Epstein-Barr Virus Encoded Latent Membrane Protein 1 Mediated The Serine Phosphorylation Of Annexin A2 Via PKC

Epstein-Barr virus (EBV) has been implicated in the pathogenesis ofseveral human malignancies, such as Burkett's and Hodgkin's as well asepithelial tumors, including nasopharyngeal carcinoma (NPC), mammarycarcinoma and gastric carcinoma. EBV-encoded latent membrane protein1 (LMP 1) was considered to be an oncoprotein, and played an importantrole in cancer pathogenesis by analyzing its genome. Three distinctfunctional domains have been identified within the C-terminal regions ofLMP1: C-terminus activating region 1, 2 and 3 (CTAR1, CTAR2 andCTAR3), which are known to constitutively activate nuclear factor kappaB (NFκB), activator protein-1 (AP-1), signal transducer and activators oftranscription (STAT) pathways by recruiting tumor necrosis factorsreceptor associated factors (TRAFs), tumor necrosis factors receptorassociated death domain (TRADD) and Janus kinase 3 (JAK3). Thesetranscription factors induce the expression and phosphorylation ofdownstream target gene, such as EGFR, cyclinD1, MMP9, survivin andso on.On the basis of previous research on a single signaling transductionpathway in NPC cells activated by Epstein-Barr-virus-encoded latentmembrane protein 1 (LMP1), our group found 25 new phosphoproteinsmediated by LMP1 and set up a signaling pathway matrix, using newtechnologies, including signalomics, 2DE and MALDT-TOF-MS. Wehave elucidated that LMP1 can increase the serine phosphorylation levelof annexin A2 by activating the protein kinase C (PKC) signalingpathway and that LMP1 induces the nuclear entry of annexin A2 in anenergy- and temperature-dependent manner.PKC is an important cellular Calcium- and Phospholipid- dependentserine/threonine protein kinase. Different isoforms of PKC locate indifferent parts of cells and demonstrate diverse biological functions.Annexin A2 is a member of Calcium-dependent Annexins family, whichis closely related to cell transformation and incidence of tumor. Theexpression and serine phosphorylation level of annexin A2 are increasedin most tumor cells. Annexin A2 exists as both 36kD monomer and 94kD heterotetramer, which consists of two molecules of annexin A2 and twomolecules of the p11 protein in cells.PKC is a newly discovered protein kinase mediated by LMP1, aswell as NFκB, JNK, JAK/STATs, MAPK and PI3K/Akt. Annexin A2 isthe downstream protein of PKC, whose phosphorylation is regarded as avery important factor of cell malignant transformation. In endothelialNPC cells, we continued our study on molecular mechanism andbiological function of phosphorylation of annexin A2 mediated by LMP1,in order to perfect this new signaling pathway. We chose LMP1-negativeCNE1 and LMP1 stably-expressing CNE1-LMP1 NPC cells as ourexperimental materials.We detected the specific PKC kinase activity of different cells usingPKC Kinase Activity Assay Kit (Non-Radioactive, Stressgen), and theserine phosphorylation of annexin A2 by IP-Western blotting usinganti-serine antibody. The data showed that the change trend of PKCkinase activity was consistent with the serine phosphorylation level ofannexin A2, with PKC activator TPA, DNAzyme Dz1 which can decreasethe expression of LMP1, PKC specific inhibitor. Thus we furtherconfirmed the existence of this new signaling pathway.PKC belongs to the "IP3 and DAG system".Phosphoinositide-specific phospholipase C (PI-PLC) is the key upstreamactivator of PKC, whose most important isoforms are PLC-βand PLC-γ.We used the specific inhibitor U73122 of PLC-βand PLC-γ, whoseinactive analog U73343 and vehicle DMSO as positive controls, to studythe influence of PI-PLC on PKC kinase activity and the serinephosphorylation of annexin A2. We found that the PI-PLC specificinhibitor U73122 can decrease cellular PKC kinase activity and the serinephosphorylation level of annexin A2. We confirmed that LMP1 mediatesthe PKC kinase activity and the serine phosphorylation of annexin A2 viaPI-PLC.We previously demonstrated that LMP1 mediated the serinephosphorylation of annexin A2 mainly via PKCαand PKCβ, using aspecific inhibitor of PKCαand PKCβ. We further study on therelationship of PKC isoforms and the serine phosphorylation of annexin A2, and found that both PKCαand PKCβcan bind directly to annexin A2.In addition, LMP1 mediates the serine phosphorylation of annexin A2 viaPKCαand PKCβ, mainly through the activation of PKCβ.Then we treated CNE1 and CNE1-LMP1 cells transfected withPKCαshRNA and PKCβshRNA plasmids, which were designed todecrease the expression of PKCαand PKCβspecifically and efficiently,and found that PKCαshRNA and PKCβshRNA can efficiently decreasethe serine phosphorylation level of annexin A2 in both cell lines. So bothPKCαand PKCβcan mediate