MOLECULAR REGULATION OF SMOOTH MUSCLE ACTOMYOSIN

Summary

Principal Investigator: Mitsuo Ikebe
Abstract: The long term objective of this research is to understand the regulation and function of myosin in smooth muscle cells. It is well accepted that the motor activity of smooth muscle myosin is regulated by phosphorylation of the regulatory light chain (RLC) at Ser19. However, it is not understood how the phosphorylation of RLC regulates the myosin motor activity. Our hypothesis is that phosphorylation of RLC changes a conformation that attenuates an inter-head interaction at RLC binding domain, and this change is transmitted to the motor domain via a long -helix shaft of the C-terminal domain of the head. We will verify this hypothesis by producing and expressing various engineered myosins and characterizing their motor function by enzymatic and mechanical assays. It is known that smooth muscle contraction is not proportional to the level of myosin phosphorylation. This can be, in part, due to the cooperative nature of the two heads of myosin; however, how the motor activity of myosin phosphorylated at a single head affects or is affected by cooperativity is not understood. Thus a major question is whether or not one head influences the motor activity of the other head. This question is best answered (and may in fact only be answerable) by using a single molecule assay system which enables us to determine ATP turnover and mechanical events simultaneously. This will be done in the proposed study. Smooth muscle cells express various myosin isoforms that show distinct function in vitro. However, the physiological significance of these isoforms for smooth muscle contraction is obscure. The critical questions are whether or not these isoforms form heterodimers, and whether or not these isoforms produce co- filaments in cells. These questions will be addressed by the use of advanced 3D fluorescence microscopy and digital imaging systems for precise colocalization analysis, the use of electron microscopy to measure filament structure and composition, and the use of immunochemistry/biochemistry/molecular biology for the detection of heterodimers. Finally, smooth muscle myosin forms a unique filament structure called "side polar" which may be critical for the contractile characteristics of smooth muscle. We will identify the molecular basis of this filament structure. The itemized specific aims are: 1) To define the structure of the 20,000 dalton light chain critical for the phosphorylation- induced activation of the myosin motor; 2) To define the heavy chain structure responsible for the regulation and function of smooth muscle myosin motor activity; 3) To define the cooperativity between the two heads of smooth muscle myosin; 4) To define whether or not myosin forms heterodimers with different isoforms; 5) To define the localization of various myosin isoforms expressed in smooth muscle cells; 6) To define the molecular basis of side polar myosin filament structure of smooth muscle myosin.
Funding Period: 1992-07-16 - 2006-06-30
more information: NIH RePORT

Top Publications

  1. pmc Novel ZIP kinase isoform lacks leucine zipper
    Norio Takamoto
    Department of Physiology, University of Massachusetts Medical School, 55 Lake Avenue North, Worcester, MA 01655, USA
    Arch Biochem Biophys 456:194-203. 2006
  2. ncbi Supervillin slows cell spreading by facilitating myosin II activation at the cell periphery
    Norio Takizawa
    Department of Cell Biology, University of Massachusetts Medical School, Worcester, MA 01605, USA
    J Cell Sci 120:3792-803. 2007
  3. pmc Kinetic and motor functions mediated by distinct regions of the regulatory light chain of smooth muscle myosin
    Shaowei Ni
    Department of Biochemistry and Molecular Biology, University of Nevada School of Medicine, Reno, NV 89557, USA
    Biochim Biophys Acta 1794:1599-605. 2009
  4. pmc Reverse conformational changes of the light chain-binding domain of myosin V and VI processive motor heads during and after hydrolysis of ATP by small-angle X-ray solution scattering
    Yasunobu Sugimoto
    Division of Biophysical Engineering, Graduate School of Engineering Science, Osaka University, Toyonaka, Osaka, Japan
    J Mol Biol 392:420-35. 2009
  5. pmc Impacts of Usher syndrome type IB mutations on human myosin VIIa motor function
    Shinya Watanabe
    Department of Physiology, University of Massachusetts Medical School, 55 Lake Avenue North, Worcester, Massachusetts 01655, USA
    Biochemistry 47:9505-13. 2008
  6. pmc Smooth muscle myosin phosphorylated at single head shows sustained mechanical activity
    Hiroto Tanaka
    PRESTO, Japan Science and Technology Agency, 3, 5 Chiyodaku, Tokyo 102 0075, Japan
    J Biol Chem 283:15611-8. 2008
  7. pmc The globular tail domain puts on the brake to stop the ATPase cycle of myosin Va
    Xiang Dong Li
    Departments of Physiology and Cell Biology, University of Massachusetts Medical School, Worcester, MA 01655, USA
    Proc Natl Acad Sci U S A 105:1140-5. 2008
  8. pmc Human myosin Vc is a low duty ratio nonprocessive motor
    Shinya Watanabe
    Department of Physiology, University of Massachusetts Medical School, Worcester, Massachusetts 01655, USA
    J Biol Chem 283:10581-92. 2008
  9. pmc The phosphorylation of myosin II at the Ser1 and Ser2 is critical for normal platelet-derived growth factor induced reorganization of myosin filaments
    Satoshi Komatsu
    Department of Physiology, University of Massachusetts Medical School, Worcester, MA 01655, USA
    Mol Biol Cell 18:5081-90. 2007
  10. ncbi cGMP-dependent relaxation of smooth muscle is coupled with the change in the phosphorylation of myosin phosphatase
    Kensei Nakamura
    University of Massachusetts Medical School, Department of Physiology, 55 Lake Avenue North, Worcester, MA 01655, USA
    Circ Res 101:712-22. 2007

