Blood Flow Metab 1993; 13(3):409-15. 94. Harada T, Seto M, Sasaki Y et al. The time course of myosin light-chain phosphorylation in blood-induced vasospasm. Neurosurgery 1995; 36(6):1178-82; discussion 1182-3, 1995. 95. Fukami M, Tani E, Takai A et al. Activity of smooth muscle phosphatases 1 and 2A in rabbit basilar artery in vasospasm. Stroke 1995; 26(12):2321-7. 96. Barker FG 2nd, Ogilvy CS. Efficacy of prophylactic nimodipine for delayed ischemic deficit after subarachnoid hemorrhage: A metaanalysis. J Neurosurg 1996; 84(3):405-14. 97. Sako M, Nishihara J, Ohta S et al. Role of protein kinase C in the pathogenesis of cerebral vasospasm after subarachnoid hemorrhage. J Cereb Blood Flow Metab 1993; 13(2):247-54. 98. Nishizawa S, Nezu N, Uemura K. Direct evidence for a key role of protein kinase C in the development of vasospasm after subarachnoid hemorrhage. J Neurosurg 1992; 76(4):635-9. 99. Oka Y, Ohta S, Todo H et al. Protein synthesis and immunoreactivities of contractile proteins in smooth muscle cells of canine basilar artery after experimental subarachnoid hemorrhage. J Cereb Blood Flow Metab 1996; 16:1335-44. 100. Doi M, Kasuya H, Weir B et al. Reduced expression of calponin in canine basilar artery after subarachnoid hemorrhage. Acta Neurochirurgica (Wien) 1997; 139(1):77-81.

CHAPTER 7

Diphosphorylation of Myosin Light Chain and Spastic Contraction of Smooth Muscle Minoru Seto and Yasuharu Sasaki

C

ontraction of smooth muscle in response to an appropriate agonist is thought to be mediated by an increase in the concentration of intracellular free calcium.1,2 A welldefined molecular target of this calcium message is the calmodulin-myosin light chain kinase (MLCK) pathway.3 It is now generally accepted that the resulting phosphorylation of 20 kDa myosin light chain (MLC20) leads to interaction of actin with myosin (cross-bridge phosphorylation), and the contraction response is initiated. However, the relationship between cytosolic calcium concentration and MLC20 phosphorylation, or that between MLC20 phosphorylation and tension, is not always constant. These relationships can be modulated by many factors which change the calcium sensitivity. The phosphorylation sites of MLC20 are located at the N terminus with the following amino acid sequence:4 acetyl-Ser-Ser-Lys-Alg-Ala-Lys-Ala-Lys-Thr-Thr-Lys-Lys-Arg-Pro-Gln-Arg-AlaThr-Ser-Asn-Val-Phe-Ala-

Ser19 is the residue that is phosphorylated by MLCK both in vitro4 and in intact muscle.5,6 At relatively high MLCK concentrations, a second site on MLC20 is phosphorylated, shown to be Thr18.7 Phosphorylation of Thr18, in addition to Ser19, in MLC20 of gizzard myosin increases the actin-activated MgATPase activity.8 Three residues are phosphorylated by PKC, Ser1, Ser2 and Thr9.9,10 Thr9 is the major phosphoamino acid resulting from phosphorylation of heavy meromyosin (HMM) by PKC. The sequential phosphorylation of turkey gizzard HMM by MLCK and PKC results in a twofold decrease in the actin-activated MgATPase activity of the HMM.11 These results mean that monophosphorylation of MLC20 on the Ser19 residue and diphosphorylation of MLC20 both on Thr18 and Ser19 residues are important for smooth muscle contraction. Monophosphorylation on the Thr18 residue of MLC20 is not produced in an in vitro system and in intact smooth muscle, so far as I know. Recently, Rho-kinase was reported to phosphorylate MLC20 on the Ser19 residue in vitro.12 However, it is not clear whether Rho-kinase directly phosphorylates MLC20 in intact smooth muscle (instead of MLCK or jointly with MLCK). Molecular Mechanisms of Smooth Muscle Contraction, edited by Kazuhiro Kohama and Yasuharu Sasaki. ©1999 R.G. Landes Company.

