PRA1 promotes the intracellular trafficking and NF-kappaB signaling of EBV latent membrane protein 1

Latent membrane protein 1 (LMP1), which is an Epstein-Barr virus (EBV)-encoded oncoprotein, induces nuclear factor-kappa B (NF-kappaB) signaling by mimicking the tumor necrosis factor receptor (TNFR). LMP1 signals primarily from intracellular compartments in a ligand-independent manner. Here, we identify a new LMP1-interacting molecule, prenylated Rab acceptor 1 (PRA1), which interacts with LMP1 for the first time through LMP1's transmembrane domain, and show that PRA1 is involved in intracellular LMP1 trafficking and LMP1-induced NF-kappaB activity. Immunofluorescence and biochemical analyses revealed that LMP1 physically interacted with PRA1 at the Golgi apparatus, and the colocalization of LMP1 and PRA1 to the Golgi was sensitive to nocodazole and brefeldin A. Coexpression of a PRA1 export mutant or knockdown of PRA1 led to redistribution of LMP1 and its associated signaling molecules to the endoplasmic reticulum and subsequent impairment of LMP1-induced NF-kappaB activation, but had no effect on CD40- and TNFR1-mediated signaling or the functional integrity of the Golgi apparatus. These novel findings provide important new insights into LMP1, and identify an unexpected new role for PRA1 in cellular signaling.     

The EBV transforming protein, latent membrane protein 1, mimics and cooperates with CD40 signaling in B lymphocytes

Latent membrane protein 1 (LMP1) is required for EBV-induced immortalization of human B cells, and expression of the protein in the absence of other viral proteins leads to an activated phenotype in B cells. It has been well documented that LMP1 causes B cells to up-regulate adhesion molecules, such as LFA-1 and ICAM-1, and coactivation molecules, such as B7-1 and CD23, as well as to activate NF-kappaB. Ligation of the endogenous B cell CD40 molecule also induces these and other activated phenotypic changes. Here, we report that expression of LMP1 also activates B cells to secrete Ig and IL-6 and rescues them from B cell receptor-mediated growth arrest analogous to CD40 signaling. Furthermore, an HLA-A2LMP1 chimeric construct demonstrates that the oligomerization of the carboxyl-terminal 200 amino acids of LMP1 is sufficient for B cell signaling. Finally, we demonstrate that LMP1 and CD40 signaling pathways interact cooperatively in inducing B cell effector functions.     

A heptad motif of leucine residues found in membrane proteins can drive self-assembly of artificial transmembrane segments

Specific interactions between alpha-helical transmembrane segments are important for folding and/or oligomerization of membrane proteins. Previously, we have shown that most transmembrane helix-helix interfaces of a set of crystallized membrane proteins are structurally equivalent to soluble leucine zipper interaction domains. To establish a simplified model of these membrane-spanning leucine zippers, we studied the homophilic interactions of artificial transmembrane segments using different experimental approaches. Importantly, an oligoleucine, but not an oligoalanine, se- quence efficiently self-assembled in membranes as well as in detergent solution. Self-assembly was maintained when a leucine zipper type of heptad motif consisting of leucine residues was grafted onto an alanine host sequence. Analysis of point mutants or of a random sequence confirmed that the heptad motif of leucines mediates self-recognition of our artificial transmembrane segments. Further, a data base search identified degenerate versions of this leucine motif within transmembrane segments of a variety of functionally different proteins. For several of these natural transmembrane segments, self-interaction was experimentally verified. These results support various lines of previously reported evidence where these transmembrane segments were implicated in the oligomeric assembly of the corresponding proteins.     

Itchy,a Nedd4 ubiquitin ligase,downregulates latent membrane protein 2A activity in B-cell signaling

Nedd4 family ubiquitin protein ligases (E3s) specifically associate with latent membrane protein 2A (LMP2A) of Epstein-Barr virus. Our previous studies analyzing LMP2A function in vitro have suggested that Nedd4 family E3s regulate LMP2A function. To determine the role of Nedd4 family E3s in LMP2A B-cell signaling, LMP2A transgenic (LMP2A(+)) mice were crossed with mice with the Itch-deficient (Itch(-/-)) background. Itchy, a mouse homologue of human AIP4, is a Nedd4 family E3 and is also the most abundant Nedd4 family E3 found in LMP2A affinity precipitates from B cells. There were significantly fewer B-cell receptor-positive B cells in spleen and bone marrow B cells in LMP2A(+) Itch(-/-) mice than in LMP2A(+) mice. In addition, LMP2A(+) Itch(-/-) bone marrow B cells formed larger colonies in cultures treated with interleukin-7 (IL-7) than control bone marrow B cells did. Finally, there was a dramatic increase in tyrosine phosphorylation of LMP2A and Syk in IL-7-cultured LMP2A(+) Itch(-/-) B cells. These results indicate that Nedd4 family E3s, in particular Itchy, downmodulate LMP2A activity in B-cell signaling.     

