ArticleLiterature Review

T cell-antigen receptor signal transduction

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Abstract

During the past year, major progress has been made in understanding proximal TCR signal-transduction events. Cbl has been identified as a negative regulator of kinases from the ZAP-70/Syk family. Studies on LAT, SLP-76, Itk and Vav have revealed their role in the activation of Ras and phospholipase-Cgamma1-Ca2+ signalling pathways. TCR-induced cytoskeletal changes involve signalling through SLP-76-Vav-Nck to activate effectors of the Rho-family of GTPases. Finally, glycolipid-enriched microdomains play a crucial role in T cell activation.

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... Such nGRE sequences were described in the promoters of HIV type 1 [23], human osteocalcin [24] and human corticotrophin releasing hormone (CRH) genes [25]. However, these nGRE sequences are less well characterised yet. ...
... Two minutes of 10 M DX treatment alone caused increased protein tyrosine-phosphorylation at the 34, 52, 59, 70 and 90 kDa molecular weight bands in whole Jurkat cell lysates, compared to the solvent-treated control (Fig. 1). Activation with monoclonal anti-CD3 antibody markedly increased the tyrosine-phosphorylation of several proteins in whole cell lysates at 25,34,38,40,52,59,70 and 90 kDa molecular weight ranges, respectively (Fig. 1). A 2 min DX pre-treatment at 10 M concentration inhibited the anti-CD3-induced tyrosine-phosphorylation the proteins of 34, 38, 40, 52, 59 and 90 kDa (Fig. 1). ...
... ZAP-70 plays a central role in the TcR signal transduction pathway, by phosphorylating a broad range of substrates, and itself is also phosphorylated on several tyrosine residues [25]. For this reason, we investigated the tyrosine-phosphorylation of ZAP-70 after DX and/or anti-CD3 treatments. ...
Thesis
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Glucocorticoids (GC) play essential role in the regulation of the maturation and activation processes in T-cells, regarding especially apoptosis mechanisms. The mutual antagonism between GC signals and T-cell receptor (TcR) derived signals concerned to be crucial in thymocyte selection. The non-genomic actions of GCs are less characterised yet, but based on literature data the existence of cross-talk mechanisms between GC and TcR signal transduction pathways are feasible. We aimed in our work to reveal rapid, non-genomic GC signal transduction mechanisms in T-cells using in vitro cultured Jurkat T-cell leukaemia as a model cell line. Our results show that glucocorticoid receptor (GR) agonist Dexamethasone (DX) causes changes in tyrosine phosphorylation pattern of resting and activated Jurkat cells. These GC effects occurred rapidly, within 5 minutes. ZAP-70 kinase has a crucial role in signal transduction originating from the TcR-CD3 complex. ZAP-70 deficient T-cells fail to respond antigenic stimuli in vitro, and ZAP-70 deficiency in humans and mice results in Severe Combined Immunodeficiency (SCID syndrome). We demonstrate that DX treatment induces rapid ZAP-70 phosphorylation in both resting and activated Jurkat cells. DX induced ZAP-70 phosphorylation is inhibited by GR antagonist (RU486) pre-treatment suggesting the process is GR dependent. However, DX fails to trigger ZAP-70 phosphorylation in p56-lck deficient JCaM-1 cells proposing the involvement of the upstream src-family kinase p56-lck. We investigated the close physical relation of GR and ZAP-70 by coimmunoprecipitation and confocal microscopy. We show, that the ligand-bound GR associates with ZAP-70 in both Jurkat cells and HeLa/trZAP-70 cells stably expressing transgenic ZAP-70. Examining the role of Hsp-90 we found, that a presumably inactive ZAP-70 fraction is associated with Hsp-90 which is most likely excluded from this signal transduction process. The association of ZAP-70 with phosphorylated ITAM tyrosines of the CD3 complex is indispensable for the appropriate transmission of signals derived from the TcR. Our results demonstrate that the association of ZAP-70 with the CD3 complex is inhibited in the DX treated samples. Based on our experimental data we suggest the following signal transduction model: GR, in the presence of its ligand, associates with ZAP-70, triggering tyrosine phosphorylation events. The alteration of tyrosine phosphorylation may influence the kinase activity of ZAP-70 or affect the clustering of other signal transduction molecules with ZAP-70. The GR association with ZAP-70 also inhibits its binding to the ITAMs of the CD3 complex. Recently GCs are widely used as immunosuppressive drugs although their sideeffects are often serious. We revealed here a new non-genomic signal transduction mechanism which may have a crucial role in GC mediated immunosuppression. The characterisation of this new non-genomic pathway may make possible the design and synthesis of new molecules which targets exclusively this immunosuppressive process. In the future this may contribute to the application of new immunosuppressive agents with more favourable side-effect spectra.
