Role of Protein Kinase D in Actin Remodeling and Cell Motiliy

描述（由申请人提供）：肿瘤细胞在趋化刺激下的定向细胞迁移是由诱导游离f -肌动蛋白刺端形成、现有纤维延伸和肌动蛋白前缘分支的机制介导的，这导致膜向刺激方向突出。这些过程的关键介质是诱导肌动蛋白切断和刺端形成的ADF/cofilin，诱导肌动蛋白成核和树突分支的WASp家族的肌动蛋白成核因子和Arp2/3复合物，以及与capping蛋白（CP）竞争的Ena/VASP。我们惊人地观察到丝氨酸/苏氨酸激酶蛋白激酶D1 （PKD1）完全抑制肌动蛋白在边缘倒钩末端的结合，这导致肌动蛋白介导的定向细胞迁移受到抑制。该提案旨在了解PKD1对迁移肿瘤细胞前沿调节肌动蛋白转换的关键过程的影响。我们的假设是PKD1在多个层面抑制肌动蛋白重塑过程。具体来说，我们假设PKD1通过调节ADF/cofilin、Arp2/3复合物和Ena/VASP介导其作用。我们提出PKD1影响所有这些关键事件，介导其对肿瘤细胞的倒刺端形成、肌动蛋白结合和细胞迁移的深刻抑制作用。本提案将研究三个特定目标：我们将确定PKD1如何调节ADF/cofilin活性（特定目标1）；确定PKD1如何调节Arp2/3功能（Specific Aim 2）；并分析pkd1介导的VASP磷酸化如何促进定向细胞运动（Specific Aim 3）。该研究的成功完成将确定PKD1在运动肿瘤细胞前沿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。

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.