Essential role of CD8 palmitoylation in CD8 coreceptor function.

To investigate the molecular basis that makes heterodimeric CD8alphabeta a more efficient coreceptor than homodimeric CD8alphaalpha, we used various CD8 transfectants of T1.4 T cell hybridomas, which are specific for H-2Kd, and a photoreactive derivative of the Plasmodium berghei circumsporozoite peptide PbCS 252-260 (SYIPSAEKI). We demonstrate that CD8 is palmitoylated at the cytoplasmic tail of CD8beta and that this allows partitioning of CD8alphabeta, but not of CD8alphaalpha, in lipid rafts. Localization of CD8 in rafts is crucial for its coreceptor function. First, association of CD8 with the src kinase p56lck takes place nearly exclusively in rafts, mainly due to increased concentration of both components in this compartment. Deletion of the cytoplasmic domain of CD8beta abrogated localization of CD8 in rafts and association with p56lck. Second, CD8-mediated cross-linking of p56lck by multimeric Kd-peptide complexes or by anti-CD8 Ab results in p56lck activation in rafts, from which the abundant phosphatase CD45 is excluded. Third, CD8-associated activated p56lck phosphorylates CD3zeta in rafts and hence induces TCR signaling and T cell activation. This study shows that palmitoylation of CD8beta is required for efficient CD8 coreceptor function, mainly because it dramatically increases CD8 association with p56lck and CD8-mediated activation of p56lck in lipid rafts.

Cutting edge: NKT cell development is selectively impaired in Fyn- deficient mice.

Most NK1.1+ T (NKT) cells express a biased TCRalphabeta repertoire that is positively selected by the monomorphic MHC class I-like molecule CD1d. The development of CD1d-dependent NKT cells is thymus dependent but, in contrast to conventional T cells, requires positive selection by cells of hemopoietic origin. Here, we show that the Src protein tyrosine kinase Fyn is required for development of CD1d-dependent NKT cells but not for the development of conventional T cells. In contrast, another Src kinase, Lck, is required for the development of both NKT and T cells. Impaired NKT cell development in Fyn-deficient mice cannot be rescued by transgenic expression of CD8, which is believed to increase the avidity of CD1d recognition by NKT cells. Taken together, our data reveal a selective and nonredundant role for Fyn in NKT cell development.

The beta1 and beta3 integrins promote T cell receptor-mediated cytotoxic T lymphocyte activation.

Recognition by CD8+ cytotoxic T lymphocytes (CTLs) of antigenic peptides bound to major histocompatibility class (MHC) I molecules on target cells leads to sustained calcium mobilization and CTL degranulation resulting in perforin-dependent killing. We report that beta1 and beta3 integrin-mediated adhesion to extracellular matrix proteins on target cells and/or surfaces dramatically promotes CTL degranulation. CTLs, when adhered to fibronectin but not CTL in suspension, efficiently degranulate upon exposure to soluble MHC.peptide complexes, even monomeric ones. This adhesion induces recruitment and activation of the focal adhesion kinase Pyk2, the cytoskeleton linker paxillin, and the Src kinases Lck and Fyn in the contact site. The T cell receptor, by association with Pyk2, becomes part of this adhesion-induced activation cluster, which greatly increases its signaling.

CTL activation is induced by cross-linking of TCR/MHC-peptide-CD8/p56lck adducts in rafts.

To investigate the role of the coreceptor CD8 and lipid rafts in cytotoxic T lymphocyte (CTL) activation, we used soluble mono-and multimeric H-2Kd-peptide complexes and cloned S14 CTL specific for a photoreactive derivative of the Plasmodium berghei circumsporozoite (PbCS) peptide 252-260 [PbCS(ABA)]. We report that activation of CTL in suspension requires multimeric Kd-PbCS(ABA) complexes co-engaging TCR and CD8. Using TCR ligand photo-cross-linking, we find that monomeric Kd-PbCS(ABA) complexes promote association of TCR/CD3 with CD8/p56lck. Dimerization of these adducts results in activation of p56lck in lipid rafts, where phosphatases are excluded. Additional cross-linking further increases p56lck kinase activity, induces translocation of TCR/CD3 and other signaling molecules to lipid rafts and intracellular calcium mobilization. These events are prevented by blocking Src kinases or CD8 binding to TCR-associated Kd molecules, indicating that CTL activation is initiated by cross-linking of CD8-associated p56lck. They are also inhibited by methyl-beta-cyclodextrin, which disrupts rafts and by dipalmitoyl phosphatidylethanolamine, which interferes with TCR signaling. Because efficient association of CD8 and p56lck takes place in rafts, both reagents, though in different ways, impair coupling of p56lck to TCR, thereby inhibiting the initial and essential activation of p56lck induced by cross-linking of engaged TCR.

