Phosphorylated claspin interacts with a phosphate-binding site in the kinase domain of Chk1 during ATR-mediated activation
Phosphorylated claspin interacts with a phosphate-binding site in the kinase domain of Chk1 during ATR-mediated activation
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DOI：
10.1074/jbc.m304551200
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发表时间：
2003-11-21
影响因子：
4.8
通讯作者：
Dunphy, WG
中科院分区：
文献类型：
--
作者：
Jeong, SY;Kumagai, A;Dunphy, WG
在含有不完全复制或紫外线损伤DNA的非洲爪哇卵提取液中，Claspin对于依赖ATR激活Chk1是必不可少的。激活形式的Claspin含有两个重复的磷酸肽基序，介导其与Chk1的结合。我们发现这些磷酸肽基序通过其N-末端的激酶域与Chk1结合。Chk1上的结合位点涉及一簇带正电的氨基酸，其中包含赖氨酸54、精氨酸129、苏氨酸153和精氨酸162。这些残基的突变强烈损害了Chk1与Claspin的相互作用能力。这些氨基酸位于Chk1的区域内，这些区域参与其催化功能的各个方面。从Claspin预测的磷酸基团在Chk1上的位置对应于各种激酶中激活环磷酸化的位置。此外，我们获得的证据表明，Chk1的C末端调节域在我们的检测条件下不与Claspin形成稳定的复合体，但在Claspin依赖的激活中仍有一定的作用。总体而言，这些结果表明，在ATR激活过程中，Claspin与Chk1的催化域中的磷酸结合位点对接。在这一过程中，磷酸化的Claspin可能会模拟Chk1的激活磷酸化。
Claspin is essential for the ATR-dependent activation of Chk1 in Xenopus egg extracts containing incompletely replicated or UV-damaged DNA. The activated form of Claspin contains two repeated phosphopeptide motifs that mediate its binding to Chk1. We show that these phosphopeptide motifs bind to Chk1 by means of its N-terminal kinase domain. The binding site on Chk1 involves a positively charged cluster of amino acids that contains lysine 54, arginine 129, threonine 153, and arginine 162. Mutagenesis of these residues strongly compromises the ability of Chk1 to interact with Claspin. These amino acids lie within regions of Chk1 that are involved in various aspects of its catalytic function. The predicted position on Chk1 of the phosphate group from Claspin corresponds to the location of activation-loop phosphorylation in various kinases. In addition, we have obtained evidence that the C-terminal regulatory domain of Chk1, which does not form a stable complex with Claspin under our assay conditions, nonetheless has some role in Claspin-dependent activation. Overall, these results indicate that Claspin docks with a phosphate-binding site in the catalytic domain of Chk1 during activation by ATR. Phosphorylated Claspin may mimic an activating phosphorylation of Chk1 during this process.