YAP精氨酸甲基化修饰调控Hippo通路及肿瘤细胞增殖

Role of YAP arginine methylation in Hippo pathway and cancer cell proliferation

目的探讨yes相关蛋白(YAP)精氨酸甲基化修饰对Hippo通路激活及肿瘤细胞增殖的影响。方法在HEK293T细胞中过表达并纯化FLAG-YAP,通过质谱鉴定YAP的精氨酸甲基化修饰及其修饰位点;Western印迹法和体外激酶反应检测YAP精氨酸甲基化修饰对YAP 127位丝氨酸(S127)磷酸化修饰的影响;免疫荧光染色法检测YAP精氨酸甲基化修饰对其核定位的影响。利用慢病毒载体系统构建敲低YAP蛋白的稳定细胞株,在敲低YAP的稳定株中分别回补YAP野生型及其修饰位点突变体,荧光实时定量PCR(q-RT-PCR)检测Hippo通路下游靶基因转录情况,MTS[3-(4,5-dimethylthiaol-2-yl)-5-(3-car-boxy-methoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazoliu-m,inner salt]实验和克隆形成实验检测YAP精氨酸甲基化修饰对肿瘤细胞增殖能力的影响。结果质谱结果显示YAP 124位精氨酸(R124)存在单甲基化修饰,并受细胞所处营养状况的调控;经体外激酶反应、Western印迹和斑点印迹杂交(Dot blot)验证,YAP R124单甲基化修饰能抑制YAP S127的磷酸化修饰;免疫荧光染色实验证实,YAP R124甲基化修饰促进YAP蛋白入核;q-RT-PCR结果表明,YAP R124甲基化修饰激活Hippo通路下游靶基因转录;MTS和克隆形成实验证实,YAP R124甲基化修饰促进肿瘤细胞的增殖。结论YAP R124单甲基化修饰能抑制YAP S127的磷酸化修饰,促进YAP蛋白进入细胞核,激活Hippo通路下游靶基因转录,进而促进肿瘤细胞增殖。

Objective To investigate the role of YAP(yes-associated protein)arginine methylation in the Hippo pathway and cancer cell proliferation.Methods YAP arginine methylation modification was analyzed by MASS SPEC.The effects of YAP methylation on its serine 127(S127)phosphorylation and nuclear translocation were examined with in vitro kinase assay and immunofluorescence staining.YAP knockdown H1299 stable cells were generated by shRNA.Wild-type YAP and its site mutants were reintroduced into the YAP knockdown cells before the downstream target genes of Hippo pathway were detected by q-RT-PCR.H1299 proliferation was examined by MTS and colony formation assays.Results YAP was mono-methylated at Arginine 124(R124).YAP R124 mono-methylation inhibited YAP S127 phosphorylation,promoted its nuclear translocation and the downstream target gene transcription,eventually promoting cell proliferation.Conclusion YAP R124 mono-methylation promotes Hippo pathway activation and cancer cell proliferation.