Cofilin / ADF Regulation in Rho GTPase Signaling

描述(由申请人提供)： 由上游信号激活的Rho GTP酶协调细胞骨架动力学 如趋化、转移、轴突引导等复杂的运动过程。Rho GTP酶控制肌动蛋白动态的一个重要机制是通过调节cofilin/ADF家族蛋白的作用。Cofilin/ADF(C/A)蛋白作用于切断F-肌动蛋白细丝和促进F-肌动蛋白解聚，这两个活性对细胞骨架重排至关重要，细胞骨架重排涉及前沿片层的形成和细胞运动。承兑汇票的行为受以下方面的监管 Ser3关键调控位点的磷酸化/去磷酸化。 我们先前已经证明，Rac和CDC42通过p21激活的激酶(PAK)和LIM激酶的下游级联作用来调节C/A的磷酸化和失活。我们现在已经确定了一种独特的磷酸酶，它可以去磷酸化从而激活C/A。结合生物化学研究，我们将利用遗传手段和RNA干扰方法来扰乱体内磷酸酶的功能。我们建议研究这种新的CA磷酸酶的性质和调节，以确定它在调节细胞骨架动态行为中的重要性。 我们已经发现了Ser3-Phospho CA和14-3-3家族蛋白之间的调控相互作用。我们将研究这一假说，除了对CA的直接调控作用外，14-3-3蛋白还充当分子间支架，将CA调节激酶和磷酸酶连接到一个协调的调节模块中。最后，我们将研究PAK1-LIMK-CA磷酸酶-CA通路在刺激细胞运动过程中的协调作用。调节这一信号通路的调控事件将被确定。CA功能的局部调控与全球调控的问题将通过对所涉及的信号分子的空间和时间动力学的实时研究来解决。建议的研究应该为我们提供关于Rho GTP酶在细胞运动过程中调节CA功能的独特信息。

DESCRIPTION (provided by applicant): Rho GTPases activated by upstream signals coordinate the cytoskeletal dynamics necessary for such complex motile processes as chemotaxis, metastasis, axon guidance, etc. One critical mechanism by which Rho GTPases control actin dynamics is via regulation of the action of cofilin/ADF family proteins. Cofilin/ADF (C/A) proteins act to sever F-actin filaments and promote F-actin depolymerization, both activities that are critical to the cytoskeletal rearrangements involved in formation of leading edge lamellae and cell motility. The action of C/A is regulated by phosphorylation/dephosphorylation of a critical regulatory site at Ser3. We have previously shown that Rac and Cdc42 regulate the phosphorylation and inactivation of C/A through the action of a downstream kinase cascade involving p21-activated kinase (PAK) and LIM kinase. We have now identified a unique phosphatase that acts to dephosphorylate and thus activate C/A. In combination with biochemical studies, we will utilize genetic means and RNA interference methodologies to disrupt phosphatase function in vivo. We propose to investigate the properties and regulation of this novel CA phosphatase in order to determine its importance in modulating cytoskeletal dynamic behavior. We have discovered regulatory interactions between Ser3-phosphoCA and 14-3-3 family proteins. We will investigate the hypothesis that, in addition to direct regulatory effects on CA, 14-3-3 proteins act as intermolecular scaffolds to link CA regulatory kinases and phosphatases into a coordinated regulatory module. Finally, we will investigate the coordinated action of the PAK1-LIMK-CA phosphatase-CA pathway during stimulated cell motility. Regulatory events modulating this signaling pathway will be determined. The question of localized vs. global regulation of CA function will be addressed by real time studies of the spatial and temporal dynamics of the signaling molecules involved. The proposed studies should give us unique information about the action of Rho GTPases to regulate CA function during cell motility.