In higher eucaryotes, the c-Ras and c-Raf-1 proto-oncogene are pivotal elements in the signal transduction pathway that mediates the growth and differentiation signals initiated by receptor and nonreceptor tyrosine kinases. Previous investigation has shown that c-Raf-1 is suited downstream of c-Ras in the pathway. But the mechanism by which the activated c-Ras leads to activation of c- Raf- 1 was completely unknown until recently. The PI and others recently discovered that activated Ras directly interacts with the aminoterminal regulatory domain of A and c- Raf-1, and the interaction requires the integrity of the Ras effector domain which is crucial for Ras-mediated transformation. These findings strongly support the conclusion that the c-Raf- 1 protein kinase protooncogene is itself an immediate downstream effector of Ras' action, through the activation of c-Raf-1's protein kinase activity. The studies proposed have two broad aims. First, I wish to understand in biochemical terms, the mechanism by which active c-Ras participates in the activation of the c-Raf- 1 protein kinase. This will be approached through biochemical reconstitution in vitro of a Ras-dependent Raf- 1 kinase activation. Current evidence suggests that accesssory factors and probably other polypeptides are required, and these elements will be identified and characterized using a combination of biochemical and expression cloning methods. A second broad goal is to characterize in detail the structural elements on c-Raf-1 that are important to a high affinity interaction that is productive for Raf kinase activation, using mutagenesis and histochemical analyses. Understanding the structural and biochemical mechanism by which these two proteins interact will provide the basis for developing entirely new strategies to interrupt the uncontrolled growth of about 30% of human cancers that express constitutively active Ras polypeptides.
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