c-Myb-binding Sites Mediate G₁/S-associated Repression of the Plasma Membrane Ca²⁺-ATPase-1 Promoter*

Abstract

We demonstrate that two Myb-binding sites of the mouse plasma membrane Ca²⁺-ATPase-1 (PMCA1) promoter are required for G₁/S cell cycle stage-associated repression of PMCA1 promoter activity. Nuclear run-on experiments revealed G₁/S-associated repression of PMCA1transcription. Ribonuclease protection assays revealed two transcription initiation sites between two Myb-binding sites in thePMCA1 promoter. Gel shift assays showed that c-Myb can bind to wild-type but not point mutated Myb binding sequences of thePMCA1 promoter. Transient transfection assays using cell cycle-synchronized vascular smooth muscle cells (VSMC) andPMCA1 promoter-luciferase constructs showed a 2-fold decrease in reporter activity at G₁/S as compared with G₀. Overexpression of wild-type c-Myb severely repressedPMCA1 promoter activity at both G₀ and G₁/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 G₁/S. Single nucleotide substitutions in the first, second, or both Myb-binding sites alleviated the G₁/S-associated repression ofPMCA1 promoter activity in transformed rat VSMC and primary mouse VSMC cultures. We conclude that c-Myb mediates G₁/S-associated transcriptional repression of thePMCA1 Ca²⁺ pump in rodent VSMC by direct binding to the PMCA1 promoter.