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.
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