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 critical for both the transactivation and biological functions of the c-Myc proteins. Through detailed phosphopeptide mapping analyses, we demonstrate that there is a cluster of four regulated and complex phosphorylation events on the N-terminal domain of Myc proteins, including Thr-58, Ser-62, and Ser-71. An apparent enhancement of Ser-62 phosphorylation occurs on v-Myc proteins having a mutation at Thr-58 which has previously been correlated with increased transforming ability. In contrast, phosphorylation of Thr-58 in cells is dependent on a prior phosphorylation of Ser-62. Hierarchical phosphorylation of c-Myc is also observed in vitro with a specific glycogen synthase kinase 3 alpha, unlike the promiscuous phosphorylation observed with other glycogen synthase kinase 3 alpha and 3 beta preparations. Although both p42 mitogen-activated protein kinase and cdc2 kinase specifically phosphorylate Ser-62 in vitro and cellular phosphorylation of Thr-58/Ser-62 is stimulated by mitogens, other in vivo experiments do not support a role for these 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 translocation of the c-Myc proteins to the nucleus is not required for phosphorylation at these sites. In addition, there appears to be an unusual block to the phosphorylation of Ser-62 during mitosis. Finally, although the enhanced transforming properties of Myc proteins correlates with the loss of phosphorylation at Thr-58 and an enhancement of Ser-62 phosphorylation, these phosphorylation events do not alter the ability of c-Myc to transactivate through the CACGTG Myc/Max binding site.
Department of Cell Biology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-2175.
B
B
Lutterbach
S R
SR
Hann
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D002642:Chick Embryo; D003370:Coturnix; D003593:Cytoplasm / Q000378:metabolism; D008934:Mitogens / Q000494:pharmacology*; D008938:Mitosis; D008969:Molecular Sequence Data; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism; D010769:Phosphothreonine / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D016271:Proto-Oncogene Proteins c-myc / Q000378:metabolism*; D015533:Transcriptional Activation
D008934:Mitogens; D016271:Proto-Oncogene Proteins c-myc; D010769:Phosphothreonine; D010768:Phosphoserine; D017346:Protein Serine-Threonine Kinases