Aberrant activation of Ras is a major contributor to human cancer. The Ras-GRF1 exchange factor, which activates Ras in response to G protein-coupled receptors, is highly expressed in central neurons and plays an essential role in the establishment of memory. Over-expression of Ras-GRF1 in human tumors implicates this exchange factor in carcinogenesis. The activity of Ras-GRF1 is controlled, at least in part, by alterations in its phosphorylation state, but a detailed picture of its regulation has not been achieved. The long-range goal of this project is to obtain a detailed understanding of the physiological and pharmacological significance of the Ras-GRF exchange factors. The objective of this proposal is to determine how the Ras-GRF1 exchange factor is controlled by phosphorylation. The central hypothesis is that Ras-GRF1 serves as a key integrator of signal transduction by regulation of its activity through both phosphorylation and interaction with calcium/calmodulin. The rationale for this proposal is that, through definition of the mechanisms that control Ras-GRF1, we will produce greater insight into a new and physiologically important pathway for Ras activation. This project will be performed in an environment that is very favorable for its successful completion through excellent collegial interactions and substantial institutional commitment. The objective of this proposal will be attained through testing the central hypothesis in three specific aims: 1).Determine the sites of regulated phosphorylation in Ras-GRFI. The hypothesis to be tested is that a combination of phosphorylation events is required for G protein-dependent activation of Ras-GRF1. 2).Define the integration of control of Ras-GRF1 by phosphorylation and calcium signaling. The ability of Ras-GRF1 to integrate multiple signals into the activation of Ras will be tested. 3).Elucidate a dual role for phosphorylation of Ras-GRFI at Serine-916. The working hypothesis is that Serine-916 is a physiologically significant site of regulated phosphorylation that contributes to both activation and down-regulation of the exchange factor. The proposed studies are innovative in that they address an important mechanism for Ras activation in the CNS that has been subject to less investigation. The results will be significant as they will provide key understanding of the control of Ras by G protein-coupled receptors, and thus may potentially lead to the identification of new therapeutic targets for intervention in cancer. The results are likely to be of fundamental importance to our comprehension of the role of the Ras/MAP kinase system in learning and memory.
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