The Interactions of E2F With pRB and With p107 Are Regulated via the Phosphorylation of pRB and p107 by a Cyclin-Dependent Kinase

Oncogene. 1995 May 4;10(9):1691-8.

Abstract

It has been postulated that the product (pRB) of the retinoblastoma gene dissociates from the E2F-pRB complex upon phosphorylation by cyclin-dependent kinase(s) (cdk). However, there is no direct evident for the regulation of formation of the E2F-pRB complex via phosphorylation by purified cdk. Therefore, we investigated the regulation of formation of this complex by phosphorylation using pRB and purified cyclin A-cdk2, cyclin E-cdk2 or cyclin D1-cdk4. Purified pRB was incubated with nuclear extracts prepared from pRB-defective cells and then subjected to gel mobility shift assays. We confirmed that unphosphorylated pRB associated with various types of E2F but pRB has been phosphorylated by cyclin A-cdk2 did not. We found that E2F-pRB complexes were disrupted as a consequence of phosphorylation by cyclin A-cdk2, and the levels of the free forms of E2Fs increased. We also found that not only the E2F-pRB complexes but also the E2F-p107 complexes were disrupted upon phosphorylation by cyclin A-cdk2. Furthermore, E2F-pRB complexes were disrupted through phosphorylation by cyclin D1-cdk4 and cyclin E-cdk2, as well as by cyclin A-cdk2. These results clearly demonstrate that the phosphorylation of pRB and p107 by cdks regulates the formation of complexes between E2F and pRB or p107.

MeSH terms

  • Base Sequence
  • Carrier Proteins*
  • Cell Cycle Proteins*
  • Cell Line
  • Cyclin-Dependent Kinases / metabolism*
  • DNA Primers / chemistry
  • DNA-Binding Proteins / metabolism
  • E2F Transcription Factors
  • Humans
  • In Vitro Techniques
  • Macromolecular Substances
  • Molecular Sequence Data
  • Nuclear Proteins / metabolism*
  • Phosphorylation
  • Retinoblastoma Protein / metabolism*
  • Retinoblastoma-Binding Protein 1
  • Retinoblastoma-Like Protein p107
  • Transcription Factor DP1
  • Transcription Factors / metabolism*

Substances

  • Carrier Proteins
  • Cell Cycle Proteins
  • DNA Primers
  • DNA-Binding Proteins
  • E2F Transcription Factors
  • Macromolecular Substances
  • Nuclear Proteins
  • RBL1 protein, human
  • Retinoblastoma Protein
  • Retinoblastoma-Binding Protein 1
  • Retinoblastoma-Like Protein p107
  • Transcription Factor DP1
  • Transcription Factors
  • Cyclin-Dependent Kinases