the serine phosphorylation of annexin A2.In order to confirm this result, we took the protein kinase phosphorylationreaction between active PKC and purified GST-annexin A2, and revealedthat all active PKCα, PKCβⅠand PKCβⅡcan serine-phosphorylateannexin A2 in vitro.We previously found that LMP1 mediated the serine phosphorylationand nuclear entry of annexin A2. The N-terminus of annexin A2 containsserine 11 and 25 sites which were demonstrated to be phosphorylated byPKC. To investigate the nucleo-cytoplasmic shuttling mechanism ofannexin A2, we constructed the single mutants pEGFP-AnxⅡS11E andS25E and the double mutant S11ES25E. These mutants can generateexpression of monomer EGFP-Annexin A2 fusion protein with a single ordouble serine amino acid mutated to glutamic acid (E) to mimic thenegative charge of a phosphate group, so that annexin A2 cannot bephosphorylated by PKC on serine 11 or serine 25. The confocal resultsshowed that the phosphorylation of serine 25 was associated with thenuclear entry of annexin A2. Then we confirmed this conclusion bycytoplasmic and nuclear Western-blotting, the aim of which is toquantitatively detect cellular distribution of EGFP-Annexin A2.We have demonstrated that PKCαshRNA and PKCβshRNAplasmids could specifically decrease the expression of PKCαand PKCβ,moreover, the serine phosphorylation level of annexin A2, and that onlythe expressing protein of pEGFP-AnxⅡWT and S11E plasmids couldenter the nucleus. The cytoplasmic and nuclear protein fromCNE1-LMP1 cells transfected with WT and/or S11E plasmid and PKCαshRNA and/or PKCβshRNA plasmid, was compared with the cytoplasmic and nuclear protein from CNE1-LMP1 cells transfected withonly WT and/or S11E plasmid. The data showed that PKCαshRNA andPKCβshRNA plasmids could decrease the nuclear entry of expressingprotein of pEGFP-AnxⅡWT and S11E plasmids, and that the serinephosphorylation of annexin A2 is associated with its nuclear entry. It issuggested that serine 25 phosphorylation of annexin A2 is associated withits nuclear entry mediated by PKCαand PKCβ.Phosphorylated annexin A2 enters the nucleus, and then promotes theactivity of DNA polymerasα, resulting in promotion of the DNAsynthesis and cell proliferation. We found that the proliferation rate ofcells transfected with pEGFP-AnXⅡWT or S11E plasmid was obviouslyhigher than that of cells transfected with S25E or S11ES25E plasmid,using CellTiter-Glo(?) Luminescent Cell Viability Assay (Promega). It issuggested that annexin A2 cannot enter the nucleus and present itsbiological function, for serine 25 site was mutated. So the serine 25phosphorylation of annexin A2 is of great importance in its biologicalfunction.Serine 25 phosphorylation of annexin A2 is closely related with itsnuclear entry, whereas serine 11 phosphorylation has no obviousinfluence. Many researchers have figured out that heterotetramer annexinA2 is phosphorylated by PKC, then dissociats from cell membrane andbecomes self-dissociation. This dissociation may be decided by serine 11phosphorylation of annexin A2, because serine 11 locates at the bindingsite of p11 (residues 1-13 of annexin A2). Whether LMP1 mediates serine11 phosphorylation of annexin A2 via PKCαand/or PKCβ, or modulatesdissociation of the heterotetramer annexin A2 and its release from cellmembrane, needs our further study.SUMMARY. Firstly, we have confirmed the existence of this newsignaling pathway, i.e., "LMP1→PI-PLC→PKC→p-ser-Annexin A2".Then we further revealed that LMP 1 increases the serine phosphorylationlevel of annexin A2 by activating the phosphoinositide-specificphospholipase C (PI-PLC)-PKCα/PKCβpathway, mainly through theactivation of the PKCβpathway. Additionally, active recombinant PKCα, PKCβⅠ, and PKCβⅡkinases are able to phosphorylate annexin A2 in vitro.Annexin A2 in the nucleus plays an important role in DNA synthesis andcell proliferation. By site-specific substitution of glutamic acid in theplace of serine 11 and 25 in N-terminus, we showed that serine 25phosphorylation of annexin A2 was associated with the nuclear entry ofannexin A2 and cell proliferation, whereas serine 11 has no obviousinfluence. It is the first demonstration of the great impotance of serine 25phosphorylation of annexin A2.We confirmed this new signaling pathway, i.e.,"LMP1→PI-PLC→PKCα/PKCβ→p-ser25-Annexin A2→the nuclearentry of Annexin A2→promote DNA synthesis and cell proliferation".We found out two new protein kinases mediated by LMP1, i.e., PI-PLCand PKC. We confirmed that LMP1 modulates the serine 25phosphorylation and nuclear entry of annexin A2 viaPI-PLC-PKCα/PKCβand influences the function of annexin A2 on cellproliferation. In conclusion, we provide new experimental evidence of theeffect of LMP1 on cell proliferation and its oncogenic function.