EB病毒潜伏膜蛋白1转化鼻咽上皮细胞的磷酸化蛋白质组学研究
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摘要
背景与目的:EB病毒(Epstein-Barr virus,EBV)是一种在人类普遍流行的γ-疱疹病毒,与许多人类肿瘤如伯基特淋巴瘤、霍奇金淋巴瘤、胃癌、鼻咽癌等的发生发展密切相关。其中EBV潜伏膜蛋白1(LMP1)被公认为病毒基因组编码的唯一具有促细胞转化作用的瘤蛋白,在鼻咽癌发生过程中可能发挥重要作用。LMP1的羧基末端(aa187～386)是LMP1致瘤作用的主要部位,包括3个活化区域(carboxy terminal activation region,CTAR):即CTAR1(194～231aa)、CTAR2(TRADD结合区351～386aa)与CTAR3(275～330aa)。研究表明,LMP1能持续活化NF-kB,AP-1,JAKs/STATs并通过这些分子激活下游信号传导通路而产生致瘤效应。由于蛋白质磷酸化是信号传导通路中最广泛最重要的翻译后修饰,而且这些通路中的许多信号分子及其磷酸化状态还远远没有阐明。因此,为了全面了解、验证LMP1介导的信号通路,阐明CTAR1、CTAR2与CTAR3在LMP1促鼻咽上皮细胞转化成瘤中所涉及的信号网络分子,本研究拟采用磷酸化蛋白质组学方法分离和鉴定LMP1转化的鼻咽上皮细胞的差异磷酸化蛋白,为EB病毒在鼻咽癌发生发展中的作用及机制提供进一步证据。
方法:采用脂质体转染法将野生型LMP1、2个突变型LMP1(TRADD位点突变和aa232～351缺失)的逆病毒载体pLNSX-LMP1~(WT)、pLNSX-LMP1~(TRADD)与pLNSX-LMP1~(Δ232-351)(以pLNSX为对照)分别导入PA317包装细胞,G418筛选2周后扩大培养,自细胞培养上清获得后续实验所需的感染性逆病毒RV-pLNSX、RV-LMP1~(WT)、RV-LMP1~(TRADD)与RV-LMP1~(Δ232-351)。然后,用浓缩的病毒分别感染人鼻咽上皮细胞NP69,汇合克隆培养,获得NP69-pLNSX、NP69-LMP1~(WT)、NP69-LMP1~(TRADD)与NP69-LMP1~(Δ232-351)4种转化细胞系。观察和检测以上转化细胞的形态、生长曲线、平板克隆、细胞周期等。同时,采用固相pH梯度双向凝胶电泳技术结合Western Blot方法分析NP69-pLNSX、NP69-LMP1~(WT)、NP69-LMP1~(TRADD)与NP69-LMP1~(Δ232-351)细胞系磷酸化蛋白质表达的差异,即在感染24小时后抽提细胞总蛋白,2-DE分别分离NP69-pLNSX、NP69-LMP1~(WT)、NP69-LMP1~(TRADD)与NP69-LMP1~(Δ232-351)细胞的总蛋白质,每组平行进行两块胶电泳,其中一块作为制备胶,另一块作为分析胶。制备胶考染显色,得到了分辨率较高、重复性较好的NP69-pLNSX、NP69-LMP1~(WT)、NP69-LMP1~(TRADD)与NP69-LMP1~(Δ232-351)细胞总蛋白质2-D胶电泳图谱;分析胶中的蛋白质转移至硝酸纤维素膜后,与抗酪氨酸磷酸化抗体孵育,进行WesternBlot分析,获得差异表达酪氨酸磷酸化蛋白质的反应图谱;将制备胶的电泳图谱和Western Blot的反应图谱进行比对分析,在制备胶上找到相应的差异酪氨酸磷酸化蛋白质点。利用MALDI-TOF-MS对差异酪氨酸磷酸化蛋白质进行鉴定,部分差异蛋白质应用免疫沉淀方法进行验证。
结果:(1)NP69-LMP1~(WT)细胞在培养过程中,逐渐由多角形、鹅卵石样典型上皮形态演变成细胞间接触减少的长梭状纤维细胞样形态,而NP69-LMP1~(TRADD)与NP69-LMP1~(Δ232-351)细胞呈近乎多角形,比较接近NP69-pLNSX细胞形态;NP69-LMP1~(WT)细胞的生长速度、S期细胞所占比例比NP69-LMP1~(TRADD)与NP69-LMP1~(Δ232-351)及载体对照组NP69-pLNSX细胞明显增加;(2)在NP69-pLNSX与NP69-LMP1~(WT)细胞比较组鉴定了12种差异表达的磷酸化蛋白质(上调有HSP27、波形蛋白等,下调有膜联蛋白A1、GTP结合蛋白等),在NP69-LMP1~(WT)与NP69-LMP1~(TRADD)细胞比较组鉴定了11种差异表达的磷酸化蛋白质,在NP69-LMP1~(WT)与NP69-LMP1~(Δ232-351)细胞比较鉴定了10种差异表达的磷酸化蛋白质。其中大部分是与细胞结构、信号传导和转录翻译相关的蛋白;(3)在这些差异蛋白质中,有10种磷酸化蛋白质体现了LMP1羧基端各功能区的特异性,如HSP27在NP69-LMP1~(WT)与NP69-LMP1~(TRADD)细胞表达增加(与NP69-pLNSX细胞比较),在NP69-LMP1~(Δ232-351)细胞却不增加等。(4)免疫沉淀验证了4种蛋白在转化细胞蛋白磷酸化表达水平的差异,结果与蛋白质组学结果一致
