A mutation causing pseudohypoaldosteronism type 1 identifies a conserved glycine that is involved in the gating of the epithelial sodium channel.

Pseudohypoaldosteronism type 1 (PHA-1) is an inherited disease characterized by severe neonatal salt-wasting and caused by mutations in subunits of the amiloride-sensitive epithelial sodium channel (ENaC). A missense mutation (G37S) of the human ENaC beta subunit that causes loss of ENaC function and PHA-1 replaces a glycine that is conserved in the N-terminus of all members of the ENaC gene family. We now report an investigation of the mechanism of channel inactivation by this mutation. Homologous mutations, introduced into alpha, beta or gamma subunits, all significantly reduce macroscopic sodium channel currents recorded in Xenopus laevis oocytes. Quantitative determination of the number of channel molecules present at the cell surface showed no significant differences in surface expression of mutant compared with wild-type channels. Single channel conductances and ion selectivities of the mutant channels were identical to that of wild-type. These results suggest that the decrease in macroscopic Na currents is due to a decrease in channel open probability (P(o)), suggesting that mutations of a conserved glycine in the N-terminus of ENaC subunits change ENaC channel gating, which would explain the disease pathophysiology. Single channel recordings of channels containing the mutant alpha subunit (alphaG95S) directly demonstrate a striking reduction in P(o). We propose that this mutation favors a gating mode characterized by short-open and long-closed times. We suggest that determination of the gating mode of ENaC is a key regulator of channel activity.

Activation of c-Jun in the nuclei of neurons of the CA-1 in thrombin preconditioning occurs via PAR-1.

Recently it has been shown that the c-Jun N-terminal kinase (JNK) plays a role in thrombin preconditioning (TPC) in vivo and in vitro. To investigate further the pathways involved in TPC, we performed an immunohistochemical study in hippocampal slice cultures. Here we show that the major target of JNK, the AP-1 transcription factor c-Jun, is activated by phosphorylation in the nuclei of neurons of the CA1 region by using phospho-specific antibodies against the two JNK phosphorylation sites. The activation is early and transient, peaking at 90 min and not present by 3 hr after low-dose thrombin administration. Treatment of cultures with a synthetic thrombin receptor agonist results in the same c-Jun activation profile and protection against subsequent OGD, both of which are prevented by specific JNK inhibitors, showing that thrombin signals through PAR-1 to JNK. By using an antibody against the Ser 73 phosphorylation site of c-Jun, we identify possible additional TPC substrates.

Internalization and homologous desensitization of the GLP-1 receptor depend on phosphorylation of the receptor carboxyl tail at the same three sites.

Homologous desensitization and internalization of the GLP-1 receptor correlate with phosphorylation of the receptor in a 33-amino acid segment of the cytoplasmic tail. Here, we identify the sites of phosphorylation as being three serine doublets located at positions 441/442, 444/445, and 451/452. The role of phosphorylation on homologous desensitization was assessed after stable expression in fibroblasts of the wild type or of mutant receptors in which phosphorylation sites were changed in various combinations to alanines. We showed that desensitization, as measured by a decrease in the maximal production of cAMP after a first exposure of the cells to GLP-1, was strictly dependent on phosphorylation. Furthermore, the number of phosphorylation sites correlated with the extent of desensitization with no, intermediate, or maximal desensitization observed in the presence of one, two, or three phosphorylation sites, respectively. Internalization of the receptor-ligand complex was assessed by measuring the rate of internalization of bound [125I]GLP-1 or the redistribution of the receptor to an endosomal compartment after agonist binding. Our data demonstrate that internalization was prevented in the absence of receptor phosphorylation and that intermediate rates of endocytosis were obtained with receptors containing one or two phosphorylation sites. Thus, homologous desensitization and internalization require phosphorylation of the receptor at the same three sites. However, the differential quantitative impairment of these two processes in the single and double mutants suggests different molecular mechanisms controlling desensitization and internalization.

