Molecular Mechanisms of Nonmuscle Myosin II Regulation

描述（由申请人提供）：有丝分裂晚期，肌动蛋白和肌球蛋白-II 在细胞赤道处短暂聚集形成收缩环 将细胞一分为二虽然肌球蛋白-II是已知的， 细胞动力学过程的保真度，分子信号和潜在的 介导其募集到收缩环的机制， 其运动活性尚未阐明。在中期， 肌球蛋白-II的轻链（RLC）在抑制 活性;然而，随着有丝分裂后期的进展，RLC是 在这些位点上去磷酸化，在激活 发动机RLC磷酸化是细胞周期依赖性的观察 表明肌球蛋白-II活性是在时间调节， 有丝分裂，并表明，组装和活动的收缩环， 依赖于通过磷酸化调节肌球蛋白II的功能。 重要的是，介导有丝分裂特异性磷酸化的激酶， RLC尚未确定。 我们已经启动了一个多学科的方法，涉及定量体外 生物化学分析和体内研究，以鉴定 磷酸化肌球蛋白-II的调节轻链（RLC）在有丝分裂过程中， 脊椎动物的非肌肉细胞。这些研究将为一个模型提供基础 描述了调节肌球蛋白-II活性的信号通路， 有助于收缩环的组装。我们的目标是：（1） 量化磷酸化对RLC抑制位点的影响， 肌球蛋白-II活性，（2）鉴定磷酸化肌球蛋白的有丝分裂激酶， 抑制位点的肌球蛋白-II RLC，（3）测试MLCK的要求， 适当的收缩环组装和细胞动力学过程的保真度， (4)确定在有丝分裂早期下调MLCK活性的机制 和（5）鉴定将MLCK靶向至所述细胞所需的分子决定簇。 收缩环

DESCRIPTION (provided by applicant): Late in mitosis, actin and myosin-II transiently accumulate at the equator of the cell to form a contractile ring that divides the cell in two. Although myosin-II is known to be required for the fidelity of the cytokinetic process, the molecular signals and underlying mechanisms that mediate its recruitment to the contractile ring and stimulate its motor activity have not been elucidated. During metaphase the regulatory light chain (RLC) of myosin-II is phosphorylated on residues inhibitory for activity; however as mitosis progresses through anaphase, the RLC is dephosphorylated on these sites and phosphorylated on residues that activate the motor. The observation that RLC phosphorylation is cell cycle dependent demonstrates that myosin-II activity is under temporal regulation during mitosis and suggests that the assembly and activity of the contractile ring depends upon the modulation of myosin-II function via phosphorylation. Importantly, the kinases that mediate mitosis-specific phosphorylation of the RLC have not been identified. We have initiated a multidisciplinary approach involving quantitative in vitro biochemical analyses and in vivo studies to identify the kinases that phosphorylate the regulatory light chain (RLC) of myosin-II during mitosis in vertebrate nonmuscle cells. These studies will provide the basis for a model describing the signaling pathways that regulate myosin-II activity and which contribute to the assembly of the contractile ring. Our objectives are: (1) quantify the effects of phosphorylation on the inhibitory sites of the RLC on myosin-II activity, (2) identify the mitotic kinase that phosphorylates the inhibitory sites on the myosin-II RLC, (3) test the requirement for MLCK in proper contractile ring assembly and the fidelity of the cytokinetic process, (4) identify the mechanisms that down regulate MLCK activity in early mitosis and (5) identify the molecular determinants required for targeting MLCK to the contractile ring.