【摘要】 背景与目的:在利用磷酸化蛋白质富集结合蛋白质组学技术分析EB病毒潜伏膜蛋白1(latent membrane protein1,LMP1)调节的磷酸化蛋白质分子时,我们发现了包括AnnexinⅠ在内的25个新的受EB病毒LMP1调节的磷酸化蛋白质分子。本实验探讨LMP1调节AnnexinⅠ磷酸化的信号通路。方法:采用鼻咽癌细胞系CNE1和LMP1稳定表达的细胞系CNE1-LMP1,Western blot检测AnnexinⅠ的蛋白表达水平;免疫沉淀结合Western blot检测AnnexinⅠ的丝氨酸和酪氨酸磷酸化;激酶分析实验检测蛋白激酶C(protein kinase C,PKC)的活性。结果:LMP1对AnnexinⅠ的蛋白表达水平没有影响,LMP1上调AnnexinⅠ的丝氨酸磷酸化水平,而对酪氨酸磷酸化水平没有影响。在CNE1细胞中PKC的相对活性为0.97±0.05,CNE1-LMP1细胞中的PKC相对活性为1.22±0.10,CNE1与CNE1-LMP1细胞之间PKC的活性差异有统计学意义(P<0.01,n=6),表明LMP1可以上调PKC活性。AnnexinⅠ的丝氨酸磷酸化水平随PKC活性的变化而变化。结论:EB病毒LMP1通过PKC信号通路调节AnnexinⅠ丝氨酸磷酸化。更多还原

【Abstract】 BACKGROUND & OBJECTIVE:Twenty-five novel phosphopro-teins,including Annexin Ⅰ,triggered by Epstein-Barr virus (EBV)-encoded latent membrane protein 1 (LMP1) were identified when we previously combined phosphorylation enrichment with proteomics technology to elucidate signaling pathway activated by LMP1. This study was to map signal transduction pathway between LMP1 and phosphorylation of AnnexinⅠ. METHODS: The expression of Annexin Ⅰ in nasopharyngeal carcinoma cell lines (stably expressing LMP1) was analyzed by Western blot. Serine-and tyrosine-phosphorylation levels of AnnexinⅠwere detected through combining immunoprecipitation with Western blot. The activity of protein kinase C (PKC) was analyzed by non-radioactive protein kinase assay. RESULTS:The protein level and tyrosine phosphorylation of Annexin Ⅰ in CNE1 and CNE1-LMP1 cells were similar; the serine phosphorylation of annexin Ⅰ was higher in CNE1-LMP1 cells than in CNE1 cells. The relative activity of PKC was significantly lower in CNE1 cells than in CNE1-LMP1 cells (0.97±0.05 vs. 1.22±0.10,P<0.01). The change of PKC activity was followed by the change of serine phosphorylation level of Annexin Ⅰ. CONCLUSION: LMP1 mediates serine phosphorylation of Annexin Ⅰ via PKC pathway.更多还原