Joint study on the function of domains of cyclins in the cell cycle

这个Monbusho国际科学研究项目是由小林英树作为首席研究员与九州大学西本武治、分子生物学系和英国国际癌症研究基金会克莱尔·霍尔实验室的蒂姆·亨特合作进行的。通过两年的合作(1992年4月-1994年3月)，获得了以下结果：(1)破坏非洲爪哇周期蛋白A和B需要结合p34；这些细胞周期蛋白在受精卵的早期分裂细胞周期中经历浓度的锯齿状振荡，因为它们在中期是稳定的，但在中期末被蛋白质分解迅速破坏。这种程序化的破坏需要在细胞周期蛋白分子的N末端附近有一个十肽基序(破坏框)。我们已经对非洲爪哇周期蛋白A进行了一些突变，并测试了它们在加入钙离子触发…后，在非洲爪哇脑脊液抑制提取物中被摧毁的能力更多的蛋白质分解。细胞周期蛋白A在细胞周期素盒或C-末端区域发生突变，从而取消与p34^cdc2&gt的结合和激活，也使细胞周期蛋白A失去了进行程序性蛋白分解的能力。因此，似乎细胞周期蛋白A必须与p34^&lt；cdc2&>结合才能被破坏。细胞周期蛋白B2似乎也是如此，但细胞周期蛋白B1的结果更难解释。非洲爪哇周期蛋白B1的一些非p34^&lt；cdc2&gt；突变体以基本正常的速率被破坏，而其他的是稳定的。我们已经证实了Glotzer等人的发现。(1991)，含有海胆Cyclin B的N-末端残基13-66的嵌合蛋白与葡萄球菌蛋白A融合后以受调控的方式降解，这取决于销毁盒的完整性。这些构建体不与p34^&lt；cdc2&gt；结合。(2)非洲爪哇周期蛋白A在发芽酵母中的表达。为了研究周期蛋白A的调控作用，我们在发芽酵母中表达了非洲爪哇周期蛋白A及其突变体。半乳糖诱导的非洲爪哇野生型和不可破坏细胞周期蛋白A的过表达抑制了酵母的生长，而缺乏cdc2结合能力的细胞周期蛋白盒突变体不能抑制酵母的生长。在G2/M过渡阶段生长受阻，酵母变大并最终死亡。(3)非洲爪哇细胞周期蛋白-cdc2复合体在细胞周期提取液中的半衰期。我们用非洲爪哇卵提取液测定了细胞周期蛋白A和细胞周期蛋白B在内源性p34^-lt；cdc2和gt之间的交换率，并细菌性表达GST-cdc2。Cyclin A/cdc2和Cyclin B/cdc2络合物的半衰期分别为4小时和15小时。与细胞周期长度相比，细胞周期蛋白A和细胞周期蛋白B均与cdc2结合较长时间。周期蛋白B似乎比周期蛋白A更紧密地结合在一起。在有丝分裂、间期或两种状态之间的转换中，这些亲和力没有显著差异。较少

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 … More 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