ATP release due to Thy-1-integrin binding induces P2X7-mediated calcium entry required for focal adhesion formation.

Thy-1, an abundant mammalian glycoprotein, interacts with αvβ3 integrin and syndecan-4 in astrocytes and thus triggers signaling events that involve RhoA and its effector p160ROCK, thereby increasing astrocyte adhesion to the extracellular matrix. The signaling cascade includes calcium-dependent activation of protein kinase Cα upstream of Rho; however, what causes the intracellular calcium transients required to promote adhesion remains unclear. Purinergic P2X7 receptors are important for astrocyte function and form large non-selective cation pores upon binding to their ligand, ATP. Thus, we evaluated whether the intracellular calcium required for Thy-1-induced cell adhesion stems from influx mediated by ATP-activated P2X7 receptors. Results show that adhesion induced by the fusion protein Thy-1-Fc was preceded by both ATP release and sustained intracellular calcium elevation. Elimination of extracellular ATP with Apyrase, chelation of extracellular calcium with EGTA, or inhibition of P2X7 with oxidized ATP, all individually blocked intracellular calcium increase and Thy-1-stimulated adhesion. Moreover, Thy-1 mutated in the integrin-binding site did not trigger ATP release, and silencing of P2X7 with specific siRNA blocked Thy-1-induced adhesion. This study is the first to demonstrate a functional link between αvβ3 integrin and P2X7 receptors, and to reveal an important, hitherto unanticipated, role for P2X7 in calcium-dependent signaling required for Thy-1-stimulated astrocyte adhesion.

Fatty acids regulate Thy-1 antigen mRNA stability in T lymphocyte precursors.

In this study, we report the effect of fatty acids on the Thy-1 antigen mRNA decay. Low serum and synthetic medium culture conditions were used to demonstrate that fatty acids, which are important metabolites involved as second messengers in signal transduction, also influence the steady-state mRNA level. Detailed analysis demonstrated that polyunsaturated lipids attached to bovine serum albumin, such as linoleic, linolenic, and arachidonic acids, modulate gene expression specifically in the S1A T lymphoma cell line by inducing a 3-5-fold increase in the steady-state Thy-1 mRNA level, concomitant with a twofold increase in cell surface expression. A similar modulation was observed in the immature CD4-CD8- T cell precursors but not in mature thymocytes. Nuclear run-on and transfection experiments indicated that the observed Thy-1 mRNA level is post-transcriptionally regulated and that the presence of the coding region is sufficient for this adaptive response. A mechanism without a requirement for protein kinase C activation, but involving Ca2+ entry, could account for this difference in Thy-1 mRNA stability.

Variations in IB1/JIP1 expression regulate susceptibility of beta-cells to cytokine-induced apoptosis irrespective of C-Jun NH2-terminal kinase signaling.

We previously reported that interleukin-1beta (IL-1beta) alone does not cause apoptosis of beta-cells, whereas when combined with gamma-interferon (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha), it exerts a distinct apoptotic effect. Studies in beta-cell lines indicated that IL-1beta reduced expression of islet brain (IB)-1/JNK interacting protein (JIP)-1, a JNK scaffold protein with antiapoptotic action. We examined whether variations in IB1/JIP-1 expression in purified primary beta-cells affect their susceptibility to cytokine-induced apoptosis. Exposure to IL-1beta for 24 h decreased cellular IB1/JIP-1 content by 66 +/- 17%; this IL-1beta effect was maintained in the presence of TNF-alpha + IFN-gamma, which did not influence IB1/JIP-1 levels by themselves. Addition of IL-1beta to TNF-alpha + IFN-gamma increased apoptosis from 20 +/- 2% to 59 +/- 5%. A similar increase in TNF-alpha + IFN-gamma-induced apoptosis was produced by adenoviral expression of antisense IB1/JIP-1 and was not further enhanced by addition of IL-1beta, indicating that IL-1beta-mediated suppression of IB1/JIP-1 in beta-cells increases their susceptibility to cytokine-induced apoptosis. However, adenovirally mediated overexpression of IB1/JIP-1 also potentiated TNF-alpha + IFN-gamma-induced apoptosis, suggesting that the antiapoptotic effect of IB1/JIP-1 depends on well-defined cellular levels. We conclude that the IB1/JIP-1 level in beta-cells can control their susceptibility to apoptosis independent of JNK signaling.