结论:(1)LMP1可能通过上调HSP27、波形蛋白、角蛋白等,下调膜联蛋白A1、GTP结合蛋白,分子伴侣TCP1等多种蛋白的磷酸化促进NP69细胞转化。(2)CTAR2可能介导LMP1对波形蛋白,ER-60,HSP70等蛋白磷酸化的上调和膜联蛋白A1,磷脂酰乙醇胺结合蛋白,蛋白酶体等蛋白磷酸化的下调而产生致瘤作用;(3)CTAR3可能介导LMP1对ER-60,角蛋白8,HSP27等蛋白磷酸化的上调和磷脂酰乙醇胺结合蛋白,蛋白酶体,膜联蛋白A2等蛋白磷酸化的下调而产生致瘤作用。
Background and Objective Epstein-Barr virus(EBV)is a prototype gamma herpes virus that infects the majority of the population worldwide and has been implicated in the pathogenesis of several human malignancies including Burkitt's and Hodgkin's lymphomas,gastric carcinoma and nasopharyngeal carcinoma(NPC).EBV-encoded latent membrane protein 1(LMP1)is considered a classic oncoprotein because of its ability to transform rodent fibroblast cell lines and drive the immortalization of primary human B-lymphocytes in vitro.So it was the first EBV latent gene found to be able to transform cell lines and alter the phenotype of cells due to its oncogenic potential.The C-terminal regions of LMP1 is the main signal activated position which can be subdivided into three essential C-terminal-activating regions(CTARs):C-terminal activation regions 1,2 and 3(CTAR 1,CTAR2 and CTAR3).Previous studies have shown that many of the oncogenic effects of LMP1 can be explained by its ability to constitutively activate nuclear factor kappa B (NF-kB),activator protein-1(AP-1),JAKs/ STATs.Due to protein phosphorylation is the most widespread and capital post-translational modification,and many signaling molecules and downstream target proteins mediated by the oncoprotein LMP1 in epithelial cells, particularly NPC cells,are largely unknown.In order to globally understand and confirm the signal pathways mediated by LMP1,to explain the molecular net involved in LMP1 CTAR1,CTAR2 and CTAR3-mediated transformation and tumorigenesis of nasopharyngeal epithelial cells,this study is to plan to separate and identify the differential phosporylated protein associated with LMP1 transforming nasopharyngeal epithelial cells by phosphoproteomics approach,and to provide further evidence for the role of EBV on the development of NPC.
