Myosin Light Chain Kinase Function in Smooth Muscle

描述（由申请人提供）：膀胱平滑肌收缩的关键信号是[Ca]i的增加，它激活Ca2+/钙调素依赖性肌球蛋白轻链激酶（MLCK），从而促进肌球蛋白调节轻链（RLC）的磷酸化并开始收缩。新出现的证据表明，与慢性膀胱梗阻相关的功能紊乱与收缩蛋白信号系统的显著变化有关。我们建议研究膀胱平滑肌收缩装置激活的必要机制，以揭示相互作用信号网络的复杂性。具体目标MLCK是膀胱平滑肌中唯一以钙依赖方式磷酸化RLC的激酶吗？表达生物传感器MLCK的转基因小鼠将被用来测定相对于[Ca2+]i、RLC磷酸化和收缩的激活对Ca2+的依赖性。蛋白质转导结构域抑制剂将用于测试特定激酶的参与。通过他莫昔芬控制的消融系统，平滑肌MLCK基因消融将仅限于平滑肌细胞。收缩反应将在离体膀胱组织和体内进行测量。具体目标2。靶向平滑肌细胞收缩区域的MLCK对RLC磷酸化是必要和充分的吗？含有肌动蛋白结合基序和肌球蛋白结合模块的基因外显子将通过敲入程序被删除，膀胱平滑肌的收缩性能将在体外和体内被表征。具体目标3。非肌球蛋白在平滑肌中的作用是什么？它是由Rho激酶和PKC选择性调节的吗？Blebbistatin，非肌球蛋白II的选择性抑制剂将用于评估非肌球蛋白对收缩特性的贡献。在不同的信号传导模式下，非肌肉RLC磷酸化将与平滑肌RLC一起被测量。我们认为非肌肉肌球蛋白可能有助于细胞膜粘附位点的稳定性，这是膀胱肌细胞通过细胞内收缩结构域进行细胞力传递所必需的。

DESCRIPTION (provided by applicant): The key signal that contracts bladder smooth muscle is an increase in [Ca]i which activates Ca2+/calmodulin-dependent myosin light chain kinase (MLCK), thereby promoting phosphorylation of myosin regulatory light chain (RLC) and initiating contraction. Emerging evidence suggests that functional derangements associated with chronic obstruction of the urinary bladder are associated with significant changes in the contractile protein signaling system. We propose to investigate mechanisms necessary for activation of the bladder smooth muscle contractile apparatus to unravel the complexities of interacting signaling networks. Specific Aim 1. Is MLCK the only kinase that phosphorylates RLC in a Ca -dependent manner in bladder smooth muscle? Transgenic mice expressing biosensor MLCK will be used to determine Ca2+-dependency of activation relative to [Ca2+]i, RLC phosphorylation and contraction. Protein transduction domain inhibitors will be used to test involvement of specific kinases. Smooth muscle MLCK gene ablation will be restricted to smooth muscle cells by a tamoxifen-controlled ablation system. Contractile responsiveness will be measured in isolated bladder tissues and in vivo. Specific Aim 2. Is MLCK targeting to the contractile domain of smooth muscle cells necessary and sufficient for RLC phosphorylation? Gene exons containing the actin-binding motifs and the myosin binding module will be deleted by knock-in procedures and contractile performance of bladder smooth muscle will be characterized in vitro and in vivo. Specific Aim 3. What are the roles ofnonmuscle myosin in smooth muscle? Is it regulated selectively by Rho kinase and PKC? Blebbistatin, a selective inhibitor of nonmuscle myosin II will be used to evaluate contributions of nonmuscle myosin to contractile properties. Nonmuscle RLC phosphorylation will be measured along with smooth muscle RLC under different modes of signaling. We consider the possibility that nonmuscle myosin may contribute to the stability of membrane adhesion sites necessary for cellular force transmission by intracellular contractile domains in bladder myocytes.