c-Myb-binding sites mediate G(1)/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-associa ted repression of PMCA1 promoter activity. Nuclear run-on experiments revea led G(1)/S-associated repression of PMCA1 transcription. Ribonuclease prote ction assays revealed two transcription initiation sites between two Myb-bi nding sites in the PMCA1 promoter. Gel shift assays showed that c-Myb can b ind to wild-type but not point mutated Myb binding sequences of the PMCA1 p romoter. Transient transfection assays using cell cycle-synchronized vascul ar smooth muscle cells (VSMC) and PMCA1 promoter-luciferase constructs show ed 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 activit y at both G(0) and G(1)/S while co-transfection of a dominant negative c-My b, 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-associa ted repression of PMCA1 promoter activity in transformed rat VSMC and prima ry mouse VSMC cultures. We conclude that c-Myb mediates G(1)/S-associated t ranscriptional repression of the PMCA1 Ca2+ pump in rodent VSMC by direct b inding to the PMCA1 promoter.