Kumagai A, Guo Z, Emami K H, Wang S X, Dunphy W G
Division of Biology, 216-76, Howard Hughes Medical Institute, California Institute of Technology, Pasadena, California 91125, USA.
J Cell Biol. 1998 Sep 21;142(6):1559-69. doi: 10.1083/jcb.142.6.1559.
We have analyzed the role of the protein kinase Chk1 in checkpoint control by using cell-free extracts from Xenopus eggs. Recombinant Xenopus Chk1 (Xchk1) phosphorylates the mitotic inducer Cdc25 in vitro on multiple sites including Ser-287. The Xchk1-catalyzed phosphorylation of Cdc25 on Ser-287 is sufficient to confer the binding of 14-3-3 proteins. Egg extracts from which Xchk1 has been removed by immunodepletion are strongly but not totally compromised in their ability to undergo a cell cycle delay in response to the presence of unreplicated DNA. Cdc25 in Xchk1-depleted extracts remains bound to 14-3-3 due to the action of a distinct Ser-287-specific kinase in addition to Xchk1. Xchk1 is highly phosphorylated in the presence of unreplicated or damaged DNA, and this phosphorylation is abolished by caffeine, an agent which attenuates checkpoint control. The checkpoint response to unreplicated DNA in this system involves both caffeine-sensitive and caffeine-insensitive steps. Our results indicate that caffeine disrupts the checkpoint pathway containing Xchk1.
我们利用非洲爪蟾卵的无细胞提取物分析了蛋白激酶Chk1在细胞周期检查点调控中的作用。重组非洲爪蟾Chk1（Xchk1）在体外可使有丝分裂诱导因子Cdc25的多个位点发生磷酸化，包括Ser-287。Xchk1催化的Cdc25在Ser-287位点的磷酸化足以使其与14-3-3蛋白结合。通过免疫去除法去除Xchk1的卵提取物，在响应未复制DNA的存在时，其经历细胞周期延迟的能力虽受到强烈影响但并未完全丧失。除Xchk1外，由于一种独特的Ser-287特异性激酶的作用，Xchk1缺失提取物中的Cdc25仍与14-3-3结合。在未复制或受损DNA存在时Xchk1高度磷酸化，而咖啡因可消除这种磷酸化，咖啡因是一种减弱检查点调控的试剂。该系统中对未复制DNA的检查点反应涉及咖啡因敏感和不敏感的步骤。我们的结果表明，咖啡因破坏了包含Xchk1的检查点途径。
