Destabilization of Kruppel-like factor 4 protein in response to serum stimulation involves the ubiquitin-proteasome pathway
Destabilization of Kruppel-like factor 4 protein in response to serum stimulation involves the ubiquitin-proteasome pathway
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DOI：
10.1158/0008-5472.can-05-2059
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发表时间：
2005-11-15
期刊：
影响因子：
11.2
通讯作者：
Tseng, CC
中科院分区：
文献类型：
--
作者：
Chen, ZY;Wang, XS;Tseng, CC
虽然锌指转录因子Kruppel样因子4（KLF 4）已被证明是细胞增殖的负调节因子，但KLF 4的翻译后修饰机制，特别是在蛋白质降解水平上的机制，知之甚少。在这里，我们表明，KLF 4蛋白水平在静止细胞是高的，但迅速下降，细胞进入增殖阶段后血清刺激。这种下降被部分逆转与MG 132，蛋白酶体抑制剂预处理。此外，KLF 4是一种不稳定的蛋白质，经历快速周转，并表现出相对较短的半衰期（t（1/2）类似于120分钟）。为了研究泛素-蛋白酶体途径参与KLF 4稳定性的调节，用蛋白酶体抑制剂处理HCT 116细胞。我们的研究结果表明，lactacystin治疗后，内源性KLF 4的水平增加的时间和剂量依赖性的方式。使用无细胞系统，在体外翻译的S-35标记的KLF 4蛋白被从指数生长的HCT 116细胞在ATP存在下制备的蛋白提取物降解。这些作用可通过MG 132预处理或用ATP-γ-S（ATP的不可水解类似物）替代ATP来防止，表明ATP是263蛋白酶体降解KLF 4所必需的。此外，当细胞用蛋白酶体抑制剂处理或用外源性泛素转染时，KLF 4受到泛素化。总的来说，这些结果表明，血清刺激后KLF 4的不稳定是介导的，至少部分是通过泛素-蛋白酶体途径。
Although the zinc finger transcription factor Kruppel-like factor 4 (KLF4) has been shown to be a negative regulator of cell proliferation, the mechanisms underlying the posttranslational modification of KLF4, especially at the level of protein degradation, are poorly understood. Here, we show that KLF4 protein levels in quiescent cells were high, but decreased rapidly as cells entered the proliferating stage following serum stimulation. This decrease was partially reversed by pretreatment with MG132, a proteasome inhibitor. Moreover, KLF4 was an unstable protein that underwent rapid turnover, and exhibited a relatively short half-life (t(1/2) similar to 120 minutes). To investigate the involvement of the ubiquitin-proteasome pathway in the regulation of the stability of KLF4, HCT116 cells were treated with proteasome inhibitors. Our results showed that, following lactacystin treatment, levels of endogenous KLF4 increased in a time- and dose-dependent manners. Using a cell-free system, in vitro -translated S-35-labeled KLF4 protein was degraded by protein extracts prepared from exponentially growing HCT116 cells in the presence of ATP. These effects were prevented by pretreatment with MG132 or replacement of ATP with ATP-gamma-S, a nonhydrolyzable analogue of ATP, suggesting that ATP is required for KLF4 degradation by the 263 proteasome. In addition, KLF4 was subject to ubiquitination when cells were treated with the proteasome inhibitor or transfected with exogenous ubiquitin. Collectively, these results indicate that destabilization of KLF4 following serum stimulation is mediated, at least in part, through a ubiquitin-proteasome pathway.