BIOCHEMISTRY OF CONTRACTILE PROTEINS

Summary

Principal Investigator: David Hartshorne
Affiliation: University of Arizona
Country: USA
Abstract: Myosin II phosphorylation is a major regulatory mechanism in smooth muscle and non-muscle cells. In smooth muscle (arteries, veins, gastrointestinal tract, uterus, etc.) it controls the contraction-relaxation cycle and is implicated in angiogenesis. In non-muscle cells it regulates essential cell functions including: motility, dynamics of cytoskeletal structure, cell division (cytokinesis) and secretion. An alteration of myosin II phosphorylation is involved in several disorders of the cardiovascular system, e.g. hypertension, cerebral and coronary vasospasm, and of other hollow organs, e.g. bronchial asthma, preterm labor and erectile dysfunction. Myosin II phosphorylation also is important in cancer cell motility and metastasis. Thus, understanding the mechanisms regulating myosin II phosphorylation is essential in treatment of these disorders and is of considerable health benefit to our society. Two key enzymes control myosin II phosphorylation: a myosin kinase (usually myosin light chain kinase) and a myosin phosphatase (MP). At constant [Ca2+]i the major factor modulating myosin II phosphorylation is regulation of MP. Both activation (associated with increased cyclic nucleotide levels) and inhibition (implicated in many of the disorders listed above) are documented. The RhoA/Rho-kinase pair is important in inhibition. Objectives of this proposal are to establish the molecular basis for regulation of MP as centered on the myosin phosphatase target subunit (MYPT1). The intent of Specific Aim 1 is to characterize several important interactions of MYPT1, with emphasis on binding of substrates, and the molecular mechanism(s) of regulation. Biochemical analyses will use isolated MP holoenzyme and purified MYPT1 and fragments. The A7r5 cell line (derived from rat aorta) will be used in the next 2 Aims as a model system to test several hypotheses. In SA 2 the effects of cyclic nucleotides, initially cGMP and subsequently cAMP, will be investigated. The objective is to establish the molecular basis for activation of MP. The role of individual isoforms will be probed by RNA interference (siRNA). The final SA characterizes MYPT1 isoforms in A7r5 cells. Subcellular localizations of isoforms will be determined and putative roles of individual isoforms assessed by siRNA. These 3 aims use combined techniques of biochemistry, molecular biology and cell biology. If successful, they will establish an understanding at a molecular level of a process essential for function in muscle and non-muscle cells, and will facilitate pharmacological intervention.
Funding Period: 1978-07-01 - 2010-04-30
more information: NIH RePORT