The C-Jun N-Terminal Kinase Inhibition in Intracerebral Hemorrhage

Background: In intracerebral hemorrhage (ICH), a subtype of stroke, the bloodentry into the brain triggers toxicity resulting in a strong loss of neurons andinflammation. Water content is also increases leading to growing intracranial pressure,which worsens neurological outcome. C-Jun N-terminal kinases (JNKs) areactivated in response to stress stimuli. Specific inhibition of JNK by a TAT-coupledpeptide (XG-102) mediates neuroprotection in several models of ischemic stroke.Recently, we have noted that the JNK pathway is also activated in a mouse modelof ICH, raising the question of the efficacy of XG-102 in this model.Method: ICH was induced in the mouse by intrastriatal injection of bacterialcollagenase (0,1U). Three hours later, animals received an i.v. injection of XG-102(100μg/kg). The neuroscore was assessed using a scale (from 0 to 9) based on 3behavioral tests performed daily. Then, mice were sacrificed at 6h, 24h, 48h and 5dafter ICH and histological studies performed.Results: XG-102 significantly improves neurological outcome at 24h (mean score:1,8±1.4 vs 3,4±1.8, p<0.01). Analysis of the lesion volume revealed a significantdecrease of the lesion area in the treated group at 48h (29±11 mm3 vs 39±5 mm3,p = 0.04). XG-102 mainly inhibits the edema component of the lesion. Indeed, asignificant decrease of the brain swelling was observed in treated animals at 48h(14±13% vs 26±9%, p=0.04) and 5d (-0,3±4.5% vs 5,1±3.6%, p=0.01).Conclusions: Inhibition of the JNK pathway by XG-102 appears to lead to asignificant decrease of the cerebral edema in the ICH model providing a furtherbeneficial effect of the XG-102 treatment. This result is of interest becausecurrently, clinical treatment for brain edema is limited. Importantly, the beneficialeffects observed with XG-102 in both stroke models open the possibility to rapidlytreat patients before identifying the stroke subtype by imaging.

Manganese-induced integrin affinity maturation promotes recruitment of alpha V beta 3 integrin to focal adhesions in endothelial cells: evidence for a role of phosphatidylinositol 3-kinase and Src.

Integrin activity is controlled by changes in affinity (i.e. ligand binding) and avidity (i.e. receptor clustering). Little is known, however, about the effect of affinity maturation on integrin avidity and on the associated signaling pathways. To study the effect of affinity maturation on integrin avidity, we stimulated human umbilical vein endothelial cells (HUVEC) with MnCl(2) to increase integrin affinity and monitored clustering of beta 1 and beta 3 integrins. In unstimulated HUVEC, beta 1 integrins were present in fibrillar adhesions, while alpha V beta 3 was detected in peripheral focal adhesions. Clustered beta 1 and beta 3 integrins expressed high affinity/ligand-induced binding site (LIBS) epitopes. MnCl(2)-stimulation promoted focal adhesion and actin stress fiber formation at the basal surface of the cells, and strongly enhanced mAb LM609 staining and expression of beta 3 high affinity/LIBS epitopes at focal adhesions. MnCl(2)-induced alpha V beta 3 clustering was blocked by a soluble RGD peptide, by wortmannin and LY294002, two pharmacological inhibitors of phosphatidylinositol 3-kinase (PI 3-K), and by over-expressing a dominant negative PI 3-K mutant protein. Conversely, over-expression of active PI 3-K and pharmacological inhibiton of Src with PP2 and CGP77675, enhanced basal and manganese-induced alpha V beta 3 clustering. Transient increased phosphorylation of protein kinase B/Akt, a direct target of PI 3K, occurred upon manganese stimulation. MnCl(2) did not alter beta 1 integrin distribution or beta1 high-affinity/LIBS epitope expression. Based on these results, we conclude that MnCl(2)-induced alpha V beta 3 integrin affinity maturation stimulates focal adhesion and actin stress fiber formation, and promotes recruitment of high affinity alpha V beta 3 to focal adhesions. Affinity-modulated alpha V beta 3 clustering requires PI3-K signaling and is negatively regulate by Src.