... Other cell-surface molecules such as CD2, CD11a/ ICAM-1, and ELAM facilitate optimal activation of endothelial and T cells by SEB (79). TCR and costimulatory receptors activate protein tyrosine kinases (PTKs), LCK and ZAP-70, resulting in phospholipase C gamma (PLCγ) activation, the release of intracellular second messengers, and subsequent protein kinase C (PKC) activation (67,80,81). Accompanying this T-cell activation is F-actin polymerization and increased intracellular Ca 2+ . ...
... Accompanying this T-cell activation is F-actin polymerization and increased intracellular Ca 2+ . PTK and PKC activation lead to mitogen-activated protein kinase (MAPK), extracellular signal regulated kinase (ERK), and cJun N-terminal kinase (JNK) pathways that activate transcriptional factors NFκB, NF-AT, and AP-1 (67,81,82). PKCθ activation leads to CARMA1, Bcl10, and MALT1 (CBM) complexes that induce NFκB transcriptional activation and controls T-cell proliferation (83). ...
... T-cell activation via the TCR-CD3 complex subsequently activates membrane proximal PTKs that phosphorylate TCR intracellular components, other cellular substrates, as well as adaptors (67,(80)(81)(82). PLCγ cleaves phosphatidylinositol 4,5-bisphosphate, generating second messengers to activate PKC and the protooncogene Ras. ...
Article
Full-text available
Staphylococcal enterotoxin B (SEB) of Staphylococcus aureus, and related superantigenic toxins produced by myriad microbes, are potent stimulators of the immune system causing a variety of human diseases from transient food poisoning to lethal toxic shock. These protein toxins bind directly to specific V regions of T-cell receptors (TCR) and major histocompatibility complex (MHC) class II on antigen-presenting cells, resulting in hyperactivation of T lymphocytes and monocytes / macrophages. Activated host cells produce excessive amounts of proinflammatory cytokines and chemokines, especially tumor necrosis factor α, interleukin 1 (IL-1), IL-2, interferon γ (IFNγ), and macrophage chemoattractant protein 1 causing clinical symptoms of fever, hypotension, and shock. Because of superantigen-induced T cells skewed towards TH1 helper cells, and the induction of proinflammatory cytokines, superantigens can exacerbate autoimmune diseases. Upon TCR / MHC ligation, pathways induced by superantigens include the mitogen-activated protein kinase cascades and cytokine receptor signaling, resulting in activation of NFκB and the phosphoinositide 3-kinase / mammalian target of rapamycin pathways. Various mouse models exist to study SEB-induced shock including those with potentiating agents, transgenic mice and an “SEB-only” model. However, therapeutics to treat toxic shock remain elusive as host response genes central to pathogenesis of superantigens have only been identified recently. Gene profiling of a murine model for SEB-induced shock reveals novel molecules upregulated in multiple organs not previously associated with SEB-induced responses. The pivotal genes include intracellular DNA / RNA sensors, apoptosis / DNA damage-related molecules, immunoproteasome components, as well as anti-viral and IFN-stimulated genes. The host-wide induction of these, and other, anti-microbial defense genes provide evidence that SEB elicits danger signals resulting in multi-organ damage and toxic shock. Ultimately, these discoveries might lead to novel therapeutics for various superantigen-based diseases.
