E3泛素连接酶WWP2通过蛋白酶体途径调控p21的稳定性

E3 ubiquitin ligase WWP2 regulates the stability of p21 through the proteasome pathway

目的 探讨含WW结构域的E3泛素蛋白连接酶2 (WWP2)调控p21稳定性的潜在作用机制。方法 通过免疫共沉淀及Western blotting检测HEK 293T细胞中WWP2与p21的结合情况。用蛋白酶体抑制剂(MG132)和放线菌酮(CHX)处理过表达或敲减WWP2 HEK 293T细胞0、4、8 h,通过Western blotting检测p21的表达情况。结果 WWP2与p21间存在结合。过表达WWP2的293T细胞中,p21的表达水平明显降低,且随WWP2表达量增加逐渐降低。敲减WWP2的293T细胞中,p21表达水平增加。随着CHX作用时间延长,对照组和实验组的p21表达水平均逐渐下降。过表达组p21半衰期较对照组缩短;敲减组p21半衰期较对照组延长。随着MG132作用时间延长,对照组和实验组p21表达水平均逐渐上升。空载组较过表达组、敲减组较对照组p21积累均更显著。结论 E3泛素连接酶WWP2可与p21结合,并通过蛋白酶体途径降解p21。

Objective To explore the potential mechanism of regulation of p21 stability by WW domain containing E3 ubiquitin protein ligase 2(WWP2). Methods The binding between WWP2 and p21 in HEK 293T cells was detected by coimmunoprecipitation and Western blotting. HEK 293T cells overexpressing WWP2 or with a knocked-down WWP2 gene were treated with the proteasome inhibitor MG132 and cycloheximide(CHX) for 0,4,and 8 h;the expression of p21 was then detected by Western blotting. Results It was observed that WWP2 interacts with p21. Additionally,cells overexpressing WWP2 showed significantly decreased expression levels of p21,which gradually decreased with the increase in WWP2 expression. Consistently,WWP2 knockdown cells showed increased levels of p21. The expression level of p21 in both the control and experimental groups decreased gradually with the prolongation of the CHX effect time. The half-life of p21 in the overexpression group was shorter than in the control group;whereas it was longer in the knockdown group than in the control group. With the prolongation of MG132 action time,the expression levels of p21 in both the control and experimental groups gradually increased. Compared with the overexpression and knockdown groups,the accumulation of p21 in the empty vector group was more significant than in the control group. Conclusion The E3 ubiquitin ligase WWP2 can bind p21 and degrade it through the proteasome pathway.