Connexin26 is regulated in rat urothelium by the scaffold protein IB1/JIP-1.

Proper function of the wall of bladder requires gap junctional communication for coordinating the responses of smooth muscle (SMC) and urothelial cells exposed to urine pressure. In the rat bladder, Cx43 is expressed by SMC and urothelial cells, whereas Cx26 expression is restricted to the epithelium. We used a model of bladder outlet obstruction, in which a ligature is placed around the urethra to increase voiding pressure. Increased fluid pressure was associated with increased Cx43 and Cx26 mRNA expression and with the activation of a signaling cascade including the transcription factor c-Jun, which is a component of the AP-1 complex. The signaling pathway of the c-Jun NH2 terminal kinase (JNK) requires the presence of the scaffold protein Islet-Brain1/c-Jun amino-terminal kinase Interacting Protein-1 (IB1/JIP-1). Under stress conditions resulting from urine retention, we have found a reduced content of IB1/JIP-1 in urothelial cells, which in turn induced a drastic increase of JNK and AP-1 binding activities. The stress-induced activation of JNK was prevented by overexpressing IB1/JIP-1, using a viral gene transfer approach, a condition which also resulted in a decrease in Cx26 mRNA. The data show that: 1) mechanical stress of urothelial cells activates in vivo JNK, as a consequence of a regulated expression of IB1/JIP-1 and 2) that urothelial Cx26 may be directly regulated by the AP-1 complex.

Subconjunctival Injection of XG-102, a c-Jun N-Terminal Kinase Inhibitor Peptide, in the Treatment of Endotoxin-Induced Uveitis in Rats.

Abstract Purpose: XG-102, a TAT-coupled dextrogyre peptide inhibiting the c-Jun N-terminal kinase, was shown efficient in the treatment of experimental uveitis. Preclinical studies are now performed to determine optimal XG-102 dose and route of administration in endotoxin-induced uveitis (EIU) in rats with the purpose of clinical study design. METHODS: EIU was induced in Lewis rats by lipopolysaccharides (LPS) injection. XG-102 was administered at the time of LPS challenge by intravenous (IV; 3.2, 35 or 355̴1;μg/injection), intravitreal (IVT; 0.08, 0.2 or 2.2̴1;μg/eye), or subconjunctival (SCJ; 0.2, 1.8 or 22̴1;μg/eye) routes. Controls received either the vehicle (saline) or dexamethasone phosphate injections. Efficacy was assessed by clinical scoring, infiltrating cells count, and expression of inflammatory mediators [inducible nitric oxide synthase (iNOS), cytokine-induced neutrophil chemoattractant-1 (CINC-1)]. The effect of XG-102 on phosphorylation of c-Jun was evaluated by Western blot. RESULTS: XG-102 demonstrated a dose-dependent anti-inflammatory effect in EIU after IV and SCJ administrations. Respective doses of 35 and 1.8̴1;μg were efficient as compared with the vehicle-injected controls, but only the highest doses, respectively 355 and 22̴1;μg, were as efficient as dexamethasone phosphate. After IVT injections, the anti-inflammatory effect of XG-102 was clinically evaluated similar to the corticoid's effect with all the tested doses. Regardless of the administration route, the lowest efficient doses of XG-102 significantly decreased the ration of phospho c-Jun/total c-Jun, reduced cells infiltration in the treated eyes, and significantly downregulated iNOS and CINC-1 expression in the retina. CONCLUSION: These results confirm that XG-102 peptide has potential for treating intraocular inflammation. SCJ injection appears as a good compromise to provide a therapeutic effect while limiting side effects.

