Processing of the Epstein-Barr virus-encoded latent membrane protein p63/LMP.

We have analyzed the processing of the Epstein-Barr virus-encoded latent membrane protein (p63/LMP) in lymphoblastoid cell lines, Burkitt's lymphoma cell lines, and rodent fibroblasts transfected with the p63/LMP gene. Pulse-chase analysis by immunoprecipitation, under denaturing conditions, reveals a half-life of 2 h. This is due to turnover in the plasma membrane with cleavage of the protein, resulting in a 25,000-molecular-weight (p25) fragment derived from the carboxy-terminal portion of LMP. This fragment is rich in proline and acidic amino acids and sheds into the cytoplasm, where it appears to accumulate, being present in a six- to sevenfold molar excess over p63/LMP in immunoprecipitation analyses. p25 is, like p63/LMP, also phosphorylated (pp25) on serine and threonine residues, in the same ratio and to approximately the same extent as the intact p63/LMP molecule. Amino acid sequence analysis and carboxy-terminal labeling suggest that p25 is derived through a single cleavage adjacent to the sequence LGAPGGGPDNGPQDPD.     

Elucidation of the c-Jun N-terminal kinase pathway mediated by Estein-Barr virus-encoded latent membrane protein 1

Epstein-Barr virus (EBV) is associated with several human diseases including infectious mononucleosis and nasopharyngeal carcinoma. EBV-encoded latent membrane protein 1 (LMP1) is oncogenic and indispensable for cellular transformation caused by EBV. Expression of LMP1 in host cells constitutively activates both the c-Jun N-terminal kinase (JNK) and NF-kappaB pathways, which contributes to the oncogenic effect of LMP1. However, the underlying signaling mechanisms are not very well understood. Based mainly on overexpression studies with various dominant-negative constructs, LMP1 was generally thought to functionally mimic members of the tumor necrosis factor (TNF) receptor superfamily in signaling. In contrast to the prevailing paradigm, using embryonic fibroblasts from different knockout mice and the small interfering RNA technique, we find that the LMP1-mediated JNK pathway is distinct from those mediated by either TNF-alpha or interleukin-1. Moreover, we have further elucidated the LMP1-mediated JNK pathway by demonstrating that LMP1 selectively utilizes TNF receptor-associated factor 6, TAK1/TAB1, and c-Jun N-terminal kinase kinases 1 and 2 to activate JNK.     

Activation of TRAF5 and TRAF6 signal cascades negatively regulates the latent replication origin of Epstein-Barr virus through p38 mitogen-activated protein kinase

Latent Epstein-Barr virus (EBV) is maintained by the virus replication origin oriP that initiates DNA replication with the viral oriP-binding factor EBNA1. However, it is not known whether oriP's replicator activity is regulated by virus proteins or extracellular signals. By using a transient replication assay, we found that a low level of expression of viral signal transduction activator latent membrane protein 1 (LMP1) suppressed oriP activity. The binding site of the tumor necrosis factor receptor-associated factor (TRAF) of LMP1 was essential for this suppressive effect. Activation of the TRAF signal cascade by overexpression of TRAF5 and/or TRAF6 also suppressed oriP activity. Conversely, blocking of TRAF signaling with dominant negative mutants of TRAF5 and TRAF6, as well as inhibition of a downstream signal mediator p38 MAPK, released the LMP1-induced oriP suppression. Furthermore, activation of TRAF6 signal cascade by lipopolysaccharides (LPS) resulted in loss of EBV from Burkitt's lymphoma cell line Akata, and inhibition of p38 MAPK abolished the suppressive effect of LPS. These results suggested that the level of oriP activity is regulated by LMP1 and extracellular signals through TRAF5- and TRAF6-mediated signal cascades.     

