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Epstein-Barr Virus Latent Membrane Protein 1 (LMP1) Activates the Phosphatidylinositol 3-Kinase/Akt Pathway to Promote Cell Survival and Induce Actin Filament Remodeling*

  • Christopher W. Dawson
    Affiliations
    Cancer Research UK Institute for Cancer Studies, The University of Birmingham Medical School, Birmingham B15 2TT, United Kingdom
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  • Giorgos Tramountanis
    Affiliations
    Cancer Research UK Institute for Cancer Studies, The University of Birmingham Medical School, Birmingham B15 2TT, United Kingdom
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  • Aristides G. Eliopoulos
    Affiliations
    Cancer Research UK Institute for Cancer Studies, The University of Birmingham Medical School, Birmingham B15 2TT, United Kingdom
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  • Lawrence S. Young
    Correspondence
    To whom correspondence should be addressed
    Affiliations
    Cancer Research UK Institute for Cancer Studies, The University of Birmingham Medical School, Birmingham B15 2TT, United Kingdom
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  • Author Footnotes
    * This work was supported by a grant from Cancer Research UK and by a Medical Research Council Career Development Award (to A. G. E.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Open AccessPublished:November 21, 2002DOI:https://doi.org/10.1074/jbc.M209840200
      The Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) is an integral membrane protein that functions as a constitutively activated member of the tumor necrosis factor receptor family. Whereas LMP1 has been shown to activate the NF-κB and mitogen-activated protein kinase pathways, these effects alone are unable to account for the profound oncogenic properties of LMP1. Here we show that LMP1 can activate phosphatidylinositol 3-kinase (PI3K), a lipid kinase responsible for activating a diverse range of cellular processes in response to extracellular stimuli. LMP1 was found to stimulate PI3K activity inducing phosphorylation and subsequent activation of Akt, a downstream target of PI3K responsible for promoting cell survival. Treatment of LMP1-expressing cells with the PI3K inhibitor LY294002 resulted in decreased cell survival. The tumor necrosis factor receptor-associated factor-binding domain of LMP1 was found to be responsible for PI3K activation. The ability of LMP1 to induce actin stress-fiber formation, a Rho GTPase-mediated phenomenon, was also dependent on PI3K activation. These data implicate PI3K activation in many of the LMP1-induced phenotypic effects associated with transformation and suggests that this pathway contributes both to the oncogenicity of this molecule and its role in the establishment of persistent EBV infection.
      EBV
      Epstein-Barr virus
      PI3K
      phosphatidylinositol 3-kinase
      NPC
      nasopharyngeal carcinoma
      TNFR
      tumor necrosis factor receptor
      TRAF
      tumor necrosis factor receptor-associated factors
      GFP
      green fluorescent protein
      TRITC
      tetramethylrhodamine isothiocyanate
      Epstein-Barr virus (EBV)1 is a ubiquitous human herpesvirus associated with the development of both lymphoid and epithelial tumors. As a common virus infection, EBV appears to have evolved to exploit the process of B cell development to persist as a lifelong asymptomatic infection. However, the virus can contribute to oncogenesis as evidenced by its frequent detection in certain tumors, namely Burkitt's lymphoma, post-transplant B cell lymphomas, Hodgkin's disease, and nasopharyngeal carcinoma (NPC), and by its unique ability to efficiently transform resting B cells in vitro into permanently growing lymphoblastoid cell lines (
      • Rickinson A.B.
      • Kieff E.
      ). These transforming effects are associated with the restricted expression of EBV genes such that only a subset of so-called latent virus proteins are expressed in virus-infected tumors and in lymphoblastoid cell lines. The full complement of eight latent genes comprising six nuclear antigens (EBNAs) and two membrane proteins (LMP1 and LMP2) are expressed only in post-transplant B cell tumors and in lymphoblastoid cell lines whereas different forms of latency are manifest in Burkitt's lymphoma (EBNA1 only) and in Hodgkin's disease and NPC (EBNA1, LMP1, and LMP2). These distinct forms of EBV latency appear to be a vestige of the pattern of latent gene expression used by the virus during the establishment of persistent infection within the B cell pool (
      • Thorley-Lawson D.A.
      ). Key to the ability of EBV to efficiently colonize memory B cells is the expression of LMP1 and LMP2 both of which provide essential survival signals. The aberrant adoption of these forms of latency can contribute to transformation as evidenced by expression of LMP1 and LMP2 in NPC and Hodgkin's disease.