Top Publications

  1. pmc Myosin loop 2 is involved in the formation of a trimeric complex of twitchin, actin, and myosin
    Daisuke Funabara
    Graduate School of Bioresources, Mie University, Tsu, Mie 514 8507, Japan
    J Biol Chem 284:18015-20. 2009
  2. ncbi Localization of myosin phosphatase target subunit and its mutants
    Yue Wu
    Muscle Biology Group, University of Arizona, Tucson, AZ 85721, USA
    J Muscle Res Cell Motil 26:123-34. 2005
  3. pmc Myosin phosphatase-targeting subunit 1 regulates mitosis by antagonizing polo-like kinase 1
    Shigeko Yamashiro
    Department of Molecular Biology and Biochemistry, Rutgers University, Piscataway, NJ 08854, USA
    Dev Cell 14:787-97. 2008
  4. pmc Myosin phosphatase target subunit: Many roles in cell function
    Fumio Matsumura
    Department of Molecular Biology and Biochemistry, Rutgers University, 604, Allison Road, Piscataway, NJ 08855, USA
    Biochem Biophys Res Commun 369:149-56. 2008
  5. pmc Unphosphorylated twitchin forms a complex with actin and myosin that may contribute to tension maintenance in catch
    Daisuke Funabara
    Graduate School of Bioresources, Mie University, Tsu, Mie 514 8507, Japan
    J Exp Biol 210:4399-410. 2007
  6. ncbi Characterization and function of MYPT2, a target subunit of myosin phosphatase in heart
    Ryuji Okamoto
    Department of Cardiology, Mie University Graduate School of Medicine, 2 174 Edobashi, Tsu, Mie 514 8507, Japan
    Cell Signal 18:1408-16. 2006
  7. ncbi Phosphorylation of Thr695 and Thr850 on the myosin phosphatase target subunit: inhibitory effects and occurrence in A7r5 cells
    Andrea Murányi
    Muscle Biology Group, Department of Nutritional Sciences, University of Arizona, 1177 E 4th Street, Shantz 627, Tucson, AZ 85721 0038, USA
    FEBS Lett 579:6611-5. 2005
  8. ncbi The targeted disruption of the MYPT1 gene results in embryonic lethality
    Ryuji Okamoto
    The First Department of Internal Medicine, Mie University School of Medicine, 2 174 Edobashi, Tsu, Mie 514 8507, Japan
    Transgenic Res 14:337-40. 2005
  9. ncbi Okadaic acid induces phosphorylation and translocation of myosin phosphatase target subunit 1 influencing myosin phosphorylation, stress fiber assembly and cell migration in HepG2 cells
    Beata Lontay
    Department of Medical Chemistry, Research Center for Molecular Medicine, Medical and Health Science Center, University of Debrecen, H 4026 Debrecen, Bem tér 18 B, Hungary
    Cell Signal 17:1265-75. 2005
  10. ncbi Overexpression of myosin phosphatase reduces Ca(2+) sensitivity of contraction and impairs cardiac function
    Hideo Mizutani
    Department of Cardiology and Nephrology, Mie University Graduate School of Medicine, Japan
    Circ J 74:120-8. 2010

Scientific Experts

  • Shigeko Yamashiro
  • David J Hartshorne
  • Ryuji Okamoto
  • Daisuke Funabara
  • Masaaki Ito
  • Hideo Mizutani
  • Ferenc Erdodi
  • Andrea Murányi
  • Shugo Watabe
  • Rika Osawa
  • Fumio Matsumura
  • Satoshi Kanoh
  • Miki Ueda
  • Noboru Suzuki
  • Nobuyuki Moriki
  • Akira Mizoguchi
  • Takeshi Nakano
  • Kyoko Imanaka-Yoshida
  • Yue Wu
  • Andrea Kiss
  • Beata Lontay
  • Satoshi Fujita
  • Katsuhisa Konishi
  • Shinji Satoh
  • Takeo Itoh
  • Masaya Taniguchi
  • Kaoru Dohi
  • Naoki Makino
  • Katsuya Onishi
  • Suechika Suzuki
  • Chieko Hamamoto
  • Akinori Inoue
  • Koji Yamamoto
  • Tetsuya Nakakuki
  • Naoki Isaka
  • Kozo Kaibuchi
  • Katsuhide Mabuchi
  • Zhaojiang Lu
  • Takaaki Kato
  • Nobuaki Takahashi
  • Terenc Tao
  • Kazushi Kimura
  • Dmitry Derkach
  • Hideki Tsumura
  • Hiromitsu Saito
  • Pal Gergely
  • Mariko Kongo