98


Fig. 7.1. Character of specific antibodies against phospholylated MLC20 on Ser19 or Thr18/Ser19 residues. A mixture of purified non-, mono-, and diphosphorylated MLC20 (60 ng each) with MLCK was separated on Glycerol-PAGE, transferred to nitrocellulose paper, and then immunostained with anti-MLC20 antibody (lane a), antibody mAb-p (lane b), and antibody pAb-p2 (lane c).

We think that diphosphorylation of MLC20, both on Thr18 and Ser19, accompanied with further increase of the actin-activated MgATPase activity, may be related to abnormal contraction of smooth muscle. In this chapter we review the mechanism of abnormal contractions, such as vasospasm, focusing on hyperphosphorylation of MLC20 including MLC20 diphosphorylation. We also describe basic and new methods for detecting the monophosphorylation of MLC20 on Ser19 and diphosphorylation of MLC20 both on Thr18 and Ser19 in smooth muscle.

Quantification Method of MLC20 Phosphorylation Glycerol-PAGE Method In our laboratory, the Glycerol-PAGE method is used for quantification of MLC20 phosphorylation. MLC20 is extracted with 8 M urea solution from an acetone-dried muscle powder and nonphosphorylated, monophosphorylated and diphosphorylated forms of MLC20 were separated by Glycerol-PAGE, followed by electrophoretic transfer of the proteins to a nitrocellulose membrane. Quantitation was made of the relative amounts of each form by an immunoblot procedure using specific anti-MLC20 rabbit polyclonal antibody.13,14 This method can easily deal with many samples at once as compared with two-dimensional gel electrophoretic methods.15 However, this method cannot quantify the MLC20 phosphorylation on Ser19, or Thr18 and Ser19, residues as distinguished from other phosphorylation sites of MLC20.

Diphosphorylation of Myosin Light Chain and Spastic Contraction of Smooth Muscle

99

Fig. 7.2. Relationship between concentration of contractile agent and extent of MLC20 phosphorylations. Comparison was made 5 minutes after the addition of various concentrations of KCl (A), PGF2α (B) and histamine (C). Percent of monophosphorylated ( ) and diphosphorylated ( ) MLC20. Each point presents the means ± SD of four or five experiments.

Phosphopeptide Maps Phosphopeptide maps differentiate MLCK-catalyzed MLC20 phosphorylation (Ser19 and Thr18/Ser19) from that catalyzed by PKC (Ser1, Ser2 and Thr9). In our laboratory, MLC20 was extracted from 32P-labeled smooth muscle and was purified using immunoprecipitation16 and SDS-PAGE. The MLC20 was digested with tosylphenylalanyl chloromethyl ketone-treated trypsin. The digested samples were subjected to high-voltage, thin-layer electrophoresis and chromatography. We used autoradiography to locate the 32P phosphopeptides. The phosphopeptides containing P-Ser19 or P-Thr18/ P-Ser19 show different mobilities from those including P-Ser1, P-Ser2 or P-Thr9. This method can quantify the MLC20 phosphorylation on Ser19, or Thr18 and Ser19, as distinguished from other phosphorylation sites of MLC20. However, this method cannot easily deal with many samples at once as compared with the Glycerol-PAGE method. Furthermore, a relatively higher concentration of radioisotope is required for this method.

