Cyclin A/CDK2 binds directly to E2F-1 and inhibits the DNA-binding activity of E2F-1/DP-1 by phosphorylation.
E2F-1, a member of the E2F transcription factor family, contributes to the regulation of the G1-to-S phase transition in higher eukaryotic cells. E2F-1 forms a heterodimer with DP-1 and binds to several cell cycle regulatory proteins, including the retinoblastoma family (RB, p107, p130) and cyclin A/CDK2 complexes. We have analyzed E2F-1 phosphorylation and its interaction with cyclin A/CDK2 complexes both in vivo and in vitro. In vitro, E2F-1 formed a stable complex with cyclin A/CDK2 but not with either subunit alone. DP-1 did not interact with cyclin A, CDK2, or the cyclin A/CDK2 complex. While the complex of cyclin A/CDK2 was required for stable complex formation with E2F-1, the kinase-active form of CDK2 was not required. However, E2F-1 was phosphorylated by cyclin A/CDK2 in vitro and was phosphorylated in vivo in HeLa cells. Two-dimensional tryptic phosphopeptide mapping studies demonstrated an overlap in the phosphopeptides derived from E2F-1 labeled in vitro and in vivo, indicating that cyclin A/CDK2 may be responsible for the majority of E2F-1 phosphorylation in vivo. Furthermore, an active DNA-binding complex could be reconstituted from purified E2F-1/DP-1 and cyclin A/CDK2. Binding studies conducted both in vitro and in vivo demonstrated that the cyclin A/CDK2-binding region resided within the N-terminal 124 amino acids of E2F-1. Because the stable association of E2F-1 with cyclin A/CDK2 in vitro and in vivo did not require a DP-1- or RB-binding domain and because the interactions could be reconstituted from purified components in vitro, we conclude that the interactions between cyclin A/CDK2 and E2F-1 are direct. Finally, we report that the DNA-binding activity of the E2F-1/DP-1 complex is inhibited following phosphorylation by cyclin A/CDK2.
Department of Physiology, Tufts University School of Medicine, Boston, Massachusetts 02111.
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M
Xu
K A
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Sheppard
C Y
CY
Peng
A S
AS
Yee
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H
Piwnica-Worms
D042846:CDC2-CDC28 Kinases; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases / Q000378:metabolism*; D016213:Cyclins / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D029721:Drosophila Proteins; D050684:E2F Transcription Factors; D050687:E2F1 Transcription Factor; D006367:HeLa Cells; D006801:Humans; D066298:In Vitro Techniques; D046911:Macromolecular Substances; D010449:Peptide Mapping; D010766:Phosphorylation; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D056465:Retinoblastoma-Binding Protein 1; D013329:Structure-Activity Relationship; D015534:Trans-Activators / Q000378:metabolism*; D050680:Transcription Factor DP1; D014157:Transcription Factors / Q000378:metabolism*
D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D016213:Cyclins; D004268:DNA-Binding Proteins; C088053:Dp transcription factor, Drosophila; D029721:Drosophila Proteins; D050684:E2F Transcription Factors; D050687:E2F1 Transcription Factor; C493753:E2F1 protein, human; D046911:Macromolecular Substances; D056465:Retinoblastoma-Binding Protein 1; C493735:TFDP1 protein, human; D015534:Trans-Activators; 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