Activation of Rac1 and the p38 mitogen-activated protein kinase pathway in response to arsenic trioxide

Arsenic trioxide induces differentiation and apoptosis of malignant cells in vitro and in vivo, but the mechanisms by which such effects occur have not been elucidated. In the present study we provide evidence that arsenic trioxide induces activation of the small G-protein Rac1 and the alpha and beta isoforms of the p38 mitogen-activated protein (MAP) kinase in several leukemia cell lines. Such activation of Rac1 and p38-isoforms results in downstream engagement of the MAP kinase-activated protein kinase-2 and is enhanced by pre-treatment of cells with ascorbic acid. Interestingly, pharmacological inhibition of p38 potentiates arsenic-dependent apoptosis and suppression of growth of leukemia cell lines, suggesting that this signaling cascade negatively regulates induction of antileukemic responses by arsenic trioxide. Consistent with this, overexpression of a dominant-negative p38 mutant (p38betaAGF) enhances the antiproliferative effects of arsenic trioxide on target cells. To further define the relevance of activation of the Rac1/p38 MAP kinase pathway in the induction of arsenic-dependent antileukemic effects, studies were performed using bone marrows from patients with chronic myelogenous leukemia. Arsenic trioxide suppressed the growth of leukemic myeloid (CFU-GM) progenitors from such patients, whereas concomitant pharmacological inhibition of the p38 pathway enhanced its growth-suppressive effects. Altogether, these data provide evidence for a novel function of the p38 MAP kinase pathway, acting as a negative regulator of arsenic trioxide-induced apoptosis and inhibition of malignant cell growth.     

Dehydroepiandrosterone negatively regulates the p38 mitogen-activated protein kinase pathway by a novel mitogen-activated protein kinase phosphatase

Dehydroepiandrosterone-sulfate, the sulfated form of dehydroepiandrosterone, is the most abundant steroid in young adults, but gradually declines with aging. In humans, the clinical application of dehydroepiandrosterone targeting some collagen diseases, such as systemic lupus erythematosus, as an adjunctive treatment has been applied in clinical trial. Here, we report that dehydroepiandrosterone may negatively regulate the mitogen-activated protein kinase pathway in humans via a novel dual specificity protein phosphatase, DDSP (dehydroepiandrosterone-enhanced dual specificity protein phosphatase). DDSP is highly homologous to LCPTP/HePTP, a tissue-specific protein tyrosine phosphatase (PTP) which negatively regulates both ERK and p38-mitogen-activated protein kinase, and is transcribed from the PTPN7 locus by alternative splicing. Although previous reports have shown that the mRNA expression of the LCPTP/HePTP gene was inducible by extracellular signals such as T-cell antigen receptor stimulation, reverse transcribed (RT)-PCR experiments using specific sets of primers suggested that the expression of LCPTP/HePTP was constitutive while the actual inducible sequence was that of DDSP. Furthermore DDSP was widely distributed among different types of human tissues and specifically interacted with p38-mitogen-activated protein kinase. This inducible negative regulation of the p38-mitogen-activated protein kinase-dependent pathway may help to clarify the broad range of dehydroepiandrosterone actions, thereby aiding the development of new preventive or adjunctive applications for human diseases.     

The p38/RK mitogen-activated protein kinase pathway regulates interleukin-6 synthesis response to tumor necrosis factor.

Tumour necrosis factor (TNF) is a pleiotropic cytokine, the activities of which include effects on gene expression, cell growth and cell death. The biological signalling mechanisms which are responsible for these TNF effects remain largely unknown. Here we demonstrate that the stress-responsive p38 mitogen-activated protein (MAP) kinase is involved in TNF-induced cytokine expression. TNF Treatment of cell activated the p38 MAP kinase pathway, as revealed by increased phosphorylation of p38 MAP kinase itself, activation of the substrate protein MAPKAP kinase-2, and culminating in the phosphorylation of the heat shock protein 27 (hsp27). Pretreatment of cells with the highly specific p38 MAP kinase inhibitor SB203580 completely blocked this TNF-induced activation of MAPKAP kinase-2 and hsp27 phosphorylation. Under the same conditions, SB203580 also completely inhibited TNF-induced synthesis of interleukin (IL)-6 and expression of a reporter gene that was driven by a minimal promoter containing two NF-Kappa B elements. However, neither TNF-induced DNA binding of TNF-Kappa B nor TNF-induced phosphorylation of its subunits was modulated by SB203580, suggesting that NF-Kappa B is not a direct target for the p38 MAP kinase pathway. Interestingly, TNF-induced cytotoxicity was not affected by SB203580, indicating that p38 MAP kinase might be an interesting target to interfere selectively with TNF-induced gene activation.     