      LMP1 is the major transforming protein of EBV behaving as a classical oncogene in rodent fibroblast transformation assays and being essential for EBV-induced B cell transformation in vitro (
      • Wang D.
      • Liebowitz D.
      • Kieff E.
      ,
      • Kaye K.M.
      • Izumi K.M.
      • Kieff E.
      ). LMP1 has pleiotropic effects when expressed in cells resulting in induction of cell surface adhesion molecules and activation antigens (
      • Wang F.
      • Gregory C.
      • Sample C.
      • Rowe M.
      • Liebowitz D.
      • Murray R.
      • Rickinson A.
      • Kieff E.
      ), up-regulation of anti-apoptotic proteins (Bcl-2, A20) (
      • Henderson S.
      • Rowe M.
      • Gregory C.
      • Croom-Carter D.
      • Wang F.
      • Longnecker R.
      • Kieff E.
      • Rickinson A.B.
      ,
      • Laherty C.D., Hu, H.M.
      • Opipari A.W.
      • Wang F.
      • Dixit V.M.
      ), and stimulation of cytokine production (interleukin-6 and interleukin-8) (
      • Eliopoulos A.G.
      • Stack M.
      • Dawson C.W.
      • Kaye K.M.
      • Hodgkin L.
      • Sihota S.
      • Rowe M.
      • Young L.S.
      ,
      • Eliopoulos A.G.
      • Gallagher N.J.
      • Blake S.M.
      • Dawson C.W.
      • Young L.S.
      ). Recent studies have demonstrated that LMP1 functions as a constitutively activated member of the tumor necrosis factor receptor (TNFR) superfamily activating a number of signaling pathways in a ligand-independent manner (
      • Gires O.
      • Zimber-Strobl U.
      • Gonnella R.
      • Ueffing M.
      • Marschall G.
      • Zeidler R.
      • Pich D.
      • Hammerschmidt W.
      ,
      • Kilger E.
      • Kieser A.
      • Baumann M.
      • Hammerschmidt W.
      ). Functionally, LMP1 resembles CD40, a member of the TNFR, and can partially substitute for CD40 in vivo providing both growth and differentiation responses in B cells (
      • Uchida J.
      • Yasui T.
      • Takaoka-Shichijo Y.
      • Muraoka M.
      • Kulwichit W.
      • Raab-Traub N.
      • Kikutani H.
      ).
      The LMP1 protein is an integral membrane protein of 63 kDa and can be subdivided into three domains: (a) a NH2-terminal cytoplasmic tail (amino acids 1–23) that tethers and orientates the LMP1 protein to the plasma membrane, (b) six hydrophobic transmembrane loops that are involved in self-aggregation and oligomerization (amino acids 24–186), and (c) a long COOH-terminal cytoplasmic region (amino acids 187–386) that possesses most of the signaling activity of the molecule. Two distinct functional domains referred to as COOH-terminal activation regions 1 and 2 (CTAR1 and CTAR2) have been identified on the basis of their ability to activate the NF-κB transcription factor pathway (
      • Huen D.S.
      • Henderson S.A.
      • Croom-Carter D.
      • Rowe M.
      ). This effect contributes to the many phenotypic consequences of LMP1 expression including the induction of various anti-apoptotic and cytokine genes. LMP1 is also able to engage the mitogen-activated protein kinase cascade resulting in activation of extracellular-regulated protein kinase, c-Jun NH2-terminal kinase, and p38 and to stimulate the JAK/signal transducers and activators of transcription pathway (
      • Eliopoulos A.G.
      • Gallagher N.J.
      • Blake S.M.
      • Dawson C.W.
      • Young L.S.
      ,
      • Kieser A.
      • Kilger E.
      • Gires O.
      • Ueffing M.
      • Kolch W.
      • Hammerschmidt W.
      ,
      • Eliopoulos A.G.
      • Young L.S.
      ,
      • Roberts M.L.
      • Cooper N.R.
      ,
      • Gires O.
      • Kohlhuber F.
      • Kilger E.
      • Baumann M.
      • Kieser A.
      • Kaiser C.
      • Zeidler R.
      • Scheffer B.
      • Ueffing M.
      • Hammerschmidt W.
      ). Many of these effects result from the ability of TNFR-associated factors (TRAFs) to interact either directly with CTAR1 or indirectly via the death domain protein TRADD to CTAR2 (
      • Rickinson A.B.
