ATR-mediated checkpoint pathways regulate phosphorylation and activation of human Chk1
ATR-mediated checkpoint pathways regulate phosphorylation and activation of human Chk1
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DOI：
10.1128/mcb.21.13.4129-4139.2001
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发表时间：
2001-07-01
影响因子：
5.3
通讯作者：
Piwnica-Worms, H
中科院分区：
文献类型：
--
作者：
Zhao, H;Piwnica-Worms, H
Chk1是一种进化上保守的蛋白激酶，其响应于检查点激活而调节细胞周期进程。在这项研究中，我们证明了阻断DNA复制或导致某些形式的DNA损伤的药物诱导人Chk1的磷酸化。磷酸化形式的Chk 1具有更高的内在蛋白激酶活性和洗脱更快的凝胶过滤柱。丝氨酸317和345被确定为在体内的磷酸化位点，ATR（ATM和Rad3相关的蛋白激酶）在体外磷酸化这两个位点。此外，Chk 1的丝氨酸317和345在体内的磷酸化是ATR依赖性的。Chk1的突变体含有丙氨酸的丝氨酸317和345的地方激活不良，在体内复制块或遗传毒性应激，不良的ATR在体外磷酸化，并没有发现在更快的洗脱洗脱馏分凝胶过滤。这些发现表明，响应于复制阻断和某些形式的遗传毒性应激的Chk1的激活涉及丝氨酸317和345的磷酸化。此外，这项研究暗示ATR作为人类细胞中Chk1的直接上游激活剂。
Chk1 is an evolutionarily conserved protein kinase that regulates cell cycle progression in response to checkpoint activation. In this study, we demonstrated that agents that block DNA replication or cause certain forms of DNA damage induce the phosphorylation of human Chk1. The phosphorylated form of Chk1 possessed higher intrinsic protein kinase activity and eluted more quickly on gel filtration columns. Serines 317 and 345 were identified as sites of phosphorylation in vivo, and ATR (the ATM- and Rad3-related protein kinase) phosphorylated both of these sites in vitro. Furthermore, phosphorylation of Chk1 on serines 317 and 345 in vivo was ATR dependent. Mutants of Chk1 containing alanine in place of serines 317 and 345 were poorly activated in response to replication blocks or genotoxic stress in vivo, were poorly phosphorylated by ATR in vitro, and were not found in faster-eluting fractions by gel filtration. These findings demonstrate that the activation of Chk1 in response to replication blocks and certain forms of genotoxic stress involves phosphorylation of serines 317 and 345. In addition, this study implicates ATR as a direct upstream activator of Chk1 in human cells.