Regulation of smooth muscle-myosin phosphatase 1 kinase

描述（由申请人提供）：平滑肌在多种生理过程中起着至关重要的作用，从通过控制周围血管直径来调节血压，到通过控制食物通过肠道的机械运动来调节消化过程，再到通过控制阴茎勃起和胎儿分娩来控制生殖功能，再到通过调节气道直径来调节肺活量。控制所有平滑肌收缩状态的主要因素是肌球蛋白轻链（myosin light chain, LC20）的磷酸化水平。在平滑肌中，LC20的稳态磷酸化是由肌球蛋白轻链激酶（MLCK）和肌球蛋白磷酸酶（SMPP-1M）的相反活性决定的。通过G蛋白偶联受体通过小GTP结合蛋白RhoA作用抑制SMPP-1M活性已被证明可导致钙致敏平滑肌。各种平滑肌对钙的敏感性的改变被假设为与许多与平滑肌功能障碍相关的疾病（如高血压、野马痉挛、性功能障碍、胃肠道疾病和青光眼）相关的潜在原因。因此，控制肌球蛋白去磷酸化状态的信号元件可能代表了与平滑肌功能障碍相关的各种人类疾病的治疗干预的有吸引力的点。最近，我们的实验室发现MYPT1激酶（MYPT1 K）是一种共定位的SMPP-1M激酶，它选择性地磷酸化磷酸酶的肌球蛋白靶向亚基（MYPT1）和抑制剂蛋白CPI17以抑制活性。将组成活性形式的MYPT1 K添加到渗透性平滑肌中，通过抑制SMPP-1M活性导致深刻的钙致敏。在平滑肌中，MYPT1K在多种钙致敏激动剂的作用下被激活，这种激活可以被Rho激酶抑制剂Y-27632阻断。这些发现表明MYPT1K介导从Rho激酶到SMPP-1M的钙敏感信号，然而，由于MYPT1K蛋白激酶在体外不是Rho激酶的直接底物，因此MYPT1K在平滑肌中的调节机制尚不清楚。在本提案中，我们将采用肌肉生理学，质谱，分子生物学，小干扰RNA和小分子化学的独特混合来确定Rho依赖的信号通路在平滑肌中激活MYPT1K的分子机制。

DESCRIPTION (provided by applicant: Smooth muscle plays an essential role in a wide variety of physiological processes, from the regulation of blood pressure by controlling vessel diameter in the periphery, to digestive processes by controlling mechanical movement of food though the gut, to reproductive functions by controlling penile erection and delivery of the fetus at birth, to regulation of lung capacity through regulation of airway diameter. A major factor governing the contractile state of all smooth muscles is the phosphorylation level of myosin light chain (LC20). In smooth muscle, steady state phosphorylation of LC20 is dictated by the opposing activities of myosin light chain kinase (MLCK) and myosin phosphatase (SMPP-1M). Inhibition of SMPP-1M activity through G protein coupled receptors acting through the small GTP binding protein RhoA has been shown to bring about calcium sensitization smooth muscle. Alterations in the sensitivity of various smooth muscles to calcium is hypothesized to be an underlying cause associated with many diseases associated with smooth muscle dysfunction such as hypertension, bronco spasm, sexual dysfunction, gastrointestinal disorders and glaucoma. Therefore signaling elements controlling myosin dephosphorylation state may represent attractive points of therapeutic intervention in a variety of human diseases associated with smooth muscle dysfunction. Recently our laboratory identified MYPT1 kinase (MYPT1 K) as a co-localizing SMPP-1M kinase that selectively phosphorylated the myosin targeting subunit (MYPT1) of the phosphatase and the inhibitor protein CPI17 to inhibit activity. Addition of constitutively active forms of MYPT1 K to permeabilized smooth muscles causes profound calcium sensitization through inhibition of SMPP-1M activity. In smooth muscle, MYPT1K is activated in response to a variety of calcium-sensitizing agonists and this activation can be blocked with the Rho kinase inhibitor Y-27632. These findings suggest MYPT1K mediates calcium-sensitizing signals from Rho kinase to SMPP-1M, however, the mechanisms by which MYPT1K is regulated in smooth muscle are unknown, since the protein kinase is not a direct substrate for Rho kinase in vitro. In this proposal we will employ a unique blend of muscle physiology, mass spectrometry, molecular biology, small interfering RNA's and small molecule chemistry to determine the molecular mechanisms by which Rho dependant signaling pathways bring about activation of MYPT1K in smooth muscle.