ILK-Actopaxin Interactions in Cell Signaling

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DESCRIPTION (provided by applicant): Integrin-mediated interaction of cells with the extracellular matrix control a diverse set of physiologic processes including cell migration during embryogenesis and metastasis, cell proliferation, tissue maintenance, differentiation and repair. Protein complexes associated with the integrin cytoplasmic domains facilitate integrin signaling and interactions with the actin cytoskeleton. This proposal will focus on the importance of recently identified interactions between the integrin-linked kinase ILK, the actin binding protein actopaxin and the molecular adapter protein paxillin in the regulation of cell adhesion, motility, proliferation and differentiation. In Aim 1 epitope-tagged mutant cDNAs of ILK and actopaxin will be transfected into fibroblasts followed by co-precipitation experiments to further characterize the ILK and actopaxin binding domains. The importance of these domains for subcellular localization will be assessed by immunofluorescence microscopy. We have recently demonstrated that actopaxin mutants perturb cell adhesion/spreading on collagen. Experiments proposed in Aim 2 will identify the molecular basis for this defect by assaying for changes in integrin function as well as effect on downstream signaling events. The role of ILK and actopaxin in mediating cell motility will be addressed using time-lapse microscopy, Boyden chamber and wound assays. In Aim 3 we will investigate the importance of ILK-actopaxin associations in regulating cell proliferation and differentiation. Sites of actopaxin phosphorylation resulting from associated cyclinB/cdc2 kinase activity will be delineated. The role of phosphorylation of these sites in regulating protein-protein interactions and cytoskeleton changes associated with transition through mitosis will be examined by co-precipitation analysis and immunofluorescence microscopy. Potential effects on proliferation and cell survival will be examined. The involvement of ILK and actopaxin interactions in regulating cell differentiation will be examined in the context of skeletal muscle myoblast differentiation. Together these experiments will address evolutionarily conserved mechanisms of protein linkages between the extracellular matrix and the actin cytoskeleton that are of importance to the understanding of cardiovascular and musculoskeletal defects and metastatic transformation.