title: c-Myb-binding sites mediate G(1)/S-associated repression of the plasma membrane Ca(2+)-ATPase-1 promoter.
abstract: We demonstrate that two Myb-binding sites of the mouse plasma membrane Ca(2+)-ATPase-1 (PMCA1) promoter are required for G(1)/S cell cycle stage-associated repression of PMCA1 promoter activity. Nuclear run-on experiments revealed G(1)/S-associated repression of PMCA1 transcription. Ribonuclease protection assays revealed two transcription initiation sites between two Myb-binding sites in the PMCA1 promoter. Gel shift assays showed that c-Myb can bind to wild-type but not point mutated Myb binding sequences of the PMCA1 promoter. Transient transfection assays using cell cycle-synchronized vascular smooth muscle cells (VSMC) and PMCA1 promoter-luciferase constructs showed a 2-fold decrease in reporter activity at G(1)/S as compared with G(0). Overexpression of wild-type c-Myb severely repressed PMCA1 promoter activity at both G(0) and G(1)/S while co-transfection of a dominant negative c-Myb, or a construct encoding an anti-c-Myb neutralizing antibody, completely abolished the repression seen at G(1)/S. Single nucleotide substitutions in the first, second, or both Myb-binding sites alleviated the G(1)/S-associated repression of PMCA1 promoter activity in transformed rat VSMC and primary mouse VSMC cultures. We conclude that c-Myb mediates G(1)/S-associated transcriptional repression of the PMCA1 Ca(2+) pump in rodent VSMC by direct binding to the PMCA1 promoter.
authors.affiliation: Centre for Cardiovascular Research, Toronto General Hospital, Toronto, Ontario, Canada.
authors.forename: T
authors.initials: T
authors.lastname: Afroze
authors.forename: M
authors.initials: M
authors.lastname: Husain
mesh_terms: D000818:Animals; D001665:Binding Sites; D000252:Calcium-Transporting ATPases / Q000235:genetics*; D027682:Cation Transport Proteins; D002462:Cell Membrane / Q000201:enzymology; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D004794:Enzyme Repression; D016193:G1 Phase / Q000235:genetics; D017930:Genes, Reporter; D007399:Interphase / Q000235:genetics*; D051379:Mice; D008969:Molecular Sequence Data; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000502:physiology*; D016297:Mutagenesis, Site-Directed; D053499:Plasma Membrane Calcium-Transporting ATPases; D017354:Point Mutation; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D020598:Proto-Oncogene Proteins c-myb / Q000378:metabolism*; D051381:Rats; D016196:S Phase / Q000235:genetics; D017384:Sequence Deletion; D014158:Transcription, Genetic
chemical_list: D027682:Cation Transport Proteins; D020598:Proto-Oncogene Proteins c-myb; D053499:Plasma Membrane Calcium-Transporting ATPases; C506954:Atp2b1 protein, mouse; C506955:Atp2b1 protein, rat; D000252:Calcium-Transporting ATPases