... Other cell surface molecules such as CD2, CD11a/ICAM-1, and ELAM facilitated optimal activation of endothelial cells and T cells by SEB [60]. TCR and costimulatory receptors activate signaling kinases, protein kinase C (PKC) and protein tyrosine kinases (PTKs) by the release of intracellular second messengers and various intracellular adaptors646566. PKC and PTK activation lead to other downstream signaling pathways including mitogen–activated protein kinase (MAPK), extracellular signal regulated kinase (ERK) and c-jun N-terminal kinase (JNK) pathways ultimately activating transcriptional factors NFκB, NF-AT, and AP-1656667. Many proinflammatory cytokine genes contain NFκB binding sites in the promotor/enhancer region and are induced by NFκB [68]. ...
... TCR and costimulatory receptors activate signaling kinases, protein kinase C (PKC) and protein tyrosine kinases (PTKs) by the release of intracellular second messengers and various intracellular adaptors646566. PKC and PTK activation lead to other downstream signaling pathways including mitogen–activated protein kinase (MAPK), extracellular signal regulated kinase (ERK) and c-jun N-terminal kinase (JNK) pathways ultimately activating transcriptional factors NFκB, NF-AT, and AP-1656667. Many proinflammatory cytokine genes contain NFκB binding sites in the promotor/enhancer region and are induced by NFκB [68]. ...
... T cell activation via the TCR-CD3 complex induces the activation of the Src family PTKs, LCK and FYN, which in turn phosphorylate tyrosine-based motifs of the TCR intracellular components and other cellular substrates646566. LCK activates another PTK, ZAP-70, which then induces tyrosine phosphorylation of the adaptors LAT (linker for activation of T cells) and SLP-76 (SH2-domain-containing leukocyte protein-76). ...
Article
Full-text available
Immunostimulating staphylococcal enterotoxin B (SEB) and related superantigenic toxins cause diseases in humans and laboratory animals by activating cells of the immune system. These toxins bind directly to the major histocompatibility complex (MHC) class II molecules on antigen-presenting cells and specific Vβ regions of T-cell receptors (TCR), resulting in hyperactivation of both T lymphocytes and monocytes/macrophages. Activated host cells produce excessive amounts of proinflammatory cytokines and chemokines, especially tumor necrosis factor α, interleukin 1 (IL-1), IL-2, interferon γ (IFNγ), and macrophage chemoattractant protein 1 causing clinical symptoms of fever, hypotension, and shock. The well-explored signal transduction pathways for SEB-induced toxicity downstream from TCR/MHC ligation and interaction of cell surface co-stimulatory molecules include the mitogen-activated protein kinase cascades and cytokine receptor signaling, culminating in NFκB activation. Independently, IL-2, IFNγ, and chemokines from activated T cells signal via the phosphoinositide 3-kinase (PI3K), the serine/threonine kinases, Akt and mammalian target of rapamycin (mTOR) pathways. This article reviews the signaling molecules induced by superantigens in the activation of PI3K/Akt/mTOR pathways leading to staphylococcal superantigen-induced toxicity and updates potential therapeutics against superantigens.
... The alternative TCR/CD3/FcRγ complex recruits and activates Syk, instead of ZAP-70 [23]. These various PTKs phosphorylate an adaptor protein LAT, ultimately resulting in the phosphorylation and activation of phospholipase C-γ (PLC-γ) [24]. PLC-γ cleaves phosphatidylinositol 4,5-bisphosphate (PIP 2 ) in the plasma membrane to generate diacylglycerol, which activates protein kinase C (PKC) and Ras-dependent pathways, and 1,4,5-inositol trisphosphate (IP 3 ), which causes entry of Ca 2+ to cytosol from two sources: the Endoplasmic reticulum (ER) and the extracellular fluid [25]. ...
