Cofilin/ADF Regulation in Rho GTPase Signaling

描述（由申请人提供）：细胞运动性有助于正常（例如伤口愈合）和病理（例如转移）细胞行为。细胞迁移能力的基础是前沿肌动蛋白的动力学，其响应于Rho GTP酶而被调节。一个这样的Rac GTP酶启动的途径至关重要的定向细胞迁移涉及cofilin依赖性肌动蛋白解聚/切断活性通过磷酸调节循环的精确空间调节。在过去的资助期间，我们的实验室确定并表征了一种新的cofilin/ADF磷酸酶，我们称之为chronophin（CIN）。我们的数据表明CIN是一个重要的调节因子的cofilin磷酸循环，从而调节肌动蛋白在细胞分裂和运动的动力学。将研究通过上游信号调节Rac依赖性肌动蛋白重塑和细胞运动来调节CIN活性。我们将使用生物化学和分子生物学的方法，沿着显微镜为基础的细胞成像，以确定信号通路，蛋白质和分子复合物，影响cofilin的调节CIN。将研究CIN在协调Rac依赖性肌动蛋白动力学和细胞运动性中的作用。我们将使用定量荧光散斑显微镜（qFSM）来分析通过CIN调制的cofilin磷酸化循环在前沿的肌动蛋白动力学的调节。此外，我们已经首次表明，我们可以应用FSM直接调查生长因子（EGF）调节的前沿肌动蛋白在癌细胞系MTLn 3的动力学。由热休克和/或缺血/ATP耗竭诱导的细胞骨架重排是心血管细胞、神经元细胞和肾近端小管细胞损伤的主要原因。这些细胞骨架反应可能具有适应性（存活）和损伤性后果。我们发现Hsp 90是CIN的内源性结合伴侣，负性调节CIN活性。我们将研究Rho GTPase-Hsp 90/CIN-cofilin信号转导在ATP耗竭介导的细胞骨架重塑中的参与。我们将研究在这些条件下Hsp 90作为CIN活性的直接调节剂的生物化学作用，并评估CIN依赖性cofilin循环如何参与应激介导的细胞骨架反应和细胞存活。与公共卫生的相关性：细胞运动在正常生理和许多疾病状态中起着重要作用。我们的研究将调查这一重要过程的新调节剂。此外，这种蛋白可能在缺血性疾病和其他应激反应中发挥重要作用。

DESCRIPTION (provided by applicant): Cell motility contributes to both normal (e.g. wound healing) and pathological (e.g. metastasis) cell behavior. Underlying the migratory abilities of cells are the dynamics of actin at the leading edge, regulated in response to Rho GTPases. One such Rac GTPase-initiated pathway crucial to directed cell migration involves the precise spatial regulation of cofilin-dependent actin depolymerizing/severing activity through a phosphoregulatory cycle. During the past grant period, our laboratory identified and characterized a novel cofilin/ADF phosphatase which we termed chronophin (CIN). Our data show CIN to be an important regulator of cofilin phosphocycling, thereby modulating actin dynamics during cell division and motility. The regulation of CIN activity by upstream signals modulating Rac-dependent actin remodeling and cell motility will be investigated. We will use biochemical and molecular biological approaches, along with microscopy-based cell imaging, to determine the signaling pathways, proteins, and molecular complexes that influence cofilin regulation by CIN. The role(s) of CIN in coordinating Rac-dependent actin dynamics and cell motility will be studied. We will use quantitative fluorescent speckle microscopy (qFSM) to analyze the regulation of actin dynamics through CIN-modulated cofilin phosphocycling at the leading edge. In addition, we have shown for the first time that we can apply FSM to directly investigate growth factor (EGF)- regulated leading edge actin dynamics in the carcinoma cell line MTLn3. Cytoskeletal rearrangements induced by heat shock and/or ischemia/ATP depletion are a major cause of injury to cardiovascular cells, neuronal cells, and renal proximal tubule cells. These cytoskeletal responses may have both adaptive (survival) and injurious consequences. We have found that Hsp90 is an endogenous binding partner of CIN and negatively regulates CIN activity. We will examine the participation of Rho GTPase-Hsp90/CIN-cofilin signaling in ATP-depletion-mediated cytoskeletal remodeling. We will investigate the biochemical role of Hsp90 as a direct regulator of CIN activity under these conditions, and assess how the CIN-dependent cofilin cycle participates in stress-mediated cytoskeletal responses and cell survival. Relevance to public health: Cell motility plays important roles in normal physiology and in numerous disease states. Our studies will investigate a novel regulator of this important process. In addition, this protein is likely to play important roles in ischemic disorders and other stress responses.