Differential regulation of E2F transactivation by cyclin/cdk2 complexes.
The mammalian transcription factor E2F plays a critical role in the expression of genes required for cellular proliferation. To understand how E2F is regulated, we have developed a reconstituted in vitro transcription assay. Using this E2F-responsive assay, we can demonstrate that E2F-mediated transcription can be directly repressed by the tumor suppressor protein pRB. This inhibition is abolished by phosphorylation of pRB with either cyclin A/cdk2 or cyclin E/cdk2. However, these cyclin/kinase complexes exhibit differences in the ability to phosphorylate E2F. Only cyclin A/cdk2 can phosphorylate E2F effectively, and this phosphorylation abolishes its ability to bind DNA and mediate trans-activation. Thus, this in vitro transcriptional assay allows activation and inactivation of E2F transcription, and our findings demonstrate how transcriptional regulation of E2F can be linked to cell cycle-dependent activation of kinases.
Dynlacht
B D
BD
Massachusetts General Hospital Cancer Center, Charlestown 02129.
Flores
O
O
Lees
J A
JA
Harlow
E
E
D042846:CDC2-CDC28 Kinases; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D002474:Cell-Free System; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases / Q000502:physiology*; D016213:Cyclins / Q000502:physiology*; D004268:DNA-Binding Proteins; D050684:E2F Transcription Factors; D006367:HeLa Cells; D006801:Humans; D008957:Models, Genetic; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000502:physiology*; D012097:Repressor Proteins / Q000502:physiology; D016160:Retinoblastoma Protein / Q000502:physiology; D056465:Retinoblastoma-Binding Protein 1; D050680:Transcription Factor DP1; D014157:Transcription Factors / Q000502:physiology*; D015533:Transcriptional Activation / Q000502:physiology*
D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D016213:Cyclins; D004268:DNA-Binding Proteins; D050684:E2F Transcription Factors; D012097:Repressor Proteins; D016160:Retinoblastoma Protein; D056465:Retinoblastoma-Binding Protein 1; D050680:Transcription Factor DP1; D014157:Transcription Factors; D017346:Protein Serine-Threonine Kinases; D042846:CDC2-CDC28 Kinases; C495894:CDK2 protein, human; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases