Direct binding of cyclin D to the retinoblastoma gene product (pRb) and pRb phosphorylation by the cyclin D-dependent kinase CDK4

Genes Dev. 1993 Mar;7(3):331-42. doi: 10.1101/gad.7.3.331.

Abstract

The product (pRb) of the retinoblastoma gene (RB-1) prevents S-phase entry during the cell cycle, and inactivation of this growth-suppressive function is presumed to result from pRb hyperphosphorylation during late G1 phase. Complexes of the cyclin-dependent kinase, cdk4, and each of three different D-type cyclins, assembled in insect Sf9 cells, phosphorylated a pRb fusion protein in vitro at sites identical to those phosphorylated in human T cells. Only D-type cyclins activated cdk4 enzyme activity, whereas cyclins A, B1, and E did not. When Sf9 cells were coinfected with baculovirus vectors encoding human pRb and murine D-type cyclins, cyclins D2 and D3, but not D1, bound pRb with high stoichiometry in intact cells. Introduction of a vector encoding cdk4, together with those expressing pRb and D-type cyclins, induced pRb hyperphosphorylation and dissociation of cyclins D2 and D3, whereas expression of a kinase-defective cdk4 mutant in lieu of the wild-type catalytic subunit yielded ternary complexes. The transcription factor E2F-1 also bound to pRb in insect cells, and coexpression of cyclin D-cdk4 complexes, but neither subunit alone, triggered pRb phosphorylation and prevented its interaction with E2F-1. The D-type cyclins may play dual roles as cdk4 regulatory subunits and as adaptor proteins that physically target active enzyme complexes to particular substrates.

Publication types

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

MeSH terms

  • Adenosine Triphosphate / metabolism
  • Animals
  • Baculoviridae / genetics
  • Cell Line
  • Cyclin D1
  • Cyclins / metabolism*
  • G1 Phase
  • Genes, Retinoblastoma*
  • Humans
  • Insecta
  • Oncogene Proteins / metabolism
  • Phosphorylation
  • Recombinant Fusion Proteins / metabolism
  • Retinoblastoma Protein / genetics
  • Retinoblastoma Protein / metabolism*
  • Transfection

Substances

  • Cyclins
  • Oncogene Proteins
  • Recombinant Fusion Proteins
  • Retinoblastoma Protein
  • Cyclin D1
  • Adenosine Triphosphate