... ZAP-70 associated with TCR/CD3 complex phosphorylates an adaptor protein LAT, resulting in the phosphorylation of PLC-γ [24]. We next measured the phosphorylation of PLC-γ1. ...
Article
Although the pH values of blood and tissues are usually maintained in a narrow range around 7.4, some diseased areas, such as cancer nests, inflammatory loci, and infarction areas, are acidified. In the present study, the effect of extracellular acidic pH on TCR signaling was examined with human acute leukemia T cell line Jurkat cells because T cell infiltration is often observed in acidic diseased areas. The phosphorylation levels of CD3-ξ ZAP-70, and PLC-γ1 induced by OKT-3, anti-CD3 antibody, were higher at pH 6.3 than those at pH 7.6. The activation of PLC-γ1 induced by OKT-3 was further increased by the co-stimulation with CD28.6, anti- CD28 antibody, at pH 7.6, but not at pH 6.3. The level of cytosolic free calcium ions was increased to a higher level by the addition of OKT-3 at pH 6.3, compared with that by the addition of OKT-3 plus CD28.6 at pH 7.6. Further addition of CD28.6 decreased the level of cytosolic free calcium ions induced by OKT-3 at pH 6.3. The Ca2+ mobilization was strongly inhibited by BTP2, a potent inhibitor of Ca2+ channels in the plasma membrane, at pH 7.6, while the inhibition was weak at pH 6.3. The Ca2+ mobilization at pH 6.3 was dependent on ZAP-70 and LAT, but not SLP-76. The activation of ERK and p38 increased as pH decreased. No activation of ERK2 in the presence of OKT-3 was observed in the Jurkat mutant deficient in ZAP-70 at pH 6.3, while ERK1 was activated by the addition of OKT-3 in this mutant. The expression of IL-2 was not induced by OKT-3 or OKT-3 plus CD28.6 at pH 6.3. These results suggest that the TCR signaling initiated by CD3 stimulation is more active at acidic pH in Jurkat cells and its pathway is different in parts under different pH conditions.
... LAT is a predominantly cytoplasmic membrane protein with two cysteine residues just below the transmembrane region and is targeted to the rafts by palmitoylation of cysteines [13,14]. LAT also contains multiple tyrosine-based motifs which, when phosphorylated by ZAP-70 or Syk, initiate the assembly with SH2 domain-containing signaling proteins such as phosphatidylinositol 3-kinase (PI 3-K), phospholipase C-+ 1 (PLC-+ 1) and Grb2, and allows subsequent extensions of the signaling scaffold [15][16][17]. Thus, it is well documented that rafts and LAT play a central role in TCR signal transduction. ...
... Several lines of evidence demonstrate that tyrosinephosphorylated LAT recruits critical signaling molecules, including PLC-+ 1, Grb2 and PI 3-K as well as Sos and SLP-Vav complex, suggesting that LAT is important in linking the activation of Syk/ZAP-70 to a number of downstream pathways [13,16,17,37,42]. Activation of PLC-+ 1 results in hydrolyzing phosphatidylinositol 4,5bisphosphate, which, in turn, increases intracellular free Ca 2+ concentrations and protein kinase C activity, resulting in enhanced cytoplasmic granule exocytosis [1,43]. ...
Article
Natural killer (NK) cells participate in both innate and adaptive immunity through the prompt secretion of cytokines and ability to lyse virally infected cells or tumor cells. Although it has been well understood that lipid rafts (rafts) and a raft-associated linker for activation of T cells (LAT) plays a central role in TCR signal transduction, there are still great gaps in our knowledge of the molecular events involved in NK cell activation. We show here that CD2 and rafts became polarized to the site of NK cell activation by CD2 cross-linking or target cell binding using confocal microscopy, and LAT and a significant amount of CD2 colocalized in raft fractions of sucrose-density gradient from an NK cell line, NK3.3. CD2 cross-linking strongly induced tyrosine phosphorylation of LAT, resulting in increased association with phosphatidylinositol 3-kinase (PI 3-K) and phospholipase C-+ 1 (PLC-+ 1). In vitro binding studies using glutathione S-transferase fusion proteins demonstrated that a large portion of the association between LAT and PI 3-K or PLC-+ 1 was mediated through their SH2 domains in tyrosine phosphorylation-dependent manner. Furthermore, disruption of lipid rafts by cholesterol depletion from cell membranes using methyl-g-cyclodextrin markedly reduced LAT tyrosine phosphorylation and NK cell functions, including cytotoxicity and granule exocytosis. These results document that modulation of raft integrity by aggregation of NK cell activating receptors, which leads to the formation of complexes of LAT with PI 3-K and PLC-+ 1, is essential for the NK cell lytic mechanisms.
... The three signals required for T-cell activation by superantigens and conventional antigens are similar even though superantigens bind outside the peptide-binding groove of MHC class II molecules. The first signal is induced upon the binding of superantigen with TCR-CD3 complex, which activates the Src family of protein tyrosine kinases (PTKs) [58][59][60]. The engagement of co-stimulatory molecules on APC and T-cells, subsequent to superantigen binding, results in a second signal that optimizes and sustains T-cell activation [61][62][63]. ...
... Co-ligation of receptors results in extensive cytoskeletal remodeling and the formation of immunological synapse, initiating signaling cascades [61,64]. PTKs, including Lck and ZAP-70, phosphorylate tyrosine-based motifs of the TCR intracellular components and other adaptors [58,59,65]. The TCR-induced kinases activate phospholipase C gamma (PLCγ) resulting in the generation of second messengers and increase in intracellular calcium levels. ...
Article
Full-text available
Staphylococcal enterotoxin B (SEB) and related bacterial toxins cause diseases in humans and laboratory animals ranging from food poisoning, acute lung injury to toxic shock. These superantigens bind directly to the major histocompatibility complex class II molecules on antigen-presenting cells and specific Vβ regions of T-cell receptors (TCR), resulting in rapid hyper-activation of the host immune system. In addition to TCR and co-stimulatory signals, proinflammatory mediators activate signaling pathways culminating in cell-stress response, activation of NFκB and mammalian target of rapamycin (mTOR). This article presents a concise review of superantigen-activated signaling pathways and focuses on the therapeutic challenges against bacterial superantigens.
... During T cell activation, members of three different cytosolic tyrosine kinase families are involved in PLC-γ1 tyrosine phosphorylation. Src family members are indirectly required, as these kinases serve to phosphorylate specific tyrosine residues within immunoreceptor tyrosine-based activation motifs (ITAMs) present on the cytoplasmic domains of T cell receptor polypeptide chains (Van Leeuwen and Samelson, 1999). Phosphorylated ITAM motifs then serve as docking sites for members of the ZAP-70/Syk tyrosine kinase family, which contain SH2 domains. ...
... LAT is a 38-kD integral membrane protein that becomes highly tyrosine phosphorylated following T cell activation. The kinase that phosphorylates LAT is thought to be ZAP-70, and, once phosphorylated, LAT associates with multiple signaling proteins that contain SH2 domains, including PLC-γ1 (Clement et al., 1999;Kennedy, et al., 1999;Van Leeuwen and Samelson, 1999). Also, the association with LAT is necessary for PLC-γ1 tyrosine phosphorylation and activation (Kennedy, et al., 1999). ...
Article
Full-text available
Phospholipase C (PLC)1 hydrolyzes phosphatidylinositol 4,5-bisphosphate to generate the second messengers, inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). IP3 induces a transient increase in intracellular free Ca2+, while DAG directly activates protein kinase C. Upon stimulation of cells with growth factors, PLC-γ1 is activated upon their association with and phosphorylation by receptor and non-receptor tyrosine kinases. In this review, we will focus on the activation mechanism and regulatory function of PLC-γ1.