Murine coronavirus replication-induced p38 mitogen-activated protein kinase activation promotes interleukin-6 production and virus replication in cultured cells

Analyses of mitogen-activated protein kinases (MAPKs) in a mouse hepatitis virus (MHV)-infected macrophage-derived J774.1 cell line showed activation of two MAPKs, p38 MAPK and c-Jun N-terminal kinase (JNK), but not of extracellular signal-regulated kinase (ERK). Activation of MAPKs was evident by 6 h postinfection. However, UV-irradiated MHV failed to activate MAPKs, which demonstrated that MHV replication was necessary for their activation. Several other MHV-permissive cell lines also showed activation of both p38 MAPK and JNK, which indicated that the MHV-induced stress-kinase activation was not restricted to any particular cell type. The upstream kinase responsible for activating MHV-induced p38 MAPK was the MAPK kinase 3. Experiments with a specific inhibitor of p38 MAPK, SB 203580, demonstrated that MHV-induced p38 MAPK activation resulted in the accumulation of interleukin-6 (IL-6) mRNAs and an increase in the production of IL-6, regardless of MHV-induced general host protein synthesis inhibition. Furthermore, MHV production was suppressed in SB 203580-treated cells, demonstrating that activated p38 MAPK played a role in MHV replication. The reduced MHV production in SB 203580-treated cells was, at least in part, due to a decrease in virus-specific protein synthesis and virus-specific mRNA accumulation. Interestingly, there was a transient increase in the amount of phosphorylation of the translation initiation factor 4E (eIF4E) in infected cells, and this eIF4E phosphorylation was p38 MAPK dependent; it is known that phosphorylated eIF4E enhances translation rates of cap-containing mRNAs. Furthermore, the upstream kinase responsible for eIF4E phosphorylation, MAPK-interacting kinase 1, was also phosphorylated and activated in response to MHV infection. Our data suggested that host cells, in response to MHV replication, activated p38 MAPK, which subsequently phosphorylated eIF4E to efficiently translate certain host proteins, including IL-6, during virus-induced severe host protein synthesis inhibition. MHV utilized this p38 MAPK-dependent increase in eIF4E phosphorylation to promote virus-specific protein synthesis and subsequent progeny virus production. Enhancement of virus-specific protein synthesis through virus-induced eIF4E activation has not been reported in any other viruses.     

Divergent roles for Ras and Rap in the activation of p38 mitogen-activated protein kinase by interleukin-1

We have found that lethal toxin from Clostridium sordellii, which specifically inactivates the low molecular weight G proteins Ras, Rap, and Rac, inhibits the activation of p38 mitogen-activated protein kinase (MAPK) by interleukin-1 (IL-1) in EL4.NOB-1 cells and primary fibroblasts. The target protein involved appeared to be Ras, because transient transfections with dominant negative RasN17 inhibited p38 MAPK activation by IL-1. Furthermore, transfections of cells with constitutively active RasVHa-activated p38 MAPK. Further evidence for Ras involvement came from the observation that IL-1 caused a rapid activation of Ras in the cells and from the inhibitory effects of the Ras inhibitors manumycin A and damnacanthal. Toxin B from Clostridium difficile, which inactivates Rac, Cdc42, and Rho, was without effect. Dominant negative versions of Rac (RacN17) or Rap (Rap1AN17) did not inhibit the response. Intriguingly, transfection of cells with dominant negative Rap1AN17 activated p38 MAPK. Furthermore, constitutively active Rap1AV12 inhibited p38 MAPK activation by IL-1, consistent with Rap antagonizing Ras function. IL-1 also activated Rap in the cells, but with slower kinetics than Ras. Our studies therefore provide clear evidence using multiple approaches for Ras as a signaling component in the activation of p38 MAPK by IL-1, with Rap having an inhibitory effect.     

Epstein-Barr virus-encoded latent membrane protein 1 modulates cyclin D1 by c-Jun/Jun B heterodimers

In our recent studies, we found that LMP1 encoded by Epstein-Barr virus could accelerate the formation of active c-Jun/Jun B heterodimer. We studied the regulation of cyclinD1 by c-Jun/Jun B heterodimers by laser scanning confocal influorescence microscopy, Western blot, luciferase activity assay, super-EMSA and flow cytometry in the Tet-on-LMP1 HNE2 cell line, in which LMP1 expression was regulated by Tet-on system. c-Jun/Jun B heterodimers induced by LMP1 could up regulate cyclin D1 promoter activity and expression. Overexpression of cyclinD1 accelerated the progression of cell cycle.     

