Specificity and regulation in a protein kinase cascade affecting the actin cytoskeleton

标题：影响肌动蛋白细胞骨架的蛋白激酶级联反应的特异性和调节 摘要 真核细胞解释细胞外和内在的线索，以影响肌动蛋白细胞骨架的重塑， 这是控制细胞形态、运动和侵袭性的关键过程。严格控制信令 因此，影响细胞骨架的途径对于正常发育和体内平衡是必不可少的。在这 我们将研究蛋白激酶信号传导的特异性和调节机制 级联聚合在cofilin/ADF（肌动蛋白解聚因子）蛋白质组的磷酸化上， 介导肌动蛋白丝重塑的分子。RHO家族GTP酶RHO、RAC和CDC 42各自 以空间受限的方式直接激活激酶（分别为ROCK、PAK 1和PAK 4）， 直接磷酸化并激活LIM激酶。LIM激酶在它们的能力方面是非常特异的， 在Ser 3处磷酸化cofilin/ADF蛋白，这通过使它们从cofilin/ADF蛋白解离而使cofilin/ADF失活。 肌动蛋白。这种信号级联反应通过多种机制受到严格控制，包括底物特异性 上游激酶和LIM激酶本身的调节，以及LIM激酶的自身调节。 这项提案的主要目标是发现这种特异性和调节的分子基础。 重要的生物学途径。在我们的初步研究中，我们已经确定了 LIM激酶1与其底物cofilin复合。因此，在目标1中，我们以此结果为基础来测试 假设LIM激酶对cofilin的精确底物特异性是由一种新的激酶定义的， 通过探测生物化学、催化和生物物理效应以及功能影响， 破坏晶体学定义的LIMK 1：cofilin界面。在我们的初步研究中， 我开始绘制LIMK 1的自动调节首尾相互作用的分子水平细节，因此在Aim 2中 我们将利用一系列的结构，生物化学和生物物理技术获得显着改善 了解这种头-尾相互作用，然后将研究靶向突变对 结构域间相互作用，体外激酶活性和细胞功能。最后，在上一个赠款期间， 我们发现了一种新的机制，通过N-末端假底物， 顺序在目的3中，我们将检验II型PAK激活是由以下直接参与介导的假设： 这种假底物序列由特定的SH 3结构域，并执行结构，生化，和细胞 SH 3-PAK 4相互作用的研究。因此，这一目标将提供II型PAK的分子水平细节 规则仍然模糊。在这个多PI提案中，Boggon和Turk实验室将进行 一个高度合作的结构导向的功能研究，以提供一个显着提高理解 控制LIM激酶介导的肌动蛋白细胞骨架和细胞外基质的分子机制， 在蛋白激酶信号传导中控制细胞结果的一般规则。

Title: Specificity and regulation in a protein kinase cascade affecting the actin cytoskeleton ABSTRACT Eukaryotic cells interpret extracellular and intrinsic cues to effect remodeling of the actin cytoskeleton, a process critical for controlling cell morphology, movement, and invasiveness. Tight control of signaling pathways impinging on the cytoskeleton is therefore essential to normal development and homeostasis. In this proposal we will investigate mechanisms underlying specificity and regulation in protein kinase signaling cascades converging on phosphorylation of the cofilin/ADF (actin-depolymerizing factor) group of proteins, key molecules that mediate remodeling of actin filaments. The RHO family GTPases RHO, RAC and CDC42 each directly activate kinases (ROCK, PAK1 and PAK4, respectively) in a spatially restricted manner that in turn directly phosphorylate and activate LIM kinases. The LIM kinases are exquisitely specific in their ability to phosphorylate cofilin/ADF proteins at Ser3, which inactivates cofilin/ADF by causing their dissociation from actin. This signaling cascade is tightly controlled through multiple mechanisms, including substrate specificity of both the upstream kinases and of LIM kinases themselves, and through autoregulation of the LIM kinases. The major goal of this proposal is to discover the molecular basis for specificity and regulation in this biologically important pathway. In our preliminary studies we have determined the X-ray crystal structure of LIM kinase 1 in complex with its substrate, cofilin. Therefore in Aim 1 we build on this result to test the hypothesis that the exquisite substrate specificity of LIM kinases for cofilin is defined by a novel kinase- substrate interaction by probing biochemical, catalytic and biophysical effects, and functional impact, of disrupting the crystallographically defined LIMK1:cofilin interface. In our preliminary studies we have also begun to map the molecular level details of the autoregulatory head-tail interaction of LIMK1, and so in Aim 2 we will utilize a range of structural, biochemical and biophysical techniques obtain a significantly improved understanding of this head-tail interaction and will then examine the effect of targeted mutations on interdomain interactions, on kinase activity in vitro, and on function in cells. Lastly, in the previous grant period we discovered a novel mechanism for autoinhibiton of type II PAKs through an N-terminal pseudosubstrate sequence. In Aim 3 we will test the hypothesis that type II PAK activation is mediated by direct engagement of this pseudosubstrate sequence by specific SH3 domains, and perform structural, biochemical, and cellular studies of SH3-PAK4 interactions. This aim will thereby provide molecular level details of type II PAK regulation that have remained obscure. In this Multi-PI proposal, the Boggon and Turk laboratories will conduct a highly collaborative structure-directed functional study to provide a significantly improved understanding of the molecular mechanisms that govern LIM kinase-mediated control of the actin cytoskeleton and of the general rules that govern cellular outcomes in protein kinase signaling.