Transforming growth factor β targeted inactivation of cyclin E:cyclin-dependent kinase 2 (Cdk2) complexes by inhibition of Cdk2 activating kinase activity
Transforming growth factor β targeted inactivation of cyclin E:cyclin-dependent kinase 2 (Cdk2) complexes by inhibition of Cdk2 activating kinase activity
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DOI：
10.1073/pnas.96.26.14961
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发表时间：
1999-12-21
影响因子：
11.1
通讯作者：
Dowdy, SF
中科院分区：
文献类型：
--
作者：
Nagahara, H;Ezhevsky, SA;Dowdy, SF
转化生长因子β（TGF-β）介导的G（1）阻滞以前已被证明特异性靶向细胞周期蛋白D：细胞周期蛋白依赖性激酶（Cdk）4/6复合物的失活。我们在这里报告，TGF-β-治疗的人肝癌细胞HepG 2停滞在G1期，但保留持续的细胞周期蛋白D：Cdk 4/6的活性和活性，低磷酸化视网膜母细胞瘤肿瘤抑制蛋白。与此观察结果一致，TGF-β处理的细胞不能诱导p15（INK 4 b），下调CDC 25 A，或增加p21（CIP 1），P27（KIP 1）和p57（KIP 2）的水平。然而，TGF-β处理导致细胞周期蛋白E：Cdk 2复合物的特异性失活，这是由于Cdk 2上激活Thr（160）磷酸化的缺失引起的。来自TGF-β处理细胞的全细胞裂解物显示Cdk 2 Thr（160）Cdk激活激酶（CAK）活性的抑制;然而，细胞周期蛋白H：Cdk 7活性（先前假定的哺乳动物CAK）没有改变。酿酒酵母含有经遗传和生物化学证实的CAK基因CAK 1，其编码与Cdk 7无关的单体44-kDa Cak 1 p蛋白，抗Cak 1 p抗体与具有CAK活性的45-kDa人蛋白交叉反应，所述CAK活性响应于TGF-β处理而特异性下调。综上所述，这些观察结果表明，TGF-β信号转导介导的G1停滞在HepG 2细胞通过靶向Cdk 2 CAK，并建议存在至少两种哺乳动物CAK：一种特异性Cdk 2和Cdk 4/6。
Transforming growth factor beta (TGF-beta)-mediated G(1) arrest previously has been shown to specifically target inactivation of cyclin D:cyclin-dependent kinase (Cdk) 4/6 complexes. We report here that TGF-beta-treated human HepG2 hepatocellular carcinoma cells arrest in G1, but retain continued cyclin D:Cdk4/6 activity and active, hypophosphorylated retinoblastoma tumor suppressor protein. Consistent with this observation, TGF-beta-treated cells failed to induce p15(INK4b), down-regulate CDC25A, or increase levels of p21(CIP1), P27(KIP1), and p57(KIP2). However, TGF-beta treatment resulted in the specific inactivation of cyclin E:Cdk2 complexes caused by absence of the activating Thr(160) phosphorylation on Cdk2, Whole-cell lysates from TGF-beta-treated cells showed inhibition of Cdk2 Thr(160) Cdk activating kinase (CAK) activity; however, cyclin H:Cdk7 activity, a previously assumed mammalian CAK, was not altered. Saccharomyces cerevisiae contains a genetically and biochemically proven CAK gene, CAK1, that encodes a monomeric 44-kDa Cak1p protein unrelated to Cdk7, Anti-Cak1p antibodies cross-reacted with a 45-kDa human protein with CAK activity that was specifically down-regulated in response to TGF-beta treatment. Taken together, these observations demonstrate that TGF-beta signaling mediates a G1 arrest in HepG2 cells by targeting Cdk2 CAK and suggests the presence of at least two mammalian CAKs: one specific for Cdk2 and one for Cdk4/6.