Nasopharyngeal carcinoma-associated Epstein-Barr virus-encoded oncogene latent membrane protein 1 potentiates regulatory T-cell function

Sequence variation in the Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) oncogene structure may affect antigen-presenting cell (APC) function of infected B cells and immune escape by EBV-specific T cells and thus contribute to the development of malignancy. Normal B cell-associated LMP1 (B-LMP1) upregulates B cell APC function through activation of the necrosis factor (NF)-kappaB subunit, RelB. We examined the ability of B-LMP1 and a nasopharyngeal carcinoma-associated LMP1 (NPC-LMP1) to modulate B cell APC function and T-cell responses. B lymphoma cells transfected with NPC-LMP1 stimulated resting T cells in mixed lymphocyte reaction less efficiently than B-LMP1 transfectants. Unexpectedly, antigen presentation to CD4(+) T helper cells was reduced owing to potentiation of regulatory T-cell function by NPC-LMP1 transfectants, which produce increased levels of interleukin-10, rendering CD4(+) T cells hyporesponsive. Thus, after primary EBV infection, T cells may escape activation by NPC-LMP1. These observations have important implications for the establishment of EBV-associated malignancy in the context of infection with tumour-associated EBV LMP1 variants.     

Epstein-Barr virus latent membrane protein 2 associates with and is a substrate for mitogen-activated protein kinase.

The latent membrane protein 2 (LMP2) of Epstein-Barr virus interferes with B-lymphocyte signal transduction through the immunoglobulin (Ig) receptor. Two isoforms of LMP2 exist and differ only in that one isoform (LMP2a) contains an N-terminal cytoplasmic domain that the other isoform does not. LMP2a is a phosphoprotein that is phosphorylated on tyrosines and serines in the cytoplasmic domain. GST1-119, a glutathione S-transferase (GST) fusion protein containing the 119 amino acids of the cytoplasmic domain, affinity precipitated serine kinase activity from BJAB cell extracts. The affinity-precipitated kinase phosphorylated LMP2a sequences, and kinase activity was increased following induction. Probing of Western immunoblots of affinity-precipitated proteins showed that the Erk1 form of mitogen-activated protein kinase (MAPK) was present. Purified MAPK phosphorylated GST fusion proteins containing the cytoplasmic domain of LMP2a and mutational analyses were used to identify S15 and S102 as the sites of in vitro phosphorylation. A polyclonal rabbit antiserum was prepared against a maltose binding protein-LMP2a cytoplasmic domain fusion protein (MBP1-119) and used to immunoprecipitate LMP2a from the in vitro-immortalized lymphoblastoid B-cell line B95-8CR. LMP2a immunoprecipitates from B95-8CR contained MAPK as a coprecipitated protein. Cross-linking surface Ig on B95-8CR cells failed to induce MAPK activity within the cells. Treatment of B95-8CR with phorbol myristate acetate (PMA) was able to bypass the Ig receptor block and activate MAPK activity. Phosphorylation of LMP2a on serine residues increased after PMA induction. The possible role for LMP2a serine phosphorylation by MAPK in the control of latency is discussed.     

Involvement of p38 mitogen-activated protein kinase in basic fibroblast growth factor-induced interleukin-6 synthesis in osteoblasts.