Detail Information

Publications11

  1. pmc Myosin loop 2 is involved in the formation of a trimeric complex of twitchin, actin, and myosin
    Daisuke Funabara
    Graduate School of Bioresources, Mie University, Tsu, Mie 514 8507, Japan
    J Biol Chem 284:18015-20. 2009
    ..The complex formation also showed a moderate Ca(2+) sensitivity in that in the presence of Ca(2+) complex formation was reduced...
  2. ncbi Localization of myosin phosphatase target subunit and its mutants
    Yue Wu
    Muscle Biology Group, University of Arizona, Tucson, AZ 85721, USA
    J Muscle Res Cell Motil 26:123-34. 2005
    ..Stable transfection of HEK 293 cells with GFP-MYPT1 was obtained. MYPT1 and its N-terminal mutants bound to retinoblastoma protein (Rb), raising the possibility that Rb is implicated in the effects caused by overexpression of MYPT1...
  3. pmc Myosin phosphatase-targeting subunit 1 regulates mitosis by antagonizing polo-like kinase 1
    Shigeko Yamashiro
    Department of Molecular Biology and Biochemistry, Rutgers University, Piscataway, NJ 08854, USA
    Dev Cell 14:787-97. 2008
    ..Taken together, our results identify a previously unrecognized role for MYPT1 in regulating mitosis by antagonizing PLK1...
  4. pmc Myosin phosphatase target subunit: Many roles in cell function
    Fumio Matsumura
    Department of Molecular Biology and Biochemistry, Rutgers University, 604, Allison Road, Piscataway, NJ 08855, USA
    Biochem Biophys Res Commun 369:149-56. 2008
    ..Open questions are whether the diverse functions of MP reflect different cellular locations and/or specific roles for the MYPT1 isoforms...
  5. pmc Unphosphorylated twitchin forms a complex with actin and myosin that may contribute to tension maintenance in catch
    Daisuke Funabara
    Graduate School of Bioresources, Mie University, Tsu, Mie 514 8507, Japan
    J Exp Biol 210:4399-410. 2007
    ..25 nm and it is suggested that the D2 site aligns with the myosin heads. It is proposed that the complex formed with the dephosphorylated D2 site of twitchin, F-actin and myosin represents a component of the mechanical linkage in catch...
  6. ncbi Characterization and function of MYPT2, a target subunit of myosin phosphatase in heart
    Ryuji Okamoto
    Department of Cardiology, Mie University Graduate School of Medicine, 2 174 Edobashi, Tsu, Mie 514 8507, Japan
    Cell Signal 18:1408-16. 2006
    ..Similarity of the two MYPT isoforms suggests common enzymatic mechanisms and regulation. Cardiac myosin is a substrate for the MYPT2 holoenzyme, but the Z-line location raises the possibility of other substrates...
  7. ncbi Phosphorylation of Thr695 and Thr850 on the myosin phosphatase target subunit: inhibitory effects and occurrence in A7r5 cells
    Andrea Murányi
    Muscle Biology Group, Department of Nutritional Sciences, University of Arizona, 1177 E 4th Street, Shantz 627, Tucson, AZ 85721 0038, USA
    FEBS Lett 579:6611-5. 2005
    ..Phosphorylation of each site on MYPT1 was detected in A7r5 cells, but Thr850 was preferred by Rho-kinase and Thr695 was phosphorylated by an unidentified kinase(s)...
  8. ncbi The targeted disruption of the MYPT1 gene results in embryonic lethality
    Ryuji Okamoto
    The First Department of Internal Medicine, Mie University School of Medicine, 2 174 Edobashi, Tsu, Mie 514 8507, Japan
    Transgenic Res 14:337-40. 2005
    ..The point of embryonic lethality is before 7.5 dpc. These findings indicate that MYPT1 is essential for mouse embryogenesis...
  9. ncbi Okadaic acid induces phosphorylation and translocation of myosin phosphatase target subunit 1 influencing myosin phosphorylation, stress fiber assembly and cell migration in HepG2 cells
    Beata Lontay
    Department of Medical Chemistry, Research Center for Molecular Medicine, Medical and Health Science Center, University of Debrecen, H 4026 Debrecen, Bem tér 18 B, Hungary
    Cell Signal 17:1265-75. 2005
    ..These changes are associated with an increased level of myosin II phosphorylation and attenuation of hepatic cell migration...
  10. ncbi Overexpression of myosin phosphatase reduces Ca(2+) sensitivity of contraction and impairs cardiac function
    Hideo Mizutani
    Department of Cardiology and Nephrology, Mie University Graduate School of Medicine, Japan
    Circ J 74:120-8. 2010
    ..In vitro, myosin phosphatase target subunit 2 (MYPT2) is a strongly suspected regulatory subunit of cardiac myosin phosphatase (MP), but there is no in-vivo evidence regarding the functions of MYPT2 in the heart...