HuR蛋白118位苏氨酸对热应激状态下的双特异性磷酸酶1的转录后调控

Post-transcriptional regulation of dual-specificity phosphatase-1 by RNA-binding protein HuR T118 in heat shock

目的探讨RNA结合蛋白HuR 118位苏氨酸对热应激状态下的双特异性磷酸酶1(DUSP1)转录后调控机制。方法构建HuR 118位苏氨酸突变体真核表达质粒,并用脂质体转染NIH 3T3细胞,Real-time PCR检测其对DUSP1 mRNA水平的影响,Western blotting检测其对DUSP1蛋白表达的效应。结果 (1)小鼠HuR不同突变体真核表达载体质粒构建成功;(2)热休克刺激后,HuRT118磷酸化明显增强;(3)过表达HuR(WT)与HuR(T118E),经过热休克刺激后,DUSP1 mRNA水平明显升高,其蛋白表达也有上调;过表达HuR(T118E)时,DUSP1的mRNA水平明显上调,而蛋白水平在热休克刺激前后均上调,其表达变化无差异而过表达HuR(T118A),热休克刺激前后DUSP1 mRNA水平及蛋白表达均下调;(4)热休克刺激下,HuR(WT)可以从细胞核内转位到细胞质并形成颗粒;当过表达HuR(T118E)时,在静息状态下,HuR(T118E)弥散分布于细胞质,热休克刺激会使其在细胞质形成明显的颗粒;而过表达HuR(T118A)时,热休克刺激并不能使其移位出核。结论 RNA结合蛋白HuR参与了DUSP1的转录后调控,可以通过其118位的苏氨酸磷酸化提高热休克刺激后的DUSP1的基因及蛋白水平。

Objective To investigate the post-transcriptional regulation of dual-specificity phosphatase-1 （DUSP1） by the RNA-binding protein HuR in heat shock. Methods The recombinant plasmids carrying wild-type （WT） HuR or its mutants at threonine 118 were constructed and transiently transfected into NIH 3T3 cells via liposome, and the changes in the expressions of DUSP1 mRNA and protein were detected by quantitative real-time PCR and Western blotting, respectively. Results Heat shock caused significantly enhanced phosphorylation of HuR at the residue T118. In 3T3 cells transfected with the plasmids carrying wild-type HuR for its over-expression showed significantly up-regulated DUSP1 mRNA and protein expressions at 24 h after transfection. Over-expression of HuR（T118A） down-regulated DUSP1 mRNA and protein expressions in cells challenged with heat shock, while HuR（T118E） over-expression significantly increased DISP1 expression at both mRNA and protein levels. After heat shock, HuR（WT） translocated from the cell nucleus to the cytoplasm to form particles. HuR（T118E） was diffusely distributed in the cytoplasm before heat shock and formed particles after heat shock. HuR（T118A） did not undergo such translocation in response to heat shock challenge. Conclusion HuR regulates DUSP1 mRNA and protein expression at the post-transcriptional level to increase its expression after heat shock by enhancing the phosphorylation HuR T118.