We previously showed that basic fibroblast growth factor (bFGF)-induced activation of protein kinase C (PKC) via phosphatidylinositol-hydrolyzing phospholipase C and phosphatidylcholine-hydrolyzing phospholipase D suppresses interleukin-6 (IL-6) synthesis by bFGF itself in osteoblast-like MC3T3-E1 cells. In the present study, we further investigated the mechanism underlying the bFGF-induced IL-6 synthesis in MC3T3-E1 cells. bFGF time-dependently induced the phosphorylation of p38 mitogen-activated protein (MAP) kinase. SB203580, a specific inhibitor of p38 MAP kinase, suppressed the bFGF-induced IL-6 synthesis dose-dependently. The phosphorylation of p38 MAP kinase by bFGF was suppressed by TMB-8, an inhibitor of intracellular Ca(2+) mobilization, or the depletion of extracellular Ca(2+) with EGTA. A23187, a Ca-ionophore, stimulated the phosphorylation of p38 MAP kinase. SB203580 inhibited the A23187-induced synthesis of IL-6. 1-Oleoyl-2-acetyl-sn-glycerol, a synthetic diacylglycerol activating PKC, reduced the bFGF-induced IL-6 synthesis. 12-O-Tetradecanoylphorbol-13-acetate, an activator of PKC, attenuated the phosphorylation of p38 MAP kinase by bFGF, but did not affect the A23187-induced phosphorylation. These results strongly suggest that bFGF-induced IL-6 synthesis is mediated via p38 MAP kinase activation in osteoblasts, and that PKC acts at a point upstream from p38 MAP kinase.     

Epstein-Barr virus nuclear antigen 2 induces expression of the virus-encoded latent membrane protein.

Infection of Epstein-Barr virus-negative human B-lymphoma cell lines with the fully transforming B95.8 Epstein-Barr virus strain was associated with complete virus latent gene expression and a change in the cell surface and growth phenotype toward that of in vitro-transformed lymphoblastoid cell lines. In contrast, the cells infected with the P3HR1 Epstein-Barr virus strain, a deletion mutant that cannot encode Epstein-Barr nuclear antigen 2 (EBNA2) or a full-length EBNA-LP, expressed EBNAs1, 3a, 3b, and 3c but were negative for the latent membrane protein (LMP) and showed no change in cellular phenotype. This suggests that EBNA2 and/or EBNA-LP may be required for subsequent expression of LMP in Epstein-Barr virus-infected B cells. Recombinant vectors capable of expressing the B95.8 EBNA2A protein were introduced by electroporation into two P3HR1-converted B-lymphoma cell lines, BL30/P3 and BL41/P3. In both cases, stable expression of EBNA2A was accompanied by activation of LMP expression from the resident P3HR1 genome; control transfectants that did not express the EBNA2A protein never showed induction of LMP. In further experiments, a recombinant vector capable of expressing the full-length B95.8 EBNA-LP was introduced into the same target lines. Strong EBNA-LP expression was consistently observed in the transfected clones but was never accompanied by induction of LMP. The EBNA2A gene transfectants expressing EBNA2A and LMP showed a dramatic change in cell surface and growth phenotype toward a pattern like that of lymphoblastoid cell lines; some but not all of these changes could be reproduced in the absence of EBNA2A by transfection of P3HR1-converted cell lines with a recombinant vector expressing LMP. These studies suggest that EBNA2 plays an important dual role in the process of B-cell activation to the lymphoblastoid phenotype; the protein can have a direct effect upon cellular gene expression and is also involved in activating the expression of a second virus-encoded effector protein, LMP.     

Activation of the p38 mitogen-activated protein kinase pathway by estrogen or by 4-hydroxytamoxifen is coupled to estrogen receptor-induced apoptosis

17beta-Estradiol (E(2)) or the antiestrogen, 4-hydroxytamoxifen (OHT), induce apoptosis in stably transfected estrogen receptor (ER)-positive HeLa-ER5 cells. p38 mitogen-activated protein kinase is implicated in cellular processes involving apoptosis. The p38 kinase inhibitor, SB203580, partially protects HeLa-ER5 cells against apoptosis induced by E(2) or by OHT. E(2) induces the p38 pathway 12-36-fold in ER-positive cell lines, while OHT induces p38 activity 2-5-fold. In an ER-positive cell line selected for resistance to E(2)-induced apoptosis, E(2) no longer induced p38, and the ER no longer bound to the estrogen response element, while OHT induced both p38 and apoptosis. In cells selected for resistance to OHT-induced apoptosis, OHT no longer induced p38, while E(2) induced p38 and apoptosis, and transactivated an estrogen response element-containing reporter gene. In MCF-7 cells, whose growth is stimulated by estrogen, E(2) did not induce p38 or apoptosis, while OHT induced both p38 and apoptosis, and SB203580 protected against OHT-induced apoptosis. This work shows that E(2) and OHT activate the p38 pathway, suggests that they use different pathways for p38 activation, and links activation of the p38 pathway to apoptosis induced by E(2) and by OHT.