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c-Myb-binding sites mediate G(1)/S-associated repression of the plasma membrane Ca(2+)-ATPase-1 promoter

T Afroze1, M Husain

  • 1Centre for Cardiovascular Research, Toronto General Hospital, Toronto, Ontario, Canada.

The Journal of Biological Chemistry
|March 18, 2000
PubMed

Insights

Two Myb-binding sites in the PMCA1 promoter are essential for repressing the Ca(2+) pump during the G(1)/S cell cycle phase. The transcription factor c-Myb directly binds these sites to mediate this repression in vascular smooth muscle cells.

Area of Science:

  • Molecular Biology
  • Cell Biology
  • Cardiovascular Research

Background:

  • The plasma membrane Ca(2+)-ATPase-1 (PMCA1) regulates intracellular calcium levels, crucial for vascular smooth muscle cell (VSMC) function.
  • Cell cycle progression influences gene expression, impacting cellular processes like calcium homeostasis.

Purpose of the Study:

  • To investigate the role of Myb-binding sites in the PMCA1 promoter in regulating its activity during the G(1)/S cell cycle transition.
  • To determine the involvement of the transcription factor c-Myb in the cell cycle-dependent repression of PMCA1.

Main Methods:

  • Nuclear run-on assays to assess PMCA1 transcription rates.
  • Ribonuclease protection assays to identify transcription initiation sites.
  • Gel shift assays to confirm c-Myb binding to PMCA1 promoter sequences.
  • Transient transfection assays with PMCA1 promoter-luciferase constructs in synchronized VSMCs.
  • Mutagenesis of Myb-binding sites and overexpression/inhibition of c-Myb.

Main Results:

  • PMCA1 transcription is repressed during the G(1)/S phase.
  • Two functional Myb-binding sites were identified in the PMCA1 promoter, located between two transcription initiation sites.
  • c-Myb directly binds to these specific Myb-binding sites.
  • PMCA1 promoter activity decreased 2-fold at G(1)/S compared to G(0) in VSMCs.
  • Overexpression of c-Myb repressed PMCA1 activity, while dominant-negative c-Myb or anti-c-Myb antibodies abolished this repression.
  • Mutating the Myb-binding sites eliminated the G(1)/S-associated repression.

Conclusions:

  • c-Myb directly binds to two specific sites within the PMCA1 promoter.
  • c-Myb mediates the G(1)/S-associated transcriptional repression of the PMCA1 Ca(2+) pump in rodent VSMCs.

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