... Tyrosine-based Activation Motifs (ITAMs) with the consensus sequence YxxI/Lx (6)(7)(8)(9)(10)(11)(12)YxxI/L, where x represents any amino acid, are typically associated with positive or activating immune response (2). In contrast, the immune system elicits negative or inhibitory response through receptors bearing the Immunoreceptor Tyrosine-based Inhibition Motifs (ITIMs) with the degenerated sequence S/I/V/LxYxxI/V/L (3). ...
... Similarly, ITAM sequences have been found to mediate inhibitory signaling (8). Indeed, even within the same cell type, an ITAM-containing receptor may mediate functions as diverse as microbial killing, antigen presentation, cytokine production, T-Cell instruction, and tissue repair (2). Moreover, exquisite binding specificities have been observed for different ITRMs. ...
Article
Full-text available
Cells of the immune system communicate with their environment through immunoreceptors. These receptors often harbor intracellular tyrosine residues, which, when phosphorylated upon receptor activation, serve as docking sites to recruit downstream signaling proteins containing the Src Homology 2 (SH2) domain. A systematic investigation of interactions between the SH2 domain and the immunoreceptor tyrosine-based regulatory motifs (ITRM), including inhibitory (ITIM), activating (ITAM) or switching (ITSM) motifs, is critical for understanding cellular signal transduction and immune function. Using the B cell inhibitory receptor CD22 as an example, we developed an approach that combines reciprocal or bidirectional phosphopeptide and SH2 domain array screens with in-solution binding assays to identify a comprehensive SH2-CD22 interaction network. Extending this approach to 194 human ITRM sequences and 78 SH2 domains led to the identification of a high-confidence immunoreceptor interactome containing 1137 binary interactions. Besides recapitulating many previously reported interactions, our study uncovered numerous novel interactions. The resulting ITRM-SH2 interactome not only helped to fill many gaps in the immune signaling network, it also allowed us to associate different SH2 domains to distinct immune functions. Detailed analysis of the NK cell ITRM-mediated interactions led to the identification of a network nucleated by the Vav3 and Fyn SH2 domains. We showed further that these SH2 domains have distinct functions in cytotoxicity. The bidirectional protein-peptide array approach described herein may be applied to the numerous other peptide-binding modules to identify potential protein-protein interactions in a systematic and reliable manner. Copyright © 2015, The American Society for Biochemistry and Molecular Biology.
... Recently, Cbl has been identified as a prominent substrate of protein tyrosine kinases (PTKs) 1 that is rapidly phosphorylated after engagement of several different receptors on hematopoietic cells. Cbl has no known catalytic function, but it contains a RING finger domain, an extensive prolinerich region, and a COOH-terminal leucine zipper (for reviews, see references [4][5][6][7][8]. Although it has been thought that Cbl contains a novel NH 2 -terminal phosphotyrosinebinding domain (9), recent crystal structure analyses indi-cate that the Cbl NH 2 -terminal region (Cbl-N) is not similar to a phosphotyrosine-binding domain (10,11). ...
... The functional importance of Cbl in hematopoietic cell signaling was initially suggested by the finding that Cbl binds to several critical signaling molecules, e.g., PTKs such as Src family PTK (12)(13)(14), Syk/Zap-70 family PTK (8,(15)(16)(17)(18), and Btk (19); adaptor molecules, including Grb2 (13,20), Crk (21)(22)(23), and Nck (24); and effector molecules, including phosphatidylinositol 3-kinase (PI3-K [16,25]) and Vav (26). The most revealing clue about the function of Cbl came from genetic studies in Caenorhabditis elegans , where SLI-1, a C . ...