The roles of PKCδ and Src kinases in the glucocorticoid induction and the TGF-β superinduction of the mouse mammary tumor virus transcription in B lymphocytes

Résumé : Le virus tumoral de la glande mammaire de la souris (MMTV) est un rétrovirus provoquant le développement de tumeurs dans les glandes mammaires des souris susceptibles femelles. Au cours de son évolution, le virus s'est adapté et s'exprime dans des cellules spécialisées. Les lymphocytes B sont les premières cellules infectées et elles sont essentielles pour la propagation de l'infection aux glandes mammaires. Dans notre étude, le virus MMTV a été utilisé afin d'examiner les voies de signalisation induites par les glucocorticoïdes (dexaméthasone (dex), une hormone stéroïdienne) et le transforming growth factor-f3 (TGF-P, une cytokine), deux molécules impliquées dans l'activation de la transcription à partir du promoteur du MMTV dans les cellules B. Le TGF-P seul n'influence pas l'activité du promoteur du MMTV. Par contre, en synergie avec dex, le TGF-P provoque une super-induction de l'expression du promoteur par rapport à une stimulation par le glucocorticoïde seul. Cette super-induction est régulée par une famille de protéines, les Smads. Ainsi, dans les lymphocytes B, l'utilisation du MMTV a permis de mettre en évidence une nouvelle synergie entre les glueocortieoïdes et le TGF-p. pans ce travail, l'utilisation d'inhibiteurs pharmacologiques et de mutants « dominant-négatifs » nous a pet mis de démontrer qu'une Protéine Kinase C delta (PKC5) active est impliquée dans la transduction du signal lors de la réponse au dex ainsi que celle au TGF-P. Néanmoins, la PKC5 est régulée différemment dans chaque voie spécifique : la voie du TGF-p nécessitait l'activation du PKC5 par diacylglycerol (DAG) et la phosphorylation de tyrosines spécifiques, alors que la voie impliquant les glucocorticoïdes ne le nécessitait pas. Nous avons aussi démontré qu'une tyrosine kinase de la famille Src est responsable de la phosphorylation des tyrosines sur la PKC5. Les essais de kinase in vitro nous ont permis de découvrir que plusieurs Src kinases peuvent phosphoryler la PKC6 dans les cellules B et qu'elles étaient constitutivement actives. Enfin, nous avons montré qu'il existe une interaction protéine - protéine induite par dex, entre le récepteur aux glucocorticoïdes (GR) et la PKC5 dans les cellules B, une association qui n'a pas été démontrée auparavant. Par ailleurs, nous avons analysé les domaines d'interactions entre PKC5 et GR en utilisant les essais de «GST pull-down». Nos résultats montrent que le domaine régulateur de la PKC5 et celui qui interagit avec l'ADN du GR sont impliqués. En résumé, nous avons trouvé que dans une lignée lymphocytaire B, le virus MMTV utilise des mécanismes pour réguler à la fois la transcription et la voie de signalisation qui sont différents de ceux utilisés dans les cellules mammaires épithéliales et les fibroblastes. Nos découvertes pourraient être utilisées comme modèles pour l'étude de gènes cellulaires impliqués dans des processus tels qu'inflammation, immunité ou cancérogénèse. Summary: Mouse Mammary Tumor Virus (MMTV) is a retrovirus that causes tumors in the mammary glands of susceptible female mice and has adapted evolutionarily to be expressed in specialized cells. The B lymphocytes are the first cells to be infected by the MMTV and are essential for the spread of infection to the mammary glands. Here, we used the MMTV as a model system to investigate the signalling cascade induced by giucocorticoids (dexamethasone, "dex", a steroid hormone), and by Transforming Growth Factor-beta (TGF-P, a cytokine) leading to its transcriptional activation in B lymphocytes. By itself, TGF-I3 does not affect the basal activity of the MMTV promoter. However, TGF-13 significantly increases glucocorticoid-induced expression, through its effectors, the Smad factors. Thus, MMTV in B cells demonstrates a novel synergism between glucocorticoids and TGF-16. In this thesis project, we present evidence, based on the use of pharmacological inhibitors and of dominant-negative mutants, that an active Protein Kinase C delta (PKC6) is required as a signal transducer for the dex response and for the TGF-P superinduction as well. The PKC6 is differentially regulated in each specific pathway: whereas the TGF-13 superinduction required PKC6 to be activated by diacylglycerol (DAG) and to be phosphorylated at specific tyrosine residues, the glueocorticoid-induced pathway did not. We also showed that a protein tyrosine kinase of the Src family is responsible for the phosphorylation of tyrosines on PKC6. By performing in vitro kinase assays, we found that several Src kinases of B cells were able to phosphorylate PKC6 and that they were constitutively active. Finally, we demonstrate a dex-dependent functional protein-protein interaction between the glucocorticoid receptor (GR) and PKC6 in B cells, an association that has not been previously described. We further analysed the interacting domains of PKG6 and GR using in vitro GST pull-down assays, whereby the regulatory domain of PKC6 and the extended DNA-binding domain of the GR were involved. In summary, we found that in B-lymphoid cell lines, MMTV uses novel mechanisms of transcriptional control and signal transduction that are different from those at work in mammary epithelial or fibroblastic cells. These findings will be used as model for cellular genes involved in cellular processes such as immune functions, inflammation, or oncogenic transformation that may have a similar pattern of regulation.

