Abstract

Regulation of the reversible Ser-19 phosphorylation of 20 kDa myosin light chain (MLC) primarily governs the extent of vascular smooth muscle contraction. Although a rise in cytoplasmic Ca/2+ acts as the main triggering mechanism for phosphorylation of MLC by activating Ca/2+- calmodulin-dependent MLC kinase, physiological membrane and cytosolic receptor ligands, such as catecholamines and nitrovasodilators, exert their effect in large part by dynamically changing the Ca/2+ sensitivity of MLC phosphorylation and contractile force. The principle objective of this proposal is to determine the molecular mechanism by which receptor activation of vascular smooth muscle modulates the contractile Ca/2+ sensitivity and to use this information to explore the molecular and cellular mechanism of cardiovascular abnormality, such as hypertension. Particularly, we wish to further elucidate the mechanism of two novel signaling pathways regulating Ca/2+ sensitivity in vascular smooth muscle: protein kinase C (PKC)-induced inhibition and protein kinase G (PKG)- induced activation of MLC phosphatase. We shall determine (1) if PKC, through phosphorylation of a novel muscle-specific protein phosphatase inhibitor protein CPI-17, increase the contractile Ca/2+ sensitivity in a manner that indicates its major effect is to inhabit MLC phosphatase, (2) if, how much and at which site(s) in situ CPI-17 is phosphorylated in response to PKC activations and other Ca/2+-sensitizing agonists, (3) if subcellular localization of CPI-17 is regulated by PKC activators and other agonists and if this translocation is modified by various inhibitors, (4) if PKG indirectly activates MLC phosphatase to decrease the Ca/2+ sensitivity and alters the phosphorylation levels/site(s) of CPI-17, (5) if cGMP changes the CPI-17 translocation and PKG is co- localized with CPI-17 and/or PKC during the Ca/2+ desensitization.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL051824-08
Application #
6330070
Study Section
Experimental Cardiovascular Sciences Study Section (ECS)
Program Officer
Lin, Michael
Project Start
1993-06-01
Project End
2002-11-30
Budget Start
2000-12-01
Budget End
2001-11-30
Support Year
8
Fiscal Year
2001
Total Cost
$290,032
Indirect Cost

  Institution

Name
Georgetown University
Department
Physiology
Type
Schools of Dentistry
DUNS #
049515844
City
Washington
State
DC
Country
United States
Zip Code
20057

  Publications

Kitazawa, Toshio (2010) G protein-mediated Ca²+-sensitization of CPI-17 phosphorylation in arterial smooth muscle. Biochem Biophys Res Commun 401:75-8
Woodsome, Terence P; Polzin, Atsuko; Kitazawa, Kazuyo et al. (2006) Agonist- and depolarization-induced signals for myosin light chain phosphorylation and force generation of cultured vascular smooth muscle cells. J Cell Sci 119:1769-80
Kitazawa, Toshio; Polzin, Atsuko N; Eto, Masumi (2004) CPI-17-deficient smooth muscle of chicken. J Physiol 557:515-28
Eto, Masumi; Kitazawa, Toshio; Brautigan, David L (2004) Phosphoprotein inhibitor CPI-17 specificity depends on allosteric regulation of protein phosphatase-1 by regulatory subunits. Proc Natl Acad Sci U S A 101:8888-93
Kitazawa, Toshio; Eto, Masumi; Woodsome, Terence P et al. (2003) Phosphorylation of the myosin phosphatase targeting subunit and CPI-17 during Ca2+ sensitization in rabbit smooth muscle. J Physiol 546:879-89
Eto, M; Kitazawa, T; Yazawa, M et al. (2001) Histamine-induced vasoconstriction involves phosphorylation of a specific inhibitor protein for myosin phosphatase by protein kinase C alpha and delta isoforms. J Biol Chem 276:29072-8
Woodsome, T P; Eto, M; Everett, A et al. (2001) Expression of CPI-17 and myosin phosphatase correlates with Ca(2+) sensitivity of protein kinase C-induced contraction in rabbit smooth muscle. J Physiol 535:553-64
Kitazawa, T; Eto, M; Woodsome, T P et al. (2000) Agonists trigger G protein-mediated activation of the CPI-17 inhibitor phosphoprotein of myosin light chain phosphatase to enhance vascular smooth muscle contractility. J Biol Chem 275:9897-900
Kitazawa, T; Takizawa, N; Ikebe, M et al. (1999) Reconstitution of protein kinase C-induced contractile Ca2+ sensitization in triton X-100-demembranated rabbit arterial smooth muscle. J Physiol 520 Pt 1:139-52
Murahashi, T; Fujita, A; Kitazawa, T (1999) Ca2+ -induced Ca2+ desensitization of myosin light chain phosphorylation and contraction in phasic smooth muscle. Mol Cell Biochem 190:91-8

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