Role of Protein Kinase D in Actin Remodeling and Cell Motiliy

描述(申请人提供)：肿瘤细胞对趋化刺激的定向迁移是由以下机制介导的：诱导形成游离的F-肌动蛋白带刺末端、现有细丝的延伸和前沿的肌动蛋白分支，从而导致膜向刺激方向突起。这些过程的关键介质是：诱导肌动蛋白断裂和带刺末端形成的ADF/cofilin，诱导肌动蛋白成核和树突分支的黄蜂家族肌动蛋白成核因子和Arp2/3复合体，以及与覆盖蛋白竞争的Ena/Vasp。我们特别注意到，丝氨酸/苏氨酸激酶D1(PKD1)在前沿的带刺末端完全抑制肌动蛋白的掺入，从而导致肌动蛋白介导的定向细胞迁移受到抑制。这项建议旨在了解PKD1在调节迁移肿瘤细胞前沿的肌动蛋白周转的关键过程中的影响。我们的假设是，PKD1在多个水平上抑制肌动蛋白重塑过程。具体地说，我们假设PKD1通过调节ADF/cofilin、Arp2/3复合体和Ena/Vasp来调节其作用。我们认为，PKD1影响所有这些关键事件，以介导其对肿瘤细胞的带刺末端形成、肌动蛋白掺入和细胞迁移的深刻抑制作用。在这个提案中，我们将研究三个具体目标：我们将确定PKD1如何调控ADF/cofilin活性(特异性目标1)；确定PKD1如何调控Arp2/3功能(特异性目标2)；以及分析PKD1介导的Vasp磷酸化如何促进定向细胞运动(特异性目标3)。这项研究的成功完成将确定PKD1在可移动肿瘤细胞前沿的肌动蛋白重塑过程中的新功能。这些知识对于开发抑制肿瘤细胞迁移和侵袭的新策略将是重要的。 公共卫生相关性：肿瘤细胞的迁移在肿瘤的扩张和转移中起着重要作用。这项建议将研究丝氨酸/苏氨酸激酶PKD1如何调节肿瘤细胞前沿的肌动蛋白动力学和抑制肿瘤细胞迁移的分子机制。

DESCRIPTION (provided by applicant): The directed cell migration of tumor cells in response to a chemotactic stimulus is mediated by mechanisms that induce formation of free F-actin barbed-ends, extensions of existing filaments and actin branching at the leading edge, which results in membrane protrusion towards the stimulus. Key mediators of these processes are ADF/cofilin, which induces actin severing and barbed end formation, the actin nucleation factors of the WASp family and the Arp2/3 complex, which induce actin nucleation and dendritic branching, as well as Ena/VASP, which competes with the capping protein (CP). We made the striking observation that the serine/threonine kinase Protein Kinase D1 (PKD1) completely inhibits actin incorporation at barbed ends at the leading edge and that this results in the inhibition of actin-mediated directed cell migration. This proposal is designed to understand the impact of PKD1 on the key processes regulating actin turnover at the leading edge of migrating tumor cells. It is our hypothesis that PKD1 inhibits actin remodeling processes at multiple levels. Specifically we hypothesize that PKD1 mediates its effects through regulation of ADF/cofilin, the Arp2/3 complex and Ena/VASP. We propose that PKD1 impacts all these key-events to mediate its profound inhibitory effects observed on barbed end formation, actin incorporation and cell migration of tumor cells. Three Specific Aims will be investigated in this proposal: We will determine how PKD1 regulates ADF/cofilin activity (Specific Aim 1); identify how PKD1 regulates Arp2/3 functions (Specific Aim 2); and analyze how PKD1-mediated phosphorylation of VASP contributes to directed cell motility (Specific Aim 3). Successful completion of this proposal will identify novel functions for PKD1 in actin remodeling processes at the leading edge of motile tumor cells. This knowledge will be important for the development of novel strategies to inhibit tumor cell migration and invasion. PUBLIC HEALTH RELEVANCE: Migration of tumor cells has an important function in tumor expansion and metastasis. This proposal will investigate the molecular mechanisms of how the serine/threonine kinase PKD1 regulates actin dynamics at the leading edge of tumor cells and inhibits tumor cell migration.