Fonction ventriculaire gauche après revascularisation pour occlusion d'une coronaire d'une durée de 1-2 heures [Left ventricular function following revascularization for occlusion of a coronary artery lasting 1 to 2 hours].

The time-lag between coronary occlusion and irreversible damage to the myocardium is ill-defined in man. In 10 patients the changes in left ventricular function have been studied after coronary occlusion during diagnostic or therapeutic cardiac catheterization of 1-2 hours' duration. Revascularization was achieved either surgically or through intracoronary streptokinase infusion. The interval between occlusion and onset of extracorporal circulation or reopening was 61 to 119 minutes. Despite enzyme elevation (CPK, CK-MB, SGOT) and appearance of Q-waves in 5 patients, no significant alteration of left ventricular function was noted on repeat cardiac catheterization 10 to 230 days after the accident. These observations, suggest that coronary occlusion of 1-2 hours' duration fails to produce significant irreversible damage to the myocardium despite electrocardiographic and enzymatic signs of myocardial infarction.

Activation mechanisms of the protease MALT1 and cleavage of one of its targets - the ribonuclease Regnase-1- in lymphocytes and lymphoma cell lines

Persistent infection induces an adaptive immune response that is mediated by T and B lymphocytes. Upon triggering with an antigen, these cells become activated and turn into fast expanding cells able to efficiently defend the host. Lymphocyte activation is controlled by a complex composed of CARMA1, BCL10 and MALT1 which regulates the NF-KB signaling pathway upon antigen triggering. Abnormally high expression or activity of either one of these three proteins can favor the development of lymphomas, while genetic defects in the pathway are associated with immunodeficiency. MALT1 was identified as a paracaspase sharing homology with other cysteine proteases, namely caspases and metacaspases. In order to be active, caspases need to dimerize. Based on their sequence similarity with MALT1, we hypothesized that dimerization might also be a mechanism of activation employed by MALT1. To address this assumption, we performed a bioinformatics modelling based on the crystal structures of several caspases. Our model suggested that the MALT1 caspase-like domain can indeed form dimers. This finding was later confirmed by several published crystal structures of MALT1. In the dimer interface of our model, we noticed the presence of charged amino acids that could potentially form salt bridges and thereby hold both monomers together. Mutation of one of these residues, E549, into alanine completely blocked the catalytic activity of MALT1. Additionally, we provided evidence for a role of E549 in promoting the MALTl-dependent growth of cells derived from diffuse large B cell lymphoma (DLBCL) of the aggressive B cell-like type (ABC). To our initial surprise, the E549A mutation showed only a partial defect in dimerization, indicating that additional residues are essential to form a stable dimer. The MALT1 crystal structures revealed a key function for E549 in stabilizing the catalytic site of the protease via its interaction with an arginine which is located next to the catalytic active cysteine. In an additional study, we discovered that MALT1 monoubiquitination is required for the catalytic activity of the protease. Interestingly, we found that the MALT1 dimer interface mutant E549A could not be monoubiquitinated. Based on these findings, we suggest that correct formation of the dimer interface is a prerequisite for monoubiquitination. In a second project, we discovered a novel target of the protease MALT1, the ribonuclease Regnase¬la It was described that the RNase activity of Regnase-1 negatively regulates immune responses. We could show that in ABC DLBCL cell lines, Regnase-1 is not only cleaved by MALT1 but also phosphorylated, at least in part, by the inhibitor of KB kinase (IKK). Both regulations appear to restrain the RNase function of Regnase-1 and thereby allow the production of pro-survival proteins. In conclusion, our studies