Method pLNSX(as controlled vector),pLNSX-LMP1~(WT)、pLNSX-LMP1~(TRADD)and pLNSX-LMP1~(Δ232-351)were transfected into the ecotropic retrovirus packaging cell line PA317 with lipofectamine, respectively.The transfected PA317 cells were selected with G418 sulfate, two weeks later,the resistant cells were collected and expanded as the virus-producing cell lines(RV-pLNSX,RV-LMP1~(WT)、RV-LMP1~(TRADD)and RV-LMP1~(Δ232-351))to use in subsequent experiment.Then,nasopharyngeal epithelium cells NP69 were infected by the concentrated retrovirus respectively.The stable transfected cell lines NP69-pLNSX, NP69-LMP1~(WT),NP69-LMP1~(TRADD)and NP69-LMP1~(Δ232-351)were observd and detected cellular morphology,cellular growth curve,colony formation and cell cycle.Meanwhile,2-DE coupled with Western Blot method were used to analysis of the differentially expressed tyrosine-phosphorylated proteins among NP69-pLNSX,NP69-LMP1~(WT), NP69-LMP1~(TRADD),NP69-LMP1~(Δ232-351)cell lines.After infected 24 hours, The total proteins of the four groups were separated by 2-DE,respectively. Each patch was separated in parallel on analytical as well as preparative 2-D gels.The preparative 2-D gels were stained by Goomassie Brilliant Blue.Well-resolved and reproducible 2-D preparative patterns of NP69-pLNSX,NP69-LMP1~(WT),NP69-LMP1~(TRADD),NP69-LMP1~(Δ232-351) cell lines were acquired.The analytical gels were electroblotted onto a nitrocellulose membrane and incubated with anti-phosphotyrosine antibody.After Western Blot analysis,2-D maps of differentially expressed tyrosine-phosphorylated proteins were acquired.After comparing the preparative maps with Western Blot maps,differentially tyrosine-phosphorylated proteins were found in corresponding preparative gels.Differentially tyrosine-phosphorylated proteins between the four cell lines were identified using MALDI-TOF-MS analysis and database searching.The differential expression levels of the partial proteins were determined by Western blot.
Result(1)The morphous of NP69-LMP1~(WT)cells gradually developed from a typical epithelial polygon,cobblestone to an elongated and fibroblastoid shape with a marked reduction in cell-cell contact.But NP69-LMP1~(TRADD)and NP69-LMP1~(Δ232-351)exhibited approximately polygonal morphous and grew in a more compact pattern with tighter cell-cell contact.The growth velocity and the proportion of S stage of NP69-LMP1~(WT)cells obviously increased than NP69-LMP1~(TRADD)、 NP69-LMP1~(Δ232-351)cells and NP69-pLNSX cells;(2)12 differentially tyrosine-phosphorylated proteins between NP69-pLNSX and NP69-LMP1~(WT)cell lines(up-regulation proteins as heat shock protein 27, vimentin ea al,down-regulation proteins as annexin A1,chaperonin cotaining TCP1 et al);11 differentially tyrosine-phosphorylated proteins between NP69-LMP1~(WT)and NP69-LMP1~(TRADD)cell lines;10 differentially tyrosine-phosphorylated proteins between NP69-LMP1~(TRADD)and NP69-LMP1~(Δ232-351)cell lines Most of them were characterized as cellular structure proteins,signal transduction and transcription and translation associated proteins.(3)Among these identified phosphoproteins,there were 10 proteins personificated the specificness of the C-terminal-activating regions of LMP1.For example, the expression of HSP27 in NP69-LMP1~(WT)and NP69-LMP1~(TRADD)cell lines was much more than NP69-pLNSX ones.However,there was no expression in NP69-LMP1~(Δ232-351)cell lines.This indicated the CTAR3 having the main contribution in this process.(4)In the transformed cells, 2 proteins were confirmed with immunoprecipitation on the level of protein expression.Both results were coincident with the results of the proteomics.
Conclusions(1)LMP1 promotes NP69 cell transforming with the up-regulation of heat shock protein 27,vimentin and CK7 and the down-regulation of annexin A1,chaperonin cotaining TCP1 and GTP binding protein et al.(2)The CTAR2 domain of LMP1 was pivotal participating in the above effects with the up-regulation of the expression phosphorylation of vimentin,ER-60,HSP70 annexin A1 and down-regulation of phosphorylation of phosphotidylethanolamin binding protein,proteasome et al to generate tumorigennesis contribution;(3)The CTAR3 domain of LMP1 was pivotal participating in the above effects with the up-regulation of the expression of phosphorylation of ER-60, cytoskeletal 8,HSP70 and down-regulation of phosphorylation of phosphotidylethanolamin binding protein,proteasome,annexin A2 et al to generate tumorigennesis contribution.
