The major focus on this proposal is to understand the mechanisms that regulate the function of the E2F transcription factor, with special emphasis on its role in tumor suppression and oncogenesis. It is clear that the retinoblastoma tumor suppressor protein exerts its growth inhibitory function at least in part through repressing the activity of E2F. Oncogenic events that disrupt the function of Rb result in the loss of the Rb-E2F interaction and increased E2F activity; further, over-expression of E2F1 itself can lead to oncogenic transformation. The experiments proposed in this application attempt to understand the biochemical mechanisms involved in regulating E2F function in response to extra-cellular stimuli. Though the steps involved in mitogenic signal transduction pathways have been elucidated, it is not clear how a signal received at the cell surface activates the cell cycle machinery. Their preliminary studies suggest that a vital signaling kinase, Raf-1, can interact with and inactivate Rb. Further, Jun Kinase (JNK) can repress E2F1 function. These observations directly link cell surface signaling with the cell cycle machinery. In this context, efforts will be made to elucidate how Jun Kinase (JNK1) regulates E2F activity and the functional consequences of such a regulation. Attempts will be made to identify the sites on E2F that is targeted by JNK1 and to evaluate whether mutations in these sites will affect the ability of E2F1 to transform cells and to induce apoptosis. Similarly, finer analysis will be conducted on the Raf-1-Rb interaction, and efforts will be made to characterize the steps involved in Raf-1 mediated repression of Rb. It will also be determined how DP1 - a dimerization partner of E2F1, is modulated by signaling pathways and what are the functional consequences of such a regulation. Finally, a novel E2F activity that is over-expressed in Burkitt's lymphoma cells will be characterized and its contribution to the oncogenic process will be assessed. It is hoped that these studies will throw new light on hitherto unknown steps involved in tumor suppression and oncogenesis.
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