STRUCTURE AND FUNCTION OF PAXILLIN

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Paxillin is a multi-domain, 68kDa phosphoprotein that localizes to specialized actin-membrane attachment sites in cultured mammalian cells and in vivo. We will test the hypothesis that paxillin functions as a scaffold/adapter protein at the plasma membrane, thereby facilitating the interplay between adhesion- and growth factor-derived signals that result in reorganization of the actin cytoskeleton and modulation of gene expression. Aim 1 will identify and characterize the functional domains of the recently identified paxillin binding proteins p42, an actin binding protein; and PKL, an ARF-GAP that links paxillin to a complex of proteins including PIX (a p21 Cdc42/Rac GEF) and the p21 activated kinase (PAK). Novel PKL and p42 binding proteins will be identified and characterized. Cloning of p42 family members is proposed. Aim 2 will evaluate the importance of paxillin interactions with these binding proteins in regulating reorganization of the actin cytoskeleton associated with activation of p21Rho- and p21ARF-family members in cultured cells stimulated by cell adhesion, soluble growth factors, or by co- expression of p21Rho or p21ARF GTPase family members. The effect of these mutants on cell motility will be determined in modified Boyden chamber assays and time-lapse video microscopy. Aim 3 will examine how paxillin, PKL and p42 impact on the biochemical signaling cascades associated with cell adhesion and growth factor stimulation. Analysis will include monitoring of changes in p21 GTPase activities using GST-p21-GTPase effector protein binding domains. Changes in phosphotyrosine content of focal adhesion proteins paxillin, PKL, FAK and CAS as well as changes in p38/JNK and ERK MAPK activation will be evaluated using phosphospecific antibodies. It is anticipated that these studies will provide insight into the molecular mechanisms by which cells interact and communicate with the extracellular environment to regulate normal and neoplastic growth, and cell motility.