Chow Jeremy P H, Siu Wai Yi, Ho Horace T B, Ma Ken Hoi Tang, Ho Chui Chui, Poon Randy Y C
Department of Biochemistry, Hong Kong University of Science and Technology, Clear Water Bay, Hong Kong.
J Biol Chem. 2003 Oct 17;278(42):40815-28. doi: 10.1074/jbc.M306683200. Epub 2003 Aug 11.
Inhibition of cyclin-dependent kinases (CDKs) by Thr14/Tyr15 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 G2 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 Thr14/Tyr15 phosphorylation in normal cell cycle and during the DNA damage checkpoint.
通过苏氨酸14/酪氨酸15磷酸化抑制细胞周期蛋白依赖性激酶（CDK）对于正常细胞周期进程至关重要，并且是多个细胞周期检查点的汇聚事件。在本研究中，我们比较了抑制性磷酸化对细胞周期蛋白A/B1-CDC2和细胞周期蛋白A/E-CDK2复合物的相对贡献。我们发现，在HeLa细胞的未受干扰细胞周期中，抑制性磷酸化在CDC2的调节中起主要作用，但对CDK2仅起次要作用。CDC2和CDK2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，CDC2和CDK2的非磷酸化突变体的表达在细胞周期早期引发了非计划的组蛋白H3磷酸化，并且具有细胞毒性。放射模拟药物造成的DNA损伤或羟基脲引起的复制阻滞刺激了细胞周期蛋白B1的积累，但同时伴随着CDC2抑制性磷酸化的增加。在DNA损伤和复制阻滞之后，所有控制S期和有丝分裂的细胞周期蛋白-CDK对都不同程度地受到磷酸化抑制。即使在DNA损伤后，非磷酸化CDC2的异位表达仍能刺激DNA复制、组蛋白H3磷酸化和细胞分裂。同样，CDK2的非磷酸化突变体而非CDK4破坏了G2期DNA损伤检查点。最后，CDC25A、CDC25B、显性负性CHK1，但不是CDC25C或显性负性WEE1，在DNA损伤后刺激了组蛋白H3磷酸化。这些数据表明，在正常细胞周期和DNA损伤检查点期间，苏氨酸14/酪氨酸15磷酸化的各种调节因子具有不同的作用。
