Degradation of APCcdc20 and APCcdh1 substrates during the second meiotic division in mouse eggs
Degradation of APCcdc20 and APCcdh1 substrates during the second meiotic division in mouse eggs
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DOI：
10.1242/jcs.01567
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发表时间：
2004-12-15
影响因子：
4
通讯作者：
Jones, KT
中科院分区：
文献类型：
--
作者：
Chang, HY;Levasseur, M;Jones, KT
通过精子诱导的Ca 2+加标刺激中期II停滞的小鼠卵完成减数分裂。Ca 2+信号导致E3连接酶后期促进复合物/细胞周期体（APC）的激活，导致减数分裂退出所必需的关键蛋白的破坏。我们显示，使用小鼠卵的蛋白质印迹，APC激活剂cdc 20和cdh 1的存在下，分别针对D盒和D盒/KEN盒底物，用于蛋白水解。我们决定通过将APC底物与GFP偶联并在第二次减数分裂停滞释放后实时检查它们的破坏来检查APC（cdc 20）和APC（cdh 1）的时间激活。D-box底物在精子诱导的Ca 2+加标开始后迅速降解，使得它们的降解在第二极体挤出时完成。相比之下，KEN盒含有底物降解时，CDK 1活性低，在极体挤出和原核形成之间的时期。观察到的表观APC（cdh 1）的活性在减数分裂II的基础上破坏外源性GFP偶联底物，然后通过观察内源性APC（cdh 1）基板的破坏证实。这些数据与精子诱导的激活的初始APC（cdc 20）激活，随后在第二极体排出后APC（cdh 1）激活的模型一致。因此，有趣的是，我们提出哺乳动物的卵子经历了具有APC（cdc 20）和APC（cdh 1）的减数分裂II，而迄今为止描述的其他物种的卵子仅具有APC（cdc 20）活性。
Metaphase II-arrested mouse eggs are stimulated to complete meiosis by sperm-induced Ca2+ spiking. The Ca2+ signal causes activation of the E3 ligase anaphase-promoting complex/cyclosome (APC), leading to the destruction of key proteins necessary for meiotic exit. We show, using western blots of mouse eggs, the presence of both APC activators cdc20 and cdh1, which target D-box and D-box/KEN-box substrates, respectively, for proteolysis. We decided to examine the temporal activation of APC(cdc20) and APC(cdh1) by coupling APC substrates to GFP and examining their destruction in real-time following release from second meiotic division arrest. D-box substrates were degraded quickly after the initiation of sperm-induced Ca2+ spiking, such that their degradation was complete by the time of second polar body extrusion. By contrast, KEN-box-containing substrates were degraded when CDK1 activity was low, during the period between polar body extrusion and pronucleus formation. This observation of apparent APC(cdh1) activity in meiosis II based on destruction of exogenous GFP-coupled substrates was then confirmed by observing destruction of endogenous APC(cdh1) substrates. These data are consistent with a model of initial APC(cdc20) activation on sperm-induced activation, followed by APC(cdh1) activation after second polar body extrusion. Interestingly, therefore, we propose that mammalian eggs undergo meiosis II with both APC(cdc20) and APC(cdh1), whereas eggs of other species so far described have APC(cdc20) activity only.