Cyclin-dependent kinases (CDKs) are essential for regulating key transitions in the cell cycle, including initiation of DNA replication, mitosis and prevention of re-replication. Here we demonstrate that mammalian CDC6, an essential regulator of initiation of DNA replication, is phosphorylated by CDKs. CDC6 interacts specifically with the active Cyclin A/CDK2 complex in vitro and in vivo, but not with Cyclin E or Cyclin B kinase complexes. The cyclin binding domain of CDC6 was mapped to an N-terminal Cy-motif that is similar to the cyclin binding regions in p21 WAF1/SDI1 and E2F-1. The in vivo phosphorylation of CDC6 was dependent on three N-terminal CDK consensus sites, and the phosphorylation of these sites was shown to regulate the subcellular localization of CDC6. Consistent with this notion, we found that the subcellular localization of CDC6 is cell cycle regulated. In G 1 , CDC6 is nuclear and it relocalizes to the cytoplasm when Cyclin A/CDK2 is activated. In agreement with CDC6 phosphorylation being specifically mediated by Cyclin A/CDK2, we show that ectopic expression of Cyclin A, but not of Cyclin E, leads to rapid relocalization of CDC6 from the nucleus to the cytoplasm. Based on our data we suggest that the phosphorylation of CDC6 by Cyclin A/CDK2 is a negative regulatory event that could be implicated in preventing re-replication during S phase and G 2 .

CDC6 is conserved during evolution and is essential and limiting for the initiation of eukaryotic DNA replication. Human CDC6 activity is regulated by periodic transcription and CDK-regulated subcellular localization. Here, we show that, in addition to being absent from nonproliferating cells, CDC6 is targeted for ubiquitin-mediated proteolysis by the anaphase promoting complex (APC)/cyclosome in G 1 . A combination of point mutations in the destruction box and KEN-box motifs in CDC6 stabilizes the protein in G 1 and in quiescent cells. Furthermore, APC, in association with CDH1, ubiquitinates CDC6 in vitro, and both APC and CDH1 are required and limiting for CDC6 proteolysis in vivo. Although a stable mutant of CDC6 is biologically active, overexpression of this mutant or wild-type CDC6 is not sufficient to induce multiple rounds of DNA replication in the same cell cycle. The APC-CDH1-dependent proteolysis of CDC6 in early G 1 and in quiescent cells suggests that this process is part of a mechanism that ensures the timely licensing of replication origins during G 1 .

The E2F family of transcription factors regulate genes, whose products are essential for progression through the mammalian cell cycle. The transcriptional activity of the E2Fs is inhibited through the specific binding of the retinoblastoma protein, pRB, and the pRB homologs p107 and p130 to their transactivation domains. Seven members of the E2F transcription factor family have been isolated so far, and we were interested in investigating the possible contribution of the various E2Fs to cell cycle control. By presenting the results of the generation of cell lines with tetracycline-controlled expression of E2F-1 and E2F-4 and microinjection of expression plasmids for all members of the E2F family, we demonstrate here that the pRB-associated E2Fs (E2F-1, E2F-2, and E2F-3) all induce S phase in quiescent rat fibroblasts when expressed alone. In contrast, the p107/p130-associated E2Fs require the coexpression of the heterodimeric partner DP-1 to promote S-phase entry and accelerate G 1 progression. Furthermore, the pRB-associated E2Fs were all able to overcome a G 1 arrest mediated by the p16 INK4 tumor suppressor protein, and E2F-1 was shown to override a G 1 block mediated by a neutralizing antibody to cyclin D1. The p16 INK4-induced G 1 arrest was not affected by expression of E2F-4, E2F-5, or DP-1 alone, but simultaneous expression of E2F-4 and DP-1 could overcome this block. Our results demonstrate that the generation of E2F activity is rate limiting for G 1 progression, is sufficient to induce S-phase entry, and overcomes a p16-mediated G 1 block, and since E2F-1, E2F-2, and E2F-3 are associated with pRB, they are the most likely downstream effectors in the p16-cyclin D-pRB pathway. Furthermore, our data suggest that the two subsets of E2Fs are regulated by distinct mechanisms and/or that they have distinct functions in cell cycle control. Since E2F-4 and E2F-5 cannot promote S-phase entry by themselves, our results may provide an explanation for the apparent lack of aberrations in p107 or p130 in human cancer.Entry into and progression through the mammalian cell cycle are highly regulated processes, which at the molecular level involve a number of positively and negatively acting proteins. Many data suggest that among the negative regulators, the prototypic tumor suppressor, pRB, is crucial for proper cell cycle control (for a review, see reference 60). The gene for pRB, RB-1, was the first mammalian tumor suppressor gene to be cloned, and as such it has attracted much attention. Mutations in the RB-1 gene have been found not only in retinoblastomas but also in a variety of human tumors such as osteosarcomas, small-cell lung carcinomas, prostate carcinomas, and cervical cancers (60). The identification of RB-1 mutations in a diversity of human tumors was the first indication that pRB may have a more global regulatory role in cell proliferation. This indication has since been substantiated by a large number of experiments, and in particular it has been demonstrated that overexpression or microinjection of wild...

