Molecular Mechanisms for Regulation of Vascular Smooth Muscle Myosin Phosphatase

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DESCRIPTION (provided by applicant): 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.