Our long-term goal is to define the signaling pathways that control directed cell motility and to understand their roles in pathological processes such as cancer. Cell motility is stimulated by binding of chemotactic stimuli to cell surface receptors and is driven by the dynamic polymerizationn of actin into filaments, a process that is spatially confined to the leading edge. The lipid second messenger phosphatidylinositol-4,5- bisphosphate (PI4,5P2) is recognized as a key signal transducer that links cell surface receptors to the actin cytoskeleton. PI4,5P2 synthesis is essential for actin assembly and cell motility and is locally upregulated at the leading edge in response to PDGF by mechanisms that remain largely obscure. To elucidate these mechanisms we will focus on one particular kinase, type I phosphatidylinositol phosphate kinase alpha (PIPKla), which catalyzes the committed step in PI4,5P2 synthesis. PIPKla localizes to the leading edge of migrating cells and induces actin polymerization in cells stimulated by PDGF. Therefore, PIPKla is a prime candidate for regulating PI4,5P2 synthesis during chemotaxis. Our data indicate that the spatial targeting and activation of PIPKla is tightly regulated by interactions with the PDGF receptor, the small GTPase Rac1, and by binding to the substrate PI4P and the lipid phosphatidic acid (PA). We propose that the coordinate interaction of PIPKla with lipid factors, PDGF receptor and Rac1 results in the specific spatial activation of PI4,5P2 synthesis required for chemotaxis. To test this hypothesis we will combine biochemical and highresolution confocal microscopy approaches in Aim 1 to define the dynamics and mechanisms of PIPKla association with the PDGF receptor complex.
Aim 2 will focus on the crosstalk and feedback mechanisms that control the spatial and temporal activation of PIPKla. We will test the hypothesis that Rac1 and PA modulate PIPKla localization and activation.
Aim 3 will test the hypothesis that PIPKla mediates PDGF-induced actin assembly and chemotaxis by using inhibitory approaches as well as by mislocalization of PIPKla. Taken together, these experiments will provide novel and detailed information about the spatial and functional relationships between PI4,5P2, the enzymes that produce them, and regulatory components that tightly control PI4,5P2 synthesis. A better understanding of these signaling mechanisms may provide novel therapeutic approaches for the treatment of human diseases.
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