HIERARCHICAL PHOSPHORYLATION AT N-TERMINAL TRANSFORMATION-SENSITIVE SITES IN C-MYC PROTEIN IS REGULATED BY MITOGENS AND IN MITOSIS

The N-terminal domain of the c-Myc protein has been reported to be cri tical for both the transactivation and biological functions of the c-M yc proteins. Through detailed phosphopeptide mapping analyses, we demo nstrate that there is a cluster of four regulated and complex phosphor ylation events on the N-terminal domain of Myc proteins, including Thr -58, Ser-62, and Ser-71. An apparent enhancement of Ser-62 phosphoryla tion occurs on v-Myc proteins having a mutation at Thr-58 which has pr eviously been correlated with increased transforming ability. In contr ast, phosphorylation of Thr-58 in cells is dependent on a prior phosph orylation of Ser-62. Hierarchical phosphorylation of c-Myc is also obs erved in vitro with a specific glycogen synthase kinase 3 alpha, unlik e the promiscuous phosphorylation observed with other glycogen synthas e kinase 3 alpha and 3 beta preparations. Although both p42 mitogen-ac tivated protein kinase and cdc2 kinase specifically phosphorylate Ser- 62 in vitro and cellular phosphorylation of Thr-58/Ser-62 is stimulate d by mitogens, other in vivo experiments do not support a role for the se kinases in the phosphorylation of Myc proteins. Unexpectedly, both the Thr-58 and Ser-62 phosphorylation events, but not other N-terminal phosphorylation events, can occur in the cytoplasm, suggesting that t ranslocation of the c-Myc proteins to the nucleus is not required for phosphorylation at these sites. In addition, there appears to be an un usual block to the phosphorylation of Ser-62 during mitosis. Finally, although the enhanced transforming properties of Myc proteins correlat es with the loss of phosphorylation at Thr-58 and an enhancement of Se r-62 phosphorylation, these phosphorylation events do not alter the ab ility of c-Myc to transactivate through the CACGTG Myc/Max binding sit e.