CD8beta endows CD8 with efficient coreceptor function by coupling T cell receptor/CD3 to raft-associated CD8/p56(lck) complexes.

The extraordinary sensitivity of CD8+ T cells to recognize antigen impinges to a large extent on the coreceptor CD8. While several studies have shown that the CD8beta chain endows CD8 with efficient coreceptor function, the molecular basis for this is enigmatic. Here we report that cell-associated CD8alphabeta, but not CD8alphaalpha or soluble CD8alphabeta, substantially increases the avidity of T cell receptor (TCR)-ligand binding. To elucidate how the cytoplasmic and transmembrane portions of CD8beta endow CD8 with efficient coreceptor function, we examined T1.4 T cell hybridomas transfected with various CD8beta constructs. T1.4 hybridomas recognize a photoreactive Plasmodium berghei circumsporozoite (PbCS) peptide derivative (PbCS (4-azidobezoic acid [ABA])) in the context of H-2K(d), and permit assessment of TCR-ligand binding by TCR photoaffinity labeling. We find that the cytoplasmic portion of CD8beta, mainly due to its palmitoylation, mediates partitioning of CD8 in lipid rafts, where it efficiently associates with p56(lck). In addition, the cytoplasmic portion of CD8beta mediates constitutive association of CD8 with TCR/CD3. The resulting TCR-CD8 adducts exhibit high affinity for major histocompatibility complex (MHC)-peptide. Importantly, because CD8alphabeta partitions in rafts, its interaction with TCR/CD3 promotes raft association of TCR/CD3. Engagement of these TCR/CD3-CD8/lck adducts by multimeric MHC-peptide induces activation of p56(lck) in rafts, which in turn phosphorylates CD3 and initiates T cell activation.

Degradation of ZAP-70 following antigenic stimulation in human T lymphocytes: role of calpain proteolytic pathway.

T cell activation by the specific Ag results in dramatic changes of the T cell phenotype that include a rapid and profound down-regulation and degradation of triggered TCRs. In this work, we investigated the fate of the TCR-associated ZAP-70 kinase in Ag-stimulated T cells. T cells stimulated by peptide-pulsed APCs undergo an Ag dose-dependent decrease of the total cellular content of ZAP-70, as detected by FACS analysis and confocal microscopy on fixed and permeabilized T cell-APC conjugates and by Western blot on total cell lysates. The time course of ZAP-70 consumption overlaps with that of zeta-chain degradation, indicating that ZAP-70 is degraded in parallel with TCR internalization and degradation. Pharmacological activation of protein kinase C (PKC) does not induce ZAP-70 degradation, which, on the contrary, requires activation of protein tyrosine kinases. Two lines of evidence indicate that the Ca2+-dependent cysteine protease calpain plays a major role in initiating ZAP-70 degradation: 1) treatment of T cells with cell-permeating inhibitors of calpain markedly reduces ZAP-70 degradation; 2) ZAP-70 is cleaved in vitro by calpain. Our results show that, in the course of T cell-APC cognate interaction, ZAP-70 is rapidly degraded via a calpain-dependent mechanism.

