The long-term goal of this proposal is to characterize the molecular mechanisms underlying potent inhibition of prostatic epithelial cell migration by Eph receptor tyrosine kinases. Cell migration is involved in several critical steps during tumor metastasis; for prostate cancer there is a direct correlation between motility in vitro and metastatic potential in vivo. Using PC-3 cells as a model system, the applicant's laboratory found that EphA2 kinase activation potently inhibited both haptotactic motility toward fibronectin and chemotactic motility toward epidermal and hepatocyte growth factors. The inhibitory effects were correlated with suppression of integrin affinity, and dephosphorylation and catalytic inactivation of focal adhesion kinase (FAK), a cytoplasmic tyrosine kinase critically involved in cell motility regulation. EphA2 physically associated with FAK and the complex was dissociated upon EphA2 activation. Shp-2 protein tyrosine phosphatase (PTPase) was transiently recruited to the activated EphA2, and represented one candidate PTPase implicated in FAK dephosphorylation. These findings defined a novel signaling pathway that negatively regulates haptotaxis and chemotaxis of prostatic epithelial cells. The focus of this proposal is to test the hypothesis that FAK is a downstream effector of EphA2 kinase.
In Specific Aim 1, the regions and sites on EphA2 and FAK responsible for their interaction will be mapped using deletion and site-directed mutagenesis. Mutants deficient in EphA2/FAK association will e used to assess the functional significance of the association in the negative regulation of cell migration b phA2 kinase.
Specific Aim 2 is designed to investigate the role of FAK dephosphorylation and Shp-2 recruitment in EphA2 signaling using molecular and genetic approaches. Given the fundamental role of cell motility in prostate cancer metastasis, completion of the proposed studies will shed light on a naturally-existing signaling pathway that turns off cell migration, and may lead to novel strategies in preventing and treating prostate cancer metastasis.
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