Phosphor-regulation of CAP1 Functions in Cell Adhesion and Migration

项目摘要 细胞迁移对于正常的生物过程包括胚胎发育和 伤口愈合以及病理反应如癌症转移。环化酶相关蛋白 (CAP)在真核生物中作为多功能肌动蛋白调节蛋白是保守的。哺乳动物CAP 1调节肌动蛋白 细胞骨架和细胞迁移，并且还涉及人类癌症的侵袭性。然而，在 细胞迁移和癌症侵袭似乎是环境依赖性的， 机制等在这种情况下，我们发现CAP 1在细胞粘附中的新功能提供了一种机制， 这可能是CAP 1在细胞迁移和癌症侵袭中的独特作用的基础。此外，我们发现， CAP的第一个调节机制，其中S307/S309的两个残基上的磷酸化 串联位点控制CAP 1与cofilin和actin的结合。我们最新的初步研究提供了 有证据表明CAP 1需要瞬时磷酸化来促进细胞肌动蛋白丝的更新， 调节整合素介导的细胞粘附。此外，细胞周期蛋白依赖性激酶5（CDK 5）和cAMP 拮抗调节CAP 1上的S307/S309磷酸化。然而，关键的差距仍然存在于分子 CAP 1通过CDK 5和cAMP进行磷酸化调节的潜在机制，以及 这种调节作用介导细胞信号以控制细胞粘附和迁移。我们提出这个项目，以填补 这些关键的差距。我们的长期目标是了解细胞信号是如何控制肌动蛋白细胞骨架， 迁移和癌症侵袭。总的假设是CDK 5和cAMP信号拮抗 调节CAP 1磷酸化以促进时空控制的磷酸化，据信这 在调节肌动蛋白动力学、细胞粘附和迁移中至关重要。我们建议实现三个具体目标： (1)进一步确定CAP 1被CDK 5磷酸化的分子机制， （2）确定CAP 1的磷酸化调节作用， 介导CDK 5和cAMP信号以控制细胞粘附和迁移;（3）确定S307/S309是否 磷酸化调节CAP 1与FAK、talin或Rap 1的结合，这可能是磷酸化的基础。 CAP 1在细胞粘附中的功能调节。该项目之所以成功，是因为 具体目标的完成将有助于我们更好地理解磷光体的作用和机制- 细胞骨架蛋白CAP 1的调节，其将CAP 1磷酸调节信号连接到肌动蛋白， 粘附和迁移的依赖性功能。预期的发现不仅将大大扩展我们的 了解CAP 1细胞功能的调节，但也可能导致靶向CAP 1和/或其 调节细胞信号以抑制癌症的侵袭性周期。这个项目具有很强的创新性，因为 代表了对现状的进一步背离，即正在进行的已经发生范式转变的研究 我们的实验室在过去的几年里，特别是对磷酸调控的CAP 1。

PROJECT SUMMARY Cell migration is important for normal biological processes including embryonic development and wound healing, as well as pathological responses such as cancer metastasis. Cyclase-Associated Protein (CAP) is conserved in eukaryotes as a versatile actin-regulating protein. Mammalian CAP1 regulates the actin cytoskeleton and cell migration, and is also implicated in the invasiveness of human cancers. However, roles in cell migration and cancer invasiveness appear to be context-dependent, with insufficiently defined mechanisms. In that vein, our finding of a novel function for CAP1 in cell adhesion provided a mechanism that likely underlies the distinct roles for CAP1 in cell migration and cancer invasiveness. Moreover, we identified the very first regulation mechanism for CAP, where phosphorylation on both residues of the S307/S309 tandem site controls CAP1 association with cofilin and actin. Our latest preliminary studies provide compelling evidence that transient phosphorylation is required for CAP1 to promote cellular actin filament turnover and regulate integrin-mediated cell adhesion. Moreover, the Cyclin-Dependent Kinase 5 (CDK5) and cAMP antagonistically regulate S307/S309 phosphorylation on CAP1. However, critical gaps remain in molecular mechanisms underlying the phosphor-regulation of CAP1 by CDK5 and cAMP, and roles and mechanisms for this regulation in mediating the cell signals to control cell adhesion and migration. We propose this project to fill these critical gaps. Our long-term goal is to understand how cell signaling controls the actin cytoskeleton, cell migration and cancer invasiveness. The overall hypothesis is that CDK5 and cAMP signals antagonistically regulate CAP1 phosphorylation to facilitate spatial-temporally controlled phosphorylation, which is believed to be critical in regulating actin dynamics, cell adhesion and migration. We propose to pursue three specific aims: (1) further determine molecular mechanisms underlying the phosphorylation of CAP1 by CDK5 and dephosphorylation induced by cAMP, respectively; (2) ascertain roles for the phosphor-regulation of CAP1 in mediating CDK5 and cAMP signals to control cell adhesion and migration; (3) determine if S307/S309 phosphorylation regulates CAP1 association with FAK, talin or Rap1, which may underlie the phosphor- regulation of CAP1 function in cell adhesion. The proposed project is significant because successful completion of the specific aims will help us much better understand roles and mechanisms of the phosphor- regulation of the cytoskeletal protein CAP1, which links CAP1 phosphor-regulatory signals to the actin- dependent functions in adhesion and migration. The expected findings will not only greatly extend our knowledge on the regulation of CAP1 cell functions, but may also lead to strategies targeting CAP1 and/or its regulatory cell signals for suppressing the invasive cycle of cancer. This project is highly innovative, because it represents a further departure from the status quo, namely the already paradigm-shifting ongoing studies in our laboratory over the last several years, particularly that on the phosphor-regulation of CAP1.