Phosphorylation of B-Myb regulates its transactivation potential and DNA binding.
The transcription factor B-Myb is a cell cycle-regulated phosphoprotein and a potent regulator of cell cycle progression. Previous studies demonstrated that B-Myb was phosphorylated at the onset of S phase, suggesting that it could be due to cyclin-dependent kinases. We identified 10 B-Myb phosphorylation sites by automated peptide radiosequencing of tryptic phosphopeptides derived from in vivo (32)P-labeled B-Myb. Each B-Myb phosphorylation site contained a phosphoserine or phosphothreonine followed by a proline, suggesting that this phosphorylation is due to a proline-directed kinase. Cyclin A-Cdk2 and cyclin E-Cdk2 complexes each phosphorylated B-Myb in a cell-free system on the same sites as in intact cells. Furthermore, the ability of B-Myb to activate a reporter plasmid was enhanced by the cotransfection of cyclin A, whereas mutagenesis of the 10 identified phosphorylation sites from B-Myb blocked the effect of cyclin A coexpression. Additional analysis revealed that the effect of phosphorylation on B-Myb transactivation potential was enhanced by phosphorylation sites in its carboxyl-terminal half. One phosphorylation site (Ser(581)) appeared to negatively regulate DNA binding, as mutation of this site enhanced the ability of B-Myb to bind a Myb-binding sequence. These data suggest that B-Myb is a target for phosphorylation by cyclin-Cdk2 and that phosphorylation of B-Myb regulates its transcriptional activity.
Johnson
T K
TK
Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, Nebraska 68198-6805, USA.
Schweppe
R E
RE
Septer
J
J
Lewis
R E
RE
D000595:Amino Acid Sequence; D000818:Animals; D018797:Cell Cycle Proteins; D002460:Cell Line; D002474:Cell-Free System; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism*; D008969:Molecular Sequence Data; D015513:Oncogene Proteins / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D015534:Trans-Activators / Q000235:genetics* / Q000378:metabolism*; D015533:Transcriptional Activation; D014162:Transfection
D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D015513:Oncogene Proteins; D015534:Trans-Activators; D004247:DNA