Saint Louis University School of Medicine, MO, USA.
Cell Cycle. 2010 Nov 15;9(22):4533-41. doi: 10.4161/cc.9.22.13789.
Cyclin E-associated CDK2 activity is required for the initiation of DNA synthesis in human cells. CDK2 activity is tightly regulated; CDK2 must be in the nucleus, bound to a cyclin, phosphorylated on T160, and dephosphorylated on T14/Y15 for complete kinase activation. Nuclear localization exposes CDK2 to activating enzymes (CAK, Cdc25A) in stimulated cells. Previous studies from our lab indicate CDK2 nuclear localization and cyclin E co-expression are insufficient to cause CDK2 activation or T160 phosphorylation in stimulated IIC9 cells; these activities still require serum stimulation and ERK kinase activity. Recent studies have implicated a role for origin of replication (ORC) licensing proteins in the activation of G1/S Cdks. In this study, we show that CDK2 associates with chromatin and Cdc6 in an ERK-dependent manner following stimulation of IIC9 CHEF cells. We show that nuclear-localized CDK2 (CDK2-NLS) ectopically expressed with cyclin E requires mitogenic stimulation and ERK activation for chromatin association, in addition to previously shown kinase activation and T160 phosphorylation in IIC9 cells. Additionally, we show that expression of Cdc6 in stimulated IIC9 cells treated with ERK inhibitor rescues CDK2-NLS chromatin association, kinase activation, and T160 phosphorylation. From the above data, we deduce ERK-dependent CDK2 activation is due in part to ERK-dependent Cdc6 expression. To examine the role of Cdc6 directly in stimulated primary human fibroblasts, we used RNA interference to attenuate the expression of Cdc6. We show that Cdc6 expression is required for CDK2 chromatin association and kinase activation in stimulated primary human fibroblasts. Additionally, we show that Cdc6 expression is required for the initiation of DNA synthesis and S phase entry in stimulated primary human fibroblasts. Ultimately, this data implicates Cdc6 expression as an important mitogen-induced mechanism in the activation of CDK2/cyclin E, the initiation of DNA synthesis, and the regulation of G1-S phase progression.
细胞周期蛋白 E 相关的 CDK2 活性对于人细胞中 DNA 合成的起始是必需的。CDK2 活性受到严格调控；CDK2 必须位于核内，与细胞周期蛋白结合，在 T160 上磷酸化，并在 T14/Y15 上去磷酸化，才能完全激活激酶。核定位使 CDK2 暴露于受刺激细胞中的激活酶（CAK、Cdc25A）。我们实验室之前的研究表明，CDK2 核定位和细胞周期蛋白 E 的共表达不足以导致受刺激的 IIC9 细胞中 CDK2 的激活或 T160 磷酸化；这些活性仍然需要血清刺激和 ERK 激酶活性。最近的研究表明，起始复制（ORC）许可蛋白在 G1/S Cdk 的激活中起作用。在这项研究中，我们显示 CDK2 在 IIC9 CHEF 细胞受到刺激后以 ERK 依赖性的方式与染色质和 Cdc6 结合。我们表明，与细胞周期蛋白 E 异位表达的核定位 CDK2（CDK2-NLS）除了先前在 IIC9 细胞中显示的激酶激活和 T160 磷酸化之外，还需要有丝分裂刺激和 ERK 激活才能与染色质结合。此外，我们表明，在用 ERK 抑制剂处理的受刺激的 IIC9 细胞中表达 Cdc6 可以挽救 CDK2-NLS 染色质结合、激酶激活和 T160 磷酸化。根据上述数据，我们推断 ERK 依赖性 CDK2 激活部分归因于 ERK 依赖性 Cdc6 表达。为了直接研究 Cdc6 在受刺激的原代人成纤维细胞中的作用，我们使用 RNA 干扰来减弱 Cdc6 的表达。我们表明，Cdc6 的表达对于受刺激的原代人成纤维细胞中 CDK2 与染色质的结合和激酶的激活是必需的。此外，我们表明，Cdc6 的表达对于受刺激的原代人成纤维细胞中 DNA 合成的起始和 S 期进入是必需的。最终，这些数据表明 Cdc6 的表达是 CDK2/细胞周期蛋白 E 激活、DNA 合成起始和 G1-S 期进展调节的重要有丝分裂诱导机制。
