The long-term goal of this project is to understand regulatory mechanisms for vascular smooth muscle contraction. Vascular smooth muscle is a target for treatment of hypertension to reduce blood pressure. Agonist stimuli induce activation of G-proteins that results in phosphorylation of myosin and contraction of smooth muscle cells. This process is governed by the inhibition of myosin phosphatase, and causes the Ca2+-independent contraction. Thus, the activity of myosin phosphatase is a determinant of the Ca2+ sensitivity of the contraction. RhoA-dependent kinase (ROCK) is believed to phosphorylate a regulatory subunit of myosin phosphatase (MYPT1) to cause inactivation of the phosphatase. However, recent studies imply that phosphorylation of MYPT1 is unchanged during the contraction, so that a critical question still remains: How is myosin phosphatase inhibited in response to agonist stimulation of smooth muscle? A novel inhibitor protein for myosin phosphatase, called CPI-17, is a candidate and highly expressed in vascular smooth muscles. The kinases activated with agonist stimuli, such as protein kinase C (PKC) and ROCK, phosphorylate CPI-17 at Thr38, which converts it into a potent inhibitor. Phosphorylation and dephosphorylation of CPI-17 reversibly occur in parallel to contraction and relaxation of the muscle. Thus, CPI-17 is a key molecule to understand the regulation of myosin phosphatase in smooth muscle. Another myosin phosphatase inhibitor in the CPI-17 family, named PHI-1, is also expressed in smooth muscle. We will ask how two inhibitors control myosin phosphatase in smooth muscle.
Aim 1 will test the hypothesis that CPI-17 and PHI-1 are phosphorylated by separate subsets of kinase signals in response to G-protein activation.
Aim 2 is to define the role of phosphorylation of CPI-17 at Ser12 and Ser128, which are recently detected in cells.
Aim 3 is to elucidate molecular mechanisms for specific inhibition of myosin phosphatase by CPI-17 by mutational/computational analyses. This project will uncover molecular mechanisms how multiple signals from G-protein activation converge onto myosin phosphatase inhibitors to control smooth muscle contraction, which is a major physiological importance in vascular biology. (Relevance) Drugs, blocking the contraction, are used for treatment of hypertension, asthma and erectile dysfunction. The results of this project will expose potential therapeutic targets for smooth muscle diseases.
|Eto, Masumi; Kirkbride, Jason A; Chugh, Rishika et al. (2013) Nuclear localization of CPI-17, a protein phosphatase-1 inhibitor protein, affects histone H3 phosphorylation and corresponds to proliferation of cancer and smooth muscle cells. Biochem Biophys Res Commun 434:137-42|
|Eto, Masumi; Brautigan, David L (2012) Endogenous inhibitor proteins that connect Ser/Thr kinases and phosphatases in cell signaling. IUBMB Life 64:732-9|
|Kim, Jee In; Urban, Mark; Young, Garbo D et al. (2012) Reciprocal regulation controlling the expression of CPI-17, a specific inhibitor protein for the myosin light chain phosphatase in vascular smooth muscle cells. Am J Physiol Cell Physiol 303:C58-68|
|Khromov, Alexander; Choudhury, Nandini; Stevenson, Andra S et al. (2009) Phosphorylation-dependent autoinhibition of myosin light chain phosphatase accounts for Ca2+ sensitization force of smooth muscle contraction. J Biol Chem 284:21569-79|
|Eto, Masumi (2009) Regulation of cellular protein phosphatase-1 (PP1) by phosphorylation of the CPI-17 family, C-kinase-activated PP1 inhibitors. J Biol Chem 284:35273-7|
|Xiao, Liqing; Eto, Masumi; Kazanietz, Marcelo G (2009) ROCK mediates phorbol ester-induced apoptosis in prostate cancer cells via p21Cip1 up-regulation and JNK. J Biol Chem 284:29365-75|
|Mori, Shunsuke; Iwaoka, Ryou; Eto, Masumi et al. (2009) Solution structure of the inhibitory phosphorylation domain of myosin phosphatase targeting subunit 1. Proteins 77:732-5|
|Kitazawa, Toshio; Semba, Shingo; Huh, Yang Hoon et al. (2009) Nitric oxide-induced biphasic mechanism of vascular relaxation via dephosphorylation of CPI-17 and MYPT1. J Physiol 587:3587-603|
|Freitas, Maria Regina; Eto, Masumi; Kirkbride, Jason A et al. (2009) Y27632, a Rho-activated kinase inhibitor, normalizes dysregulation in alpha1-adrenergic receptor-induced contraction of Lyon hypertensive rat artery smooth muscle. Fundam Clin Pharmacol 23:169-78|
|Kim, Jee In; Young, Garbo D; Jin, Li et al. (2009) Expression of CPI-17 in smooth muscle during embryonic development and in neointimal lesion formation. Histochem Cell Biol 132:191-8|
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