方法:采用脂质体转染法将野生型LMP1、2个突变型LMP1(TRADD位点突变和aa232～351缺失)的逆病毒载体pLNSX-LMP1~(WT)、pLNSX-LMP1~(TRADD)与pLNSX-LMP1~(Δ232-351)(以pLNSX为对照)分别导入PA317包装细胞,G418筛选2周后扩大培养,自细胞培养上清获得后续实验所需的感染性逆病毒RV-pLNSX、RV-LMP1~(WT)、RV-LMP1~(TRADD)与RV-LMP1~(Δ232-351)。然后,用浓缩的病毒分别感染人鼻咽上皮细胞NP69,汇合克隆培养,获得NP69-pLNSX、NP69-LMP1~(WT)、NP69-LMP1~(TRADD)与NP69-LMP1~(Δ232-351)4种转化细胞系。观察和检测以上转化细胞的形态、生长曲线、平板克隆、细胞周期等。同时,采用固相pH梯度双向凝胶电泳技术结合Western Blot方法分析NP69-pLNSX、NP69-LMP1~(WT)、NP69-LMP1~(TRADD)与NP69-LMP1~(Δ232-351)细胞系磷酸化蛋白质表达的差异,即在感染24小时后抽提细胞总蛋白,2-DE分别分离NP69-pLNSX、NP69-LMP1~(WT)、NP69-LMP1~(TRADD)与NP69-LMP1~(Δ232-351)细胞的总蛋白质,每组平行进行两块胶电泳,其中一块作为制备胶,另一块作为分析胶。制备胶考染显色,得到了分辨率较高、重复性较好的NP69-pLNSX、NP69-LMP1~(WT)、NP69-LMP1~(TRADD)与NP69-LMP1~(Δ232-351)细胞总蛋白质2-D胶电泳图谱;分析胶中的蛋白质转移至硝酸纤维素膜后,与抗酪氨酸磷酸化抗体孵育,进行WesternBlot分析,获得差异表达酪氨酸磷酸化蛋白质的反应图谱;将制备胶的电泳图谱和Western Blot的反应图谱进行比对分析,在制备胶上找到相应的差异酪氨酸磷酸化蛋白质点。利用MALDI-TOF-MS对差异酪氨酸磷酸化蛋白质进行鉴定,部分差异蛋白质应用免疫沉淀方法进行验证。
结果:(1)NP69-LMP1~(WT)细胞在培养过程中,逐渐由多角形、鹅卵石样典型上皮形态演变成细胞间接触减少的长梭状纤维细胞样形态,而NP69-LMP1~(TRADD)与NP69-LMP1~(Δ232-351)细胞呈近乎多角形,比较接近NP69-pLNSX细胞形态;NP69-LMP1~(WT)细胞的生长速度、S期细胞所占比例比NP69-LMP1~(TRADD)与NP69-LMP1~(Δ232-351)及载体对照组NP69-pLNSX细胞明显增加;(2)在NP69-pLNSX与NP69-LMP1~(WT)细胞比较组鉴定了12种差异表达的磷酸化蛋白质(上调有HSP27、波形蛋白等,下调有膜联蛋白A1、GTP结合蛋白等),在NP69-LMP1~(WT)与NP69-LMP1~(TRADD)细胞比较组鉴定了11种差异表达的磷酸化蛋白质,在NP69-LMP1~(WT)与NP69-LMP1~(Δ232-351)细胞比较鉴定了10种差异表达的磷酸化蛋白质。其中大部分是与细胞结构、信号传导和转录翻译相关的蛋白;(3)在这些差异蛋白质中,有10种磷酸化蛋白质体现了LMP1羧基端各功能区的特异性,如HSP27在NP69-LMP1~(WT)与NP69-LMP1~(TRADD)细胞表达增加(与NP69-pLNSX细胞比较),在NP69-LMP1~(Δ232-351)细胞却不增加等。(4)免疫沉淀验证了4种蛋白在转化细胞蛋白磷酸化表达水平的差异,结果与蛋白质组学结果一致
结论:(1)LMP1可能通过上调HSP27、波形蛋白、角蛋白等,下调膜联蛋白A1、GTP结合蛋白,分子伴侣TCP1等多种蛋白的磷酸化促进NP69细胞转化。(2)CTAR2可能介导LMP1对波形蛋白,ER-60,HSP70等蛋白磷酸化的上调和膜联蛋白A1,磷脂酰乙醇胺结合蛋白,蛋白酶体等蛋白磷酸化的下调而产生致瘤作用;(3)CTAR3可能介导LMP1对ER-60,角蛋白8,HSP27等蛋白磷酸化的上调和磷脂酰乙醇胺结合蛋白,蛋白酶体,膜联蛋白A2等蛋白磷酸化的下调而产生致瘤作用。
Background and Objective Epstein-Barr virus(EBV)is a prototype gamma herpes virus that infects the majority of the population worldwide and has been implicated in the pathogenesis of several human malignancies including Burkitt's and Hodgkin's lymphomas,gastric carcinoma and nasopharyngeal carcinoma(NPC).EBV-encoded latent membrane protein 1(LMP1)is considered a classic oncoprotein because of its ability to transform rodent fibroblast cell lines and drive the immortalization of primary human B-lymphocytes in vitro.So it was the first EBV latent gene found to be able to transform cell lines and alter the phenotype of cells due to its oncogenic potential.The C-terminal regions of LMP1 is the main signal activated position which can be subdivided into three essential C-terminal-activating regions(CTARs):C-terminal activation regions 1,2 and 3(CTAR 1,CTAR2 and CTAR3).Previous studies have shown that many of the oncogenic effects of LMP1 can be explained by its ability to constitutively activate nuclear factor