Specific Antibodies Against Phosphorylated MLC20 on Ser19 or Thr18/Ser19 Monophosphorylated peptide at Ser19 (peptide 1) and diphosphorylated peptide at Thr18/Ser19 (peptide 2) of partial sequence of MLC20 were synthesized and coupled with keyhole limpet hemocyanin. Peptide 1 was injected into BALB/C mice and monoclonal antibody (mAb-p) against monophosphorylated MLC was prepared.17 Peptide 2 was injected into rabbits and a polyclonal antibody (pAb-p2) against diphosphorylated MLC20 was prepared.17 As shown in Figure 7.1, mAb-p only recognized monophosphorylated MLC20 which was prepared using MLCK, and pAb-p2 only recognized MLC20 diphosphorylated by MLCK. These antibodies did not recognize PKC-dependent mono- or diphosphorylated MLC20. The method18,19 of SDS-PAGE or Glycerol-PAGE followed by immunoblot using these antibodies differentiately quantifies MLCK-catalyzed MLC20 phosphorylation (Ser19 and

100

Molecular Mechanisms of Smooth Muscle Contraction Fig. 7.3. Concentration/response curves for MLC20 Diphosphorylation in response to calyculin A with PGF2a. Cultured smooth muscle cells (SM3) were stimulated with various concentration of PGF2α in the presence ( ) or in the absence ( ) of 10 nM calyculin A. The extent of diphosphorylated MLC20 2 minutes after stimulation is shown as means ± SD from four independent experiments.

Thr18/Ser19) from that catalyzed by PKC (Ser1, Ser2 and Thr9). Furthermore, this method can easily deal with many samples at once as compared with phosphopeptide maps.

Diphosphorylation of MLC20 in Intact Smooth Muscle Diphosphorylation of MLC20 has been observed in intact smooth muscles such as carbachol- or oxytocin-stimulated rat uterus,20 carbachol-stimulated bovine trachea,5 endothelin-1-stimulated lamb tracheal muscle.21 Tryptic phosphopeptide mapping showed that the sites of the diphosphorylation were Thr18 and Ser19, which are MLCK-catalyzed sites.5 Diphosphorylation of MLC20 was also observed in phorbol dibutyrate-treated carotid artery.22 In this case, tryptic phosphopeptide mapping revealed peptides characteristic for both MLCK and PKC phosphorylation. Only phorbol esters may induce phosphorylation of MLC20 on Ser1, Ser2 or Thr9 sites, and other physiological stimulants may induce mainly MLCK-catalyzed phosphorylation of MLC20 in intact smooth muscle. The extent of diphosphorylation was reported not to exceed 10-15% of total MLC20 upon stimulation of intact arteries with general physiological stimulants. Thus, it was generally accepted that the contribution of MLC20 diphosphorylation on Thr18 and Ser19 to the contraction of smooth muscle was not very great. However, we found that the diphosphorylation of MLC20 was induced by specific agents, and that the rate of force generation was accelerated with increased diphosphorylation of MLC20.23 Figure 7.2 shows the relationship between concentration of contractile agent and extent of MLC20 phosphorylation in rabbit aortic strips. A comparison was made 5 minutes after the addition of the contractile agent, in which the diphosphorylated form was induced maximally by Prostaglandin F2-alpha (PGF2α). Only the monophosphorylated form was induced by K+ or by histamine, even at a high dose that led to the maximal phosphorylation (Figure 7.2A, C) In contrast, the diphosphorylated form was induced by PGF2α, even at low concentration (Figure 7.2B). The amount of incorporated phosphate stimulated with 30 µM PGF2α was 0.46 mol MLC20, which is nearly equal to that seen with 100 µM histamine (0.47 mol MLC20). However, the diphosphorylated form was not induced by histamine. We

Diphosphorylation of Myosin Light Chain and Spastic Contraction of Smooth Muscle

101

Table 7.1. Relationships between MLC20 phosphorylation and maximal rate of force generation Agonists

KCI (40mM) PFG2α (30µM) Histamine (7µM)

Monophosphorylated MLC20 (%)

Diphosphorylated MLC20 (%)

Total Phosphorylated MLC (%)

