Reorganization of the Actin Cytoskeleton by Phosphatases

磷酸酶重组肌动蛋白细胞骨架

• 批准号: 7119328
• 负责人: DAVID L. BRAUTIGAN
• 金额: $ 21.1万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-05-25 至 2010-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7119328
• 关键词: actins; biological signal transduction; cell migration; cell morphology; crosslink; cytoskeleton; enzyme complex; immunoprecipitation; phosphoprotein phosphatase; phosphorylation; protein isoforms; protein localization; small interfering RNA

Dynamic reorganization of the actin cytoskeleton is the basis for cell morphology and migration. In different cellular locales F-actin accessory proteins undergo transient Ser/Thr phosphorylation and act as molecular transducers, switching on/off to control F-actin. Multiple upstream kinases phosphorylate these accessory proteins in response to signals. The opposing phosphatases are complexes of PP1 catalytic and regulatory subunits physically targeted and under stringent negative control. Phosphorylation of PP1 subunits and phosphoinhibitor proteins couple the activity of kinases with PP1 complexes, producing reciprocal changes that generate the on/off switching behavior. The goal of this Project is to understand how signaling pathways control dynamic reorganization of the cytoskeleton, through regulation of F-actin accessory proteins by specialized forms of protein Ser/Thr phosphatase PPI. this Project will study two different PP1 complexes. One complex is PP1C8 plus the regulatory-targeting subunit called MYPT1 (myosin phosphatase targeting subunit), which binds to actomyosin-II in stress fibers and uses ankryin-repeats to bind the F-actin-anchoring proteins ezrin/radixin/moesin (ERM), and the actin-capping protein adducin. MYPT1 phosphorylation regulates the PP1 activity, but the consequences on the F-actin cytoskeleton have not been examined. The other PP1 complex, discovered by this project, is composed of PP1Ccalpha plus tensin, an F-actin anchoring phosphoprotein concentrated in mature focal adhesions. Specific Aim 1 will test the hypothesis that the activity of MYPT1-PP1C toward accessory proteins such as ERM proteins and adducin alters cortical F-actin dynamics. Wild type MYPT1 and MYPT1 mutated in its regulatory phosphorylation sites will be over-expressed with PP1C8 as dominant active and inactive phosphatases. Alternatively, endogenous MYPT1 will be depleted by siRNA silencing. The phosphorylation of ERM proteins and adducin will be monitored in parallel with changes in F-actin distribution, cell morphology and cell migration. Specific Aim 2 will test the hypothesis that the C terminal PTB domain in tensin directly binds PP1Ccalpha and functions as a regulatory-targeting subunit. The tensin binding of PP1C is proposed to be in competition with P-Tyr ligands, modulating recruitment of PP1 in time and space. Experiments will test whether the tensin.PP1C complex serves to reduce Ser/Thr phosphorylation in specific substrates and thereby alter turnover of fibrillar adhesions and modify cell motility. This Project connects signaling pathways to cortical F-actin and focal adhesions that are critical to cell polarity, and cell migration, which requires PI3K-dependent formation of polyphosphoinositides. Our experimental plan involves use of microscopic imaging and depends on collaborative interactions with other Projects.

肌动蛋白细胞骨架的动态重组是细胞形态和迁移的基础。在不同的细胞位置，F-肌动蛋白辅助蛋白经历瞬时的Ser/Thr磷酸化，作为分子转导，开启/关闭以控制F-肌动蛋白。多个上游激酶在信号反应中使这些辅助蛋白磷酸化。相反的磷酸酶是PP1催化和调节亚基的复合体，具有物理靶向性和严格的阴性对照。PP1亚基和磷酸抑制蛋白的磷酸化将激酶的活性与PP1复合体偶联，产生相互作用的变化，从而产生开关行为。本项目的目标是了解信号通路如何通过特殊形式的丝氨酸/苏氨酸磷酸酶PPI调节F-肌动蛋白辅助蛋白来控制细胞骨架的动态重组。本项目将研究两种不同的PP1复合体。其中一个复合体是PP1C8加上被称为MYPT1(肌球蛋白)的调控靶向亚基 磷酸酶靶向亚基)，它与应激纤维中的肌动蛋白-II结合，并使用ankryin重复序列来结合F-肌动蛋白锚定蛋白Ezrin/Radisin/moesin(ERM)和肌动蛋白封帽蛋白adducin。MYPT1的磷酸化调节PP1的活性，但对F-肌动蛋白细胞骨架的影响尚未被研究。该项目发现的另一个PP1复合体是由PP1Ccalpha和tensin组成的，tensin是一种F-肌动蛋白锚定磷酸蛋白，集中在成熟的局灶性粘连中。特定目标1将测试MYPT1-PP1C对辅助蛋白(如ERM蛋白和内收蛋白)的活性改变皮质F-肌动蛋白动态的假设。野生型MYPT1和MYPT1在其调节磷酸化位点突变后将过度表达，PP1C8为显性活性和非活性 磷酸酶。或者，内源性MYPT1将被siRNA沉默耗尽。ERM蛋白和内收蛋白的磷酸化将与F-肌动蛋白分布、细胞形态和细胞迁移的变化同步监测。特定目的2将测试这一假设，即张力蛋白中的C末端PTB结构域直接与PP1Ccalpha结合，并作为调控靶向亚基发挥作用。PP1C的张力蛋白结合被认为是与P-Tyr配体竞争的，调节PP1在时间和空间上的招募。实验将测试tensin.PP1C复合体是否有助于减少特定底物上的丝氨酸/苏氨酸磷酸化，从而改变纤维粘连的周转和改变细胞的运动性。该项目将信号通路连接到皮质F-肌动蛋白和对细胞极性至关重要的焦点粘连，以及细胞迁移，这需要依赖于PI3K的聚磷脂酰肌醇的形成。我们的实验计划涉及使用显微成像，并依赖于与其他项目的协作互动。