Leiss D, Félix M A, Karsenti E
Cell Biology Program, EMBL, Heidelberg, FRG.
J Cell Sci. 1992 Jun;102 ( Pt 2):285-97. doi: 10.1242/jcs.102.2.285.
Cell cycle progression is controlled by changes in kinase activity of homologs of the fission yeast protein p34cdc2. The p34cdc2 kinase is activated by its association with a cyclin subunit, followed by post-translational modifications. Here, we show that in Xenopus eggs stimulated to enter the early embryonic cell cycle by an electric shock, part of the p34cdc2 becomes associated with subcellular fractions as the eggs progress towards mitosis. This occurs as a result of cyclin accumulation because most of the B-type cyclins and some of the A-type cyclins are found in the particulate fraction. Moreover, as soon as cyclins are degraded, p34cdc2 is released in the soluble fraction. The p34cdc2-cyclin complex can be solubilised by 80 mM beta-glycerophosphate (in the standard MPF extraction buffer) or by high salt concentrations. The post-translational modifications leading to cdc2 kinase activation by cyclin occur in the insoluble form. Following fractionation of egg extracts by sucrose gradient centrifugation, the p34cdc2-cyclin B complex is found in several fractions, but especially in two discrete peaks. We present evidence that in the slow-sedimenting peak the p34cdc2-cyclin B complex is associated with the 60 S subunit of monoribosomes. It could be targeted in this fashion to substrates such as ribosomal proteins and maybe to cytoskeletal proteins, since ribosomes bind to microtubules and are present in the spindle. The p34cdc2-cyclin B complex is also found in a faster-migrating fraction containing various membranous structures, including Golgi stacks. Therefore, as observed by immunofluorescence in other systems, it seems that cyclin subunits target p34cdc2 to specific cellular sites and this is certainly important for its function. In addition, we present preliminary evidence suggesting that some component present in the ribosome-containing fraction is required for activation of the p34cdc2-cyclin B complex.
细胞周期进程受裂殖酵母蛋白p34cdc2同源物激酶活性变化的控制。p34cdc2激酶通过与细胞周期蛋白亚基结合而被激活，随后发生翻译后修饰。在此，我们表明，在通过电击刺激进入早期胚胎细胞周期的非洲爪蟾卵中，随着卵向有丝分裂进展，部分p34cdc2与亚细胞组分结合。这是细胞周期蛋白积累的结果，因为大多数B型细胞周期蛋白和一些A型细胞周期蛋白存在于颗粒组分中。此外，一旦细胞周期蛋白降解，p34cdc2就会释放到可溶性组分中。p34cdc2 - 细胞周期蛋白复合物可以用80 mMβ - 甘油磷酸（在标准MPF提取缓冲液中）或高盐浓度溶解。导致细胞周期蛋白激活cdc2激酶的翻译后修饰以不溶性形式发生。通过蔗糖梯度离心对卵提取物进行分级分离后，p34cdc2 - 细胞周期蛋白B复合物存在于几个组分中，但特别是在两个离散峰中。我们提供的证据表明，在沉降较慢的峰中，p34cdc2 - 细胞周期蛋白B复合物与单核糖体的60 S亚基结合。它可能以这种方式靶向底物，如核糖体蛋白，也可能靶向细胞骨架蛋白，因为核糖体与微管结合并存在于纺锤体中。p34cdc2 - 细胞周期蛋白B复合物也存在于一个迁移较快的组分中，该组分包含各种膜结构，包括高尔基体堆栈。因此，正如在其他系统中通过免疫荧光观察到的那样，细胞周期蛋白亚基似乎将p34cdc2靶向特定的细胞位点，这对其功能肯定很重要。此外，我们提供的初步证据表明，含核糖体组分中存在的某些成分是激活p34cdc2 - 细胞周期蛋白B复合物所必需的。
