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J Biol Chem. 1999 Dec 17;274(51):36741-9.

Phosphorylation of B-Myb regulates its transactivation potential and DNA binding.

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Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, Nebraska 68198-6805, USA.


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.

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