Wohlbold Lara, Larochelle Stéphane, Liao Jack C-F, Livshits Geulah, Singer Juliet, Shokat Kevan M, Fisher Robert P
Molecular Biology Program, Memorial Sloan-Kettering Cancer Center, New York, New York 10021, USA.
Cell Cycle. 2006 Mar;5(5):546-54. doi: 10.4161/cc.5.5.2541. Epub 2006 Mar 1.
The cyclin-dependent kinases (CDKs) that drive the eukaryotic cell cycle must be phosphorylated within the activation segment (T-loop) by a CDK-activating kinase (CAK) to achieve full activity. Although a requirement for CDK-activating phosphorylation is conserved throughout eukaryotic evolution, CAK itself has diverged between metazoans and budding yeast, and fission yeast has two CAKs, raising the possibility that additional mammalian enzymes remain to be identified. We report here the characterization of PNQALRE (also known as CCRK or p42), a member of the mammalian CDK family most similar to the cell-cycle effectors Cdk1 and Cdk2 and to the CAK, Cdk7. Although PNQALRE/CCRK was recently proposed to activate Cdk2, we show that the monomeric protein has no intrinsic CAK activity. Depletion of PNQALRE by >80% due to RNA interference (RNAi) impairs cell proliferation, but fails to arrest the cell cycle at a discrete point. Instead, both the fraction of cells with a sub-G(1) DNA content and cleavage of poly(ADP-ribose) polymerase (PARP) increase. PNQALRE knockdown did not diminish Cdk2 T-loop phosphorylation in vivo or decrease CAK activity of a cell extract. In contrast, depletion of Cdk7 by RNAi causes a proportional decrease in the ability of an extract to activate recombinant Cdk2. Our data do not support the proposed function of PNQALRE/CCRK in activating CDKs, but instead reinforce the notion of Cdk7 as the major, and to date the only, CAK in mammalian cells.
驱动真核细胞周期的细胞周期蛋白依赖性激酶（CDK）必须被CDK激活激酶（CAK）在激活片段（T环）内磷酸化才能达到完全活性。尽管CDK激活磷酸化的需求在整个真核生物进化过程中是保守的，但CAK本身在后生动物和芽殖酵母之间已经发生了分化，裂殖酵母有两种CAK，这增加了仍有待鉴定其他哺乳动物酶的可能性。我们在此报告了PNQALRE（也称为CCRK或p42）的特性，它是哺乳动物CDK家族的成员，与细胞周期效应因子Cdk1和Cdk2以及CAK Cdk7最为相似。尽管最近有人提出PNQALRE/CCRK可激活Cdk2，但我们发现该单体蛋白没有内在的CAK活性。由于RNA干扰（RNAi）导致PNQALRE耗竭>80%会损害细胞增殖，但无法在离散点使细胞周期停滞。相反，DNA含量低于G1期的细胞比例以及聚（ADP - 核糖）聚合酶（PARP）的裂解均增加。PNQALRE敲低在体内并未减少Cdk2 T环磷酸化，也未降低细胞提取物的CAK活性。相比之下，RNAi介导的Cdk7耗竭会导致提取物激活重组Cdk2的能力成比例下降。我们的数据不支持PNQALRE/CCRK在激活CDK方面的拟议功能，而是强化了Cdk7作为哺乳动物细胞中主要且迄今为止唯一的CAK这一概念。