      • Kieff E.
      ). The binding of TRAFs to the multimerized cytoplasmic tails of LMP1 provides a platform for the assembly and activation of upstream signaling molecules including the NIK and Tpl-2 mitogen-activated protein kinase kinase kinases (
      • Sylla B.S.
      • Hung S.C.
      • Davidson D.M.
      • Hatzivassiliou E.
      • Malinin N.L.
      • Wallach D.
      • Gilmore T.D.
      • Kieff E.
      • Mosialos G.
      ,
      • Eliopoulos A.G.
      • Davies C.
      • Blake S.S.
      • Murray P.
      • Najafipour S.
      • Tsichlis P.N.
      • Young L.S.
      ). The precise mechanisms responsible for signal initiation from these multiprotein complexes remain unknown. The region between CTAR1 and CTAR2 (so-called CTAR3) has been suggested to be responsible for the JAK/signal transducers and activators of transcription pathway although other data refute this finding and deletion of this region has no effect of the efficiency of B cell transformation (
      • Gires O.
      • Kohlhuber F.
      • Kilger E.
      • Baumann M.
      • Kieser A.
      • Kaiser C.
      • Zeidler R.
      • Scheffer B.
      • Ueffing M.
      • Hammerschmidt W.
      ,
      • Izumi K.M.
      • McFarland E.C.
      • Riley E.A.
      • Rizzo D.
      • Chen Y.
      • Kieff E.
      ).
      The expression of LMP1 in NPC is associated with increased metastatic spread, an effect that is also reflected in the ability of LMP1 to induce increased cell motility and invasive growth when expressed in epithelial cells in vitro (
      • Fahraeus R.
      • Chen W.
      • Trivedi P.
      • Klein G.
      • Obrink B.
      ,
      • Hu L-F.
      • Chen F.
      • Zhen Q-F.
      • Zhang Y-W.
      • Luo Y.
      • Zheng X.
      • Winberg G.
      • Ernberg I.
      • Klein G.
      ,
      • Kim K.R.
      • Yoshizaki T.
      • Miyamori H.
      • Hasegawa K.
      • Horikawa T.
      • Furukawa M.
      • Harada S.
      • Seiki M.
      • Sato H.
      ,
      • Horikawa T.
      • Yoshizaki T.
      • Sheen T.S.
      • Lee S.Y.
      • Furukawa M.
      ,
      • Horikawa T.
      • Sheen T.S.
      • Takeshita H.
      • Sato H.
      • Furukawa M.
      • Yoshizaki T.
      ). Thus it appears that the transforming ability of LMP1 may be regulated by novel signaling pathways that, in addition to the NF-κB and mitogen-activated protein kinase cascades, may account for the ability of this molecule to influence cell motility and to provide a profound survival advantage. One pathway that fulfils these criteria is that mediated by phosphatidylinositol 3-kinase (PI3K), which via generation of specific phospholipids, activates a diverse range of cellular processes including cell growth, motility, adhesion, and survival (
      • Vivanco I.
      • Sawyers C.L.
      ).
      The PI3K family of enzymes is activated by a wide range of extracellular growth and mitogenic stimuli including ligands of the TNFR family such as TNF-α and CD40, thus suggesting that LMP1 may also target this pathway (
      • Andjelic S.
      • Hsia C.
      • Suzuki H.
      • Kadowaki T.
      • Koyasu S.
      • Liou H-C.
      ,
      • Kilpatrick L.E.
      • Lee J.Y.
      • Haines K.M.
      • Campbell D.E.
      • Sullivan K.E.
      • Korchak H.M.
      ). PI3K-generated phospholipids are responsible for activating the Akt (PKB) kinase thereby promoting cell survival and for regulating the Rho family of small GTPases resulting in effects on the actin cytoskeleton and on cell signaling. Here we demonstrate that LMP1 can activate the PI3K/Akt pathway and that this effect is responsible for LMP1-induced actin polymerization, morphological transformation, and also contributes to cell survival. Furthermore, we show that the region of LMP1 previously identified as being essential for EBV-induced B cell transformation, CTAR1, is also responsible for PI3K activation.