Scientific Experts

  • Gary J Kargacin
  • Xiang Dong Li
  • Satoshi Komatsu
  • Mitsuo Ikebe
  • Shinya Watanabe
  • Reiko Ikebe
  • Kazuaki Homma
  • Osamu Sato
  • Yasuhiko Koga
  • Shaowei Ni
  • Yasunobu Sugimoto
  • Nobuhisa Umeki
  • Hiroto Tanaka
  • Norio Takizawa
  • Kensei Nakamura
  • Taketoshi Kambara
  • Norio Takamoto
  • Nobutaka Saeki
  • Hirofumi Onishi
  • Christine R Cremo
  • Feng Hong
  • Kevin C Facemyer
  • Katsuzo Wakabayashi
  • JONATHAN E BAKER
  • Paul D Brewer
  • Howard D White
  • Hideo Higuchi
  • Toshio Yanagida
  • Tomonobu M Watanabe
  • Junya Awata
  • Elizabeth J Luna
  • Hiroyasu Sakai
  • Naohisa Niiro
  • Shigeru Komaba
  • Hiroshi Tokuo

Detail Information

Publications19

  1. pmc Novel ZIP kinase isoform lacks leucine zipper
    Norio Takamoto
    Department of Physiology, University of Massachusetts Medical School, 55 Lake Avenue North, Worcester, MA 01655, USA
    Arch Biochem Biophys 456:194-203. 2006
    ..These results suggest that the newly found hZIPK-S as well as the long isoform play an important role in the regulation of myosin phosphorylation...
  2. ncbi Supervillin slows cell spreading by facilitating myosin II activation at the cell periphery
    Norio Takizawa
    Department of Cell Biology, University of Massachusetts Medical School, Worcester, MA 01605, USA
    J Cell Sci 120:3792-803. 2007
    ..Thus, the membrane protein SV apparently contributes to myosin II assembly during cell spreading by modulating myosin II regulation by L-MLCK...
  3. pmc Kinetic and motor functions mediated by distinct regions of the regulatory light chain of smooth muscle myosin
    Shaowei Ni
    Department of Biochemistry and Molecular Biology, University of Nevada School of Medicine, Reno, NV 89557, USA
    Biochim Biophys Acta 1794:1599-605. 2009
    ....
  4. pmc Reverse conformational changes of the light chain-binding domain of myosin V and VI processive motor heads during and after hydrolysis of ATP by small-angle X-ray solution scattering
    Yasunobu Sugimoto
    Division of Biophysical Engineering, Graduate School of Engineering Science, Osaka University, Toyonaka, Osaka, Japan
    J Mol Biol 392:420-35. 2009
    ..The results suggest that some additional alterations or elements are required for MVI-S1 to take maximal working strokes along the actin filament...
  5. pmc Impacts of Usher syndrome type IB mutations on human myosin VIIa motor function
    Shinya Watanabe
    Department of Physiology, University of Massachusetts Medical School, 55 Lake Avenue North, Worcester, Massachusetts 01655, USA
    Biochemistry 47:9505-13. 2008
    ..Taken together, the results suggest that the mutations responsible for USH1B cause the complete loss of the actin-activated ATPase activity or the reduction of duty ratio of myosin VIIa...
  6. pmc Smooth muscle myosin phosphorylated at single head shows sustained mechanical activity
    Hiroto Tanaka
    PRESTO, Japan Science and Technology Agency, 3, 5 Chiyodaku, Tokyo 102 0075, Japan
    J Biol Chem 283:15611-8. 2008
    ..We propose that the "latch state" can be explained by the motor characteristics of SHPMII that is present during the sustained phase of contraction...
  7. pmc The globular tail domain puts on the brake to stop the ATPase cycle of myosin Va
    Xiang Dong Li
    Departments of Physiology and Cell Biology, University of Massachusetts Medical School, Worcester, MA 01655, USA
    Proc Natl Acad Sci U S A 105:1140-5. 2008
    ..We propose that binding of the GTD to the motor domain prevents the movement of the converter/lever arm during ATP hydrolysis cycle, thus inhibiting the chemical cycle of the motor domain...
  8. pmc Human myosin Vc is a low duty ratio nonprocessive motor
    Shinya Watanabe
    Department of Physiology, University of Massachusetts Medical School, Worcester, Massachusetts 01655, USA
    J Biol Chem 283:10581-92. 2008
    ..Our findings suggest that myosin Vc fulfills its function as a cargo transporter by different mechanisms from other myosin V isoforms...
