Bartosch, Birke; (1999) The stability and turnover of Xenopus cyclin E. Doctoral thesis (Ph.D.), University College London.

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Abstract

Cyclins and cyclin dependent kinases (Cdk) are key regulators of the cell cycle. Cyclin E binds to and activates Cdk2, and is required for cells to enter S-phase. In somatic cell cycles, restriction of the activity of cyclin E/Cdk2 to the G1/S transition is mediated by cell cycle stage-specific expression of cyclin E, whose levels rise late in G1 and decline rapidly once cells have entered S-phase. During the first twelve cell cycles of the frog Xenopus laevis, however, cyclin E is extremely stable. After the twelfth division, the midblastula transition occurs, the cell cycle slows down, and cyclin E protein is rapidly degraded. The aim of these studies was to investigate the control of cyclin E stability before and after MBT. Measurements of the half-life of cyclin E showed that it was very stable in Xenopus embryos and egg extracts, suggesting that the disappearance of cyclin E at the midblastula transition is due to proteolysis, which is activated at this stage of development. Binding to Cdk2 appears to be required for this proteolysis, as mutant versions of cyclin E, unable to form stable complexes with Cdk2, failed to be degraded at the MBT. Deletion of residues in the N-terminus of cyclin E stabilized the protein, although these versions still bound to Cdk2 and formed active protein kinases. Cyclin E is phosphorylated on several sites in vitro. Two major sites were identified as serine 67 and threonine 76. Mutation of all six canonical Cdk sites in cyclin E to alanine did not interfere with its activity or localization but stabilized the protein when expressed in monkey COS-1 cells. Extensive efforts to develop a cell-free system from frog-eggs and embryos, which could degrade cyclin E, were not successful.

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