Cheong Jit Kong, Gunaratnam Lakshman, Hsu Stephen I-Hong
Renal Division and Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115, USA.
J Biol Chem. 2008 Apr 25;283(17):11661-76. doi: 10.1074/jbc.M708365200. Epub 2008 Mar 3.
Overexpression of the proto-oncogene TRIP-Br2 (SERTAD2) has been shown to induce E2F activity and promote tumorigenesis, whereas ablation of TRIP-Br2 arrests cell proliferation. Timely degradation of many cell cycle regulators is fundamental to the maintenance of proper cell cycle progression. Here we report novel mechanism(s) that govern the tight regulation of TRIP-Br2 levels during cell cycle progression. TRIP-Br2 was observed to be a short-lived protein in which the expression level peaks at the G(1)/S boundary. TRIP-Br2 accumulated in cells treated with 26 S proteasome inhibitors. Co-immunoprecipitation studies revealed that TRIP-Br2 forms ubiquitin conjugates. In silico analysis identified a putative leucine-rich nuclear export signal (NES) motif that overlaps with the PHD-Bromo interaction domain in the acidic C-terminal transactivation domain (TAD) of TRIP-Br2. This NES motif is highly conserved in widely divergent species and in all TRIP-Br family members. TRIP-Br2 was shown to be stabilized in G(2)/M phase cells through nuclear entrapment, either by deletion of the acidic C-terminal TAD, which includes the NES motif, or by leptomycin B-mediated inhibition of the CRM1-dependent nuclear export machinery. Mutation of leucine residue 238 of this NES motif abolished the interaction between CRM1 and TRIP-Br2, as well as the nuclear export of TRIP-Br2 and its subsequent 26 S proteasome-dependent degradation. These data suggest that CRM1-mediated nuclear export may be required for the proper execution of ubiquitin-proteasome-dependent degradation of TRIP-Br2.
原癌基因TRIP - Br2（SERTAD2）的过表达已被证明可诱导E2F活性并促进肿瘤发生，而TRIP - Br2的缺失会使细胞增殖停滞。许多细胞周期调节因子的及时降解是维持细胞周期正常进程的基础。在此，我们报告了在细胞周期进程中控制TRIP - Br2水平严格调控的新机制。观察到TRIP - Br2是一种半衰期短的蛋白质，其表达水平在G(1)/S边界达到峰值。TRIP - Br2在经26S蛋白酶体抑制剂处理的细胞中积累。免疫共沉淀研究表明TRIP - Br2形成泛素缀合物。计算机分析确定了一个推定的富含亮氨酸的核输出信号（NES）基序，该基序与TRIP - Br2酸性C末端反式激活域（TAD）中的PHD - 溴结构域相互作用域重叠。这个NES基序在广泛不同的物种和所有TRIP - Br家族成员中高度保守。通过缺失包含NES基序的酸性C末端TAD或通过雷帕霉素B介导的对CRM1依赖性核输出机制的抑制，TRIP - Br2在G(2)/M期细胞中通过核滞留而稳定。该NES基序的亮氨酸残基238突变消除了CRM1与TRIP - Br2之间的相互作用，以及TRIP - Br2的核输出及其随后的26S蛋白酶体依赖性降解。这些数据表明CRM1介导的核输出可能是TRIP - Br2泛素 - 蛋白酶体依赖性降解正确执行所必需的。