Peptide modification or blocking of CD8, resulting in weak TCR signaling, can activate CTL for Fas- but not perforin-dependent cytotoxicity or cytokine production.

This study describes a form of partial agonism for a CD8+ CTL clone, S15, in which perforin-dependent killing and IFN-gamma production were lost but Fas (APO1 or CD95)-dependent cytotoxicity preserved. Cloned S15 CTL are H-2Kd restricted and specific for a photoreactive derivative of the Plasmodium berghei circumsporozoite peptide PbCS 252-260 (SYIPSAEKI). The presence of a photoactivatable group in the epitope permitted assessment of TCR-ligand binding by TCR photoaffinity labeling. Selective activation of Fas-dependent killing was observed for a peptide-derivative variant containing a modified photoreactive group. A similar functional response was obtained after binding of the wild-type peptide derivative upon blocking of CD8 participation in TCR-ligand binding. The epitope modification or blocking of CD8 resulted in an > or = 8-fold decrease in TCR-ligand binding. In both cases, phosphorylation of zeta-chain and ZAP-70, as well as calcium mobilization were reduced close to background levels, indicating that activation of Fas-dependent cytotoxicity required weaker TCR signaling than activation of perforin-dependent killing or IFN-gamma production. Consistent with this, we observed that depletion of the protein tyrosine kinase p56(lck) by preincubation of S15 CTL with herbimycin A severely impaired perforin- but not Fas-dependent cytotoxicity. Together with the observation that S15 CTL constitutively express Fas ligand, these results indicate that TCR signaling too weak to elicit perforin-dependent cytotoxicity or cytokine production can induce Fas-dependent cytotoxicity, possibly by translocation of preformed Fas ligand to the cell surface.

SOCS-1 protein prevents Janus Kinase/STAT-dependent inhibition of beta cell insulin gene transcription and secretion in response to interferon-gamma.

In the pathogenesis of type I diabetes mellitus, activated leukocytes infiltrate pancreatic islets and induce beta cell dysfunction and destruction. Interferon (IFN)-gamma, tumor necrosis factor-alpha and interleukin (IL)-1 beta play important, although not completely defined, roles in these mechanisms. Here, using the highly differentiated beta Tc-Tet insulin-secreting cell line, we showed that IFN-gamma dose- and time-dependently suppressed insulin synthesis and glucose-stimulated secretion. As described previously IFN-gamma, in combination with IL-1 beta, also induces inducible NO synthase expression and apoptosis (Dupraz, P., Cottet, S., Hamburger, F., Dolci, W., Felley-Bosco, E., and Thorens, B. (2000) J. Biol. Chem. 275, 37672--37678). To assess the role of the Janus kinase/signal transducer and activator of transcription (STAT) pathway in IFN-gamma intracellular signaling, we stably overexpressed SOCS-1 (suppressor of cytokine signaling-1) in the beta cell line. We demonstrated that SOCS-1 suppressed cytokine-induced STAT-1 phosphorylation and increased cellular accumulation. This was accompanied by a suppression of the effect of IFN-gamma on: (i) reduction in insulin promoter-luciferase reporter gene transcription, (ii) decrease in insulin mRNA and peptide content, and (iii) suppression of glucose-stimulated insulin secretion. Furthermore, SOCS-1 also suppressed the cellular effects that require the combined presence of IL-1 beta and IFN-gamma: induction of nitric oxide production and apoptosis. Together our data demonstrate that IFN-gamma is responsible for the cytokine-induced defect in insulin gene expression and secretion and that this effect can be completely blocked by constitutive inhibition of the Janus kinase/STAT pathway.

Zoledronate sensitizes endothelial cells to tumor necrosis factor-induced programmed cell death: evidence for the suppression of sustained activation of focal adhesion kinase and protein kinase B/Akt.

