The cell-cycle regulated transcription factor B-Myb is phosphorylated by cyclin A/Cdk2 at sites that enhance its transactivation properties.
Expression of the B-Myb transcription factor is upregulated during late G1 phase of the cell cycle by an E2F-dependent transcriptional mechanism. B-Myb is specifically phosphorylated during S phase, suggesting that a cyclin-dependent kinase (Cdk) regulates its activity. Consistent with this notion, the S phase-specific cyclin A/Cdk2 was found previously to enhance B-Myb transactivation activity in cotransfected cells. In this study we provide evidence that B-Myb is a direct physiological target for cyclin A/Cdk2. We demonstrate that B-Myb is an in vitro 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 cycling U-2 OS cells. Inhibition of endogenous Cdk2 by dominant negative Cdk2 attenuated phosphorylation of Thr447, Thr490 and Thr497, but had no effect upon Ser581 modification. B-Myb transactivation activity was significantly reduced in a mutant containing amino acid substitutions at all four identified cyclin A/Cdk2 sites and was constitutively low in Saos-2 cells where endogenous cyclin A/Cdk2 activity was unable to phosphorylate ectopically expressed B-Myb. These data indicate that phosphorylation by cyclin A/Cdk2 is directly involved in enhancing B-Myb transactivation activity and that levels of endogenous cyclin A/Cdk2 activity may contribute to cell line-specific B-Myb function.
Saville
M K
MK
Section of Virology and Cell Biology and Ludwig Institute for Cancer Research, Imperial College School of Medicine, St Mary's Campus, London, UK.
Watson
R J
RJ
D001859:Bone Neoplasms / Q000473:pathology; D042846:CDC2-CDC28 Kinases; D018797:Cell Cycle Proteins; D019925:Cyclin A / Q000378:metabolism*; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D016193:G1 Phase; D005786:Gene Expression Regulation; D006801:Humans; D012516:Osteosarcoma / Q000473:pathology; D010766:Phosphorylation; D010768:Phosphoserine / Q000032:analysis; D010769:Phosphothreonine / Q000032:analysis; D011499:Protein Processing, Post-Translational; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D016196:S Phase; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D015534:Trans-Activators / Q000737:chemistry / Q000378:metabolism*; D015533:Transcriptional Activation; D014162:Transfection; D014407:Tumor Cells, Cultured
D018797:Cell Cycle Proteins; D019925:Cyclin A; D004268:DNA-Binding Proteins; C064352:MYBL2 protein, human; D011993:Recombinant Fusion Proteins; D015534:Trans-Activators; D010769:Phosphothreonine; D010768:Phosphoserine; D017346:Protein Serine-Threonine Kinases; D042846:CDC2-CDC28 Kinases; C495894:CDK2 protein, human; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases