? Activation of growth signaling cascades by epidermal growth factor (EGF) and other mitogens is a key step in cell growth and development as well as the generation of neural and other epithelial-derived tumors. Among the main mediators of signals transmitted by tyrosine kinase receptors such as the EGF receptor are the mitogenactivated protein kinases (MAPKs), a superfamily of highly homologous proline-directed serine/threonine kinases that participate in the transduction of growth and differentiation-promoting signals. The Ras/Raf kinase cascade that leads to activation of ERK1 and ERK2, two members of the ERK subfamily of MAPKs, is required for cell growth and thus is an important target of therapeutic agents for tumor treatment. Recent studies from our laboratory have revealed a new mechanism for the regulation of Raf-1 by growth factors. Under nonstimulatory conditions, Raf-1 can form a complex with a Raf Kinase Inhibitory Protein (RKIP/PEBP). An evolutionarily conserved protein that is present from bacteria to man, RKIP prevents Raf- 1 from downstream signaling. We have shown that different growth factors activate specific members of the protein kinase C (PKC) family that, in turn, cause release of RKIP, enabling activation of Raf and the MAPK signaling cascade. More recent results have implicated RKIP in mitotic progression. We therefore hypothesize that RKIP is a key regulator of mitogenesis. The goal of this proposal is to characterize further the physiological functions of RKIP, elucidate the mechanisms by which RKIP functions, and define the structural interactions that underlie these mechanisms. Specifically, we plan to (1) Determine the role(s) of RKIP at different stages of the cell cycle in regulating cell growth; (2) Determine the mechanism(s) by which RKIP regulates MAP kinase signaling and mitotic progression; and (3) Identify the key functional domains in RKIP. The studies proposed here will increase our understanding of the regulation of these key events in cell proliferation, and should lead to the development of new reagents that can inhibit growth of neural and other epithelial-derived tumors. ? ?
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