Animal cells depend on signals from the extracellular matrix and growth factors to survive, proliferate, and find their position in the organism. De-regulation of these signals is a hallmark of malignant cell transformation. SHEP1 is the prototype of a novel family of cytoplasmic proteins that likely coordinate survival and migratory signals from extracellular matrix receptors (integrins) and growth factor receptor tyrosine kinases. SHEP family proteins contain a bifunctional domain that binds both Ras proteins that positively regulate integrins and the docking protein p130Cas (Cas), which transmits signals downstream of integrins. In addition, SHEP proteins bind activated receptor tyrosine kinases. Our goal is to examine the molecular mechanisms underlying SHEP 1 function in intracellular signaling. SHEP 1 activates the c-Jun N-terminal kinase (JNK) and the nuclear factor (NF)-kB transcription factor. Through these pathways, which are involved in adhesion-dependent cell survival, SHEP1 may contribute to making cells more resistant to environmental insults such as detachment from the extracellular matrix and exposure to cytotoxic agents. Furthermore, SHEP 1 promotes the formation of membrane ruffles, suggesting a role in cell migration and invasion. Cell culture models will be used to examine how SHEP 1 activates JNK and NF-kB and whether the SHEP 1-Cas complex regulates cell adhesion and motility by linking integrins and receptor tyrosine kinases. X-ray crystallography and NMR spectroscopy will define the SHEP1 protein interfaces involved in Cas and Ras proteins binding, which we hypothesize to be critical for SHEP1 function. In a complementary approach, phage display will identify peptides that inhibit SHEP1 binding to Cas and Ras proteins by targeting these interfaces. Finally, conditional inactivation of the SHEP 1 gene in conjunction with mouse mammary tumor models will elucidate the role of SHEP 1 in cell survival/proliferation and migration/invasion in vivo in normal tissues and in tumors. This multidisciplinary approach is made possible by collaborations among the program laboratories and the resources provided by the cores. This project may yield new insight into the signaling networks that protect cells from apoptosis and promote motility and invasion, and elucidate the role of SHEP family proteins in these networks.
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