Cell cycle-dependent regulation of the Skp2 promoter by GA-binding protein.
Skp2 is the F-box protein component of an SCF-type ubiquitin ligase that interacts specifically with p27(Kip1) and thereby promotes its ubiquitylation and degradation. The abundance of Skp2 mRNA oscillates in a cell cycle-dependent manner, being maximal in S and G(2) 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 approximately 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 GABPbeta increased Skp2 promoter activity, and suppression of GABPalpha or GABPbeta 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 G(1) to S phase.
Imaki
Hiroyuki
H
Departments of Molecular and Cellular Biology, Medical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka, Fukuoka 812-8582, Japan.
Nakayama
Keiko
K
Delehouzee
Sophie
S
Handa
Hiroshi
H
Kitagawa
Masatoshi
M
Kamura
Takumi
T
Nakayama
Keiichi I
KI
D016475:3T3 Cells; D000818:Animals; D001483:Base Sequence; D002453:Cell Cycle / Q000235:genetics / Q000502:physiology; D018797:Cell Cycle Proteins / Q000096:biosynthesis / Q000235:genetics*; D004247:DNA / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D051268:GA-Binding Protein Transcription Factor; D005786:Gene Expression Regulation; D005809:Genes, Regulator; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics; D044786:S-Phase Kinase-Associated Proteins; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism / Q000502:physiology*
D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D051268:GA-Binding Protein Transcription Factor; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D044786:S-Phase Kinase-Associated Proteins; D014157:Transcription Factors; D004247:DNA