Modification of Cepsilon mRNA expression by EBV-encoded latent membrane protein 1

To evaluate the effect of expression of latent membrane protein (LMP) 1 encoded by Epstein-Barr virus (EBV) on Cepsilon mRNA expression, mRNA levels were examined by RT-PCR or Northern blot analysis upon transient transfection of LMP1 in the splenocytes derived from Brown-Norway rats with or without immunization with 2,4-dinitrophenyl-conjugated Ascaris suum antigen. Splenocytes were transfected with LMP1 expression vector, pSG5-LMP1, using lipofection method. Cepsilon mRNA levels were considerably increased by transfection with pSG5-LMP1 in the splenocytes derived from the nonimmunized rats; however, Cepsilon mRNA levels were decreased in the splenocytes derived from the immunized rats. Cepsilon mRNA expression in IgE-producing cells are modulated by LMP1, which might depend on the differentiation status of B cells upon exposure to allergen.     

The IFT-A complex regulates Shh signaling through cilia structure and membrane protein trafficking

Hectd1 mutant mouse embryos exhibit the neural tube defect exencephaly associated with abnormal cranial mesenchyme. Cellular rearrangements in cranial mesenchyme are essential during neurulation for elevation of the neural folds. Here we investigate the molecular basis of the abnormal behavior of Hectd1 mutant cranial mesenchyme. We demonstrate that Hectd1 is a functional ubiquitin ligase and that one of its substrates is Hsp90, a chaperone protein with both intra- and extracellular clients. Extracellular Hsp90 enhances migration of multiple cell types. In mutant cranial mesenchyme cells, both secretion of Hsp90 and emigration of cells from cranial mesenchyme explants were enhanced. Importantly, we show that this enhanced emigration was highly dependent on the excess Hsp90 secreted from mutant cells. Together, our data set forth a model whereby increased secretion of Hsp90 in the cranial mesenchyme of Hectd1 mutants is responsible, at least in part, for the altered organization and behavior of these cells and provides a potential molecular mechanism underlying the neural tube defect.     

MUC1 induced by Epstein-Barr virus latent membrane protein 1 causes dissociation of the cell-matrix interaction and cellular invasiveness via STAT signaling

Disruption of cellular adhesion is an essential pathobiologic step leading to tumor dissemination. Mucin 1 (MUC1) is a mucinous glycoprotein expressed at the surfaces of epithelial cells in many tissues and their carcinomas. MUC1 plays crucial roles in tumor invasion and metastasis, especially in opposing cell adhesion. We have shown that virus infection, specifically by the human tumor virus Epstein-Barr virus (EBV) induces a spectrum of cellular invasiveness and metastasis factors. Here we show that expression of MUC1 is increased in diverse latently EBV-infected cell lines that express latent membrane protein 1 (LMP1), the main viral oncoprotein, and that the level of MUC1 was suppressed by expression of a dominant-negative mutant of LMP1. Expression of LMP1 in EBV-negative nasopharyngeal cell lines induces expression of MUC1 through activation of the MUC1 promoter via binding of STAT1 and STAT3. Finally, LMP1 reduces cell adhesion ability, which is restored by inhibition of MUC1 expression with MUC1 small interfering RNA (siRNA). In addition, LMP1 increases cell invasiveness, which is suppressed by MUC1 siRNA. Thus, LMP1 induces MUC1, a factor important in an early step of detachment and release of tumor cells, which along with induction of other invasiveness and angiogenic factors may combine to act in a complex sequential process that culminates in metastasis of EBV-infected tumor cells.     

Probing phosphoinositide functions in signaling and membrane trafficking

The inositol phospholipids (PIs) comprise a family of eight species with different combinations of phosphate groups arranged around the inositol ring. PIs are among the most versatile signaling molecules known, with key roles in receptor-mediated signal transduction, actin remodeling and membrane trafficking. Recent studies have identified effector proteins and specific lipid-binding domains through which PIs signal. These lipid-binding domains can be used as probes to further our understanding of the spatial and temporal control of individual PI species. New layers of complexity revealed by the use of such probes include the occurrence of PIs at intracellular locations, the identification of phosphatidylinositol signaling hotspots and the presence of non-membrane pools of PIs in cell nuclei.     