Article
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Accumulating evidence indicates that the Cbl protein plays a negative role in immune receptor signaling; however, the mode of Cbl action in B cell receptor (BCR) signaling still remains unclear. DT40 B cells deficient in Cbl showed enhanced BCR-mediated phospholipase C (PLC)- g 2 activation, thereby leading to increased apoptosis. A possible explanation for the in- volvement of Cbl in PLC- g 2 activation was provided by findings that Cbl interacts via its Src homology 2 (SH2) domain with B cell linker protein (BLNK) after BCR ligation. BLNK is a critical adaptor molecule for PLC- g 2 tyrosine phosphorylation through its binding to the PLC- g 2 SH2 domains. As a consequence of the interaction between Cbl and BLNK, the BCR-induced recruitment of PLC- g 2 to BLNK and the subsequent PLC- g 2 tyrosine phosphorylation were inhibited. Thus, our data suggest that Cbl negatively regulates the PLC- g 2 pathway by inhibit- ing the association of PLC- g 2 with BLNK.
... Together, the data indicate that these regulatory protens are not a factor here affecting the antiviral efficacy of the CTL clones and that antiviral efficacy of the same epitope-specific CTLs is determined by TCR clonotypes. The ability of the TCR to initiate and sustain intracellular signal transduction events is essential to ensure productive T-cell activation and induction of antiviral activities [30,31]. To define the possible mechanisms responsible for these differences among the TCR clonotypes, we evaluated a panel of cytokine production (CD107a, IFN-γ, IL-2, MIP-1β, and TNF-α) for these clones by flow cytometry (Figure 3b). ...
... The properties of CTLs, such as IFN-γ expression, lysis, proliferation, simultaneous expression of multiple cytokines, currently measured following peptide stimulation, have been associated with a decline in plasma viremia but has not been causally linked to viral control [1,4,5,7]. Another critical qualifying component of the virus-specific CTLs is the selection of CTL-bearing TCRs that interact with HLA/peptide complex to initiate and sustain intracellular signal transduction events to ensure productive T cell activation and induction of antiviral activities [30,31]. Functional differences between TCR variants that recognize the same HLA/peptide complex have not been well defined. ...
Article
Increasing evidence suggests that the immune control of viral replication by HIV-1-specific cytotoxic T lymphocytes (CTLs) is relevant to the selection of human leukocyte antigen (HLA) allele-restricted antigen-specific CTL repertoire. But the underlying factors accounting for CTL functional difference from T cell receptor (TCR) clonal diversity are currently unclear. Here we found that CTL repertoire specific for HLA-B*27-restricted HIV-1 Gag p24 epitope KK10 (KRWIILGLNK, residues 263-272) selected multiple TCR clonotypes. By dissection of the bulk KK10-specific CTL compartment, differential potency and breadth of KK10-specific CTL clones to inhibit HIV-1 replication was defined due to the distinct TCR usage. Superior control of viral replication of wild-type HIV-1 isolates was observed by the TCR-clonotypic CTLs characteristic of higher ability to produce MIP-1β. A unique TCR-equipped KK10- specific CTLs efficiently controlled wide-type HIV-1 isolates and broadly cross-recognized viral variants. These data suggest that clonally diverse CTL responses to a viral epitope increase the likelihood that multiple divergent strains of viruses may be recognized by cross-reactive CTLs through immunization with only a single viral epitope sequence.
... Two defining points in the field of antigen receptor signal transduction came from the discovery of immunoreceptor tyrosine-based activation motifs (ITAMs), characterised by tandem YxxL sequences, within the signalling chains of these receptors (Reth, 1989) and from the observation that antigen receptor engagement induces multiple cytoplasmic protein tyrosine phosphorylation events. The mechanisms underlying antigen receptor signalling have been extensively characterised through both genetic and biochemical approaches (Campbell, 1999;Clements et al., 1999;Kinet, 1999;Kurosaki, 1999;van Leeuwen and Samelson, 1999 One of the earliest events following antigen receptor engagement is the phosphorylation of tyrosine residues within the signalling chain ITAM motifs of these receptors by activated Src family kinases such as Lyn, Fyn, BIk and/or Lck. ...
... The guanine nucleotide binding protein p2l'^®® rapidly accumulates in its active, (DeFranco, 1997;Kurosaki, 1999;van Leeuwen and Samelson, 1999). ...
