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[Cancer Research 63, 4607-4613, August 1, 2003]
© 2003 American Association for Cancer Research

Molecular Biology and Genetics

Cell Cycle-dependent Regulation of the Skp2 Promoter by GA-binding Protein1

Hiroyuki Imaki, Keiko Nakayama, Sophie Delehouzee, Hiroshi Handa, Masatoshi Kitagawa2, Takumi Kamura and Keiichi I. Nakayama3

Departments of Molecular and Cellular Biology [H. I., M. K., T. K., K. I. N.] and Molecular Genetics [K. N., K. I. N.], Medical Institute of Bioregulation, Kyushu University, Fukuoka, Fukuoka 812-8582; CREST, Japan Science and Technology Corporation, Kawaguchi, Saitama 332-0012 [H. I., K. N., M. K., T. K., K. I. N.]; and Faculty of Bioscience and Biotechnology, Tokyo Institute of Technology, Yokohama, Kanagawa 226-8501 [S. D., H. H.], Japan

Skp2 is the F-box protein component of an SCF-type ubiquitin ligase that interacts specifically with p27Kip1 and thereby promotes its ubiquitylation and degradation. The abundance of Skp2 mRNA oscillates in a cell cycle-dependent manner, being maximal in S and G2 phases. The regulation of Skp2 transcription was investigated by cloning the promoter region of the mouse gene and determination of its activity in a luciferase reporter assay. Deletion analysis identified a minimal ~0.3-kb promoter region with marked transcriptional activity and a 105-bp essential sequence within this region. Electrophoretic mobility shift assays indicated the presence in nuclear extracts of proteins that bind to this sequence. Site-directed mutagenesis revealed that the core binding motif, CACTTCCG, which is similar to that of GA-binding protein (GABP), is essential for Skp2 transcription. "Supershift" analysis indicated that the protein-probe complexes detected by electrophoretic mobility shift assays contain GABP. Endogenous GABP bound to Skp2 promoter element in a cell cycle-dependent manner. Furthermore, overexpression of GABPß increased Skp2 promoter activity, and suppression of GABP{alpha} or GABPß by a small interfering RNA resulted in the reduction of Skp2 promoter activity. These data suggest that the cell cycle-dependent binding of GABP to the Skp2 promoter plays an important role in the regulation of Skp2 expression and cell cycle progression from G1 to S phase.




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Copyright © 2003 by the American Association for Cancer Research.