      DISCUSSION

      We have shown that LMP1 can activate the PI3K/Akt pathway and that this is responsible for some of the phenotypic effects of LMP1 associated with cell transformation. This result is not surprising given the similarities between LMP1 signaling and that elicited from the TNFR family, members of which can activate PI3K. Thus, TNF-α has been shown to activate PI3K and Akt and this effect was reported to be required for activation of NF-κB (
      • Ozes O.N.
      • Mayo L.D.
      • Gustin J.A.
      • Pfeffer S.R.
      • Pfeffer L.M.
      • Donner D.B.
      ,
      • Reddy S.A.
      • Huang J.H.
      • Liao W.S.
      ). Inhibition of PI3K was found to potentiate TNF-α-induced apoptosis, an effect reminiscent of that observed in this study when LMP1-expressing cells were treated with the LY294002 inhibitor. LMP1 most closely resembles an activated CD40 in its phenotypic effects and LMP1 can partially correct the B cell development defect in CD40-deficient mice (
      • Uchida J.
      • Yasui T.
      • Takaoka-Shichijo Y.
      • Muraoka M.
      • Kulwichit W.
      • Raab-Traub N.
      • Kikutani H.
      ). It has thus been suggested that this ability to mimic CD40 is required for EBV to efficiently colonize the B cell pool and establish persistence in the healthy host (
      • Thorley-Lawson D.A.
      ). Previous studies have demonstrated that CD40 ligation can activate PI3K and Akt in B cells and that this effect is important for both cell proliferation and survival (
      • Andjelic S.
      • Hsia C.
      • Suzuki H.
      • Kadowaki T.
      • Koyasu S.
      • Liou H-C.
      ,
      • Arron J.R.
      • Vologodskaia M.
      • Wong B.R.
      • Naramura M.
      • Kim N., Gu, H.
      • Choi Y.
      ). Mice deficient in the p85 subunit of PI3K are severely impaired in B cell development with reduced proliferative responses to CD40 ligation (
      • Fruman D.A.
      • Snapper S.B.
      • Yballe C.M.
      • Alt F.W.
      • Cantley L.C.
      ,
      • Suzuki H.
      • Terauchi Y.
      • Fujiwara M.
      • Aizawa S.
      • Yazaki Y.
      • Kadowaki T.
      • Koyasu S.
      ). Taken together these data highlight the role of the PI3K pathway in B cell growth and suggest that the ability of LMP1 to activate this pathway contributes to EBV persistence in B cells.
      An understanding of the signaling capacity of LMP1 is key to defining its role in EBV-induced oncogenesis and in identifying pathways that may be of general significance in the transformation process. As the only EBV-encoded protein with the characteristics of a classical oncogene, the signaling pathways engaged by this molecule are clearly crucial in effecting B cell transformation and in inducing a plethora of phenotypic effects relevant to cellular transformation. Whereas previous work has focused on the contribution of the NF-κB and mitogen-activated protein kinase pathways to LMP1-induced effects, we now demonstrate that the PI3K/Akt pathway is also activated by LMP1 and that this may account for a some of the more profound oncogenic properties of the protein. This observation supports our previous work suggesting that certain aspects of LMP1 behavior in epithelial cells resemble those induced by an activated ras gene that also functions via activation of PI3K (
      • Dawson C.W.
      • Rickinson A.B.
      • Young L.S.
      ). Using co-immunoprecipitation experiments we have demonstrated that LMP1 associates with the p85 regulatory subunit of PI3K, thus showing that PI3K is recruited to, and forms part of, an LMP1 signaling complex. That this association results in PI3K activation is evidenced by increased basal lipid kinase activity in cells stably expressing LMP1 and by the ability of inducible LMP1 expression to also stimulate PI3K activity. Although PI3K associates with the LMP1 signaling complex, the exact nature of this interaction is currently unknown. The finding that a dominant-negative form of p85, deleted for the amino-terminal Src homology 2 domain, is able to block PI3K-mediated effects in Swiss 3T3 fibroblasts suggests that the LMP1-p85 association may be mediated through the amino-terminal Src homology 2 domain. Interestingly, unlike most growth factor receptors, LMP1 lacks putative YXXL motifs within its carboxyl terminus suggesting that the LMP1-p85 interaction may be mediated through a novel p85 domain, or is facilitated indirectly through an as yet unidentified adaptor protein(s). Use of the chimeric CD2-LMP1-(192–386) molecule demonstrated that the cytosolic carboxyl terminus of LMP1 and not the amino terminus and transmembrane spanning regions was responsible for PI3K and Akt activation. To further define the LMP1 domains responsible for PI3K activation, a GFP-tagged GRP1 fusion protein that has high affinity and specificity for phosphatidylinositol 3,4,5-triphosphate was used in microinjection studies (
      • Gray A.
