The overall picture which has emerged from numerous studies of c-myc expression during differentiation and development is that c-myc is associated with proliferation with some notable exceptions. These studies suggest a tightly regulated and complex role for c-Myc in differentiation and development; however, few of these studies have examined the c-Myc proteins. Recent reports and our preliminary studies reveal the continued presence of c-myc protein after differentiation of several different cell types. The goal of this proposal is to determine if there are changes in the synthesis, stability, localization and modification of the c-myc proteins during differentiation and growth inhibition and if alterations in synthesis and phosphorylation of c-myc can effect these processes. There are two major forms of c-myc which initiate at two distinct sites. Preliminary studies reveal that c-myc 1 can be synthesized independently of c-myc 2 (the exon 2 ATG-initiated protein) depending on the proliferation state. In addition c-myc 1 is differentially stabilized and localized in murine erythroleukemia cells. Therefore, it is possible that c-myc 1 may have a different role than c-myc 2 during differentiation and growth, perhaps as an antagonist to c-myc 2. The other major known characteristic of c-myc proteins is that they are all phosphorylated. Preliminary studies indicate that a major phosphorylation site in c-myc is in a highly conserved region which is frequently found to have mutations in 'activated"""""""" c-myc genes. This suggests that phosphorylation at this site may play a role in c-myc function. To test these hypotheses, changes in c- myc protein during differentiation of several cell types will be examined. Any observed differences will be compared with changes during growth inhibition due to TGF beta. To directly determine if c-myc synthesis or phosphorylation have roles in differentiation or growth inhibition, cell lines which overexpress c-myc mutants having altered initiation sites or phosphorylation sites will be developed. Finally, transgenic mice will be established with these c-myc mutants under the control of tissue-specific enhancers to examine the effects of altered synthesis or phosphorylation of c-myc protein in specific tissues during mouse embryogenesis. These studies should establish if there are any major differences in c-myc proteins during differentiation versus growth inhibition and help determine the role of any such differences on c-Myc function in differentiation and growth inhibition.
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