THE CELL-CYCLE-REGULATED TRANSCRIPTION FACTOR B-MYB IS PHOSPHORYLATEDBY CYCLIN A CDK2 AT SITES THAT ENHANCE ITS TRANSACTIVATION PROPERTIES/

Expression of the B-Myb transcription factor is upregulated during lat e G(1) phase of the cell cycle by an E2F-dependent transcriptional mec hanism. B-Myb is specifically phosphorylated during S phase, suggestin g that a cyclin-dependent kinase (Cdk) regulates its activity. Consist ent with this notion, the S phase-specific cyclin A/Cdk2 was found pre viously to enhance B-Myb transactivation activity in cotransfected cel ls. In this study we provide evidence that B-Myb is a direct physiolog ical target for cyclin A/Cdk2. We demonstrate that B-Myb is an ill vit ro substrate for cyclin A/Cdk2, but not for cyclin D1/Cdk4 or cyclin E /Cdk2. By mutating candidate Cdk2 phosphorylation sites, we show that B-Myb is phosphorylated at Thr447, Thr490, Thr497 and Ser581 by cyclin A/Cdk2 in vitro and that these sites are also phosphorylated in cycli ng U-2 OS cells. Inhibition of endogenous Cdk2 by dominant negative Cd k2 attenuated phosphorylation of Thr447, Thr490 and Thr497, but had no effect upon Ser581 modification. B-Myb transactivation activity was s ignificantly reduced in a mutant containing amino acid substitutions a t all four identified cyclin A/Cdk2 sites and was constitutively low i n Saos-2 cells where endogenous cyclin A/Cdk2 activity was unable to p hosphorylate ectopically expressed B-Myb. These data indicate that pho sphorylation by cyclin A/Cdk2 is directly involved in enhancing B-Myb transactivation activity and that levels of endogenous cyclin A/Cdk2 a ctivity may contribute to cell line-specific B-Myb function.