HEC, a novel nuclear protein rich in leucine heptad repeats specifically involved in mitosis.

The protein encoded by the human gene HEC (highly expressed in cancer) contains 642 amino acids and a long series of leucine heptad repeats at its C-terminal region. HEC protein is expressed most abundantly in the S and M phases of rapidly dividing cells but not in terminal differentiated cells. It localizes to the nuclei of interphase cells, and a portion distributes to centromeres during M phase. Inactivation of HEC by microinjection of specific monoclonal antibodies into cells during interphase severely disturbs the subsequent mitoses. Disordered sister chromatid alignment and separation, as well as the formation of nonviable cells with multiple, fragmented micronuclei, are common features observed. These results suggest that the HEC protein may play an important role in chromosome segregation during M phase.     

Roles of TNF receptor-associated factor 3 in signaling to B lymphocytes by carboxyl-terminal activating regions 1 and 2 of the EBV-encoded oncoprotein latent membrane protein 1

TNFR-associated factor (TRAF)3, an adaptor protein that binds the cytoplasmic domains of both CD40 and the EBV-encoded oncoprotein latent membrane protein (LMP)1, is required for positive signaling by LMP1 but not CD40 in B lymphocytes. The present study further investigated how TRAF3 participates in LMP1 signaling. We found that TRAF3 mediates signaling both through direct interactions with the C-terminal activating region (CTAR)1 of LMP1 and through indirect interactions with the CTAR2 region of LMP1 in mouse B cells. Notably, our results demonstrated that the CTAR2 region appears to inhibit the recruitment of TRAF1 and TRAF2 to membrane rafts by the CTAR1 region. Additionally, the absence of TRAF2 in B cells resulted in only a modest reduction in CTAR1-mediated signals and no detectable effect on CTAR2-mediated signals. CTAR1 and CTAR2 cooperated to achieve the robust signaling activity of LMP1 when recruited to the same membrane microdomains in B cells. Interestingly, TRAF3 deficiency completely abrogated the cooperation between CTAR1 and CTAR2, supporting the hypothesis that TRAF3 participates in the physical interaction between CTAR1 and CTAR2 of LMP1. Together, our findings highlight the central importance of TRAF3 in LMP1-mediated signaling, which is critical for EBV persistent infection and EBV-associated pathogenesis.     

Epstein-barr virus latent membrane protein 1: Structure and functions

The Epstein-Barr virus latent membrane protein (LMP) 1 is a versatile protein that has profound effects on target cells through its effect on constitutive cellular proteins, e.g. TRAFs, TRADD, RIP, JAK3, BRAM1, and p85. LMP1 can stimulate or inhibit signaling pathways, resulting in transformation of rodent fibroblast cell lines, blockade of differentiation in epithelial cells, upregulation of anti-apoptotic proteins, production of cytokines, upregulation of cell surface markers, upregulation of DNA methyltransferase activity, and downregulation of cell adhesion molecules and cyclin-dependent kinases. Overall, this results in greater transformation and survival in LMP1-expressing cells. Within nasopharyngeal carcinoma biopsy tissues, a naturally occurring LMP1 variant has been identified as having a 10-amino acid deletion in the C-terminus that seems to confer greater transformation potential than non-deleted LMP1. The role of LMP1 as a viral oncogene and its interaction with cellular factors are discussed.     

