Regulation of Ras-Dependent Signal Transduction Pathways

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The Ras pathway is a critical signal transduction cascade involved in regulating cellular proliferation, differentiation, and survival. Members of the Raf serine/threonine kinase family are key intermediates in this pathway, functioning to relay signals from activated Ras to the downstream protein kinases, MEK and ERK. Three Raf proteins are found in mammalian cells, Raf-1, A-Raf and B-Raf. As might be expected for proteins so centrally involved in cell signaling, the Raf kinases also contribute to oncogenic transformation and cancer. For example, mutation or amplification of upstream regulators of Raf, such as receptor tyrosine kinases and Ras, frequently induces deregulated signaling through the Raf/MEK/ERK cascade in tumors harboring these alleles. Moreover, constitutively active Raf proteins can themselves cause cell transformation. In particular, mutation of the B-Raf family member is observed in 67% of malignant melanomas as well as in many colorectal, ovarian and papillary thyroid carcinomas. During this past fiscal year, our research identified CK2 as an upstream protein kinase that contributes to Raf activation. Our studies has also revealed that the oncogenic potential of the B-Raf kinase can be altered by specific phosphorylation events and protein interactions. Together, these findings identify potential targets for therapeutic intervention in tumors with constitutive Ras- or Raf-dependent signaling. Another conserved component of the Ras pathway is KSR1, a protein that acts as a molecular scaffold to facilitate signal transmission through the ERK cascade. During the past fiscal year, our studies provided novel insight into the endogenous scaffolding role of KSR1 within the nervous system. This work demonstrated that KSR1 functions biochemically in the hippocampus to scaffold the components of the ERK cascade, specifically regulating the cascade when a membrane fraction of ERK is activated via a PKC-dependent pathway but not via a cAMP/PKA-dependent pathway. Consistent with these findings, mice lacking KSR1 were found to have deficits in associative learning and certain typses of synaptic plasticity. In this fiscal year, we also discovered that KSR1 undergoes caspase-dependent cleavage in apoptotic cells and that cleavage destroys the scaffolding function of the KSR1 and generates a stable C-terminal fragment (CTF) that can inhibit ERK activation. These findings indicate that cleavage of the KSR1 scaffold is another mechanism whereby caspases down-regulate ERK survival signaling to promote cellular apoptosis.