kappa B (NF-kB),activator protein-1(AP-1),JAKs/ STATs.Due to protein phosphorylation is the most widespread and capital post-translational modification,and many signaling molecules and downstream target proteins mediated by the oncoprotein LMP1 in epithelial cells, particularly NPC cells,are largely unknown.In order to globally understand and confirm the signal pathways mediated by LMP1,to explain the molecular net involved in LMP1 CTAR1,CTAR2 and CTAR3-mediated transformation and tumorigenesis of nasopharyngeal epithelial cells,this study is to plan to separate and identify the differential phosporylated protein associated with LMP1 transforming nasopharyngeal epithelial cells by phosphoproteomics approach,and to provide further evidence for the role of EBV on the development of NPC.
Method pLNSX(as controlled vector),pLNSX-LMP1~(WT)、pLNSX-LMP1~(TRADD)and pLNSX-LMP1~(Δ232-351)were transfected into the ecotropic retrovirus packaging cell line PA317 with lipofectamine, respectively.The transfected PA317 cells were selected with G418 sulfate, two weeks later,the resistant cells were collected and expanded as the virus-producing cell lines(RV-pLNSX,RV-LMP1~(WT)、RV-LMP1~(TRADD)and RV-LMP1~(Δ232-351))to use in subsequent experiment.Then,nasopharyngeal epithelium cells NP69 were infected by the concentrated retrovirus respectively.The stable transfected cell lines NP69-pLNSX, NP69-LMP1~(WT),NP69-LMP1~(TRADD)and NP69-LMP1~(Δ232-351)were observd and detected cellular morphology,cellular growth curve,colony formation and cell cycle.Meanwhile,2-DE coupled with Western Blot method were used to analysis of the differentially expressed tyrosine-phosphorylated proteins among NP69-pLNSX,NP69-LMP1~(WT), NP69-LMP1~(TRADD),NP69-LMP1~(Δ232-351)cell lines.After infected 24 hours, The total proteins of the four groups were separated by 2-DE,respectively. Each patch was separated in parallel on analytical as well as preparative 2-D gels.The preparative 2-D gels were stained by Goomassie Brilliant Blue.Well-resolved and reproducible 2-D preparative patterns of NP69-pLNSX,NP69-LMP1~(WT),NP69-LMP1~(TRADD),NP69-LMP1~(Δ232-351) cell lines were acquired.The analytical gels were electroblotted onto a nitrocellulose membrane and incubated with anti-phosphotyrosine antibody.After Western Blot analysis,2-D maps of differentially expressed tyrosine-phosphorylated proteins were acquired.After comparing the preparative maps with Western Blot maps,differentially tyrosine-phosphorylated proteins were found in corresponding preparative gels.Differentially tyrosine-phosphorylated proteins between the four cell lines were identified using MALDI-TOF-MS analysis and database searching.The differential expression levels of the partial proteins were determined by Western blot.
