Molecular Mechanisms for Regulation of Vascular Smooth Muscle Myosin Phosphatase

描述(由申请者提供)：本项目的长期目标是了解血管平滑肌收缩的调节机制。血管平滑肌是治疗高血压、降低血压的靶点。激动剂刺激诱导G蛋白的激活，导致肌球蛋白的磷酸化和平滑肌细胞的收缩。这一过程受肌球蛋白磷酸酶抑制的支配，并导致钙非依赖性的收缩。因此，肌球蛋白磷酸酶的活性是收缩对钙的敏感性的决定因素。RhoA依赖的激酶(ROCK)被认为使肌球蛋白磷酸酶(MYPT1)的一个调节亚基磷酸化，导致磷酸酶失活。然而，最近的研究表明，MYPT1的磷酸化在收缩过程中没有变化，因此一个关键的问题仍然存在：肌球蛋白磷酸酶是如何被激动剂刺激的平滑肌所抑制的？一种新的肌球蛋白磷酸酶抑制蛋白，称为CPI-17，是一个候选蛋白，在血管平滑肌中高表达。在激动剂刺激下，如蛋白激酶C(PKC)和ROCK激活，在Thr38处使CPI-17磷酸化，从而将其转化为有效的抑制物。CPI-17的磷酸化和去磷酸化可逆地与肌肉的收缩和松弛平行发生。因此，CPI-17是了解肌球蛋白磷酸酶在平滑肌中的调节的关键分子。CPI-17家族中的另一种肌球蛋白磷酸酶抑制物，名为PHI-1，也在平滑肌中表达。我们将询问两种抑制剂是如何控制平滑肌中的肌球蛋白磷酸酶的。目标1将检验这一假说，即CPI-17和PHI-1在G蛋白激活后被不同的激酶信号亚群磷酸化。目的2是确定最近在细胞中检测到的CPI-17在Ser12和Ser128处的磷酸化作用。目的3通过突变/计算分析阐明CPI-17特异性抑制肌球蛋白磷酸酶的分子机制。该项目将揭示G蛋白激活的多种信号如何汇聚到肌球蛋白磷酸酶抑制剂上以控制平滑肌收缩的分子机制，这在血管生物学中具有重要的生理意义。(相关)阻断收缩的药物用于治疗高血压、哮喘和勃起功能障碍。该项目的结果将揭示平滑肌疾病的潜在治疗靶点。

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.