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Cell cycle-dependent regulation of the Skp2 promoter by GA-binding protein

Hiroyuki Imaki1, Keiko Nakayama, Sophie Delehouzee

  • 1Departments of Molecular and Cellular Biology, Medical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka, Fukuoka 812-8582, Japan.

Cancer Research
|August 9, 2003
PubMed

Insights

Skp2 (Skip complex 2) expression, crucial for cell cycle progression, is regulated by GA-binding protein (GABP) binding to its promoter. This interaction controls Skp2 transcription, impacting cell cycle advancement from G1 to S phase.

Area of Science:

  • Molecular Biology
  • Cell Biology
  • Biochemistry

Background:

  • Skp2 (Skip complex 2) is an F-box protein within an SCF-type ubiquitin ligase complex.
  • Skp2 targets p27(Kip1) for ubiquitylation and degradation, influencing cell cycle control.
  • Skp2 mRNA levels oscillate, peaking during S and G2 phases of the cell cycle.

Purpose of the Study:

  • To investigate the transcriptional regulation of the mouse Skp2 gene.
  • To identify key regulatory elements and transcription factors involved in Skp2 gene expression.
  • To elucidate the role of Skp2 regulation in cell cycle progression.

Main Methods:

  • Cloning and functional analysis of the mouse Skp2 promoter using luciferase reporter assays.
  • Deletion analysis to identify essential promoter regions and a core binding motif.
  • Electrophoretic mobility shift assays (EMSA) and supershift analysis to identify DNA-binding proteins.
  • Site-directed mutagenesis to confirm the functional importance of the binding motif.
  • Small interfering RNA (siRNA) to suppress GABP expression and assess its impact on Skp2 promoter activity.

Main Results:

  • A minimal 0.3-kb promoter region with high transcriptional activity was identified, containing a 105-bp essential sequence.
  • Electrophoretic mobility shift assays revealed proteins binding to this essential sequence, identified as GA-binding protein (GABP).
  • Site-directed mutagenesis confirmed a CACTTCCG motif as critical for Skp2 transcription.
  • Endogenous GABP binding to the Skp2 promoter was cell cycle-dependent.
  • Overexpression of GABPbeta enhanced Skp2 promoter activity, while siRNA-mediated suppression of GABPalpha or GABPbeta reduced it.

Conclusions:

  • The cell cycle-dependent binding of GABP to the Skp2 promoter is a key mechanism regulating Skp2 expression.
  • GABP plays a significant role in controlling Skp2 transcription and, consequently, cell cycle progression from G1 to S phase.

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