Bresnahan W A, Boldogh I, Ma T, Albrecht T, Thompson E A
Department of Microbiology, University of Texas Medical Branch, Galveston 77550, USA.
Cell Growth Differ. 1996 Oct;7(10):1283-90.
The experiments described in this report were undertaken to define the parameters that regulate cyclin E/cyclin-dependent kinase 2 (Cdk2) kinase activity in mitotically quiescent, serum-starved fibroblastic cells and in cells that had been stimulated to enter the cell cycle and progress through G1 into S phase. We have analyzed the expression of cyclin E and Cdk2, the extent to which these two proteins form complexes, and the enzymatic activity of cyclin E/cdk2 kinase. Particular attention was focused upon subcellular localization and the effect of compartmentalization on the association between cyclin E and Cdk2. In addition, we have examined the interaction of cyclin E/Cdk2 complexes with two well-characterized inhibitors of Cdk2 kinase activity, Cip1 and Kip1. This represents the first report in which all of these parameters have been measured simultaneously in a single, normal diploid cell line. In G0 cells, there is abundant cyclin E and Cdk2, yet there is little or no detectable Cdk2-dependent histone H1 kinase activity. After serum stimulation, there is a rapid increase in the amount of cyclin E that is bound to Cdk2, although there is no significant change in the abundance of either the cyclin or the Cdk. Immunocytochemical data indicate that cyclin E, Cip1, and Kip1 are located within the nuclei of cell in G0, but very little Cdk2 is observed within the nuclei of serum-starved cells. Cdk2 rapidly enters the nucleus upon serum stimulation. The abundance of the cyclin E/Cdk2 complex increases to the extent that the binding capacity of Cip1 is exceeded about 8-12 h after serum stimulation. The abundance of Kip1 decreases at the same time that the Cip1 threshold is exceeded, so that cyclin E/Kip1-containing complexes decrease by 90% within 8-12 h. Cyclin E/Cdk2 kinase activity begins to increase rapidly thereafter, reaching a maximum level about 16 h after serum stimulation. We have been unable to detect histone H1 kinase activity in complexes that contain cyclin E bound to Kip1 or Cip1. We conclude that compartmentalization is the predominant barrier to activation of cyclin E-dependent kinases in quiescent cells. Cip1 and Kip1 serve to prevent premature activation of cyclin E/Cdk2 complexes that form during G0 or early G1.
本报告中描述的实验旨在确定调控有丝分裂静止、血清饥饿的成纤维细胞以及已被刺激进入细胞周期并从G1期进入S期的细胞中细胞周期蛋白E/细胞周期蛋白依赖性激酶2（Cdk2）激酶活性的参数。我们分析了细胞周期蛋白E和Cdk2的表达、这两种蛋白形成复合物的程度以及细胞周期蛋白E/Cdk2激酶的酶活性。特别关注亚细胞定位以及分隔化对细胞周期蛋白E和Cdk2之间结合的影响。此外，我们研究了细胞周期蛋白E/Cdk2复合物与两种已充分表征的Cdk2激酶活性抑制剂Cip1和Kip1的相互作用。这是第一份在单一正常二倍体细胞系中同时测量所有这些参数的报告。在G0期细胞中，存在大量的细胞周期蛋白E和Cdk2，但几乎没有或无法检测到Cdk2依赖性组蛋白H1激酶活性。血清刺激后，与Cdk2结合的细胞周期蛋白E的量迅速增加，尽管细胞周期蛋白或Cdk的丰度没有显著变化。免疫细胞化学数据表明，细胞周期蛋白E、Cip1和Kip1位于G0期细胞的细胞核内，但在血清饥饿细胞的细胞核内几乎观察不到Cdk2。血清刺激后，Cdk2迅速进入细胞核。细胞周期蛋白E/Cdk2复合物的丰度增加，以至于在血清刺激后约8 - 12小时超过了Cip1的结合能力。在超过Cip1阈值的同时，Kip1的丰度下降，因此含细胞周期蛋白E/Kip1的复合物在8 - 12小时内减少了90%。此后，细胞周期蛋白E/Cdk2激酶活性开始迅速增加，在血清刺激后约16小时达到最高水平。我们无法在含有与Kip1或Cip1结合的细胞周期蛋白E的复合物中检测到组蛋白H1激酶活性。我们得出结论，分隔化是静止细胞中细胞周期蛋白E依赖性激酶激活的主要障碍。Cip1和Kip1用于防止在G0期或G1早期形成的细胞周期蛋白E/Cdk2复合物过早激活。
