Regulation of mammalian CDC6 by CDK phosphorylation and proteasome dependent degradation.

The mammalian cell cycle is regulated by periodic gene transcription, kinase activities and protein degradation. A human cDNA encoding a protein homologous to S. cerevisiae Cdc6p and S. pombe Cdc18 was identified. Mammalian CDC6 is essential for DNA replication, and the obtained data demonstrate that the mammalian CDC6 protein is regulated by several mechanisms. The subcellular localization of mammalian CDC6 is cell cycle regulated controlled by Cyclin A/CDK2 phosphorylation. The CDC6 protein interacts directly with the Cyclin A/CDK2 complex via a Cy-motif in the N-terminal of CDC6 and phosphorylation of hCDC6 results in relocation of hCDC6 from the nucleus to the cytoplasm during S- phase. The protein level of mammalian CDC6 is growth and cell cycle regulated. The CDC6 protein is induced by stimulation of serum starved fibroblasts and the CDC6 protein level remains high throughout S-phase and G2. As cells progress through mitosis the amount of CDC6 protein declines abruptly. Mammalian CDC6 is an unstable protein. The level of CDC6 protein increases in the presence of proteasome inhibitors and, furthermore, poly-ubiquitinated CDC6 was identified in vivo, demonstrating that CDC6 is targeted for degradation by ubiquitination in mammalian cells. The instability of CDC6 was shown to be dependent on the N- terminal region. A peptide sequence with homology to destruction box sequences found in substrates of the anaphase promoting complex was identified in hCDC6 and shown to mediate the degradation of hCDC6 in quiescent cells. These data suggest that the mammalian CDC6 protein is highly regulated. In GO and early G1 accumulation of the mammalian CDC6 protein is prevented by ubiquitin mediated degradation. During S-phase and G2 mammalian CDC6 is retained in the cytoplasm by Cyclin A/CDK2 phosphorylation

Cell Cycle-Regulated Expression of Mammalian CDC6 Is Dependent on E2F

The E2F transcription factors are essential regulators of cell growth in multicellular organisms, controlling the expression of a number of genes whose products are involved in DNA replication and cell proliferation. In Saccharomyces cerevisiae, the MBF and SBF transcription complexes have functions similar to those of E2F proteins in higher eukaryotes, by regulating the timed expression of genes implicated in cell cycle progression and DNA synthesis. The CDC6 gene is a target for MBF and SBF-regulated transcription. S. cerevisiae Cdc6p induces the formation of the prereplication complex and is essential for initiation of DNA replication. Interestingly, the Cdc6p homolog in Schizosaccharomyces pombe, Cdc18p, is regulated by DSC1, the S. pombe homolog of MBF. By cloning the promoter for the human homolog of Cdc6p and Cdc18p, we demonstrate here that the cell cycle-regulated transcription of this gene is dependent on E2F. In vivo footprinting data demonstrate that the identified E2F sites are occupied in resting cells and in exponentially growing cells, suggesting that E2F is responsible for downregulating the promoter in early phases of the cell cycle and the subsequent upregulation when cells enter S phase. Our data also demonstrate that the human CDC6 protein (hCDC6) is essential and limiting for DNA synthesis, since microinjection of an anti-CDC6 rabbit antiserum blocks DNA synthesis and CDC6 cooperates with cyclin E to induce entry into S phase in cotransfection experiments. Furthermore, E2F is sufficient to induce expression of the endogenous CDC6 gene even in the absence of de novo protein synthesis. In conclusion, our results provide a direct link between regulated progression through G(1) controlled by the pRB pathway and the expression of proteins essential for the initiation of DNA replication

Cell cycle– and cell growth–regulated proteolysis of mammalian CDC6 is dependent on APC–CDH1

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)