Stimulation of the Raf/MEK/ERK cascade is necessary and sufficient for activation and Thr-160 phosphorylation of a nuclear-targeted CDK2
Stimulation of the Raf/MEK/ERK cascade is necessary and sufficient for activation and Thr-160 phosphorylation of a nuclear-targeted CDK2
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DOI：
10.1074/jbc.m207425200
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发表时间：
2002-12-06
影响因子：
4.8
通讯作者：
Baldassare, JJ
中科院分区：
文献类型：
--
作者：
Lents, NH;Keenan, SM;Baldassare, JJ
细胞周期蛋白依赖性激酶2的活性是真核细胞周期G（1）-S期进程所必需的。在这项研究中，我们通过构建一个组成性靶向细胞核的CDK 2来研究CDK 2-cyclin E的激活。CDK 2的活化需要通过核定位的CDK活化激酶去除两个抑制性磷酸（Thr-14和Tyr-15）并添加一个活化性磷酸（Thr-160），其被认为是组成性活性的。令人惊讶的是，缺乏抑制性磷酸化位点的核定位CDK 2-NLS和CDK 2-NLS（A14、F15）需要血清才能变得活跃，尽管与表达的细胞周期蛋白E复合。我们发现，通过用U 0126（一种选择性MEK抑制剂）治疗或显性负性ERK表达来抑制有丝分裂原介导的ERK活化，显著降低了Thr-160的磷酸化和CDK 2-NLS构建体的酶活性。与ERK在Thr-160磷酸化中的作用一致，组成型活性Raf-1的表达诱导血清停滞细胞中CDK 2-NLS的Thr-160磷酸化，这种作用可被U 0126处理阻断。综上所述，这些数据显示ERK在G1期细胞周期进程中的新作用：除了刺激细胞周期蛋白D1表达和CDK 2核转位的作用外，ERK还调节CDK 2-细胞周期蛋白E的Thr-160磷酸化。
The activity of cyclin-dependent kinase 2 is required for G(1)-S-phase progression of the eukaryotic cell cycle. In this study, we examine the activation of CDK2-cyclin E by constructing a CDK2 that is constitutively targeted to the nucleus. Activation of CDK2 requires the removal of two inhibitory phosphates (Thr-14 and Tyr-15) and the addition of one activating phosphate (Thr-160) by a nuclear localized CDK-activating kinase, which is thought to be constitutively active. Surprisingly, nuclear localized CDK2-NLS and CDK2-NLS(A14,F15), which lacks the inhibitory phosphorylation sites, require serum to become active, despite complexing with expressed cyclin E. We show that inhibition of mitogen-mediated ERK activation by treatment with U0126, a selective MEK inhibitor, or expression of dominant-negative ERK markedly reduces the phosphorylation of Thr-160 and enzymatic activity of both CDK2-NLS constructs. Consistent with a role for ERK in Thr-160 phosphorylation, expression of constitutively active Raf-1 induces Thr-160 phosphorylation of CDK2-NLS in serum-arrested cells, an effect that is blocked by treatment with U0126. Taken together, these data show a new role for ERK in G1 cell cycle progression: In addition to its role in stimulating cyclin D1 expression and nuclear translocation of CDK2, ERK regulates Thr-160 phosphorylation of CDK2-cyclin E.