p62蛋白在食管鳞癌细胞中核浆易位的机制研究

Study on the mechanism of nucleo-plasma translocation of p62 protein in esophageal squamous cell carcinoma

目的:探讨p62蛋白在食管鳞癌细胞中核浆易位的影响因素及其可能机制。方法:采用核输出抑制剂KPT330处理食管鳞癌细胞KYSE70、KYSE150和KYSE510后,利用免疫荧光技术结合激光共聚焦显微镜分别观察抑制剂处理前后食管鳞癌细胞中p62蛋白的定位情况。利用液相色谱串联质谱(LC-MS/MS)磷酸化蛋白质组技术鉴定食管鳞癌细胞中p62蛋白的磷酸化位点。定点突变技术用于构建p62不同磷酸化状态的突变体。通过免疫共沉淀实验(Co-IP)、邻位连接实验(PLA)分析p62与GSK-3β的相互作用情况。结果:使用核输出抑制处理后,免疫荧光结合激光共聚焦显微镜观察发现,p62积聚在细胞核中。质谱鉴定结果显示,食管鳞癌细胞中p62蛋白存在T269/S272/S328/S332这4个位点的磷酸化,进一步发现T269/S272位点磷酸化、且同时S328/S332去磷酸化可使胞浆中的p62转移至细胞核中。Co-IP和PLA实验结果显示,p62与GSK-3β存在直接的相互作用。结论:食管鳞癌细胞中p62的核浆易位与其S328/S332磷酸化状态相关,且可能受GSK-3β激酶调控。

OBJECTIVE:To explore possible mechanisms that mediated the translocation of p62 protein from nuclei into cytoplasm in esophageal squamous cell carcinoma.METHODS:After treatments with KPT330 in KYSE70,KYSE150 and KYSE510 ESCC cell lines,localizations of p62 were observed by immunofluorescence assay combined with confocal laser microscopy as compared with the untreated group.LC-MS/MS mass spectrometry was used to identify the phosphorylation sites of p62 in esophageal cancer cells.Site-directed mutagenesis was performed to construct mutants with different phosphorylation status of p62.Co-IP and PLA experiments were conducted to analyze interactions between p62 and GSK3β.p62 accumulated in the nucleus after treatment with nuclear export inhibitor.RESULTS:The results of mass spectrometry showed phosphorylation of p62 protein at four sites of T269/S272/S328/S332 in esophageal cancer cells.By transfecting different phosphorylated mutants of p62 into KYSE30 cells and KYSE150 cells with endogenous knockdown of p62,phosphorylation at T269/S272 and dephosphorylation of S328/S332 contributed to the transfer of p62 from cytoplasm to nucleus.Co-IP and PLA results showed direct interactions between p62 and GSK-3β.CONCLUSION:Our results indicates that nucleo-plasmic translocation of p62 in esophageal cancer cells was related to the phosphorylation status of S328/S332 and might be regulated by GSK-3β kinase.