Focal adhesions (FAs), the sites where cells in culture adhere most tightly to the underlying extracellular matrix (ECM), provide a model for analyzing the interaction of cells with the ECM. The goals of this proposal are to characterize the links between the ECM and the actin cytoskeleton in FAs, and to identify the signalling pathways at these sites of cell-ECM interaction. How integrin-mediated cell adhesion stimulates the tyrosine kinase (TK) p125FAK will be investigated. Binding assays will be used to determine whether p125FAK interacts with integrin cytoplasmic domains, and kinase assays will reveal whether integrin binding stimulates the activity of p125FAK. TK inhibitors that prevent FA formation will be examined for their action on p125FAK in vitro. Microinjection of antibodies against p125FAK will be employed as an alternative way to test the functions of this enzyme. The cloned SH2 domain of tensin will be used to determine whether tensin binds FA proteins that become tyrosine phosphorylated in response to adhesion. Is elevated tyrosine phosphorylation in FAs critical for anchorage- dependent growth? Is adhesion-induced expression of growth-associated genes blocked by inhibitors of p125FAK? Potential signalling proteins, such as PI-3 kinase and phospholipase Cgamma, will be assessed for adhesion-induced tyrosine phosphorylation. The src family of TKs will be studied for activation in response to ECM adhesion. FA assembly and tyrosine phosphorylation will be examined in cells that lack pp60c-src. The protein that binds the myristylated N-terminus of the src protein will be isolated, cloned and sequenced; its interaction with FA components will be studied. Proteins binding to the cytoplasmic domains of integrin alpha and beta subunits will continue to be investigated, using synthetic peptides to map binding sites. Proteins binding to the 47 kD talin domain will be identified, and the interactions and functions of the new FA protein, aciculin, will be explored. FA disassembly will be studied as cells enter mitosis and under conditions where motility is promoted. Does the mitotic phosphorylation of the beta1 integrin cytoplasmic domain inhibit its binding of talin, alpha-actinin or p125FAK? FA proteins that become phosphorylated as FAs disassemble will be studied and the effects of these phosphorylations on protein interactions determined. Does tyrosine dephosphorylation have a role in FA disassembly or merely accompany it? Phosphatase inhibitors will be used to inhibit dephosphorylation and to determine whether this prevents FA disassembly. These studies should provide an understanding of the structural organization of FAs, the regulation of their assembly and disassembly, and their role in signalling from the ECM.
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