title: S-Phase entry upon ectopic expression of G1 cyclin-dependent kinases in the absence of retinoblastoma protein phosphorylation.
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.
authors.affiliation: Department of Biochemistry Baylor College of Medicine Houston, Texas 77030, USA.
authors.forename: X
authors.initials: X
authors.lastname: Leng
authors.forename: L
authors.initials: L
authors.lastname: Connell-Crowley
authors.forename: D
authors.initials: D
authors.lastname: Goodrich
authors.forename: J W
authors.initials: JW
authors.lastname: Harper
mesh_terms: D000818:Animals; D042846:CDC2-CDC28 Kinases; D002460:Cell Line; D019938:Cyclin D1 / Q000378:metabolism; D019927:Cyclin E / Q000378:metabolism; D056746:Cyclin G; D051357:Cyclin-Dependent Kinase 2; D051358:Cyclin-Dependent Kinase 4; D018844:Cyclin-Dependent Kinases / Q000378:metabolism*; D016213:Cyclins / Q000378:metabolism*; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011518:Proto-Oncogene Proteins; D016160:Retinoblastoma Protein / Q000378:metabolism*; D016196:S Phase; D014162:Transfection
chemical_list: D019927:Cyclin E; D056746:Cyclin G; D016213:Cyclins; D011518:Proto-Oncogene Proteins; D016160:Retinoblastoma Protein; D019938:Cyclin D1; D017346:Protein Serine-Threonine Kinases; D042846:CDC2-CDC28 Kinases; D051357:Cyclin-Dependent Kinase 2; D051358:Cyclin-Dependent Kinase 4; D018844:Cyclin-Dependent Kinases