B-Myb repressor function is regulated by cyclin A phosphorylation and sequences within the C-terminal domain.
B-Myb is a widely expressed member of the myb oncogene family that has been shown to act as either an activator or repressor of gene transcription in a cell-type-specific fashion. For example, in aortic smooth muscle cells B-Myb represses transcription of the alpha2(V) collagen gene. Recently, phosphorylation of B-Myb by cyclin A was shown to enhance greatly its ability to transactivate. Here, we have tested the effects of cyclin A on the ability of B-Myb to repress. We report that coexpression of cyclin A abolished repression of the alpha2(V) collagen promoter, whereas a dominant-negative cyclin-dependent kinase 2 (cdk2) enhanced repression by ectopic and endogenous B-Myb protein. Mutation of 10 of 22 putative cyclin A sites, which greatly reduces the effects of cyclin A on transactivation by B-Myb, had no effect on the ability of cyclin A to alleviate B-Myb-mediated repression of alpha2(V) collagen promoter activity. Furthermore, the stability of the mutant B-Myb protein was largely unaffected by cyclin A, although ectopic expression of cyclin A enhanced the rate of decay of wild-type B-Myb protein. Thus, the mechanisms of repression and activation appear distinct, for example, mediated by different critical phosphorylation sites or protein-protein interactions. B-Myb mutants with either deletion of aa 374-581 (B-Myb-Mut3) or C-terminal truncation beyond aa 491 (B-Myb-491) positively regulated alpha2(V) collagen promoter activity, and were not affected by cyclin A. Thus, our findings indicate that the ability of B-Myb to function as a repressor of matrix promoter activity is abolished by cyclin A, and maps the sites mediating negative regulation by B-Myb to the region between aa 491 and 582.
Petrovas
Constantinos
C
Department of Biochemistry, Boston University Medical School, MA 02118-2394, USA.
Jeay
Sébastien
S
Lewis
Robert E
RE
Sonenshein
Gail E
GE
D000818:Animals; D001859:Bone Neoplasms / Q000473:pathology; D002417:Cattle; D018797:Cell Cycle Proteins; D024062:Collagen Type V / Q000096:biosynthesis / Q000235:genetics; D019925:Cyclin A / Q000737:chemistry / Q000502:physiology*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D005260:Female; D005786:Gene Expression Regulation / Q000235:genetics; D006801:Humans; D051379:Mice; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000378:metabolism; D015513:Oncogene Proteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D012516:Osteosarcoma / Q000473:pathology; D010766:Phosphorylation; D011401:Promoter Regions, Genetic / Q000235:genetics; D025941:Protein Interaction Mapping; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000502:physiology; D012097:Repressor Proteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D017384:Sequence Deletion; D015534:Trans-Activators / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D015533:Transcriptional Activation / Q000235:genetics; D014162:Transfection; D014407:Tumor Cells, Cultured
D018797:Cell Cycle Proteins; D024062:Collagen Type V; D019925:Cyclin A; D004268:DNA-Binding Proteins; C064352:MYBL2 protein, human; C499306:Mybl2 protein, mouse; D015513:Oncogene Proteins; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D015534:Trans-Activators