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The functions and proteolysis of cyclin A and cyclin F during mitosis

Authors Fung, Tsz Kan
Issue Date 2006
Summary The cell cycle is driven by the oscillating activity of a class of protein called cyclin-dependent kinases (CDKs). By definition, CDKs are activated by binding to cyclins. The oscillating activity of CDKs is predominantly conferred by the periodic expression of cyclins. Cyclin B-CDC2 is a M-phase-promoting factor that induces the entry into mitosis. Cyclin A is also believed to be essential for mitotic entry, as disruption of cyclin A causes a G2 arrest. However, the precise role of cyclin A in mitosis is still obscure. To decipher the function of cyclin A, I have suppressed the expression of cyclin A by short-hairpin RNA (shRNA) in HeLa cells. Down-regulation of cyclin A induces a G2 phase arrest through a checkpoint-independent inactivation of cyclin B-CDC2 by inhibitory phosphorylation. Deregulation of WEE1, but not the PLK1-CDC25 axis, can override this arrest, suggesting that cyclin A-CDK may tip the balance of the cyclin B-CDC2 bistable system by initiating the inactivation of WEE1. In contrast, knock-down of cyclin B inhibits mitosis without inactivating cyclin A-CDK, indicating that cyclin A-CDK acts upstream of cyclin B-CDC2. Even when ectopically expressed, cyclin A cannot replace cyclin B in driving mitosis, indicating the specific role of cyclin B as a component of MPF. These observations show that cyclin A cannot form MPF independent of cyclin B and underscore the critical role of cyclin A as a trigger for MPF activation. After the entry into mitosis, degradation of the mitotic cyclins is required for the onset of anaphase. Proteolysis of cyclin B is dependent on the destruction box (D-box) motif at the N-terminal region of the protein and involves the ubiquitin-proteasome pathway. However, the degradation mechanism of cyclin A is still poorly understood. I found that the D-box is not absolutely required for cyclin A proteolysis. I found that in addition to the D-box, the region between residues 123-157 also contributed to the ubiquitination and degradation of human cyclin A. Indeed, removal of the bulk of the N-terminal regulatory domain was needed to completely stabilize cyclin A and eliminate ubiquitination. A putative second RxxL motif around residue 138 played only a minor role in cyclin A degradation. I also applied a novel technique to map the ubiquitin-acceptor sites in cyclin A, which are located near the D-box. Cyclin F is an essential protein but its functions remain elusive. Cyclin F oscillates in the cell cycle with a similar pattern as cyclin A and cyclin B. I found that similar to cyclin A, cyclin F is degraded when the spindle-assembly checkpoint is activated and accumulates when the DNA damage checkpoint is activated. Cyclin F is a very unstable protein throughout much of the cell cycle. Unlike other cyclins, degradation of cyclin F is independent of ubiquitination and proteasome-mediated pathways. Interestingly, proteolysis of cyclin F is likely to involve metalloproteases. Rapid destruction of cyclin F does not require the N-terminal F-box motif but requires the COOH-terminal PEST sequences. Finally, I found that the PEST region alone is sufficient to interfere with the degradation of cyclin F and confer instability when fused to cyclin A. These data show that although cyclin F is degraded at similar time as the mitotic cyclins, the underlying mechanisms are entirely distinct.
Note Thesis (Ph.D.)--Hong Kong University of Science and Technology, 2006
Language English
Format Thesis
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