Ataxia telangiectasia-mutated phosphorylates Chk2 in vivo and in vitro
Ataxia telangiectasia-mutated phosphorylates Chk2 in vivo and in vitro
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DOI：
10.1073/pnas.190030497
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发表时间：
2000-09-12
影响因子：
11.1
通讯作者：
Elledge, SJ
中科院分区：
文献类型：
--
作者：
Matsuoka, S;Rotman, G;Elledge, SJ
蛋白激酶Chk 2是芽殖酵母Rad 53和裂殖酵母Cds 1检查点激酶的哺乳动物同源物，在电离辐射（IR）引起DNA损伤时被磷酸化和激活。紫外线照射，并复制块的羟基脲（HU）。Chk 2的磷酸化和激活依赖于共济失调毛细血管扩张突变（ATM）对IR的响应，而当细胞暴露于UV或HU时，Chk 2的磷酸化不依赖于ATM。我们在试管中展示。ATM磷酸化Chk 2上的Ser-Gln/Thr-Gln（SQ/TQ）簇结构域（SCD），该结构域含有7个SQ/TQ基序，其中Thr 68是ATM体外磷酸化的主要位点。ATM-和Rad 3-相关还磷酸化Thr 68，除了Thr 26和Ser 50，其在体外不被ATM磷酸化到显著程度。在体内，Thr 68以ATM依赖性方式响应于IR而磷酸化，但不响应于UV或HU。Thr 68被Ala取代可降低IR后Chk 2的磷酸化和激活程度，SQ/TQ基序的全部突变阻断了IR后Chk 2的所有磷酸化和激活。Chk 2在IR反应中被ATM直接磷酸化，并且Chk 2受SCD磷酸化的调节。
The protein kinase Chk2, the mammalian homolog of the budding yeast Rad53 and fission yeast Cds1 checkpoint kinases, is phosphorylated and activated in response to DNA damage by ionizing radiation (IR). UV irradiation, and replication blocks by hydroxyurea (HU). Phosphorylation and activation of Chk2 are ataxia telangiectasia-mutated (ATM) dependent in response to IR, whereas Chk2 phosphorylation is ATM-independent when cells are exposed to UV or HU. Here we show that in vitro. ATM phosphorylates the Ser-Gln/Thr-Gln (SQ/TQ) cluster domain (SCD) on Chk2, which contains seven SQ/TQ motifs, and Thr68 is the major in vitro phosphorylation site by ATM. ATM- and Rad3-related also phosphorylates Thr68 in addition to Thr26 and Ser50, which are not phosphorylated to a significant extent by ATM in vitro. In vivo, Thr68 is phosphorylated in an ATM-dependent manner in response to IR, but not in response to UV or HU. Substitution of Thr68 with Ala reduced the extent of phosphorylation and activation of Chk2 in response to IR, and mutation of all seven SQ/TQ motifs blocked all phosphorylation and activation of Chk2 after IR. These results suggest that in vivo. Chk2 is directly phosphorylated by ATM in response to IR and that Chk2 is regulated by phosphorylation of the SCD.