Result(1)The morphous of NP69-LMP1~(WT)cells gradually developed from a typical epithelial polygon,cobblestone to an elongated and fibroblastoid shape with a marked reduction in cell-cell contact.But NP69-LMP1~(TRADD)and NP69-LMP1~(Δ232-351)exhibited approximately polygonal morphous and grew in a more compact pattern with tighter cell-cell contact.The growth velocity and the proportion of S stage of NP69-LMP1~(WT)cells obviously increased than NP69-LMP1~(TRADD)、 NP69-LMP1~(Δ232-351)cells and NP69-pLNSX cells;(2)12 differentially tyrosine-phosphorylated proteins between NP69-pLNSX and NP69-LMP1~(WT)cell lines(up-regulation proteins as heat shock protein 27, vimentin ea al,down-regulation proteins as annexin A1,chaperonin cotaining TCP1 et al);11 differentially tyrosine-phosphorylated proteins between NP69-LMP1~(WT)and NP69-LMP1~(TRADD)cell lines;10 differentially tyrosine-phosphorylated proteins between NP69-LMP1~(TRADD)and NP69-LMP1~(Δ232-351)cell lines Most of them were characterized as cellular structure proteins,signal transduction and transcription and translation associated proteins.(3)Among these identified phosphoproteins,there were 10 proteins personificated the specificness of the C-terminal-activating regions of LMP1.For example, the expression of HSP27 in NP69-LMP1~(WT)and NP69-LMP1~(TRADD)cell lines was much more than NP69-pLNSX ones.However,there was no expression in NP69-LMP1~(Δ232-351)cell lines.This indicated the CTAR3 having the main contribution in this process.(4)In the transformed cells, 2 proteins were confirmed with immunoprecipitation on the level of protein expression.Both results were coincident with the results of the proteomics.
Conclusions(1)LMP1 promotes NP69 cell transforming with the up-regulation of heat shock protein 27,vimentin and CK7 and the down-regulation of annexin A1,chaperonin cotaining TCP1 and GTP binding protein et al.(2)The CTAR2 domain of LMP1 was pivotal participating in the above effects with the up-regulation of the expression phosphorylation of vimentin,ER-60,HSP70 annexin A1 and down-regulation of phosphorylation of phosphotidylethanolamin binding protein,proteasome et al to generate tumorigennesis contribution;(3)The CTAR3 domain of LMP1 was pivotal participating in the above effects with the up-regulation of the expression of phosphorylation of ER-60, cytoskeletal 8,HSP70 and down-regulation of phosphorylation of phosphotidylethanolamin binding protein,proteasome,annexin A2 et al to generate tumorigennesis contribution.
引文
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[2] Chi LM, Yu JS, Chang YS. Identification of protein kinase CK2 as a potent kinase of Epstein-Barr virus latent membrane protein 1. Biochemical and Biophysical Research Communications, 2002 294: 586-591.
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[4] Li HP, Chang YS. Epstein-Barr virus latent membrane protein 1: structure and function. J Biomed Sci, 2003 10: 490-504.
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[2] Chi LM, Yu JS, Chang YS. Identification of protein kinase CK2 as a potent kinase of Epstein-Barr virus latent membrane protein 1. Biochemical and Biophysical Research Communications, 2002 294: 586-591.
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[5] Yan GR, Luo W, Lu ZX, et al. Epstein-Bar virus latent membrane protein 1 mediates phosphorylation and nuclear translocation of annexin A2 by activating PKC pathway. Cell Signalling, 2007 19: 341-348.
[6] Imen N, Fanny BM, Christophe LC, et al. Latent membrane protein 1 regulates STAT1 through NF-KB-dependent interferon secretion in Epstein-Barr viru-immortalized B cell. Journal of Virology, 2005 4: 4936-4943.
[7] Zheng H, Li LL Hu DS, et al. Role of Epstein-Barr virus encoded latent membrane protein 1 in the carcinogenesis of nasopharyngeal carcinoma. Cell Molecular Immunology, 2007 4(3): 185-195.
[8] Micah 1, Efthimios P, Teruhito Y, et al. Epstein-Barr virus latent membrane protein 1 activation of NF-KB through IRAKI and TRAF6. Proc Natl Acad Sci USA, 2003 10: 15595-15600.
