S-Phase entry upon ectopic expression of G1 cyclin-dependent kinases in the absence of retinoblastoma protein phosphorylation

Curr Biol. 1997 Sep 1;7(9):709-12. doi: 10.1016/s0960-9822(06)00301-0.

Abstract

In mammalian cells, the retinoblastoma protein (Rb) is thought to negatively regulate progression through the G1 phase of the cell cycle by its association with the transcription factor E2F [1-3]. Rb-E2F complexes suppress transcription of genes required for DNA synthesis ([4], reviewed in [3,5]), and the prevailing view is that phosphorylation of Rb by complexes of cyclin-dependent kinases (Cdks) and their regulatory cyclin subunits, and the subsequent release of active E2F, is required for S-phase entry [1-3]. This view is based, in part, on the fact that ectopic expression of cyclin-Cdks leads to Rb phosphorylation and that this modification correlates with S-phase entry [6-8]. In Drosophila, however, cyclin E expression can bypass a requirement for E2F, suggesting that cyclins may activate replication independently of the Rb/E2F pathway [9]. We sought to examine whether Rb phosphorylation is a prerequisite for S-phase entry in Rb-deficient SAOS-2 osteosarcoma cells, using a commonly used cotransfection assay [6-8,10]. We find that a G1 arrest in SAOS-2 cells mediated by an Rb mutant lacking all 14 consensus Cdk phosphorylation sites is bypassed by coexpressing G1-specific E-type or D-type cyclin-Cdk complexes, and that injection of purified cyclin-Cdks during G1 accelerates S-phase entry. Our results indicate that Rb phosphorylation is not essential for S-phase entry when G1 cyclin-Cdks are overexpressed, and that other substrates of these kinases can be rate-limiting for the G1 to S-phase transition. These data also reveal that the SAOS-2 cotransfection assay is complicated by Rb-independent effects of the coexpressed Cdks.

Publication types

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

MeSH terms

  • Animals
  • CDC2-CDC28 Kinases*
  • Cell Line
  • Cyclin D1 / metabolism
  • Cyclin E / metabolism
  • Cyclin G
  • Cyclin-Dependent Kinase 2
  • Cyclin-Dependent Kinase 4
  • Cyclin-Dependent Kinases / metabolism*
  • Cyclins / metabolism*
  • Phosphorylation
  • Protein-Serine-Threonine Kinases / metabolism
  • Proto-Oncogene Proteins*
  • Retinoblastoma Protein / metabolism*
  • S Phase*
  • Transfection

Substances

  • Cyclin E
  • Cyclin G
  • Cyclins
  • Proto-Oncogene Proteins
  • Retinoblastoma Protein
  • Cyclin D1
  • Protein-Serine-Threonine Kinases
  • CDC2-CDC28 Kinases
  • Cyclin-Dependent Kinase 2
  • Cyclin-Dependent Kinase 4
  • Cyclin-Dependent Kinases