Molecular Analysis Of Signal Transduction by Integrins

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DESCRIPTION(adapted from applicant's abstract): The long-term goals of the present proposal are to understand signal transduction mechanisms in integrin-mediated cell migration. We have been focusing on identifying intracellular signaling molecules involved in the regulation of cell migration. Using a stable CHO cell transfection model, we identified the adaptor molecule Cas as a mediator of FAK signaling in cell migration. Furthermore, we showed that FAK interaction with PI3K is required for stimulation of cell migration and that the FAK/Src complex formation and subsequent Cas phosphorylation is not sufficient. We also found FAK interaction with another adaptor molecule Grb7 and provided evidence of potential function of the interaction in the regulation of cell migration. Further studies suggested that Grb7 recruitment to the focal contacts and its phosphorylation by FAK play a role in the stimulation of cell migration by Grb7 and its interaction with FAK. Finally, we have cloned a novel protein inhibitor FIP200 for FAK family kinases using yeast two hybrid screening and showed that FIP200 could inhibit FAK activity in vitro and in vivo. In this proposal, we plan to determine the molecular mechanisms by which Grb7 and FIP200 participate in signaling pathways in the regulation of cell migration. In Aim l, we will first determine the critical domains and key residues of Grb7 in its regulation of cell migration using site-directed mutagenesis. We will then examine the role of these domains in the regulation of Grb7 phosphorylation by FAK. Lastly, we will directly map the Grb7 phosphorylation sites by FAK. In Aim 2, we will examine the relationships and possible cooperations of FAK downstream signaling pathways through FAK/Src/Cas, FAK/PI3K and FAK/Grb7 complexes. We will determine the subcellular localization of the activated complexes. We will also create and analyze the role of FAK mutants with selective binding to these downstream signaling molecules. In the last Aim, we will investigate the mechanisms and regulation of FIP200 inhibition of FAK signaling pathways in cell migration. We will determine the functional domains of FIP200 using mutational approaches. We will also examine FIP200 inhibition of FAK downstream signaling pathways using stable inducible cell lines, study the regulation and functions of the endogenous FIP200/FAK complex in cell migration. These studies will enhance our understanding of the molecular mechanisms of signal transduction by integrins, which are critical factors in cell migration and invasiveness in biological processes such as embryonic development, wound healing and cancer.