Bisphosphonates are potent inhibitors of osteoclast function widely used to treat conditions of excessive bone resorption, including tumor bone metastases. Recent evidence indicates that bisphosphonates have direct cytotoxic activity on tumor cells and suppress angiogenesis, but the associated molecular events have not been fully characterized. In this study we investigated the effects of zoledronate, a nitrogen-containing bisphosphonate, and clodronate, a non-nitrogen-containing bisphosphonate, on human umbilical vein endothelial cell (HUVEC) adhesion, migration, and survival, three events essential for angiogenesis. Zoledronate inhibited HUVEC adhesion mediated by integrin alphaVbeta3, but not alpha5beta1, blocked migration and disrupted established focal adhesions and actin stress fibers without modifying cell surface integrin expression level or affinity. Zoledronate treatment slightly decreased HUVEC viability and strongly enhanced tumor necrosis factor (TNF)-induced cell death. HUVEC treated with zoledronate and TNF died without evidence of enhanced annexin-V binding, chromatin condensation, or nuclear fragmentation and caspase dependence. Zoledronate inhibited sustained phosphorylation of focal adhesion kinase (FAK) and in combination with TNF, with and without interferon (IFN) gamma, of protein kinase B (PKB/Akt). Constitutive active PKB/Akt protected HUVEC from death induced by zoledronate and TNF/IFNgamma. Phosphorylation of c-Src and activation of NF-kappaB were not affected by zoledronate. Clodronate had no effect on HUVEC adhesion, migration, and survival nor did it enhanced TNF cytotoxicity. Taken together these data demonstrate that zoledronate sensitizes endothelial cells to TNF-induced, caspase-independent programmed cell death and point to the FAK-PKB/Akt pathway as a novel zoledronate target. These results have potential implications to the clinical use of zoledronate as an anti-angiogenic or anti-cancer agent.

Characterization of the phosphorylation sites involved in G protein-coupled receptor kinase- and protein kinase C-mediated desensitization of the alpha1B-adrenergic receptor.

Catecholamines as well as phorbol esters can induce the phosphorylation and desensitization of the alpha1B-adrenergic receptor (alpha1BAR). In this study, phosphoamino acid analysis of the phosphorylated alpha1BAR revealed that both epinephrine- and phorbol ester-induced phosphorylation predominantly occurs at serine residues of the receptor. The findings obtained with receptor mutants in which portions of the C-tail were truncated or deleted indicated that a region of 21 amino acids (393-413) of the carboxyl terminus including seven serines contains the main phosphorylation sites involved in agonist- as well as phorbol ester-induced phosphorylation and desensitization of the alpha1BAR. To identify the serines invoved in agonist- versus phorbol ester-dependent regulation of the receptor, two different strategies were adopted, the seven serines were either substituted with alanine or reintroduced into a mutant lacking all of them. Our findings indicate that Ser394 and Ser400 were phosphorylated following phorbol ester-induced activation of protein kinase C, whereas Ser404, Ser408, and Ser410 were phosphorylated upon stimulation of the alpha1BAR with epinephrine. The observation that overexpression of G protein-coupled kinase 2 (GRK2) could increase agonist-induced phosphorylation of Ser404, Ser408, and Ser410, strongly suggests that these serines are the phosphorylation sites of the alpha1BAR for kinases of the GRK family. Phorbol ester-induced phosphorylation of the Ser394 and Ser400 as well as GRK2-mediated phosphorylation of the Ser404, Ser408, and Ser410, resulted in the desensitization of alpha1BAR-mediated inositol phosphate response. This study provides generalities about the biochemical mechanisms underlying homologous and heterologous desensitization of G protein-coupled receptors linked to the activation of phospholipase C.

The role of the TRAF-interacting protein in proliferation and differentiation.