further highlight and explain the importance of the catalytic activity of MALT1 for the activation of lymphocytes and provide additional knowledge for the development of specific drugs targeting the catalytic activity of MALT1 for immunomodulation and treatment of lymphomas.195; SUMMARY IN FRENCH PhD Thesis Katrin Cabalzar 2 SUMMARY IN FRENCH Une infection persistante induit une réponse immunitaire adaptative par l'intermédiaire des lymphocytes T et B. Quand elles reconnaissent l'antigène, ces cellules sont activées et se multiplient très rapidement pour défendre efficacement l'hôte. L'activation des lymphocytes est transmise par un complexe composé de trois protéines, CARMA1, BCL10 et MALT1, qui régule la voie de signalisation NF-KB lorsque l'antigène est reconnu. L'expression ou l'activité anormalement élevée de l'une de ces trois protéines peut favoriser le développement de lymphomes, tandis que des défauts génétiques de cette voie de signalisation sont associés à l'immunodéficience. MALT1 a été identifiée comme étant une paracaspase qui partage des séquences homologues avec d'autres protéases à cystéine, comme les caspases et les métacaspases. Pour être actives, les caspases ont besoin de dimériser. Etant donné leur similarité de séquence avec MALT1, nous avons supposé que la dimérisation pouvait aussi être un mécanisme d'activation utilisé par MALT1. Pour vérifier cette hypothèse, nous avons conçu un modèle bioinformatique à partir des structures cristallographiques de plusieurs caspases. Et notre modèle a suggéré que le domaine catalytique de MALT1 était effectivement capable de former des dimères. Cette découverte a été confirmée plus tard par des publications qui montrent des structures cristallographiques dimériques de MALT1. Dans l'interface du dimère de notre modèle, nous avons remarqué la présence d'acides aminés chargés qui pouvaient former des liaisons ioniques et ainsi réunir les deux monomères. La mutation de l'un de ces résidus, E549, pour une alanine, a complètement inhibé l'activité catalytique de MALT1. De plus, nous avons mis en évidence un rôle d'E549 dans la croissance dépendante de MALT1, des cellules dérivées de lymphomes B diffus à grandes cellules (DLBCL) de sous-type cellules B actives (ABC). Dans un premier temps nous avons été surpris de constater que cette mutation révélait seulement un défaut partiel de dimérisation, ce qui indique que des acides aminés supplémentaires sont indispensables pour former un dimère stable. Les structures cristallographiques de MALT1 ont révélé un rôle primordial d'E549 dans la stabilisation du site catalytique de la protéase via son interaction avec une arginine qui se trouve à côté de la cystéine du site actif. Dans une autre étude, nous avons découvert que la monoubiquitination de MALT1 est requise pour l'activité catalytique de la protéase. A remarquer que nous avons trouvé que le mutant E549A de l'interface dimère de MALT1 n'a pas pu être monoubiquitiné. Sur la base de ces résultats, nous suggérons que la formation correcte de l'interface du dimère est une condition préalable pour la monoubiquitination. Dans un second projet, nous avons découvert une nouvelle cible de la protéase MALT1, la ribonucléase Regnase-1. Il a été décrit que l'activité RNase de Regnase-1 régulait négativement les réponses immunitaires. Nous avons pu montrer que dans les lignées cellulaires ABC DLBCL, la Regnase-1 n'était pas seulement clivée par MALT1 mais également phosphorylée, au moins en partie, par la kinase de l'inhibiteur de KB (IKK). Les deux régulations semblent supprimer la fonction RNase de Regnase-1 et permettre ainsi la stabilisation de certains ARN messagers et la production de protéines favorisant la survie. En conclusion, nos études mettent en évidence le rôle-clé de la dimérisation de MALT1 et expliquent l'importance de l'activité catalytique de MALT1 pour l'activation des lymphocytes. Ainsi, nos résultats apportent des connaissances supplémentaires pour le développement de médicaments spécifiques ciblant l'activité catalytique de MALT1, qui pourraient être utiles pour modifier les réponses immunitaires et traiter des lymphomes.

