Regulation of smooth muscle-myosin phosphatase 1 kinase

说明(申请人提供：从通过控制外周血管直径来调节血压，到通过控制食物在肠道中的机械运动来控制消化过程，通过控制出生时阴茎的勃起和分娩来实现生殖功能，再到通过调节呼吸道直径来调节肺活量，平滑肌在各种生理过程中都扮演着重要的角色。肌球蛋白轻链(LC20)的磷酸化水平是控制所有平滑肌肉收缩状态的一个主要因素。在平滑肌中，肌球蛋白轻链激酶(MLCK)和肌球蛋白磷酸酶(SMPP-1M)的相对活性决定了LC20的稳态磷酸化。通过小的GTP结合蛋白RhoA作用于G蛋白偶联受体，从而抑制SMPP-1M的活性，从而导致钙增敏血管。各种平滑肌对钙敏感性的改变被认为是许多与平滑肌功能障碍相关的疾病的潜在原因，如高血压、支气管痉挛、性功能障碍、胃肠道疾病和青光眼。因此，控制肌球蛋白去磷酸化状态的信号元件可能代表着多种人类疾病与平滑肌功能障碍相关的治疗干预的吸引点。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，它选择性地将磷酸酶的肌球蛋白靶向亚基(MYPT1)和抑制蛋白CPI17磷酸化以抑制活性。通过抑制SMPP-1M的活性，在通透性的平滑肌中加入具有结构性活性的MYPT1K，可引起深刻的钙增敏作用。在平滑肌中，MYPT1K被各种钙增敏激动剂激活，这种激活可以被Rho激酶抑制剂Y-27632阻断。这些发现表明，MYPT1K介导了Rho激酶对SMPP-1M的钙敏感信号，然而，MYPT1K在平滑肌中的调节机制尚不清楚，因为该蛋白激酶在体外不是Rho激酶的直接底物。在这项研究中，我们将运用肌肉生理学、质谱学、分子生物学、小干扰RNA和小分子化学的独特组合来确定Rho依赖的信号通路导致MYPT1K在平滑肌中激活的分子机制。

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.