Rate of force generation mg/sec

38.8±1.0 35.8±1.6 41.5±1.5

0 4.9±2.4 0

38.3±1.0 40.9±2.3 41.5±1.5

11.7±1.0 24.2±1.1 8.8±0.6

Rabbit aortic strips were stimulated with three types of contractile agents and comparison were made 1 minute after the addition of the drugs. The rate of force generation was maximal at 1 minute and the amount of total phosphorylated MLC20 induced by each agonist was practically equal; however, the maximal rate of force generation stimulated with PFGα (30µM) was twice that seen in KC1 (40mM) or Histamine (7µM). Results are expressed as mean ± SEM of four or five experiments.

confirmed that the phosphorylation sites of diphosphorylated MLC20 induced by PGF2α in rabbit aortic arteries were mainly Thr18 and Ser19, using pAb-p2. These results suggest that the amount of diphosphorylation of MLC20 formation may not simply correlate with the amount of monophosphorylation of MLC20 in intact smooth muscle. The rates of force generation were measured 1 minutes after stimulation with 40 mM K+, 30 µM PGF2α and 7 µM histamine (Table 7.1). At this point the rate of force generation was maximal and the amount of total phosphorylated MLC20 was practically equal. However, the rate of force generation in the case of PGF2α stimulation was about double that seen with K+ or histamine. Thus, we think that the diphosphorylated form of MLC20, specifically induced by certain agents such as PGF2α (one of the candidates for spasmogen) modifies the mode of contraction of smooth muscle.

Mechanism for the Formation of MLC20 Diphosphorylation in Smooth Muscle The level of MLC20 phosphorylation is determined by a balance between MLC20 phosphorylation by MLCK and diphosphorylation by MLC phosphatase.3,24 We have previously suggested that the generdation of diphosphorylated MLC20 may be caused in part by inhibition of MLC20 phosphatase in smooth muscle cells. The concentration/response curves of MLC20 diphosphorylation with PGF2α and the phosphatase inhibitor Calyculin A are shown in Figure 7.3.16 Cultured smooth muscle cells from rabbit aorta (SM3)25 were stimulated with vrious concentrations of PGF2α in the presence or absence of 10 nM Calyculin A and the extent of MLC20 diphosphorylation was measured 2 minutes after the stimulation. Calyculin A (10 nM) alone did not increase MLC20 diphosphorylation in SM3. However, Calyculin A (10 nM) shifted the PGF2α concentration/response curves of MLC20 diphosphorylation to the left and upwards. We also found that direct increase of intracellular calcium concentration of SM3 by the calcium ionophore ionomycine did not change the PGF2α concentration/response curves of MLC20 diphosphorylation (unpublished data). Noda et al reported that in permeabilized porcine aortic smooth muscle cells, the increase in intracellular calcium levels caused monophosphorylation of MLC20 alone, whereas additional treatment with GTPγS, which is thought to inactivate MLC20 phosphatase, caused mono- and diphosphorylation of MLC20.26 These results suggest that inhibition of MLC20 phosphatase activity is important for induction of MLC20 diphosphorylation. The contribution of intracellular calcium concentration to the formation of MLC20 diphosphorylation may be minimal in smooth muscle.

102


Fig.7.4. Time course of MLC20 monophosphorylation (A) and Diphosphorylation (B) of the swine coronary artery in response to serotonin (1 µM)—interleukin-1β-treated spastic coronary segment ( ) and control segment ( ). Each point presents the means ± SD of four or six experiments. * p

Molecular Mechanisms of Smooth Muscle Contraction (Molecular Biology Intelligence Unit)

Translation Mechanisms (Molecular Biology Intelligence Unit)

Endosomes (Molecular Biology Intelligence Unit)

Calreticulin (Molecular Biology Intelligence Unit)

p53 (Molecular Biology Intelligence Unit)

Molecular Mechanisms of Phagocytosis (Medical Intelligence Unit)

Biochemistry of Smooth Muscle Contraction

Biochemistry of Smooth Muscle Contraction

Discovering Biomolecular Mechanisms with Computational Biology (Molecular Biology Intelligence Unit)

Molecular Biology of the Parathyroid (Molecular Biology Intelligence Unit)

Discovering Biomolecular Mechanisms with Computational Biology (Molecular Biology Intelligence Unit)

