| Research Abstract |
This Monbusho International Scientific Research Program was carried out by Hideki Kobayashi as a head investigator in collaboration with Takeharu Nishimoto, Kyushu University, Department of Molecular Biology and Tim Hunt, ICRF Clare Hall Laboratories, UK. Through this collaboration for two years(April 1992-March 1994), the following results are obtained.
(1) Destruction of Xenopus cyclins A and B requires binding to p34^<cdc2>.
Mitotic cyclins are activating subuntis of p34^<cdc2>. These cyclins undergo saw-tooth oscillations in concentration during the early cleavage cell cycles of fertilized eggs because are stable during interphase, but rapidly destroyed by proteolysis at the end of metaphase. This programmed destruction requires a decapeptide motif(the destruction box)near the N-terminus of the cyclin molecule. We have made a number of mutations of Xenopus cyclin A and tested them for their ability to be destroyed in Xenopus CSF-arrested extracts after addition of Ca^<2+> to trigger
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proteolysis. Mutations of cyclin A within the cyclin box or the C-terminal domain that abolish the binding to and activation of p34^<cdc2> also abolishes the ability of cyclin A to undergo programmed proteolysis. It thus appears that cyclin A must bind to p34^<cdc2> in order to be destroyed. This also seems to be true for cyclin B2, but the results for cyclin B1 are more difficult to explain. Some non-p34^<cdc2>-binding mutants of Xenopus cyclin B1 are destroyed at essentially normal rates, whereas others are stable. And we have confirmed the findings of Glotzer et al. (1991) that chimeric proteins containing N- terminal residues 13-66 of sea urchin cyclin B fused to staphylococcal protein A are degraded in a regulated manner that depends on the integrity of the destruction box. These constructs do not bind to p34^<cdc2>.
(2) The expression of Xenopus cyclin A in the budding yeast Saccharomyces cervisiae.
To investigate the regulatory role of cyclin A, we have expressed Xenopus cyclin A and its mutant in the budding yeast. The growth of yeast inhibited by galactose-induced overexpression of Xenopus wild-type and indestructible cyclin A, but not by the cyclin box mutant lacking cdc2 binding ability. Growth is arrested at the G2/M transition and the yeast become larger in size and eventually die. These results suggest that Xenopus cyclin A, which can bind cdc2, perturbs cell cycle progession at the G2/M boundary.
(3) The half life of cyclin-cdc2 complexes in Xenopus cell cycle extracts.
We have measured the exchange rate of cyclin A and cyclin B between endogenous p34^<cdc2> and bacterialy expressed GST-cdc2 using Xenopus egg extracts. The half lives of the cyclin A/cdc2 and cyclin B/cdc2 complexes are estimated as 4 hours and 15 hours, respectively. As compared with the length of the cell cycle, cyclin A and cyclin B both remain bound together with cdc2 for a long time. Cyclin B appears to be somewhat more tightly bound than cyclin A. There is no significant difference in these affinities in mitosis, interphase or the transition between two states. Less
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