B. Lutterbach et Sr. Hann, HIERARCHICAL PHOSPHORYLATION AT N-TERMINAL TRANSFORMATION-SENSITIVE SITES IN C-MYC PROTEIN IS REGULATED BY MITOGENS AND IN MITOSIS, Molecular and cellular biology, 14(8), 1994, pp. 5510-5522
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.