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

Petersen, Birgit Otzen (1999). Regulation of mammalian CDC6 by CDK phosphorylation and proteasome dependent degradation. PhD thesis The Open University.



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.

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