Editors' ChoiceCell Cycle

Safe from Degradation

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Science's STKE  27 Sep 2005:
Vol. 2005, Issue 303, pp. tw344
DOI: 10.1126/stke.3032005tw344

DNA replication is initiated through the assembly of prereplication complexes (pre-RCs); replication does not occur in quiescent cells and is limited to once per cell cycle because, outside of the G1 phase of the cell cycle, inhibitors prevent assembly of the pre-RC. During G1, APCCdh1 (the form of the anaphase-promoting complex E3 ubiquitin ligase that contains Cdh1) targets the pre-RC assembly inhibitors geminin and cyclin A for degradation. Paradoxically, however, APCCdh1 targets Cdc6, which is essential to pre-RC assembly, for destruction as well (see Ayad). Mailand and Diffley noted that, in cultured human cells treated with the cyclin-dependent kinase (CDK) inhibitor roscovitine, Cdc6 abundance was reduced, whereas the abundance of geminin and cyclin A was unaffected. Knockdown of Cdk2 with small interfering RNA (siRNA) also decreased Cdc6 abundance. Roscovitine accelerated Cdc6 degradation, as did depletion of Cdk2 or cyclins E1 and E2, whereas overexpression of cyclin E (which forms an active CDK complex with Cdk2) promoted Cdc6 accumulation. Mutational analysis indicated that the effects of cyclin E on Cdc6 stability depended on Cdc6 phosphorylation. Cdc6 phosphorylation inhibited its APCCdh1-dependent ubiquitination and degradation; further, whereas wild-type Cdc-6 or an unphosphorylatable mutant coimmunoprecipitated with Cdh1, a phospho-mimicking Cdc6 mutant did not. In serum-starved (noncycling) cells, Cdc6, geminin, and cyclin A were undetectable. After serum stimulation, and reentry into the cell cycle, Cdc6 abundance increased 3 to 4 hours before the other APCCdh1 targets became detectable. When Cdc6 was detected, it was phosphorylated, and cyclin E-associated kinase activity appeared simultaneously with Cdc6. Thus, the authors propose that, after entry into the cell cycle, cyclin E-Cdk2-dependent phosphorylation of Cdc6 allows it to accumulate (and promote pre-RC assembly) before inactivation of APCCdh1 and the ensuing accumulation of pre-RC assembly inhibitors such as geminin and cyclin A.

N. Mailand, J. F. X. Diffley, CDKs promote DNA replication origin licensing in human cells by protecting Cdc6 from APC/C-dependent proteolysis. Cell 122, 915-926 (2005). [PubMed]

N. G. Ayad, CDKs give Cdc6 a license to drive into S phase. Cell 122, 825-827 (2005). [PubMed]

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