Differential contribution of inhibitory phosphorylation of CDC2 and CDK2 for unperturbed cell cycle control and DNA integrity checkpoints
Differential contribution of inhibitory phosphorylation of CDC2 and CDK2 for unperturbed cell cycle control and DNA integrity checkpoints
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DOI：
10.1074/jbc.m306683200
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发表时间：
2003-10-17
影响因子：
4.8
通讯作者：
Poon, RYC
中科院分区：
文献类型：
--
作者：
Chow, JPH;Siu, WY;Poon, RYC
Thr（14）/Tyr（15）磷酸化对细胞周期蛋白依赖性激酶（CDK）的抑制对于正常细胞周期进程至关重要，并且是几个细胞周期检查点的会聚事件。在这项研究中，我们比较了细胞周期蛋白A/B1-CDC 2和细胞周期蛋白A/E-CDK 2复合物抑制磷酸化的相对贡献。我们发现，抑制性磷酸化在调节CDC 2中起着重要作用，但在HeLa细胞未受干扰的细胞周期中，CDK 2仅起次要作用。CDC 2和CDK 2抑制性磷酸化的相对重要性可能反映了它们不同的细胞功能。尽管如此，CDC 2和CDK 2的非磷酸化突变体的表达在细胞周期的早期触发了非预定的组蛋白H3磷酸化，并且具有细胞毒性。DNA损伤的拟放射性药物或复制块由羟基脲刺激细胞周期蛋白B1的积累，但伴随着增加的抑制性磷酸化的CDC 2。在DNA损伤和复制阻断后，所有控制S期和有丝分裂的cyclin-CDK对都受到不同程度的磷酸化抑制。即使在DNA损伤后，非磷酸化CDC 2的异位表达也能刺激DNA复制、组蛋白H3磷酸化和细胞分裂。类似地，CDK 2的非磷酸化突变体，而不是CDK 4，破坏了G（2）DNA损伤检查点。最后，CDC 25 A，CDC 25 B，一个显性负性CHK 1，而不是CDC 25 C或显性负性WEE 1，刺激组蛋白H3磷酸化后DNA损伤。这些数据表明，在正常细胞周期和DNA损伤检查点期间，Thr（14）/Tyr（15）磷酸化的各种调节剂的贡献不同。
Inhibition of cyclin-dependent kinases (CDKs) by Thr(14)/Tyr(15) phosphorylation is critical for normal cell cycle progression and is a converging event for several cell cycle checkpoints. In this study, we compared the relative contribution of inhibitory phosphorylation for cyclin A/B1-CDC2 and cyclin A/E-CDK2 complexes. We found that inhibitory phosphorylation plays a major role in the regulation of CDC2 but only a minor role for CDK2 during the unperturbed cell cycle of HeLa cells. The relative importance of inhibitory phosphorylation of CDC2 and CDK2 may reflect their distinct cellular functions. Despite this, expression of nonphosphorylation mutants of both CDC2 and CDK2 triggered unscheduled histone H3 phosphorylation early in the cell cycle and was cytotoxic. DNA damage by a radiomimetic drug or replication block by hydroxyurea stimulated a buildup of cyclin B1 but was accompanied by an increase of inhibitory phosphorylation of CDC2. After DNA damage and replication block, all cyclin-CDK pairs that control S phase and mitosis were to different degrees inhibited by phosphorylation. Ectopic expression of nonphosphorylated CDC2 stimulated DNA replication, histone H3 phosphorylation, and cell division even after DNA damage. Similarly, a nonphosphorylation mutant of CDK2, but not CDK4, disrupted the G(2) DNA damage checkpoint. Finally, CDC25A, CDC25B, a dominant-negative CHK1, but not CDC25C or a dominant-negative WEE1, stimulated histone H3 phosphorylation after DNA damage. These data suggest differential contributions for the various regulators of Thr(14)/Tyr(15) phosphorylation in normal cell cycle and during the DNA damage checkpoint.