p38/MKP-1-regulated AKT coordinates macrophage transitions and resolution of inflammation during tissue repair

Repair of damaged tissue requires the coordinated action of inflammatory and tissue-specific cells to restore homeostasis, but the underlying regulatory mechanisms are poorly understood. In this paper, we report new roles for MKP-1 (mitogen-activated protein kinase [MAPK] phosphatase-1) in controlling macrophage phenotypic transitions necessary for appropriate muscle stem cell¿dependent tissue repair. By restricting p38 MAPK activation, MKP-1 allows the early pro- to antiinflammatory macrophage transition and the later progression into a macrophage exhaustion-like state characterized by cytokine silencing, thereby permitting resolution of inflammation as tissue fully recovers. p38 hyperactivation in macrophages lacking MKP-1 induced the expression of microRNA-21 (miR-21), which in turn reduced PTEN (phosphatase and tensin homologue) levels, thereby extending AKT activation. In the absence of MKP-1, p38-induced AKT activity anticipated the acquisition of the antiinflammatory gene program and final cytokine silencing in macrophages, resulting in impaired tissue healing. Such defects were reversed by temporally controlled p38 inhibition. Conversely, miR-21¿AKT interference altered homeostasis during tissue repair. This novel regulatory mechanism involving the appropriate balance of p38, MKP-1, miR-21, and AKT activities may have implications in chronic inflammatory degenerative diseases.

Protein kinase C-zeta mediates retinal degeneration in response to TNF.

Tumor necrosis factor-alpha (TNF) has been implicated in retinal ganglion cells (RGC) degeneration in glaucoma. Atypical protein kinase C (PKC) zeta is involved in cell protection against various stresses. The aim of this study was to investigate the potential proapoptotic effects of intravitreal injections of TNF with or without PKCzeta specific inhibitor on the rat retina. TNF was injected in the vitreous of rat eyes alone or in combination with specific PKCzeta inhibitor. PKCzeta and NF-kappaB were studied by immunohistochemistry and western-blotting analysis on retina, and apoptosis quantified by the TUNEL assay. While low basal PKCzeta was observed in the control eyes, TNF induced intense expression of PKCzeta mostly in bipolar cells processes. PKCzeta staining became nuclear when TNF was coinjected with PKCzeta inhibitor. TNF alone did not induce apoptosis in the retina. Coinjection of the PKCzeta-specific inhibitor and TNF, however, induced apoptosis in the inner nuclear and ganglion cell layers. The PKCzeta-specific inhibitor unmasks retinal cells to TNF cytotoxicity showing a link between the proapoptotic effects of TNF and the antiapoptotic PKCzeta signaling pathway.

Ubiquitination of the Epstein-Barr virus-encoded latent membrane protein 1 depends on the integrity of the TRAF binding site.

The latent membrane protein 1 (LMP1) encoded by the Epstein-Barr virus functions as a constitutively activated receptor of the tumor necrosis factor receptor family. LMP1 is a short-lived protein that is ubiquitinated and degraded by the proteasome. We have previously shown that LMP1 recruits the adapter protein tumor necrosis factor receptor-associated factor 3 (TRAF3) to lipid rafts. To test if TRAFs are involved in LMP1's ubiquitination, we have mutated the LMP1 CTAR1 site that has been identified as a TRAF binding site. We show that the CTAR1 mutant (CTAR1(-)) is expressed after transfection at a similar level to wild-type LMP1, and behaves as wild-type LMP1 with respect to membrane localization. However, CTAR1(-) does not bind TRAF3. We demonstrate that ubiquitination of CTAR1(-) is significantly reduced when compared to wild-type LMP1. In addition, the expression of wild-type LMP1 induces the ubiquitination, an effect that is significantly reduced when the CTAR1(-) is expressed. Taken together, our results suggest that TRAF proteins are involved in the ubiquitination of LMP1, and that their binding to LMP1 may facilitate their own ubiquitination.