OVEREXPRESSION OF C-MYC AND CELL IMMORTALIZATION ALTERS C-MYC PHOSPHORYLATION

Using an extensive series of deletion and site-specific mutation const ructs, we have identified five new phosphorylation sites in c-Myc in t he N-terminal transactivation domain and near the C-terminal DNA bindi ng/heterodimerization domain. We have also found that Thr-58 phosphory lation is regulated by specific cellular events. When c-Myc is overexp ressed in cells Thr-58 phosphorylation was greatly enhanced in the ove rexpressed, exogenous c-Myc as compared with the endogenous protein. I n contrast, an inhibition of Thr-58 phosphorylation and an enhancement of Serine 62 phosphorylation was observed in c-Myc from immortalized cells compared with primary cells. No significant changes in c-Myc pho sphorylation were found when transformed and nontransformed cells were compared. Finally, mutations at these phosphorylation sites, either i ndividually or in combination with previously described sites, did not affect the ability of c-Myc to transactivate through the CACGTG Myc/M ax DNA binding sites. These results further suggest that either the mo lecular role for c-Myc phosphorylation does not involve modulating tra nscriptional activity of c-Myc or that the CACGTG site does not repres ent a physiological promoter element.