[9] Devergne O, Hatzivassiliou E, Izumi KM, et al. Association of TRAF1, TRAF2, and TRAF3 with an Epstein-Barr virus LMP1 domain important for B-lymphocyte transformation: role in NF-κB activation. Mol Cell Biol, 1996 16: 7098-7108.
[10] Wang C, Ai M, Ren W, et al. Epstein-Barr virus encoded latent membrane protein 1 induces TRAF1 expression to promote anti-apoptosis activity via NF-κB signaling pathway in nasopharyngeal carcinoma. Chin Med J, 2003 116: 1022-1028.
[11] Chrystalla TC, Adele H, Penelope T, et al. Latent membrane protein 1-induced EGFR signalling is negatively regulated by TGFa prior to neoplasia. Carcinogenesis, 2007 28(4): 1839-1848.
[12] Wang CX, Li XY, Gu HH, et al. Preliminarily study on the activation of TRAFs mediated by Epstein-Barr virus encoded LMP-1 in Nasopharyngeal Carcinoma. Virologica Sinica, 2001 16: 6-10.
[13] Yoon JS, Kai YJ, Vishal S, et al. IL-1 receptor-associated kinase 1 is critical for latent membrane protein 1 induced p65/RelA serine 536 phosphorylation and NF-KB activation. Proc Natl Acad Sci USA, 2005 12: 2689-2694.
[14] Liming W, Hiroyasu N, Zhengguo W. The C-terminal activating region 2 of the Epstein-Barr virus encoded latent membrane protein 1 activates NF-KB through TRAF6 and TAK1. The J of Biological Chemistry. 2005, 281(4): 2162-2169.
[15] David S, Chrystalla C, Joanna B. Epstein-Barr Virus Latent Membrane Protein 1 (CAO) Up-regulates VEGF and TGFA Concomitant with Hyperlasia, with Subsequent Up-regulation of p16 and MMP9. Cancer Res, 2005 65(19): 8826-8835.
[16] Zhao XR, Deng L, Weng XX, et al. Further study on cyclin D1 expression and function in human nasopgaryngeal carcinoma cell line. Zhonghua Zhong Liu ZaZhi. 2001 23: 373-375.
[17] Aristides GE, Lawrence SY. Activation of the cJun N-terminal kinase (JNK) pathway by the Epstein-Barr virus-encoded latent membrane protein 1 (LMP1). Oncogene 1998 16: 1731-1742.
[18] Jun W, Luguo S, Jennifer W, et al. Elucidation of the c-Jun N-Terminal Kinase Pathway Mediated by Epstein-Barr Virus-Encoded Latent Membrane Protein 1. Molecular and cellular biology, 2004 24(1): 192-199.
[19] Song X, Tao YG, Zeng L, et al. Latent membrane protein 1 encoded by Epstein-Barr virus modulates directly and synchronously cyclin D1 and p16 by newly forming a c-Jun/Jun B heterodimer in nasopharyngeal carcinoma cell line. Virus Res, 2005 113: 89-99.
[20] Aristides G, Elyse R, Sarah MS. TRAF1 is a critical regulator of JNK signaling by the TRAF-binding domain of the Epstein-Barr virus-encoded latent infection membrane protein 1 but not CD40. Journal of virology, 2003 77(2): 1316-1328.
[21] Noriyuki U, Taisuke K, Hideki S, et al. TAK1 is a component of the Epstein-Barr virus LMP1 complex and and is essential for activation of JNK but not of NF-KB. The J of Biological Chemistry, 2006 12: 7863-7872.
[22] Chen H, Hutt-Fletcher L, Cao L, Hayward SD. A positive autoregulatory loop of LMP1 expression and STAT activation in epithelial cells latently infected with Epstein-Barr virus. J Virol, 2003 77: 4139-4148.
[23] Kathy HY, Katherine M, Rachel H, et al. EBV Latent Membrane Protein 1 Activates Akt, NFjB, and Stat3 in B Cell Lymphomas. Plos Pathog 3(11): 0001-0015.
[24] Timothy RG, Jennifer MM. The Epstein-Barr Virus-Encoded LMP-1 Oncoprotein Negatively Affects Tyk2 Phosphorylation and Interferon Signaling in Human B Cells. Journal of virologr, 2006 80(23): 11638-11650.
