Division of Biochemistry, Aichi Cancer Center Research Institute, 1-1 Kanokoden, Chikusa-Ku, Nagoya, Aichi 464-8681, Japan.
Mol Biol Cell. 2012 Apr;23(8):1582-92. doi: 10.1091/mbc.E11-10-0883. Epub 2012 Feb 22.
The ataxia telangiectasia mutated- and rad3-related kinase (ATR)/Chk1 pathway is a sentinel of cell cycle progression. On the other hand, the Ras/mitogen-activated protein kinase/90-kDa ribosomal S6 kinase (p90 RSK) pathway is a central node in cell signaling downstream of growth factors. These pathways are closely correlated in cell proliferation, but their interaction is largely unknown. Here we show that Chk1 is phosphorylated predominantly at Ser-280 and translocated from cytoplasm to nucleus in response to serum stimulation. Nonphosphorylated Chk1-Ser-280 mutation attenuates nuclear Chk1 accumulation, whereas the phosphomimic mutation has a reverse effect on the localization. Treatment with p90 RSK inhibitor impairs Chk1 phosphorylation at Ser-280 and accumulation at the nucleus after serum stimulation, whereas these two phenomena are induced by the expression of the constitutively active mutant of p90 RSK in serum-starved cells. In vitro analyses indicate that p90 RSK stoichiometrically phosphorylates Ser-280 on Chk1. Together with Chk1 phosphorylation at Ser-345 by ATR and its autophosphorylation at Ser-296, which are critical for checkpoint signaling, Chk1-Ser-280 phosphorylation is elevated in a p90 RSK-dependent manner after UV irradiation. In addition, Chk1 phosphorylation at Ser-345 and Ser-296 after UV irradiation is also attenuated by the treatment with p90 RSK inhibitor or by Ser-280 mutation to Ala. These results suggest that p90 RSK facilitates nuclear Chk1 accumulation through Chk1-Ser-280 phosphorylation and that this pathway plays an important role in the preparation for monitoring genetic stability during cell proliferation.
共济失调毛细血管扩张突变相关激酶（ATR）/细胞周期检查点激酶 1（Chk1）通路是细胞周期进程的监测器。另一方面，Ras/丝裂原活化蛋白激酶/90kDa 核糖体 S6 激酶（p90RSK）通路是生长因子下游细胞信号转导的中心节点。这些通路在细胞增殖中密切相关，但它们的相互作用在很大程度上是未知的。在这里，我们发现 Chk1 在受到血清刺激时主要在丝氨酸 280 位磷酸化并从细胞质转位到细胞核。非磷酸化的 Chk1-Ser-280 突变削弱了核 Chk1 的积累，而磷酸模拟突变则对定位产生相反的影响。p90RSK 抑制剂处理会损害血清刺激后 Chk1 在 Ser-280 位的磷酸化和在核内的积累，而这两种现象是由在血清饥饿细胞中表达组成性激活的 p90RSK 突变体诱导的。体外分析表明，p90RSK 在 Chk1 的 Ser-280 位上进行化学计量磷酸化。与 ATR 对 Chk1 的 Ser-345 磷酸化及其自身在 Ser-296 位的磷酸化一起，这对于检查点信号转导至关重要，Chk1-Ser-280 磷酸化在 UV 照射后以 p90RSK 依赖的方式升高。此外，UV 照射后 Chk1 的 Ser-345 和 Ser-296 磷酸化也被 p90RSK 抑制剂处理或 Ser-280 突变为 Ala 所减弱。这些结果表明，p90RSK 通过 Chk1-Ser-280 磷酸化促进核 Chk1 积累，并且该通路在细胞增殖过程中监测遗传稳定性的准备中发挥重要作用。
