De Smedt V, Crozet N, Jessus C
Laboratoire de Physiologie de la Reproduction, ESA-CNRS 7080, Université Pierre et Marie Curie, Paris, France.
Microsc Res Tech. 1999 Apr 1;45(1):13-30. doi: 10.1002/(SICI)1097-0029(19990401)45:1<13::AID-JEMT2>3.0.CO;2-R.
The G2-M transition of the cell cycle is under the control of the M-phase promoting factor (MPF) formed of cdc2 kinase and cyclin B. The Xenopus prophase-blocked oocyte contains a stockpile of cyclin B2-cdc2 complexes that are maintained inactive by a double inhibitory phosphorylation on Thr-14 and Tyr-15 of cdc2. Free cdc2 molecules that are not associated with cyclin, are present in excess as compared to cyclin B2-associated cdc2. This pool of free cdc2 is permanently recruited to associate with neosynthetized cyclin B2 in the resting prophase oocyte, to feed up the pre-MPF stockpile. During re-entry into meiosis, free cdc2 could generate with newly synthesized cyclin B a small level of active MPF, that could serve as starter to initiate the conversion of pre-MPF into MPF. It was, therefore, of high interest to investigate whether free cdc2 interacts with other proteins and what could be its intracellular localization. To address these questions, we developed an in vitro system of membrane vesicles. We demonstrate here that free cdc2 is recovered in association with the external layer of membrane vesicles, whereas cyclin B2-associated cdc2 is not. Cyclin is able to associate in vitro with cdc2-containing membrane vesicles. This association does not induce the inhibitory cdc2 phosphorylations. However, it does not lead to active complexes, suggesting that membrane vesicles prevent cdc2 activation. C-Raf1, another kinase activated during reentry into meiosis, is also totally recovered in association with the membrane vesicles.
细胞周期的G2-M转换受由细胞分裂周期蛋白依赖性激酶2（cdc2激酶）和细胞周期蛋白B形成的M期促进因子（MPF）控制。非洲爪蟾处于前期阻滞的卵母细胞中含有大量细胞周期蛋白B2-cdc2复合物，这些复合物通过cdc2第14位苏氨酸（Thr-14）和第15位酪氨酸（Tyr-15）的双重抑制性磷酸化而保持无活性。与和细胞周期蛋白B2结合的cdc2相比，未与细胞周期蛋白结合的游离cdc2分子过量存在。在静止的前期卵母细胞中，这部分游离cdc2会持续与新合成的细胞周期蛋白B2结合，以补充前MPF储备。在重新进入减数分裂过程中，游离cdc2可与新合成的细胞周期蛋白B产生少量活性MPF，该活性MPF可作为启动子，启动前MPF向MPF的转化。因此，研究游离cdc2是否与其他蛋白质相互作用以及其细胞内定位是什么具有重要意义。为了解决这些问题，我们开发了一种膜泡体外系统。我们在此证明，游离cdc2与膜泡外层结合，而与细胞周期蛋白B2结合的cdc2则不然。细胞周期蛋白能够在体外与含有cdc2的膜泡结合。这种结合不会诱导抑制性cdc2磷酸化。然而，它不会产生活性复合物，这表明膜泡可阻止cdc2激活。另一种在重新进入减数分裂过程中被激活的激酶C-Raf1也完全与膜泡结合。
