Shiromizu Takashi, Goto Hidemasa, Tomono Yasuko, Bartek Jiri, Totsukawa Go, Inoko Akihito, Nakanishi Makoto, Matsumura Fumio, Inagaki Masaki
Division of Biochemistry, Aichi Cancer Center Research Institute, Nagoya, Aichi 464-8681, Japan.
Genes Cells. 2006 May;11(5):477-85. doi: 10.1111/j.1365-2443.2006.00955.x.
Chk1 is phosphorylated at Ser317 and Ser345 by ATR in response to stalled replication and genotoxic stresses. This Chk1 activation is thought to play critical roles in the prevention of premature mitosis. However, the behavior of Chk1 in mitosis remains largely unknown. Here we reported that Chk1 was phosphorylated in mitosis. The reduction of this phosphorylation was observed at the metaphase-anaphase transition. Two-dimensional phosphopeptide mapping revealed that Chk1 phosphorylation sites in vivo were completely overlapped with the in vitro sites by cyclin-dependent protein kinase (Cdk) 1 or by p38 MAP kinase. Ser286 and Ser301 were identified as novel phosphorylation sites on Chk1. Treatment with Cdk inhibitor butyrolactone I induced the reduction of Chk1-S301 phosphorylation, although treatment with p38-specific inhibitor SB203580 or siRNA did not. In addition, ionizing radiation (IR) or ultraviolet (UV) light did not induce Chk1 phosphorylation at Ser317 and Ser345 in nocodazole-arrested mitotic cells. These observations imply the regulation of mitotic Chk1 function through Chk1 phosphorylation at novel sites by Cdk1.
在应对复制停滞和基因毒性应激时，Chk1会被ATR磷酸化于Ser317和Ser345位点。这种Chk1激活被认为在预防有丝分裂过早发生中起关键作用。然而，Chk1在有丝分裂中的行为在很大程度上仍不清楚。在此我们报道Chk1在有丝分裂中被磷酸化。在中期-后期转换时观察到这种磷酸化的减少。二维磷酸肽图谱显示，体内Chk1磷酸化位点与细胞周期蛋白依赖性蛋白激酶（Cdk）1或p38丝裂原活化蛋白激酶在体外的磷酸化位点完全重叠。Ser286和Ser301被鉴定为Chk1上的新磷酸化位点。用Cdk抑制剂丁内酯I处理可诱导Chk1-S301磷酸化的减少，而用p38特异性抑制剂SB203580或小干扰RNA处理则不会。此外，在诺考达唑阻滞的有丝分裂细胞中，电离辐射（IR）或紫外线（UV）光不会诱导Chk1在Ser317和Ser345位点的磷酸化。这些观察结果表明通过Cdk1在新位点对Chk1进行磷酸化来调控有丝分裂Chk1的功能。