Muscle Development in Drosophilia (Molecular Biology Intelligence Unit)

Immunobiology of Carbohydrates (Molecular Biology Intelligence Unit)

Molecular Basis Of Epithelial Appendage Morphogenesis (Molecular Biology Intelligence Unit)

Ubiquitin and Disease (Molecular Biology Intelligence Unit)

HOX Gene Expression (Molecular Biology Intelligence Unit)

Cell-Cell Channels (Molecular Biology Intelligence Unit)

Lipocalins (Molecular Biology Intelligence Unit (Unnumbered).)

Protein Kinase C (Molecular Biology Intelligence Unit)

Rho GTPases (Molecular Biology Intelligence Unit)

Tight Junctions (Molecular Biology Intelligence Unit)

VEGF in Development (Molecular Biology Intelligence Unit)

Branching Morphogenesis (Molecular Biology Intelligence Unit)

Ceramide Signaling (Molecular Biology Intelligence Unit)

The Nucleolus (Molecular Biology Intelligence Unit)

HOX Gene Expression (Molecular Biology Intelligence Unit)

The Molecular Mechanisms of Axenfeld-Rieger Syndrome (Medical Intelligence Unit)

Lysosomal Pathways of Protein Degradation (Molecular Biology Intelligence Unit)

Proteasomes : The World of Regulatory Proteolysis (Molecular Biology Intelligence Unit)

The Biogenesis of Cellular Organelles (Molecular Biology Intelligence Unit)

Regulation of G1 Phase Progression (Molecular Biology Intelligence Unit)

Molecular Mechanisms of Smooth Muscle Contraction (Molecular Biology Intelligence Unit)

Translation Mechanisms (Molecular Biology Intelligence Unit)

Endosomes (Molecular Biology Intelligence Unit)

Calreticulin (Molecular Biology Intelligence Unit)

p53 (Molecular Biology Intelligence Unit)

Molecular Mechanisms of Phagocytosis (Medical Intelligence Unit)

Biochemistry of Smooth Muscle Contraction

Biochemistry of Smooth Muscle Contraction

Discovering Biomolecular Mechanisms with Computational Biology (Molecular Biology Intelligence Unit)

Molecular Biology of the Parathyroid (Molecular Biology Intelligence Unit)

Discovering Biomolecular Mechanisms with Computational Biology (Molecular Biology Intelligence Unit)

Muscle Development in Drosophilia (Molecular Biology Intelligence Unit)

Immunobiology of Carbohydrates (Molecular Biology Intelligence Unit)

Molecular Basis Of Epithelial Appendage Morphogenesis (Molecular Biology Intelligence Unit)

Ubiquitin and Disease (Molecular Biology Intelligence Unit)

HOX Gene Expression (Molecular Biology Intelligence Unit)

Cell-Cell Channels (Molecular Biology Intelligence Unit)

Lipocalins (Molecular Biology Intelligence Unit (Unnumbered).)

Protein Kinase C (Molecular Biology Intelligence Unit)

Rho GTPases (Molecular Biology Intelligence Unit)

Tight Junctions (Molecular Biology Intelligence Unit)

VEGF in Development (Molecular Biology Intelligence Unit)

Branching Morphogenesis (Molecular Biology Intelligence Unit)

Ceramide Signaling (Molecular Biology Intelligence Unit)

The Nucleolus (Molecular Biology Intelligence Unit)

HOX Gene Expression (Molecular Biology Intelligence Unit)

The Molecular Mechanisms of Axenfeld-Rieger Syndrome (Medical Intelligence Unit)

Lysosomal Pathways of Protein Degradation (Molecular Biology Intelligence Unit)

Proteasomes : The World of Regulatory Proteolysis (Molecular Biology Intelligence Unit)

The Biogenesis of Cellular Organelles (Molecular Biology Intelligence Unit)

Regulation of G1 Phase Progression (Molecular Biology Intelligence Unit)

Recommend Documents