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.