Developmental downregulation of Xenopus cyclin E is phosphorylation and nuclear import dependent and is mediated by ubiquitination.

Cyclins are regulatory subunits that bind to and activate catalytic Cdks. Cyclin E associates with Cdk2 to mediate the G1/S transition of the cell cycle. Cyclin E is overexpressed in breast, lung, skin, gastrointestinal, cervical, and ovarian cancers. Its overexpression correlates with poor patient prognosis and is involved in the etiology of breast cancer. We have been studying how cyclin E is normally downregulated during development in order to determine if disruption of similar mechanisms could either contribute to its overexpression in cancer, or be exploited to decrease its expression. In Xenopus laevis embryos, cyclin E protein level is high and constant until its abrupt destabilization by an undefined mechanism after the 12th cell cycle, which corresponds to the midblastula transition (MBT) and remodeling of the embryonic to the adult cell cycle. Since degradation of mammalian cyclin E is regulated by the ubiquitin proteasome system and is phosphorylation dependent, we examined the role of phosphorylation in Xenopus cyclin E turnover. We show that similarly to human cyclin E, phosphorylation of serine 398 and threonine 394 plays a role in cyclin E turnover at the MBT. Immunofluorescence analysis shows that cyclin E relocalizes from the cytoplasm to the nucleus preceding its degradation. When nuclear import is inhibited, cyclin E stability is markedly increased after the MBT. To investigate whether degradation of Xenopus cyclin E is mediated by the proteasomal pathway, we used proteasome inhibitors and observed a progressive accumulation of cyclin E in the cytoplasm after the MBT. Ubiquitination of cyclin E precedes its proteasomal degradation at the MBT. These results show that cyclin E destruction at the MBT requires both phosphorylation and nuclear import, as well as proteasomal activity.