      • Van Der Kaay J.
      • Downes C.P.
      ). This approach showed that the CTAR1 domain of LMP1 is responsible for mediating PI3K activation. This finding identifies the TRAF binding region of LMP1 as the mediator of PI3K activation and suggests that PI3K recruitment to LMP1 is mediated through a TRAF molecule or an as yet unidentified adaptor protein.
      The ability of LMP1 to activate PI3K helps to explain a number of the phenotypic consequences of its expression in different cellular environments. PI3K plays a key role in regulating actin cytoskeletal organization and cell shape remodeling by regulating the activity of the small Rho GTPases (
      • Bar-Sagi D.
      • Hall A.
      ,
      • Cantrell D.A.
      ). We recently reported that LMP1 induces filopodial extensions associated with lamellopodia and stress fibers in Swiss 3T3 cells (
      • Puls A.
      • Eliopoulos A.G.
      • Nobes C.D.
      • Bridges T.
      • Young L.S.
      • Hall A.
      ). In this report we show that LMP1 can induce robust actin stress-fiber formation that is dependent on the small Rho GTPases and on PI3K. Through the use of a panel of LMP1 mutants, we have identified CTAR1 as the region of LMP1 essential for actin stress-fiber formation and this correlates with the ability of CTAR1 to mediate PI3K activation. In our earlier study (
      • Puls A.
      • Eliopoulos A.G.
      • Nobes C.D.
      • Bridges T.
      • Young L.S.
      • Hall A.
      ) we demonstrated that Cdc42-induced filopodia formation was mediated through the transmembrane spanning regions of LMP1 whereas here, Rho-mediated stress-fiber formation requires CTAR1-generated signals. Although, Cdc42-induced filopodia formation was not investigated in this current study, the possibility that LMP1 may activate both Cdc42 and Rac/Rho independently through distinct mechanisms clearly warrants further examination. An important consequence of these effects on actin filament remodeling and cell shape change is the ability of LMP1 to induce morphological transformation in Rat-1 cells. Interesting, this phenotype could be reversed by the LY294002 PI3K inhibitor suggesting that PI3K-generated signals are an important component in LMP1-mediated cell transformation. Future studies will elucidate the precise mechanisms involved in this process and the relative contributions of the small Rho GTPases. The significance of these observations for the function of LMP1 in EBV-infected epithelial cells and B cells is worthy of consideration. Previous studies have demonstrated that LMP1 expression in NPC is associated with more advanced tumors and with increased metastatic spread (
      • Hu L-F.
      • Chen F.
      • Zhen Q-F.
      • Zhang Y-W.
      • Luo Y.
      • Zheng X.
      • Winberg G.
      • Ernberg I.
      • Klein G.
      ,
      • Horikawa T.
      • Yoshizaki T.
      • Sheen T.S.
      • Lee S.Y.
      • Furukawa M.
      ,
      • Horikawa T.
      • Sheen T.S.
      • Takeshita H.
      • Sato H.
      • Furukawa M.
      • Yoshizaki T.
      ). These observations concur with in vitro data showing that LMP1 induces morphological transformation as well as a more motile and invasive phenotype in Madin-Darby canine kidney cells (
      • Kim K.R.
      • Yoshizaki T.
      • Miyamori H.
      • Hasegawa K.
      • Horikawa T.
      • Furukawa M.
      • Harada S.
      • Seiki M.
      • Sato H.
      ).
      C. W. Dawson, G. Tramountanis, A. G. Eliopoulos, and L. S. Young, unpublished observations.
      Interestingly these effects of LMP1 were mapped to the CTAR1 region that we have now shown to be responsible for PI3K activation. The activation and migration of B cells also requires small Rho GTPase-dependent cytoskeletal changes that are crucial for the formation of germinal centers (
      • Tarlinton D.
      ,
      • Westerberg L.
      • Greicius G.
      • Snapper S.B.
      • Aspenstrom P.
      • Severinson E.
      ). Thus the ability of LMP1 to induce actin reorganization is likely to contribute to the establishment of EBV persistence in the memory B cell pool.
      The role of Akt in cellular growth transformation is now clearly established (
      • Testa J.R.
