Checkpoint激酶1（Chk1）和Chk2是细胞DNA损伤反应中的效应激酶，其功能受损与肿瘤发生密切相关。先前的研究揭示了Chk1和Chk2的几种底物蛋白，但是为了了解它们的肿瘤抑制功能，识别其他靶标仍然很重要。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Chk1和Chk2的新底物。通过全基因组肽数据库搜索选择潜在的候选物,并通过体外激酶测定进行检查。ST5,HDAC5,PGC-1alpha,PP2APR130,FANCG,GATA3，细胞周期蛋白G，Rad51D和MAD1a被新鉴定为Chk1和/或Chk2的体外底物。其中,进一步分析HDAC5和PGC-1a以证实筛选结果。全长蛋白质的免疫沉淀激酶测定和靶基序的定点诱变分析表明，HDAC5和PGC-1α至少在体外是Chk1和/或Chk2的特异性靶标。
Checkpoint kinase 1 (Chk1) 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 Chk1 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-1alpha, 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-1a 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-1alpha were specific targets for Chk1 and/or Chk2 at least in vitro.
