S-PHASE ENTRY UPON ECTOPIC EXPRESSION OF G1 CYCLIN-DEPENDENT KINASES IN THE ABSENCE OF RETINOBLASTOMA PROTEIN-PHOSPHORYLATION

In mammalian cells, the retinoblastoma protein (Rb) is thought to nega tively regulate progression through the G1 phase of the cell cycle by its association with the transcription factor E2F [1-3]. Rb-E2F comple xes suppress transcription of genes required for DNA synthesis ([4], r eviewed in [3,5]), and the prevailing view is that phosphorylation of Rb by complexes of cyclin-dependent kinases (Cdks) and their regulator y cyclin subunits, and the subsequent release of active E2F, Bs requir ed for S-phase entry [1-3], This view is based, in part, on the fact t hat ectopic expression of cyclin-Cdks leads to Rb phosphorylation and that this modification correlates with S-phase entry [6-8], In Drosoph ila, however, cyclin E expression can bypass a requirement for E2F, su ggesting 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 ce lls, 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 c onsensus Cdk phosphorylation sites is bypassed by coexpressing G1-spec ific E-type or D-type cyclin-Cdk complexes, and that injection of puri fied cyclin-Cdks during G1 accelerates S-phase entry, Our results indi cate that Rb phosphorylation is not essential for S-phase entry when G 1 cyclin-Cdks are overexpressed, and that other substrates of these ki nases can be rate-limiting for the G1 to S-phase transition, These dat a also reveal that the SAOS-2 cotransfection assay is complicated by R b-independent effects of the coexpressed Cdks.