c-Myb-binding sites mediate G1/S-associated repression of the plasma membrane Ca2+-ATPase-1 promoter

We demonstrate that two Myb-binding sites of the mouse plasma membrane Ca2+-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 a-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 Ca2+ pump in rodent VSMC by direct binding to the PMCA1 promoter.