  9. pmc The phosphorylation of myosin II at the Ser1 and Ser2 is critical for normal platelet-derived growth factor induced reorganization of myosin filaments
    Satoshi Komatsu
    Department of Physiology, University of Massachusetts Medical School, Worcester, MA 01655, USA
    Mol Biol Cell 18:5081-90. 2007
    ....
  10. ncbi cGMP-dependent relaxation of smooth muscle is coupled with the change in the phosphorylation of myosin phosphatase
    Kensei Nakamura
    University of Massachusetts Medical School, Department of Physiology, 55 Lake Avenue North, Worcester, MA 01655, USA
    Circ Res 101:712-22. 2007
    ..This results in the decrease in MLC phosphorylation and smooth muscle relaxation...
  11. ncbi Myosin Va becomes a low duty ratio motor in the inhibited form
    Osamu Sato
    Department of Physiology, University of Massachusetts Medical School, Worcester, Massachueetts 01655, USA
    J Biol Chem 282:13228-39. 2007
    ....
  12. ncbi Localization of telokin at the intercalated discs of cardiac myocytes
    Gary J Kargacin
    Department of Physiology and Biophysics, University of Calgary, Calgary, Alta, Canada T2N 4N1
    Arch Biochem Biophys 456:151-60. 2006
    ....
  13. ncbi The globular tail domain of myosin Va functions as an inhibitor of the myosin Va motor
    Xiang Dong Li
    Department of Physiology, University of Massachusetts Medical School, Worcester, Massachusetts 01655, USA
    J Biol Chem 281:21789-98. 2006
    ....
  14. ncbi Drosophila myosin VIIA is a high duty ratio motor with a unique kinetic mechanism
    Shinya Watanabe
    Department of Physiology, University of Massachusetts Medical School, 55 Lake Avenue North, Worcester, MA 01655, USA
    J Biol Chem 281:7151-60. 2006
    ..9. Therefore, myosin VIIA is classified to be a high duty ratio motor. The present results suggested that myosin VIIA can be a processive motor to serve cargo trafficking in cells once it forms a dimer structure...
  15. ncbi A unique ATP hydrolysis mechanism of single-headed processive myosin, myosin IX
    Taketoshi Kambara
    Department of Physiology, University of Massachusetts Medical School, Worcester, Massachusetts 01655, USA
    J Biol Chem 281:4949-57. 2006
    ..After ATP hydrolysis, Pi is quickly released to form a strong actin binding form, and a power stroke takes place...
  16. ncbi Myosin X is a high duty ratio motor
    Kazuaki Homma
    Department of Physiology, University of Massachusetts Medical School, Worcester, MA 01655, USA
    J Biol Chem 280:29381-91. 2005
    ..The duty ratio calculated from our kinetic model, 0.6, was consistent with the duty ratio, 0.7, obtained from comparison of K(m ATPase) and K(m motility). Our results suggest that myosin X is a high duty ratio motor...
  17. ncbi Activation of myosin Va function by melanophilin, a specific docking partner of myosin Va
    Xiang Dong Li
    Department of Physiology, University of Massachusetts Medical School, Worcester, Massachusetts 01655, USA
    J Biol Chem 280:17815-22. 2005
    ..The present finding raises the idea that myosin motors are switched upon their binding to the cargo molecules, thus avoiding the waste of ATP consumption...
  18. ncbi BIG1 is a binding partner of myosin IXb and regulates its Rho-GTPase activating protein activity
    Nobutaka Saeki
    Department of Physiology, University of Massachusetts Medical School, 55 Lake Ave, Worcester, Massachusetts 01655, USA
    J Biol Chem 280:10128-34. 2005
    ..The findings raise a concept that the myosin transports the signaling molecule as a cargo that functions as a regulator for the myosin molecule...
  19. ncbi p116Rip decreases myosin II phosphorylation by activating myosin light chain phosphatase and by inactivating RhoA
    Yasuhiko Koga
    Department of Physiology, University of Massachusetts Medical School, Worcester, Massachusetts 01655, USA
    J Biol Chem 280:4983-91. 2005
    ..Based upon these findings, we propose that p116Rip is an important regulatory component that controls the RhoA signaling pathway, thus regulating MLCP activity and myosin phosphorylation in cells...