Direct immunosuppressive effects of EBV-encoded latent membrane protein 1

In neoplastic cells of EBV-positive lymphoid malignancies latent membrane protein (LMP1) is expressed. Because no adequate cellular immune response can be detected against LMP1, we investigated whether LMP1 had a direct effect on T lymphocyte activation. In this study we show that nanogram amounts of purified recombinant LMP1 (rLMP1) strongly suppresses activation of T cells. By sequence alignment two sequences (LALLFWL and LLLLAL) in the first transmembrane domain of LMP1 were identified showing strong homology to the immunosuppressive domain (LDLLFL) of the retrovirus-encoded transmembrane protein p15E. The effects of rLMP1 and LMP1-derived peptides were tested in T cell proliferation and NK cytotoxicity assays and an Ag-induced IFN-gamma release enzyme-linked immunospot assay. LMP1 derived LALLFWL peptides showed strong inhibition of T cell proliferation and NK cytotoxicity, while acetylated LALLFWL peptides had an even stronger effect. In addition, Ag-specific IFN-gamma release was severely inhibited. To exert immunosuppressive effects in vivo, LMP1 has to be excreted from the cells. Indeed, LMP1 was detected in supernatant of EBV-positive B cell lines (LCL), and differential centrifugation in combination with Western blot analysis of the pellets indicated that LMP1 is probably secreted by LCL in the form of exosomes. The amount of secreted LMP1 in B cell cultures is well below the immunosuppressive level observed with rLMP1. Our results demonstrate direct immunosuppressive properties of LMP1 (fragments) and suggest that EBV-positive tumor cells may actively secrete LMP1 and thus mediate immunosuppressive effects on tumor-infiltrating lymphocytes. Moreover, we demonstrate, for the first time, that transmembrane protein-mediated immunosuppression is not solely restricted to RNA tumor viruses, but can also be found in DNA tumor viruses.     

Epstein-Barr virus latent membrane protein 1 (LMP1) C-terminal-activating region 3 contributes to LMP1-mediated cellular migration via its interaction with Ubc9

Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1), the principal viral oncoprotein and a member of the tumor necrosis factor receptor superfamily, is a constitutively active membrane signaling protein that regulates multiple signal transduction pathways via its C-terminal-activating region 1 (CTAR1) and CTAR2, and also the less-studied CTAR3. Because protein sumoylation among other posttranslational modifications may regulate many signaling pathways induced by LMP1, we investigated whether during EBV latency LMP1 regulates sumoylation processes that control cellular activation and cellular responses. By immunoprecipitation experiments, we show that LMP1 interacts with Ubc9, the single reported SUMO-conjugating enzyme. Requirements for LMP1-Ubc9 interactions include enzymatically active Ubc9: expression of inactive Ubc9 (Ubc9 C93S) inhibited the LMP1-Ubc9 interaction. LMP1 CTAR3, but not CTAR1 and CTAR2, participated in the LMP1-Ubc9 interaction, and amino acid sequences found in CTAR3, including the JAK-interacting motif, contributed to this interaction. Furthermore, LMP1 expression coincided with increased sumoylation of cellular proteins, and disruption of the Ubc9-LMP1 CTAR3 interaction almost completely abrogated LMP1-induced protein sumoylation, suggesting that this interaction promotes the sumoylation of downstream targets. Additional consequences of the disruption of the LMP1 CTAR3-Ubc9 interaction revealed effects on cellular migration, a hallmark of oncogenesis. Together, these data demonstrate that LMP1 CTAR3 does in fact function in intracellular signaling and leads to biological effects. We propose that LMP1, by interaction with Ubc9, modulates sumoylation processes, which regulate signal transduction pathways that affect phenotypic changes associated with oncogenesis.     

Negative regulation of Epstein-Barr virus latent membrane protein 1-mediated functions by the bone morphogenetic protein receptor IA-binding protein,BRAM1

The latent membrane protein 1 (LMP1) of Epstein-Barr virus causes cellular transformation and activates several intracellular signals, including NF-kappaB and c-Jun N-terminal kinase. Using yeast two-hybrid screening with the LMP1 C-terminal sequence as bait, we demonstrate that BRAM1 (bone morphogenetic protein receptor-associated molecule 1) is an LMP1-interacting protein. BRAM1 associates with LMP1, both in vitro and in vivo, as revealed by confocal microscopy, glutathione S-transferase pull-down, and co-immunoprecipitation assays. This association mainly involves the C-terminal half of BRAM1 comprising the MYND domain and the CTAR2 region of LMP1, which is critical in LMP1-mediated signaling pathways. We show that BRAM1 interferes with LMP1-mediated NF-kappaB activation but not the JNK signaling pathway. Because the CTAR2 region interacts with the tumor necrosis factor (TNF-alpha receptor-associated death domain protein, it is interesting to find that BRAM1 also interferes with NF-kappaB activation mediated by TNF-alpha. BRAM1 interferes LMP1-mediated and TNF-alpha-induced NF-kappaB activation by targeting IkappaBalpha molecules. Moreover, BRAM1 inhibits the resistance of LMP1-expressing cells to TNF-alpha-induced cytotoxicity. We therefore propose that the BRAM1 molecule associates with LMP1 and functions as a negative regulator of LMP1-mediated biological functions.