Temporal regulation of cyclin A-p107 and p33cdk2 complexes binding to a human thymidine kinase promoter element important for G1-S phase transcriptional regulation.
The cyclins are an extensive family of proteins whose cell cycle-dependent synthesis is postulated to control multiple events during the cell cycle. The synthesis of A-type cyclins begins at the start of S phase. In mammalian cells, association with the cdc-type kinases suggests that cyclin A complexes are important for DNA replication and regulating other DNA-bound substrates required for S phase. We report here that a 25-bp promoter element previously shown to be important for the G1-S activation of the human thymidine kinase (htk) promoter in growth-stimulated cells is a cellular target of cyclin A and the p33cdk2 complexes. Though the p33cdk2 and other nuclear factor complexes exhibit constitutive binding to the htk G1-S regulatory domain, the binding activity of a cyclin A/p107 protein complex is greatly enhanced when the cells enter S phase, correlating with the increase in the tk mRNA levels and the replication of DNA. The binding activity of the cyclin A complex is maintained throughout S phase. Mutation of the DNA sequences on either half of the 25-bp protein binding site results in the loss of its ability to compete efficiently in vitro for the htk complexes, including that of cyclin A-containing complex. The loss of high-affinity binding for the htk complexes also substantially reduces the S-phase regulation of the htk promoter in vivo. Our results support the hypothesis that a cyclin A complex, in association with the p33cdk2 kinase, mediates the S-phase-regulated transcription of the htk promoter in growth-stimulated cells.
Li
L J
LJ
Department of Biochemistry, University of Southern California School of Medicine, Los Angeles 90033.
Naeve
G S
GS
Lee
A S
AS
D001483:Base Sequence; D042846:CDC2-CDC28 Kinases; D002453:Cell Cycle / Q000235:genetics* / Q000502:physiology; D002460:Cell Line; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics / Q000378:metabolism; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases; D016213:Cyclins / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism; D016193:G1 Phase / Q000235:genetics; D005786:Gene Expression Regulation; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D011401:Promoter Regions, Genetic; D011494:Protein Kinases / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012045:Regulatory Sequences, Nucleic Acid; D016196:S Phase / Q000235:genetics; D013937:Thymidine Kinase / Q000235:genetics* / Q000378:metabolism; D013997:Time Factors; D014158:Transcription, Genetic; D014162:Transfection
D016213:Cyclins; D004268:DNA-Binding Proteins; D009838:Oligodeoxyribonucleotides; D011993:Recombinant Fusion Proteins; D015500:Chloramphenicol O-Acetyltransferase; D011494:Protein Kinases; D013937:Thymidine Kinase; D017346:Protein Serine-Threonine Kinases; D042846:CDC2-CDC28 Kinases; C495894:CDK2 protein, human; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases