Research On The Mechanism By Which EB Virus Encoded LMP1 Regulates Op18/Stathmin Signaling

The theory on the regulation of signaling transduction and cell cycle considers that the aberrant activation of signaling pathway of regulated network can induce tumorigenesis,and the activation of various oncogene and oncoprotein signaling pathways which leads to the disruption of cell cycle check points and the preferred growth of cancer cells.Epstein-Barr virus(EBV)is closely associated with nasopharyngeal carcinoma(NPC),EBV infection is mainly characterized by the expression of typeⅡlatent proteins including latent membrane proteins (LMP)1,2.Of which LMP1 is an EBV encoded oncogenic protein that is able to induce tumorigenesis such as promoting cellular transformation of B lymphocytes and epithelial cells,and immortalized cells to neoplastic cells.Previous studies on LMP1 regulation demonstrated that EBV-encoded LMP1 abnormally activated nuclear factor-kappa B (NF-κB),activator protein 1(AP-1),and signal transducer and activator of transcription(STAT)signaling pathways by phosphorylating epidermal growth factor receptor(EGFR),c-Jun N-terminal kinase (JNK),Janus kinase(JAK),and other moleculars.Activation of these signaling pathways by LMP1 has been linked to cell cycle progression, to cell growth and proliferation,but the downstream target proteins regulated by LMP1 have not been thoroughly identified.Using phosphate metal affinity chromatography(PMAC)to enrich phosphoproteins,combined with matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry(MALDI-TOF-MS)analysis, this study showed that phosphorylation levels of 25 novel proteins for LMP1 expression were changed obviously,including Op18/stathmin, which are possible downstream target molecules of LMP1.On the basis of these results,a suppositional signaling network for LMP1 regulation was established through prediction via bioinformatics.This study focused on Op18/stathmin,a small-molecule phosphoprotein in the complicated suppositional signaling network regulated by LMP1.Op18/stathmin is genetically a highly conserved, small cytosolic phosphoprotein,which is expressed at high levels in tumors such as leukemia and breast carcinomas,so also named oncoprotein 18.Op18/stathmin has been hypothesized to relay the integration of diverse cell signals to regulate microtubule dynamics. Microtubule dynamic equilibrium is crucial to cell morphological stabilization,substance transportation,cell division and proliferation, tumor migration and invasion,and so on.Similarly,Op18/stathmin, as a main regulator of microtubule dynamics,plays a significant role in maintaining cell biological characteristics,the phosphorylation/ dephosphorylation of Op18/stathmin is tightly linked to cell cycle progression.The suppositional signaling network on LMP1 regulation suggests that LMP1 maybe regulate Op18/stathmin signaling pathway by MAPK or cdc2 mediation.MAPK is a type of Ser/Thr kinases which can combine with wild type or mutant Op18/stathmin.MAPK family mainly consists of three subfamilies,extracelluar signal regulated protein kinase(Erk),Jun N-terminus kinase(JNK)and p38.Studies had testified that Op18/stathmin is a good substrate of MAPK for both Set25 and Set38 of Op18/stathmin's phosphorylation sites with a proline residue.MAPK prefers to phosphorylate Ser25 and Ser38 of Op18/stathmin.Activated p38,Erk,JNK all can phosphorylate Op18/stathmin at Ser25/Ser38.Cdc2 is a crucial kinase to start M phase event during cell cycle progression,and a positive regulator promoting cell cycle processes. Cdc2 activation depends on cyclin B1 production,accumulation, degradation and cell cycle progression.It is confirmed that cdc2 can directly phosphorylate Ser25 and Ser38 of Op18/stathmin,the expression of cdc2 kinase target site-deficient mutant of Op18/stathmin (Op18-Ser25A,38A)resulted in rapid accumulation of cells at G2 phase of cell cycle,and a serious defect of mitotic chromosome segregation.This study focused on these two new pathways of cdc2 and MAPK mediation via which LMP1 regulates Op18/stathmin signaling.These established pathways not only perfect the LMP1 regulation network,but so provide new insight elucidating the molecular mechanism of LMP1 that leads to carcinogenesis.1.Effects of LMP1 regulation on the expression and the phosphorylation of Op18/stathminCNE1 is an LMP1-negative poorly differentiated NPC cell line,the CNE1-LMP1 cell line stably expressing LMP1 was established in our laboratory;Tet-on-LMP1 HNE2 is a NPC cell line in which the expression of LMP1 would be turned on by Doxycline;NP69 series cell lines including NP69,NP69-PLNSX and NP69-LMP1 cells derived from a newly established,SV40T-immortalized nasopharyngeal(NP) cells,of which NP69-LMP1 is a NP cell line expressing LMP1. Western-blot analysis showed that Op18/stathmin expression was not affected by LMP1 among CNE1,CNE1-LMP1 and NP69 series cell lines.Tet-on-LMP1 HNE2 cells,in which the expression of LMP1 was increased with Doxycline addition in a dose-dependent manner,western blot analysis performed in Tet-on-LMP1 HNE2 cells treated with Doxycline at different dose and time points showed that the expression of Op18/stathmin was not affected with LMP1 induction in time and dose-dependent manners.This study also demonstrated that LMP1 enhanced the phosphorylation of Op18/stathmin between CNE1 and CNE1-LMP1 cells.The level of phosphorylation of Op18/stathmin was also markedly up-regulated by LMP1 in the Tet-on-LMP1 cells treated with Doxycline.Analysis of the status of Op18/stathmin phosphorylation of NP69 series cells,which derived from immortalized nasopharyngeal (NP)cells,showed that the change of phosphorylation of Op18/stathmin was not obvious by LMP1 induction.All these results demonstrated that LMP1 could regulate Op18/stathmin signaling through promoting the phosphorylation of Op18/stathmin,but the mechanism by which LMP1 regulates Op18/stathmin signaling may be different between NPC cells and normal epithelial cells.2.Regulation of MAPK mediated Op18/stathmin signaling pathway triggered by EBV encoded LMP1 in nasopharyngeal carcinoma cellsTo testify whether LMP1 can regulate Op18/stathmin signaling by MAPK mediation,we still chose CNE1 and CNE1-LMP1 cell models, took multiple methods including cell synchronization with colcemid, flow cytometric analysis,CO-IP-Western-blot and Extraction of solubilized and polymerized microtubulin to examine the change of MAPK activity,the interaction of MAPK with Op18/stathmin,the status of microtubule dynamics and the correlativity of cell cycle progression for LMP1.FACS analysis showed that the majority of cells untreated with cocemid stayed at G1/S.LMP1 enhanced the levels of the phosphorylation of p38,ERK,JNK/MAPK,of which the change of phosphorylation of ERK/MAPK was the most obvious when these cells stayed at G1/S phase;On the contrary,LMP1 negatively regulated the phosphorylation of MAPK in these cells stayed at G2/M phase. Similarly,the down-regulation of the levels of ERK/MAPK phosphorylation was the most obvious in MAPK family.The activity of Op18/stathmin destabilizing microtubules was analyzed,the recombinant carrier targeted Op18/stathmin was constructed to study the status of microtubule dynamics when the experession of Op18/stathmin was inhibited.This study showed that RNAi effectively inhibited the transcription and expression of Op18/stathmin,resulted in the decrease of levels of solublized microtubulin.All evidences indicated that Op18/stathmin can regulate microtubule dynamics and promotes microtubule depolymerization in NPC.Inhibition of LMP1 expression by LMP1-specific DNAzyme DZ1 attenuated the phosphorylation of MAPK and the interaction of MAPK with Op18/stathmin,and the levels of the polymerized tubulin was markedly down-regulated,which was accordant with the regulation of Op18/stathmin dephosphorylation.When PD98059 selectively blocked the ERK/MAPK signaling in CNE1-LMP1 cells,the phosphorylation of Op18/stathmin was weakened with the ERK/MAPK signaling repression,and the solublized tubulin was increased.These results indicated that LMP1 positively regulates Op18/stathmin signaling by MAPK mediation at G1/S phase,but at G2/M phase,LMP1 negatively regulates Op18/stathmin signaling by MAPK mediation;LMP1 mainly depends on ERK/MAPK pathway to regulate Op18/stathmin signaling in MAPK family;There are multiple possibilities on the mechanism by which LMP1 negatively regulates Op18/stathmin signaling,such as cdc2,p53 involved in.3.LMP1 regulates Op18/stathmin signaling pathway by cdc2 mediation at G2/M phase in nasopharyngeal carcinoma cellsThe signaling of Op18/stathmin is closely associated with cell cycle progression,and cdc2 is a positive regulation factor of cell cycle.We inspected the activity change of cdc2 induced by LMP1 at M phase and the possibility that LMP1 regulates Op18/stathmin signaling by cdc2 mediation.By these ways such as extraction of solublized and polymerized microtubulin,cell synchronization,cdc2 kinase activity assay, co-immunoprecipitation analysis and immunofluorescence staining, studies showed that LMP1 didn't affect the expression of cdc2 and Op18/stathmin whether at different phase;LMP1 upregulated the levels of phosphorylation of cdc2 on threonine 161 and cdc2 kinase activity, the levels of phosphorylated Op18/stathmin was greatly enhanced at M phase related to G1/S phase.Inhibition LMP1 expression by LMP1-specific DNAzyme DZ1 attenuated the interaction of cdc2 with Op18/stathmin and promoted microtubule depolymerization,cdc2 kinase activity was also weakened;the inhibitor of CDK1 Purvalanol A effectively inhibited cdc2 kinase activation and the phosphorylation of Op18/stathmin,and promoted microtubule depolymerization.All these results indicated that LMP1 regulates cdc2 signaling by cdc2 mediation at M phase. Summary:Using stratges including cell synchronization,solublized microtubulin and polymerized microtubulin extraction, immunofluorescence staining,CO-IP-Western-blot analysis and signaling blockage to explore the regulation of Op18/stathmin signaling induced by LMP1,we gained some innovative findings as follows:1)Studies firstly demonstrated that LMP1 doesn't affect the expression of Op18/stathmin,but enhanced the phosphorylation of Op18/stathmin;the status of Op18/stathmin phosphorylation of NP69 series cell lines which derived from immortalized nasopharyngeal(NP) cells showed that the change of phosphorylation of Op18/stathmin was not obvious by LMP1 induction,which indicated that the mechanism by which LMP1 regulates Op18/stathmin signaling in NPC isn't the same with normal tissues.2)It is first to find that the activity of MAPK induced by LMP1 is associated with cell cyle progression.At G1/S phase,LMP1 positively regulates the activation of MAPK,on the contrary,negatively regulates MAPK activity at G2/M phase;ERK/MAPK is the mainly pathways regulated by LMP1 in MAPK family.RNAi showed that Op18/stathmin regulated microtubule dynamics and promoted microtubules depolymerization in NPC.LMP1 promoted the interaction of MAPK with Op18/stathmin and microtubule polymerization at G1/S phase.LMP1 regulated Op18/stathmin signaling by MAPK mediation mainly acts to keep interphase microtubule stable.3)It is first to demonstrate that LMP1 regulates Op18/stathmin signaling by cdc2 mediation at mitotic phase.LMP1 up-regulated the phosphorylation of cdc2 on threonine161 and cdc2 kinase activity,but doesn't change the expression of cdc2.LMP1 promotes the phosphorylation of op18/stathmin and spindle assembly,enhances the interaction cdc2 with Op18/stathmin,cdc2 mediated Op18/stathmin signaling regulated by LMP1 mainly functions at mitotic phase,which is associated with mitotic spindles assembly.All above provide experimental proof on LMP1 regulated Op18/stathmin signaling by cdc2 and MAPK mediation.The regulation influences the equilibria of microtubule dynamics which is associated with cell cycle,and is characterized by time-phase speciality.The mechanism by which LMP1 regulates Op18/stathmin signaling is different at different phase.At G1/S phase,LMP positively regulates Op18/stathmin signaling by MAPK mediation,which mainly keep interphase microtubule stable;At M phase,LMP1 up-regulates cdc2 kinase activity to regulate Op18/stathmin signaling,which mainly promotes spindle formation.These results reveal two new pathways via which LMP1 regulates Op18/stathmin signaling by cdc2 and MAPK mediation.These new pathways not only perfect the LMP1 regulation network,but also provide new insights elucidating the molecular mechanism of LMP1 that leads to tumorigenesis.