The E2F transcription factors are essential for regulating the correct timing of activation of several genes whose products are implicated in cell proliferation and DNA replication. The E2Fs are targets for negative regulation by the retinoblastoma protein family, which includes pRB, p107, and p130, and they are in a pathway that is frequently found altered in human cancers. There are five members of the E2F family, and they can be divided into two functional subgroups. Whereas, upon overexpression, E2F-1, -2, and -3 induce S phase in quiescent fibroblasts and override G 1 arrests mediated by the p16 INK4A tumor suppressor protein or neutralizing antibodies to cyclin D1, E2F-4 and -5 do not. Using E2F-1 and E2F-4 as representatives of the two subgroups, we showed here, by constructing a set of chimeric proteins, that the amino terminus of E2F-1 is sufficient to confer S-phase-inducing potential as well as the ability to efficiently transactivate an E2F-responsive promoter to E2F-4. We found that the E2F-1 amino terminus directs chimeric proteins to the nucleus. Surprisingly, a short nuclear localization signal derived from simian virus 40 large T antigen could perfectly substitute for the presence of the E2F-1 amino terminus in these assays. Thus, nuclearly localized E2F-4, when overexpressed, displayed biological activities similar to those of E2F-1. Furthermore, we showed that nuclear localization of endogenous E2F-4 is cell cycle regulated, with E2F-4 being nuclear in the G 0 and early G 1 phases and mainly cytoplasmic after the pRB family members have become phosphorylated. We propose a novel mechanism for the regulation of E2F-dependent transcription in which E2F-4 regulates transcription only from G 0 until mid-to late G1 phase whereas E2F-1 is active in late G 1 and S phases, until it is inactivated by cyclin A-dependent kinase in late S phase.E2F was originally defined as a cellular activity required for the transactivation of the adenovirus E2 promoter by the E1A oncoproteins (34). E1A binds directly to pRB, the product of the retinoblastoma susceptibility gene, and to two pRB relatives, p107 and p130 (40). These proteins, often referred to as pocket proteins, are regulators of the E2F family of transcription factors. Five E2F family members have so far been isolated by virtue of their ability to bind directly to pocket proteins and by homology cloning (38). The affinity of the E2Fs toward pocket proteins and DNA is greatly enhanced by their binding to one of two heterodimeric partners, DP-1 and DP-2/3 (38). The DNA tumor virus oncoproteins E1A, human papillomavirus E7, and simian virus 40 (SV40) large T antigen all regulate E2F-dependent transcription by binding and dissociating the pocket proteins from the E2F heterodimers (4).The E2Fs are believed to regulate the correct timing of transcription of several genes whose products are required for DNA replication (dyhydrofolate reductase, DNA polymerase ␣, and thymidine kinase) and progression through the cell cycle (cyclin A, cyclin E, CDC2, E2F-1, B-Myb...