Specific regulation of E2F family members by cyclin-dependent kinases.
The transcription factor E2F-1 interacts stably with cyclin A via a small domain near its amino terminus and is negatively regulated by the cyclin A-dependent kinases. Thus, the activities of E2F, a family of transcription factors involved in cell proliferation, are regulated by at least two types of cell growth regulators: the retinoblastoma protein family and the cyclin-dependent kinase family. To investigate further the regulation of E2F by cyclin-dependent kinases, we have extended our studies to include additional cyclins and E2F family members. Using purified components in an in vitro system, we show that the E2F-1-DP-1 heterodimer, the functionally active form of the E2F activity, is not a substrate for the active cyclin D-dependent kinases but is efficiently phosphorylated by the cyclin B-dependent kinases, which do not form stable complexes with the E2F-1-DP-1 heterodimer. Phosphorylation of the E2F-1-DP-1 heterodimer by cyclin B-dependent kinases, however, did not result in down-regulation of its DNA-binding activity, as is readily seen after phosphorylation by cyclin A-dependent kinases, suggesting that phosphorylation per se is not sufficient to regulate E2F DNA-binding activity. Furthermore, heterodimers containing E2F-4, a family member lacking the cyclin A binding domain found in E2F-1, are not efficiently phosphorylated or functionally down-regulated by cyclin A-dependent kinases. However, addition of the E2F-1 cyclin A binding domain to E2F-4 conferred cyclin A-dependent kinase-mediated down-regulation of the E2F-4-DP-1 heterodimer. Thus, both enzymatic phosphorylation and stable physical interaction are necessary for the specific regulation of E2F family members by cyclin-dependent kinases.
Dynlacht
B D
BD
Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts 02138, USA. dynlacht@biosun.harvard.edu
Moberg
K
K
Lees
J A
JA
Harlow
E
E
Zhu
L
L
D042846:CDC2-CDC28 Kinases; D002352:Carrier Proteins; D002453:Cell Cycle; D018797:Cell Cycle Proteins; D002474:Cell-Free System; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases / Q000378:metabolism; D016213:Cyclins / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D050684:E2F Transcription Factors; D050687:E2F1 Transcription Factor; D010449:Peptide Mapping; D010748:Phosphopeptides / Q000032:analysis; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011994:Recombinant Proteins; D056465:Retinoblastoma-Binding Protein 1; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D014157:Transcription Factors / Q000502:physiology*
D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D016213:Cyclins; D004268:DNA-Binding Proteins; D050684:E2F Transcription Factors; D050687:E2F1 Transcription Factor; D010748:Phosphopeptides; D011994:Recombinant Proteins; D056465:Retinoblastoma-Binding Protein 1; D014157:Transcription Factors; D017346:Protein Serine-Threonine Kinases; D042846:CDC2-CDC28 Kinases; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases