Intrinsic kinase activity and SQ/TQ domain of Chk2 kinase as well as N-terminal domain of Wip1 phosphatase are required for regulation of Chk2 by Wip1
Intrinsic kinase activity and SQ/TQ domain of Chk2 kinase as well as N-terminal domain of Wip1 phosphatase are required for regulation of Chk2 by Wip1
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DOI：
10.1074/jbc.m600403200
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发表时间：
2006-08-25
影响因子：
4.8
通讯作者：
Minami, Yasuhiro
中科院分区：
文献类型：
--
作者：
Yoda, Akinori;Xu, Xiao Zhou;Minami, Yasuhiro
抗肿瘤的Chk2激酶在DNA损伤诱导的细胞周期检查点调控中起着至关重要的作用。最近，我们已经证明了Chk2与致癌的Wip1(PPM1D)磷酸酶有关，并且Wip1在DNA损伤反应中通过使激活的Chk2(Fujimoto，H.，Onishi，N.，Kato，N.，Takekawa，M.，Xu，X.Z.，Kosugi，A.，Kondo，T.，Imamura，M.，Oishi，I.，Yoda，A.，和Minami，Y.)去磷酸化Thr-68而起到负调控作用。13、1170-1180)。在这里，我们利用Chk2和Wip1的一系列缺失或氨基酸替代的突变蛋白对Chk2和Wip1进行了结构功能分析。我们发现Chk2和Wip1的核定位是它们在培养细胞中结合所必需的，而Chk2的丝氨酸-谷氨酰胺(SQ)/苏氨酸-谷氨酰胺(TQ)结构域(包含Thr-68)和Wip1的N-末端结构域(约由100个氨基酸组成)是两个分子结合的必要条件和充分条件。然而，研究发现，全长Chk2和Wip1之间的联系需要Chk2的内在激酶活性，而不是Wip1的磷酸酶活性。有趣的是，我们还发现突变的Wip1蛋白单独携带Wip1的N末端结构域或缺乏内在的磷酸酶活性，对野生型Wip1的功能表现出显性的负面影响，即这两个突变体中的任何一个的异位表达都抑制了Wip1对Chk2中Thr-68的去磷酸化和Wip1的抗凋亡功能。这些结果为开发针对致癌Wip1磷酸酶的新型抗癌药物提供了分子基础。
The anti-oncogenic Chk2 kinase plays a crucial role in DNA damage-induced cell cycle checkpoint regulation. Recently, we have shown that Chk2 associates with the oncogenic Wip1 (PPM1D) phosphatase and that Wip1 acts as a negative regulator of Chk2 during DNA damage response by dephosphorylating phosphorylated Thr-68 in activated Chk2 (Fujimoto, H., Onishi, N., Kato, N., Takekawa, M., Xu, X. Z., Kosugi, A., Kondo, T., Imamura, M., Oishi, I., Yoda, A., and Minami, Y. (2006) Cell Death Differ. 13, 1170 - 1180). Here, we performed structure-function analyses of Chk2 and Wip1 by using a series of deletion or amino acid-substituted mutant proteins of Chk2 and Wip1. We show that nuclear localization of both Chk2 and Wip1 is required for their association in cultured cells and that the serine-glutamine(SQ)/threonine-glutamine (TQ) domain of Chk2, containing Thr-68, and the N-terminal domain of Wip1, comprising about 100 amino acids, are necessary and sufficient for the association of both molecules. However, it was found that an intrinsic kinase activity of Chk2, but not phosphatase activity of Wip1, is required for the association of full-length Chk2 and Wip1. Interestingly, we also show that the mutant Wip1 proteins, bearing the N-terminal domain of Wip1 alone or lacking an intrinsic phosphatase activity, exhibit dominant negative effects on the functions of the wild-type Wip1, i.e. ectopic expression of either of these Wip1 mutants inhibits dephosphorylation of Thr-68 in Chk2 by Wip1 and anti-apoptotic function of Wip1. These results provide a molecular basis for developing novel anti-cancer drugs, targeting oncogenic Wip1 phosphatase.