Cdk2-dependent and -independent pathways in E2F-mediated S phase induction.
The transcription factor E2F plays an important role in G(1) to S phase transition in the higher eukaryotic cell cycle. Although a number of E2F-inducible genes have been identified, the biochemical cascades from E2F to the S phase entry remain to be investigated. In this study, we generated stably transfected mouse NIH3T3 cells that express exogenous human E2F-1 under the control of a heavy metal-inducible metallothionein promoter and analyzed the molecular mechanism of the E2F-1-mediated initiation of chromosomal DNA replication. Ectopic E2F-1 expression in cells arrested in G(0)/G(1) by serum deprivation enabled them to progress through G(1) and to enter S phase. During the G(1) progression, mouse cyclin E, but little of cyclin D1, was induced to express, which subsequently activated Cdk2. Experiments using the Cdk inhibitory proteins p27, p18, and p19 proved that the activity of Cdk2, but not of Cdk4, was required for S phase entry mediated by E2F-1. Minichromosome maintenance proteins (MCM) 4 and 7, the components of the DNA-replication initiation complex (RC), were constitutively expressed during the cell cycle, although the MCM genes are well known E2F-inducible genes. However, tight association of these two proteins with chromatin depended upon ectopic E2F-1 expression. In contrast, the Cdc45 protein, another RC component, which turned out to be a transcriptional target of E2Fs, was induced to express and subsequently bound to chromatin in response to E2F-1. Experiments utilizing a chemical Cdk-specific inhibitor, butyrolactone I, revealed that Cdk2 activity was required only for chromatin binding of the Cdc45 proteins, and not for the expression of Cdc45 or chromatin binding of MCM4 and -7. These results indicate that at least two separate pathways function downstream of E2F to initiate S phase; one depends upon the activity of Cdk2 and the other does not.
Arata
Y
Y
Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0101, Japan.
Fujita
M
M
Ohtani
K
K
Kijima
S
S
Kato
J Y
JY
D016475:3T3 Cells; D015107:4-Butyrolactone / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D001483:Base Sequence; D042846:CDC2-CDC28 Kinases; D002352:Carrier Proteins; D018797:Cell Cycle Proteins / Q000378:metabolism; D002843:Chromatin / Q000378:metabolism; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases / Q000378:metabolism*; D016213:Cyclins / Q000494:pharmacology*; D004261:DNA Replication / Q000235:genetics; D004268:DNA-Binding Proteins / Q000378:metabolism; D050684:E2F Transcription Factors; D050687:E2F1 Transcription Factor; D006801:Humans; D007399:Interphase; D051379:Mice; D064148:Minichromosome Maintenance Complex Component 4; D064168:Minichromosome Maintenance Complex Component 7; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D056465:Retinoblastoma-Binding Protein 1; D016196:S Phase; D050680:Transcription Factor DP1; D014157:Transcription Factors / Q000378:metabolism*; D015533:Transcriptional Activation; D014162:Transfection
C533431:Arid4a protein, mouse; C113571:CDC45 protein, human; D002352:Carrier Proteins; C491876:Cdc45 protein, mouse; D018797:Cell Cycle Proteins; D002843:Chromatin; D016213:Cyclins; D004268:DNA-Binding Proteins; D050684:E2F Transcription Factors; D050687:E2F1 Transcription Factor; C493753:E2F1 protein, human; C493754:E2f1 protein, mouse; D009687:Nuclear Proteins; D012333:RNA, Messenger; D056465:Retinoblastoma-Binding Protein 1; D050680:Transcription Factor DP1; D014157:Transcription Factors; C082868:butyrolactone I; D017346:Protein Serine-Threonine Kinases; D042846:CDC2-CDC28 Kinases; C495894:CDK2 protein, human; C495893:Cdk2 protein, mouse; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases; C099243:MCM4 protein, human; C101624:MCM7 protein, human; C491086:Mcm7 protein, mouse; D064148:Minichromosome Maintenance Complex Component 4; D064168:Minichromosome Maintenance Complex Component 7; D015107:4-Butyrolactone