Ubiquitination of proteins is a post-translational modification, which decides on the cellular fate of the protein. Addition of ubiquitin moieties to proteins is carried out by the sequential action of three enzymes: E1, ubiquitin-activating enzyme; E2, ubiquitin-conjugating enzyme; and E3, ubiquitin ligase. The TRAF-interacting protein (TRAIP, TRIP, RNF206) functions as Really Interesting New Gene (RING)-type E3 ubiquitin ligase, but its physiological substrates are not yet known. TRAIP was reported to interact with TRAF [tumor necrosis factor (TNF) receptor-associated factors] and the two tumor suppressors CYLD and Syk (spleen tyrosine kinase). Ectopically expressed TRAIP was shown to inhibit nuclear factor-kappa B (NF-κB) signalling. However, recent results suggested a role for TRAIP in biological processes other than NF-κB regulation. Knock-down of TRAIP in human epidermal keratinocytes repressed cellular proliferation and induced a block in the G1/S phase of the cell cycle without affecting NF-κB signalling. TRAIP is necessary for embryonal development as mutations affecting the Drosophila homologue of TRAIP are maternal effect-lethal mutants, and TRAIP knock-out mice die in utero because of aberrant regulation of cell proliferation and apoptosis. These findings underline the tight link between TRAIP and cell proliferation. In this review, we summarize the data on TRAIP and put them into a larger perspective regarding the role of TRAIP in the control of tissue homeostasis.

Tyrosine phosphorylation is an early and specific event involved in primary keratinocyte differentiation.

Very little is known about early molecular events triggering epithelial cell differentiation. We have examined the possible role of tyrosine phosphorylation in this process, as observed in cultures of primary mouse keratinocytes after exposure to calcium or 12-O-tetradecanoylphorbol-13-acetate (TPA). Immunoblotting with phosphotyrosine-specific antibodies as well as direct phosphoamino acid analysis revealed that induction of tyrosine phosphorylation occurs as a very early and specific event in keratinocyte differentiation. Very little or no induction of tyrosine phosphorylation was observed in a keratinocyte cell line resistant to the differentiating effects of calcium. Treatment of cells with tyrosine kinase inhibitors prevented induction of tyrosine phosphorylation by calcium and TPA and interfered with the differentiative effects of these agents. These results suggest that specific activation of tyrosine kinase(s) may play an important regulatory role in keratinocyte differentiation.

Polar gradients of the DYRK-family kinase Pom1 couple cell length with the cell cycle.

Cells normally grow to a certain size before they enter mitosis and divide. Entry into mitosis depends on the activity of Cdk1, which is inhibited by the Wee1 kinase and activated by the Cdc25 phosphatase. However, how cells sense their size for mitotic commitment remains unknown. Here we show that an intracellular gradient of the dual-specificity tyrosine-phosphorylation regulated kinase (DYRK) Pom1, which emanates from the ends of rod-shaped Schizosaccharomyces pombe cells, serves to measure cell length and control mitotic entry. Pom1 provides positional information both for polarized growth and to inhibit cell division at cell ends. We discovered that Pom1 is also a dose-dependent G2-M inhibitor. Genetic analyses indicate that Pom1 negatively regulates Cdr1 and Cdr2, two previously described Wee1 inhibitors of the SAD kinase family. This inhibition may be direct, because in vivo and in vitro evidence suggest that Pom1 phosphorylates Cdr2. Whereas Cdr1 and Cdr2 localize to a medial cortical region, Pom1 forms concentration gradients from cell tips that overlap with Cdr1 and Cdr2 in short cells, but not in long cells. Disturbing these Pom1 gradients leads to Cdr2 phosphorylation and imposes a G2 delay. In short cells, Pom1 prevents precocious M-phase entry, suggesting that the higher medial Pom1 levels inhibit Cdr2 and promote a G2 delay. Thus, gradients of Pom1 from cell ends provide a measure of cell length to regulate M-phase entry.

Cell entry of Lassa virus induces tyrosine phosphorylation of dystroglycan.

The extracellular matrix (ECM) receptor dystroglycan (DG) serves as a cellular receptor for the highly pathogenic arenavirus Lassa virus (LASV) that causes a haemorrhagic fever with high mortality in human. In the host cell, DG provides a molecular link between the ECM and the actin cytoskeleton via the adapter proteins utrophin or dystrophin. Here we investigated post-translational modifications of DG in the context of LASV cell entry. Using the tyrosine kinase inhibitor genistein, we found that tyrosine kinases are required for efficient internalization of virus particles, but not virus-receptor binding. Engagement of cellular DG by LASV envelope glycoprotein (LASV GP) in human epithelial cells induced tyrosine phosphorylation of the cytoplasmic domain of DG. LASV GP binding to DG further resulted in dissociation of the adapter protein utrophin from virus-bound DG. This virus-induced dissociation of utrophin was affected by genistein treatment, suggesting a role of receptor tyrosine phosphorylation in the process.