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首页> 外文期刊>Molecular and Cellular Biology >S and G2 Phase Roles for Cdk2 Revealed by Inducible Expression of a Dominant-Negative Mutant in Human Cells
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S and G2 Phase Roles for Cdk2 Revealed by Inducible Expression of a Dominant-Negative Mutant in Human Cells

机译：通过在人类细胞中的显性负突变体的诱导表达揭示了Cdk2的S和G2阶段作用。

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Cyclin-dependent kinase 2 (Cdk2) is essential for initiation of DNA synthesis in higher eukaryotes. Biochemical studies inXenopus egg extracts and microinjection studies in human cells have suggested an additional function for Cdk2 in activation of Cdk1 and entry into mitosis. To further examine the role of Cdk2 in human cells, we generated stable clones with inducible expression of wild-type and dominant-negative forms of the enzyme (Cdk2-wt and Cdk2-dn, respectively). Both exogenous proteins associated efficiently with endogenous cyclins. Cdk2-wt had no apparent effect on the cell division cycle, whereas Cdk2-dn inhibited progression through several distinct stages. Cdk2-dn induction could arrest cells at the G1/S transition, as previously observed in transient expression studies. However, under normal culture conditions, Cdk2-dn induction primarily arrested cells with S and G2/M DNA contents. Several observations suggested that the latter cells were in G2 phase, prior to the onset of mitosis: these cells contained uncondensed chromosomes, low levels of cyclin B-associated kinase activity, and high levels of tyrosine-phosphorylated Cdk1. Furthermore, Cdk2-dn did not delay progression through mitosis upon release of cells from a nocodazole block. Although the G2arrest imposed by Cdk2-dn was similar to that imposed by the DNA damage checkpoint, the former was distinguished by its resistance to caffeine. These findings provide evidence for essential functions of Cdk2 during S and G2 phases of the mammalian cell cycle.
机译：细胞周期蛋白依赖性激酶2（Cdk2）对于启动高等真核生物的DNA合成至关重要。爪蟾卵提取物的生化研究和在人体细胞中的显微注射研究表明，Cdk2在激活Cdk1和进入有丝分裂中具有附加功能。为了进一步检查Cdk2在人细胞中的作用，我们生成了具有可诱导表达的酶的野生型和显性负性形式的稳定克隆（分别为Cdk2-wt和Cdk2-dn）。两种外源蛋白均与内源性细胞周期蛋白有效结合。 Cdk2-wt对细胞分裂周期没有明显影响，而Cdk2-dn通过几个不同的阶段抑制进展。如先前在瞬时表达研究中所观察到的，Cdk2-dn诱导可将细胞停滞在G 1 / S过渡。然而，在正常培养条件下，Cdk2-dn诱导主要阻滞具有S和G 2 / M DNA含量的细胞。一些观察结果表明，后者的细胞在有丝分裂发生之前处于G 2 期：这些细胞包含未凝聚的染色体，低水平的细胞周期蛋白B相关激酶活性和高水平的酪氨酸磷酸化Cdk1。此外，Cdk2-dn在诺考达唑阻滞释放细胞后不会延迟有丝分裂的进程。尽管Cdk2-dn施加的G 2 逮捕与DNA损伤检查站施加的逮捕类似，但前者的特点是对咖啡因具有抗性。这些发现为Cdk2在哺乳动物细胞周期的S和G 2 阶段的基本功能提供了证据。

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