Latent Membrane Protein 1 Encoded By Epstein-Barr Virus Regulates P53 Function

Nasopharyngeal carcinoma (NPC) has a remarkably distinctive ethnic andgeographic distribution, more than 80% of which were reported from China,Southeast Asia, and some other Asian countries. A unique feature of NPC is itsstrong association with Epstein-Barr Virus (EBV). Latent membrane protein 1(LMP1) is the only one with oncogenic properties among EBV encodedproteins. Our previous studies have been focused on signaling transductionpathways mediated by LMP1. Based on the LMP1-inducible expressionsystem, we elucidated that LMP1 was involved in multiple biologicalfunctions, such as cell proliferation, apoptosis, invasion and metastasis,through initiating NF-κB, JNK/AP-1 and STAT signaling pathwayThe p53 tumor suppressor becomes stabilized and activated in response todiverse cellular stresses, such as DNA damage, hypoxia and oncogeneactivation. Activation of p53 can cause either a cell cycle arrest or apoptosis,both at which are largely mediated by activation of p53-responsive targetgenes such as MDM2, p21. Over half of human tumors are associated withp53 mutations, indicating its pivotal role as a tumor suppressor protein.Oncogenic viral proteins such as large T antigen of papovaviruses, adenovirusE1B protein, hepatitis C virus core protein and hepatitis B virus X protein, have been shown to interact with p53 and alter its functions through distinctmechanism. But in NPC, unlike most human tumors, nearly 100% are wildtype for the p53 tumor suppressor. Analysis of the expression of the p53protein by immunohistochemistry on NPC biopsies indicates that p53accumulation is significantly correlated to LMP1. Overexpression of p53seems to occur at an early stage in the development of NPC and associatedwith advanced disease stage, poor response to therapy. In LMP1 transgenicmice, p53 protein is detected at high level in the transgenic lymphomas and nomutation is found. These data suggest that p53 has the functional activation inthe carcinogenesis of NPC.p53 mutation is frequently observed in the NPC cell lines, which suggests thatmutation of the p53 gene may confer growth advantage to the tumor cells tobe established in culture. So choosing immortalized normal nasopharyngealcell line with no p53 mutation will provide a good model to study theregulation of LMP1 to p53 in the carcinogenesis of NPC. With collaborationwith Prof. Tsao in Hong Kong University, we introduced the immortalizednasopharyngeal epithelial cell model: NP69, NP69 transfected with pLNSXvector, and NP69 transfected with LMP1. We firstly confirmed that there werelots of free, wide-type p53 in immortalized NP69 cell model. Our data showedthat LMP1 could promote p53 nuclear accumulation, up-regulatetranscriptional activity and the expression of p53. These results provide a good model for us to study the function of p53 in the carcinogenesis of NPC.p53 is regulated by post-translational modifications including phosphorylation,acetylation, sumoylation and ubiquitination events that contribute both tostabilizing p53 and to conversing p53 from a latent to an active transcriptionfactor. Among them, phosphorylation has been studied most intensively andproposed to play a critical role in the stabilization and activation of p53. Butthe precise mechanisms of this signaling and its regulation by LMP1 are notwell understood. The Mitogen-Activated Protein Kinase (MAPK) pathwaystransduce a variety of external signals, leading to a wide range of cellularresponses, including growth, differentiation, inflammation and apoptosis.Previously, we and others found that LMP1 caused activation of MAP kinases.These data led us to test whether p53 may be a target for phosphorylation byMAP kinases, which will be the first time to elucidate the activation of p53modulated by LMP1 from post-translational level.Interestingly, we found that p53 could be activated and phosphorylated clearlyat serine 15, serine 20, serine 392 and threonine 81 induced by LMP1. Theevidence that, LMP1-induced phosphorylation of p53 at serine 15 was directlyby ERKs; at serine 20 and threonine 81 by JNK; at serine 392 and Ser 15 byp38 kinase, was provided. MAP kinases play a critical role in LMP1-inducedphosphorylation of p53 at multiple sites, and that JNK signaling pathway mediated p53 Ser 20 phosphorylation is more important than others. Theinduction of p53 transcription activity, trans-activation and stability by LMP1depends on the activities of MAP kinases and their phosphorylation of p53.These results strongly suggest that MAP kinases play a direct role inLMP1-induced phosphorylation of p53 at multiple sites, which provide anovel view for us to understand the mechanism of the activation of p53 in thecarcinogenesis of NPC.Ubiquitination is an emerging mechanism implicated in a variety ofnonproteolytic cellular functions. Protein ubiquitination exhibits inducibility,reversibility and recognition by specialized domains, which closely associatedwith protein phosphorylation and enable Ub to act as a signaling deviceinvolving in signaling events. The role of different linkages in polyubiquitinshas begun to be elucidated in recent years. Linkage through Lys 48 (Ubk48) ismainly used for targeting to the proteasome, and Lys 63 (Ubk63) linkagesseem to play important roles in DNA damage tolerance, inflammatoryresponse, the endocytic pathway, and ribosomal protein synthesis.Previous data showed that LMP1 decreased the binding of MDM2 to p53, so itis possible that LMP1 regulate p53 function via ubiquitination. Our datasuggested that LMP1 could up-regulate p53 phosphorylation via MPAKsignaling cascades, and induced Ub molecular Ubk63 to bind with p53, which was corrected with the p53 stability and activation. Among them, theregulation of p53 Ser20 via JNK pathway was more important than others. Wealso found that the phosphorylation of p53 inhibited the combination of p53 toUb molecular Ubk48, involving in the p53 stability. Thus, the data show thatUb molecular Ubk48 and Ubk63 could bind p53 to play different function inthe carcinogenesis of NPC.In summary, our study has indicated that EB virus encoded oncogenic proteinLMP1 can regulate p53 constitutive phosphorylation; confirm the key kinaseof p53 phosphorylation modulated by LMP1—MAP Kinase cascades, providethe work model, our work suggest that p53 is an important transcriptionalfactor, its activation play a key role in maintaining EBV latent infection,promote cell transformation and regulate of cell cycle. Furthermore, we haveestablished the relationship among LMP1, MAP Kinase and p53; testified thatp53 phosphorylation is the important modification manner in inducing p53transcriptional activity, transactivation and stability. Moreover, we stated thatLMP1 mediates the Ubk63 binding to p53 and inhibit Ubk48 combining withp53, which is involved in the biological function of p53.Therefore, this study elucidate the regulation of EB virus LMP1 to p53 frompost-translational level—phosphorylation and ubiquitination, which providedus with a novel view of comprehensive understanding in p53 function activated by LMP1 and the oncogenic function of LMP1 in the carincogenesisof NPC, which will lead to the identification of novel targets for drugdiscovery and provide a basis for gene therapy.