Phosphoproteomcis Analysis Of Transformation Potential Of The Epstein-Barr Virus-encoded Latent Membrane Protein 1 In Nasopharyngeal Epithelial Cells NP69

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^TRADDand pLNSX-LMP1^Δ232-351were 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^TRADDand 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^TRADDand NP69-LMP1^Δ232-351were 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^Δ232-351cell 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^WTcells 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^TRADDand NP69-LMP1^Δ232-351exhibited 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^WTcells obviously increased than NP69-LMP1^TRADD, NP69-LMP1^Δ232-351cells and NP69-pLNSX cells;(2)12 differentially tyrosine-phosphorylated proteins between NP69-pLNSX and NP69-LMP1^WTcell 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^WTand NP69-LMP1^TRADDcell lines;10 differentially tyrosine-phosphorylated proteins between NP69-LMP1^TRADDand NP69-LMP1^Δ232-351cell 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^WTand NP69-LMP1^TRADDcell lines was much more than NP69-pLNSX ones.However,there was no expression in NP69-LMP1^Δ232-351cell 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.