Hormones that couple to the second messenger cyclic AMP (cAMP) have cell-type specific effects on cell growth and differentiation. These effects mirror cAMP's activation of the mitogen-activated protein (MAP) kinase (also called extracellular signal-regulated kinase, or ERK) cascade, the principal mediator of stimulated growth in normal and malignant cells. Work from our previous funding period has established that the cell-type specificity of cAMP is dictated by the expression of the MAP kinase kinase kinase B-Raf. B-Raf has received recent attention for its extremely high rate of mutation/activation in human malignancies and its primary role in specific human cancers. The relationship between B-Rafs oncogenic potential and its role in normal hormonal signaling is unknown. Both B-Raf and the better-studied Raf-1 can activate ERKs in response to signals from the small G protein Ras, but only B-Raf can activate ERKs in response to signals from the related G protein Rap1. Rap1 has been shown to potentiate malignant progression in some B-Raf- expressing cells via its activation of ERKs. We have previously identified Rap 1/B-Raf as a critical mediator of cAMP's activation of ERKs. A major theme of this application is to demonstrate that Rapt regulation of ERKs via cAMP is dictated by the choice of Rap1 guanine nucleotide exchange factor (GEF) utilized.
In Specific Aim 1, we will examine the biochemical mechanism by which cAMP activates Rap1/B-Raf/ERK and test the hypothesis that protein kinase A (PKA), the tyrosine kinase Src, and the RaplGEF C3G are required.
In Specific Aim 2, we will test the hypothesis that cAMP activates at least two distinct pools of Rap1, each mediated by a distinct RaplGEF, only one of which is capable of activating B-Raf/ERK signaling pathways. Novel cellular principles that underscore this segregation of signaling will be tested. We will determine the biochemical basis for this specificity of Rap1 for B-Raf in Specific Aim 3. In this aim, we will also establish novel mouse models to test the hypothesis that B-Raf has irreplaceable functions in vivo.
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