FUNCTIONAL STUDIES OF FOCAL ADHESION KINASE

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DESCRIPTION: Integrin-mediated adhesion to the extracellular matrix (ECM) profoundly affect cell shape, motility, survival, proliferation, and differentiation. These behavioral properties are critical for normal growth and development, and impact upon many human pathological processes including tumor growth and metastasis, atherogenesis, thrombosis and hemostasis, inflammation, and cutaneous wound repair. The broad long-term objective of this project is to understand the biochemical signaling mechanisms by which ligand-engaged integrins influence cell behavior, thereby revealing new therapeutic targets for the treatment of adhesion-influenced disease processes. The three specific aims address the general hypothesis that the focal adhesion tyrosine kinase "FAK" is a key signaling molecule affecting integrin-mediated changes in cell behavior. Aim 1 will determine the regulatory mechanisms involved in a FAK-enhanced cell migration response. The effect of inducible FAK expression on the ability of cells to migrate toward defined serum components in a Boyden assay will be assessed, and the FAK functional properties required for the response will be examined by measuring migration rates of cells expressing FAK mutants deficient in specific signaling activities. Aim 2 will examine the role of FAK kinase domain activation loop phosphorylation in integrin signaling responses. The mechanism of FAK activation will be defined by determining the ability of mutant FAK variants, deficient in either activation loop phosphorylation or interactions with Src family kinases, to become fully phosphorylated and catalytically active in response to call adhesion. Monoclonal antibodies that specifically recognize the phosphorylated FAK activation loop will be employed to reveal the dynamics and regionalization of activated FAK during the course of cell spreading and migration. Aim 3 will examine the role of serine phosphorylation on FAK's signaling properties and adhesion-stimulated cellular responses. The major site of FAK serine phosphorylation in adherent fibroblasts will be determined by a phosphopeptide mapping strategy, and the potential role of this phosphorylation event in regulating FAK's signaling capacity and functional properties will be assessed through expression and analysis of the phosphoaccentor serine FAK mutant.