Cyclin A/CDK2 binds directly to E2F-1 and inhibits the DNA-binding activity of E2F-1/DP-1 by phosphorylation

Mol Cell Biol. 1994 Dec;14(12):8420-31. doi: 10.1128/mcb.14.12.8420-8431.1994.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • CDC2-CDC28 Kinases*
  • Carrier Proteins*
  • Cell Cycle Proteins*
  • Cyclin-Dependent Kinase 2
  • Cyclin-Dependent Kinases / metabolism*
  • Cyclins / metabolism*
  • DNA-Binding Proteins / metabolism*
  • Drosophila Proteins*
  • E2F Transcription Factors
  • E2F1 Transcription Factor
  • HeLa Cells
  • Humans
  • In Vitro Techniques
  • Macromolecular Substances
  • Peptide Mapping
  • Phosphorylation
  • Protein Binding
  • Protein Serine-Threonine Kinases / metabolism*
  • Retinoblastoma-Binding Protein 1
  • Structure-Activity Relationship
  • Trans-Activators / metabolism*
  • Transcription Factor DP1
  • Transcription Factors / metabolism*

Substances

  • Carrier Proteins
  • Cell Cycle Proteins
  • Cyclins
  • DNA-Binding Proteins
  • Dp transcription factor, Drosophila
  • Drosophila Proteins
  • E2F Transcription Factors
  • E2F1 Transcription Factor
  • E2F1 protein, human
  • Macromolecular Substances
  • Retinoblastoma-Binding Protein 1
  • TFDP1 protein, human
  • Trans-Activators
  • Transcription Factor DP1
  • Transcription Factors
  • Protein Serine-Threonine Kinases
  • CDC2-CDC28 Kinases
  • CDK2 protein, human
  • Cyclin-Dependent Kinase 2
  • Cyclin-Dependent Kinases