The role of Cdc2 feedback loop control in the DNA damage checkpoint in mammalian cells.
The role of Cdc2 feedback loop control in the DNA damage checkpoint in mammalian cells.
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发表时间：
1997-11
期刊：
影响因子：
11.2
通讯作者：
Randy Yat Choi Poon;Man Sang Chau;Katsumi Yamashita;Tony Hunter
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作者：
Randy Yat Choi Poon;Man Sang Chau;Katsumi Yamashita;Tony Hunter
DNA 损伤会使细胞周期蛋白依赖性激酶 (CDK) 失活并阻止细胞周期。 DNA 损伤后，G1-S CDK 受到 p53 依赖性 p21Cip1/Waf1 诱导机制的抑制；但 Cdc2 是如何被抑制的却不太明显。我们发现 G2 中 DNA 损伤检查点产生的信号比纺锤体微管组装检查点产生的信号占主导地位，因为诺考达唑或紫杉醇捕获细胞中存在的高 Cdc2 活性因 DNA 损伤而降低。 Cdc2、Thr14 和 Tyr15 中抑制残基的磷酸化与 DNA 损伤后 Cdc2 的失活同时发生。然而，由于 Thr14/Tyr15 磷酸化通过反馈环路进行调节，因此对该结果的解释并不简单。因此，它们的磷酸化原则上只能由 Cdc2 活性的抑制引起。与此一致的是，当丁内酯-I 抑制 Cdc2 激酶活性时，会诱导 Thr14/Tyr15 磷酸化。考虑到这些并发症，我们对 DNA 损伤后调节 Cdc2 的机制进行了更严格的分析。咖啡因通过引起 Cdc2 去磷酸化来逆转 DNA 损伤诱导的 Cdc2 抑制，即使用丁内酯-I 阻断 Cdc2 反馈环，这种去磷酸化仍然发生。这些数据表明，DNA 损伤检查点部分通过 Thr14/Tyr15 磷酸化发挥作用，其机制与 Cdc2 活性无关，并且这种磷酸化可以通过涉及 Thr14/Tyr15 蛋白激酶和磷酸酶的 Cdc2 反馈环路来增强。 DNA损伤后，Wee1Hu Tyr15蛋白激酶的激酶活性没有改变，但Cdc25C的磷酸酶活性降低。因此，Cdc25C 活性的降低可能部分解释了 DNA 损伤诱导的 Thr14/Tyr15 磷酸化增加。
DNA damage inactivates cyclin-dependent kinases (CDKs) and arrests the cell cycle. Following DNA damage, the G1-S CDKs are inhibited by a mechanism involving p53-dependent induction of p21Cip1/Waf1; but how the Cdc2 is inhibited is less apparent. We found that the signal generated by the DNA damage checkpoint in G2 was dominant over that from the spindle microtubule-assembly checkpoint, because the high Cdc2 activity present in nocodazole or Taxol-arrested cells was reduced by DNA damage. Phosphorylation of the inhibitory residues in Cdc2, Thr14, and Tyr15 coincided with the inactivation of Cdc2 after DNA damage. Interpretation of this result, however, was not straightforward due to the regulation of Thr14/Tyr15 phosphorylation by feedback loops; hence, their phosphorylation can in principle result merely from the inhibition of Cdc2 activity. Consistent with this, Thr14/Tyr15 phosphorylation was induced when Cdc2 kinase activity was inhibited with butyrolactone-I. Given these complications, we undertook a more critical analysis of the mechanisms that regulate Cdc2 after DNA damage. Caffeine reversed the DNA damage-induced inhibition of Cdc2 by causing dephosphorylation of Cdc2, and this dephosphorylation still occurred even when the Cdc2 feedback loops were blocked with butyrolactone-I. These data suggest that the DNA damage checkpoint in part acts through Thr14/Tyr15 phosphorylation by a mechanism independent of Cdc2 activity, and this phosphorylation can be accentuated by the Cdc2 feedback loops involving Thr14/Tyr15 protein kinases and phosphatases. The kinase activity of the Wee1Hu Tyr15 protein kinase was unaltered after DNA damage, but the phosphatase activity of Cdc25C was reduced. Thus, the decrease in Cdc25C activity may in part account for the DNA damage-induced increase in Thr14/Tyr15 phosphorylation.