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.
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