MECHANISM OF RAF1 ACTIVATION

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DESCRIPTION: Raf-1 is a physiological kinase upstream of MEK 1/2, whose downstream target, MAPK/Erk was first identified as an insulin-responsive kinase. Raf-1 is a major downstream effector of the proto-oncogene c-Ras and its activation is crucial for the regulation of proliferation, differentiation and other cellular functions. Despite this, knowledge of the detailed mechanism of Raf activation is still incomplete. Studies from a number of laboratories have suggested that extracellular signals, such as insulin, IGF and EGF initially interacting with its amino-terminal regulatory domain. Findings by others and these investigators suggest that this binding opens the tight structure of inactive Raf- and makes it accessible to as yet unidentified activators, which lock Raf in an active state by phosphorylation and possibly other mechanisms. We have recently shown that 14-3-3 is required for this activation process, either by mediating dimerization of Raf-1 or causing it to interact with other protein factors that assist in the assembly of an activatable Raf activation. The proposed studies will examine this model using biochemical and molecular approaches. The specific aims are as follows: 1. To elucidate the mechanism by which oligomerization/dimerization contributes to the activation of Raf-1. 2. To explore the role of 14-3-3 in the regulation of Raf kinase activity. 3. To identify sequences on Raf that are critical for its activation. The focus will be on identifying a new phospho-peptide that appear on the peptide map of Raf-1 when EGF activates it. 4. To identify the kinase that phosphorylates the sequences described in aim 3. These studies should provide new insights into the mechanism by which Raf-1 is activated in responses to changes in its extracellular and intracellular milieu. They should also expend our understanding of the role 14-3-3 and of dimerization in cellular signal transduction.