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

AbstractIn 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

Doc1 mediates the activity of the anaphase-promoting complex by contributing to substrate recognition

The anaphase-promoting complex (APC) is a multisubunit E3 ubiquitin ligase that targets specific cell cycle-related proteins for degradation, regulating progression from metaphase to anaphase and exit from mitosis. The APC is regulated by binding of the coactivator proteins Cdc20p and Cdh1p, and by phosphorylation. We have developed a purification strategy that allowed us to purify the budding yeast APC to near homogeneity and identify two novel APC-associated proteins, Swm1p and Mnd2p. Using an in vitro ubiquitylation system and a native gel binding assay, we have characterized the properties of wild-type and mutant APC. We show that both the D and KEN boxes contribute to substrate recognition and that coactivator is required for substrate binding. APC lacking Apc9p or Doc1p/Apc10 have impaired E3 ligase activities. However, whereas Apc9p is required for structural stability and the incorporation of Cdc27p into the APC complex, Doc1p/Apc10 plays a specific role in substrate recognition by APC–coactivator complexes. These results imply that Doc1p/Apc10 may play a role to regulate the binding of specific substrates, similar to that of the coactivators

p53-dependent inhibition of cyclin-dependent kinase activities in human fibroblasts during radiation-induced G1 arrest

International audiencegamma-Irradiation of human diploid fibroblasts in the G1 interval caused arrest of the cell cycle prior to S phase. This cell cycle block was correlated with a lack of activation of both cyclin E-Cyclin-dependent kinase 2 (Cdk2) and cyclin A-Cdk2 kinases and depended on wild-type p53. Although the accumulation of cyclin A was strongly inhibited in gamma-irradiated cells, cyclin E accumulated and bound Cdk2 at normal levels but remained in an inactive state. We found that both whole-cell lysates and inactive cyclin E-Cdk2 complexes prepared from irradiated cells contained an activity capable of inactivating cyclin E-Cdk2 complexes. The protein responsible for this activity was shown to be p21CIP1/WAF1, recently described as a p53-inducible Cdk inhibitor. Our data suggest a model in which ionizing radiation confers G1 arrest via the p53-mediated induction of a Cdk inhibitor protein