      • Bellacosa A.
      ). Akt has emerged as a critical signaling molecule that regulates a variety of cellular processes including cell growth, proliferation, and apoptosis. Although Akt is implicated in the regulation of various metabolic events, it is its role in regulating aspects of cell survival that has received most attention. Akt targets and inactivates a number of pro-apoptotic molecules associated with the induction of apoptosis (
      • Coffer P.J.
      • Jin J.
      • Woodgett J.R.
      ). These include the pro-apoptotic Bcl-2 family member, Bad, caspase 9, and GSK3 among others (
      • Lawlor M.A.
      • Alessi D.R.
      ). The ability of Akt to modulate these key effector molecules explains how cytokines and growth factors are able to promote cell survival in response to growth factor or serum withdrawal. The recent finding that Akt is able to induce NF-κB activation by directly phosphorylating and activating I-κB kinase suggests that in addition to directly inactivating pro-apoptotic effector molecules, Akt may contribute to the suppression of apoptosis by activating NF-κB. Although this phenomenon appears to be cell type-specific, it points to the ability of Akt to provide an additional level of protection from apoptotic stimuli through the activation of NF-κB. Our findings that PI3K inhibition results in robust apoptosis in LMP1-expressing cells implies that LMP1-generated signals activate signal transduction pathways that generate pro-apoptotic signals and that the PI3K/Akt pathway serves to counteract this effect. The recent demonstration that PI3K negatively regulates the activity of c-Jun NH2-terminal kinase and signal transducers and activators of transcription (
      • Ivanov V.N.
      • Krasilnikov M.
      • Ronai Z.
      ) points to a role for these factors in LMP1-associated cytotoxicity. It is possible that PI3K/Akt plays a role in LMP1-mediated NF-κB activation and that the apoptosis observed in LY294002-treated cells is a result of NF-κB down-regulation. However, our preliminary work indicates that LY294002 treatment does not affect the ability of LMP1 to induce NF-κB in both Rat-1 fibroblasts or in HeLa cells stably expressing LMP1 (data not shown). These data suggest that in addition to the ability of LMP1 to up-regulate Bcl-2 in B cells (
      • Henderson S.
      • Rowe M.
      • Gregory C.
      • Croom-Carter D.
      • Wang F.
      • Longnecker R.
      • Kieff E.
      • Rickinson A.B.
      ), Akt may provide another anti-apoptotic pathway activated by LMP1 that contributes to the survival of EBV-infected cells. This possibility is supported by recent work demonstrating that PI3K plays a role in both the survival and proliferation of EBV-transformed B cells (
      • Brennan P.
      • Mehl A.M.
      • Jones M.
      • Rowe M.
      ). These studies also identify PI3K as an attractive target for the development of novel approaches for the treatment of EBV-associated tumors.
      Given the profound effects of activating PI3K on cell survival and proliferation, it is not surprising that a diverse range of viruses have evolved to target this pathway for the efficient promotion of virus infection and replication. This is particularly evident for tumor viruses where oncoproteins such as polyoma middle T antigen, SV40 large T antigen, and the KSHV-encoded GPCR have all been shown to activate the PI3K/Akt pathway. Indeed, PI3K activity was first discovered in a complex with polyoma middle T antigen and the c-src tyrosine kinase (
      • Whitman M.
      • Kaplan D.R.
      • Schaffhausen B.
      • Cantley L.
      • Roberts T.M.
      ). That PI3K and Akt activation are critical for polyoma middle T antigen-mediated cellular transformation is borne out by the findings that mutation of the putative p85 binding site in polyoma middle T antigen abrogates its ability to transform rodent fibroblast cell lines and to protect from apoptosis induced by serum withdrawal (
      • Dahl J.
      • Jurczak A.
      • Cheng L.A.
      • Baker D.C.
      • Benjamin T.L.
      ,
      • Summers S.A.
      • Lipfert L.
      • Birnbaum M.J.
      ). Unlike these other oncoproteins, LMP1 does not possess intrinsic tyrosine kinase activity nor has it been shown to associate with any known tyrosine kinase(s). Thus, the mechanism by which LMP1 recruits and activates PI3K is clearly a focus for future work as is a more detailed examination of the functional contribution of PI3K activation to LMP1-induced transformation.

      Acknowledgments

      We thank Professor Mike Wakelam for help and advice.

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