Identification of novel substrates for human checkpoint kinase Chk1 and Chk2 through genome-wide screening using a consensus Chk phosphorylation motif
Identification of novel substrates for human checkpoint kinase Chk1 and Chk2 through genome-wide screening using a consensus Chk phosphorylation motif
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DOI：
10.1038/emm.2007.23
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发表时间：
2007-04-30
影响因子：
12.8
通讯作者：
Yun, Jeanho
中科院分区：
文献类型：
--
作者：
Kim, Myoung-Ae;Kim, Hyun-Ju;Yun, Jeanho
检查点激酶1（Checkpoint Kinase 1，Chk 1）和Chk 2是细胞DNA损伤反应中的效应激酶，其功能的损伤与肿瘤发生密切相关。以前的研究揭示了Chk 1和Chk 2的几种底物蛋白，但为了了解它们的肿瘤抑制功能，识别其他靶点仍然很重要。在这项研究中，我们使用先前通过定向肽文库方法确定的底物靶基序筛选Chkl和Chk 2的新底物。通过全基因组肽数据库搜索选择潜在候选物，并通过体外激酶测定进行检查。ST 5、HDAC 5、PGC-1 α、PP 2A PR 130、FANCG、GATA 3、细胞周期蛋白G、Rad 51 D和MAD 1a是新鉴定的Chk 1和/或Chk 2的体外底物。其中，进一步分析HDAC 5和PGC-1 α以证实筛选结果。全长蛋白的免疫沉淀激酶测定和靶基序的定点突变分析证明HDAC 5和PGC-1 α至少在体外是Chk 1和/或Chk 2的特异性靶。
Checkpoint kinase 1 (Chkl) and Chk2 are effector kinases in the cellular DNA damage response and impairment of their function is closely related to tumorigenesis. Previous studies revealed several substrate proteins of Chk1 and Chk2, but identification of additional targets is still important in order to understand their tumor suppressor functions. In this study, we screened novel substrates for Chkl and Chk2 using substrate target motifs determined previously by an oriented peptide library approach. The potential candidates were selected by genome-wide peptide database searches and were examined by in vitro kinase assays. ST5, HDAC5, PGC-1 alpha, PP2A PR130, FANCG, GATA3, cyclin G, Rad51D and MAD1a were newly identified as in vitro substrates for Chk1 and/or Chk2. Among these, HDAC5 and PGC-1 alpha were further analyzed to substantiate the screening results. Immunoprecipitation kinase assay of full-length proteins and site-directed mutagenesis analysis of the target motifs demonstrated that HDAC5 and PGC-1 alpha were specific targets for Chk1 and/or Chk2 at least in vitro.