Hamada K, Takuwa N, Zhou W, Kumada M, Takuwa Y
Department of Cardiovascular Biology, Faculty of Medicine, University of Tokyo, Bunkyo-ku, Japan.
Biochim Biophys Acta. 1996 Jan 10;1310(1):149-56. doi: 10.1016/0167-4889(95)00148-4.
Serum stimulation of human diploid fibroblast IMR-90 cells leads to phosphorylation of p33CDK2 at Thr160 and activation of CDK2 kinase, a necessary event for G1/S transition. We report that serum stimulation causes a gradual, sustained increase in the activity of CDK-activating kinase (CAK) that phosphorylates CDK2 at Thr160, which starts by 5 h after serum stimulation and reaches the maximal plateau level at around the G1/S boundary. In this cell type addition of phorbol-12, 13-dibutyrate 5 h but not 16 h after serum stimulation completely inhibits CDK2 kinase activation and DNA synthesis. Phorbol ester treatment does not reduce the protein level of p33CDK2, but does inhibit serum-stimulated increases in the CAK activity and CDK2 phosphorylation at Thr160. The suppression of the CAK activity by the phorbol ester is accompanied by decreases in the message levels of both CDK7 and cyclin H, the catalytic and the positive regulatory subunit of CAK, respectively. These results indicate that in IMR-90 cells activation of protein kinase C in the late G1 phase causes cell cycle arrest before the G1/S boundary at least in part through downregulation of CAK and CAK-mediated CDK2 phosphorylation and activation.
血清刺激人二倍体成纤维细胞IMR - 90会导致p33CDK2在苏氨酸160位点磷酸化以及CDK2激酶激活，这是G1/S期转换的必要事件。我们报告血清刺激会导致CDK激活激酶（CAK）活性逐渐、持续增加，CAK使CDK2在苏氨酸160位点磷酸化，该过程在血清刺激后5小时开始，在G1/S边界附近达到最大平台水平。在这种细胞类型中，血清刺激后5小时而非16小时添加佛波醇 - 12,13 - 二丁酸酯会完全抑制CDK2激酶激活和DNA合成。佛波醇酯处理不会降低p33CDK2的蛋白水平，但会抑制血清刺激引起的CAK活性增加以及CDK2在苏氨酸160位点的磷酸化。佛波醇酯对CAK活性的抑制伴随着CDK7和细胞周期蛋白H（分别为CAK的催化亚基和正调节亚基）的信使水平降低。这些结果表明，在IMR - 90细胞中，G1期晚期蛋白激酶C的激活至少部分通过下调CAK以及CAK介导的CDK2磷酸化和激活导致细胞周期在G1/S边界之前停滞。