[25] Maike B, Nicole H, Gerald N. Expression of Epstein-Barr virus (EBV)-encoded latent membrane proteins and STAT3 activation in nasopharyngeal carcinoma. Virchows Arch, 2006 449: 513-519
[26] Honglin C, Lindsey HF, Liang C, et al. A Positive Autoregulatory Loop of LMP1 Expression and STAT Activation in Epithelial Cells Latently Infected with Epstein-Barr Virus. Journal of virologr, 2003 77(7): 4139-4148.
[27] Guangrong Y, Wei L, Zhongxin L, et al. Epstein-Barr virus latent membrane protein 1 mediates phosphorylation and nuclear translocation of annexin A2 by activating PKC pathway. Cellular Signalling, 2007 19: 341-348.
[28] Lili L, Lili G, Yongguang T, et al. Latent membrane protein 1 of Epstein-Barr virus regulates p53 phosphorylation through MAP kinases. Cancer Letters, 2007 255:219-231.
[29] Nakayama J, Tahara H, Tahara E, et al. Telomerase activation by hTRT in human normal fibroblasts and hepatocellular carcinomas. Nat Genet. 1998 18: 65-68.
[30] Ding L, Li LL, Yang J, et al. Epstein-Barr virus encoded latent membrane protein 1 modulates nuclear translocation of telomerase reverse transcriptase protein by activating nuclear factor-κB p65 in human nasopharyngeal carcinoma cells. Int J Biochem Cell Biol. 2005 37: 1881-1889.
[31] Fqing T, Zhi H, Liqun Y, et al. Epstein-Barr virus LMP1 initiates cell proliferation and apoptosis inhibition via regulating expression of Survivin in nasopharyngeal carcinoma. Exp Oncol. 2005 27: 96-101.
[32] Tang F, Wang H, Yin L, et al. Epstein-Barr virus LMP1 triggers the expression of apoptosis protein survivin via NFkB and AP-1 signaling pathway in nasopharyngeal carcinoma. Exp Oncol. 2003 25: 28-32.
[33] Brendan ND, Leonard C. Edelstein PM, et al. Nuclear Factor-B-Dependent Activation of the Antiapoptotic blf-1 Gene by the Epstein-Barr Virus Latent Membrane Protein 1 and Activated CD40 Receptor. Journal of virologr, 2004 78(4): 1800-1816.
[34] Ngan L, Mark LS, Bill S. High Physiological Levels of LMP1 Result in Phosphorylation of eIF2 in Epstein-Barr Virus-Infected Cells. Journal of virologr, 2004 78(4): 1657-1664.
[35] Wu HY, Tseng VS, Liao PC. Mining phosphopeptide signals in liquid chromatography-mass spectrometry data for protein phosphorylation analysis. J Proteome Res, 2007 6(5): 1812-1821.
[36] Roberto R, Stefano G, Georg CT. Phosphoproteome Analysis. Bioscience Reports, 2005,25(1/2): 30-44。
[37] Wu HY, Tseng VS, Liao PC. Mining phosphopeptide signals in liquid chromatography-mass spectrometry data for protein phosphorylation analysis. J Proteome Res,2007,6(5):1812-1821.
[38]Shiaw LW,Jeongkwon K,Russell W,et al.Dynamic profiling of the post-translational modification and interaction partners of epidermal growth factor receptor signaling after stimulation by epidermal growth factor using extended range proteomics analysis(ERPA).Mol Cell proteomics,2006,5(9):1610-1627.
[39]Feng S,Ye M,Zhou H,et al.Immobilized zirconium ion affinity chromatography for specific enrichment of phosphopeptides in phosphoproteome analysis.Mol Cell Proteomics,2007,6(9):1656-65
[40]Gronborg M,Kristiansen TZ,Stensballe A,et al.A mass spectrometry-based proteomic approach for identification of serine/threonine-phosphorylated proteins by enrichment with phosphospecific antibodies:identification of a novel protein,frigg,as a protein kinase A substrate.Mol Cell Proteomics,2002,1(7):517-527.
[41]张琼.EB病毒潜伏性膜蛋白1转化鼻咽上皮细胞的蛋白质组学研究.硕士学位论文,2007.
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