Thesis
Protein kinase C (PKC) enzymes have been implicated in a wide range of biological responses in vivo, including cell morphology, differentiation and proliferation. A related kinase is Protein kinase D (PKD), also known as PKCμ. The catalytic domain of PKD is distinct from that of PKC enzymes, although it contains a conserved C1 domain that binds phorbol esters and diacylglycerol (DAG) in a manner similar to classical and novel PKCs. The experiments described here demonstrate that the pharmacological agent bryostatin 1, like phorbol esters, stimulates PKD activity through a novel PKC-dependent signalling pathway in fibroblasts. Importantly, physiological antigen receptor triggering also induces PKC-dependent activation of PKD, in T and B cell lines, in mast cells and in peripheral blood-derived T lymphocytes. Significantly, PKD activity is dynamically regulated by both positive and negative signals from antigen receptors in B lymphocytes. The second cysteine-rich motif within the C1 domain of PKD is the major binding site for phorbol esters, both in vitro and in vivo. However, direct phorbol ester/DAG binding to PKD is dispensable for its activation, as shown by analysis of a PKD C1 domain mutant, consistent with the proposed role for classical and novel PKC enzymes in the regulation of PKD activity. A functional C1 domain is however required for the translocation of PKD from the cytosol to the plasma membrane of intact cells in response to antigen receptor ligation or phorbol ester treatment. Strikingly, PKD only briefly translocates to the plasma membrane of B lymphocytes and mast cells following receptor stimulation, before returning to the cytosol within minutes of the initiation of antigen receptor signalling. In contrast, a sustained phase of PKD activity is observed in antigen receptor-activated B lymphocytes which is maintained over a period of hours. These data suggests that both direct and indirect DAG signalling pathways contribute to the regulation of PKD in vivo and indicate that PKD may disseminate DAG signals away from the plasma membrane into the cell interior during sustained responses to antigen receptor engagement.
... A major subset of these cells was found to express NK1.1, previously associated with NK cells, and to have the potential to secrete high levels of IL-4, IFN-␥ and tumor necrosis factor (TNF). A population of NK1.1 ϩ CD4 ϩ T cells was also identified with similar characteristics [1][2][3] . Subsequent reports showed that both populations predominantly express TCR V␣14J␣281 and that their development was dependent on the MHC class I-like, ␤ 2microglobulin-associated molecule, CD1d. ...
Article
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Recent studies have identified protein kinase Cθ (PKCθ), a member of the Ca2+-independent PKC family, as an essential component of the T-cell synapse that cooperates with calcineurin to activate the interleukin-2 (IL-2) gene. Several selective functions of PKCθ involved in the activation and survival of T cells are reviewed herein. Among these, the nuclear factor-κB (NF-κB) signaling cascade appears to be the most critical target of PKCθ in the T-cell receptor/CD28 costimulatory pathway that leads to T-cell activation.
... IL-2 expression is a hallmark of T-cell proliferation and differentiation after T cell activation [32] and HIV-1 infection effectively increase the promoter activity of IL-2 through activation of TCR signaling [13]. As in previous studies, the IL-2 levels increased markedly in acutely HIV-1-infected T cells treated with stimulus but were resistant to stimulation in latently infected cells (Fig. 4). ...
... However, BLNK might not be expressed in mast cells because a model mast cell line RBL-1 does not express BLNK (48). Similar functions may be played by other linker proteins, such as SLP-76, a BLNK homologue in T cells, LAT (49,50), and Clnk (51), which are expressed in mast cells. SLP-76 was shown to be required for FcRI-induced mast cell activation (52). ...
Article
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Two protein-tyrosine kinases, Bruton's tyrosine kinase (Btk) and Syk, and members of the protein kinase C (PKC) subfamily of serine/threonine kinases play crucial roles in signal transduction through antigen receptors in B lymphocytes and high-affinity IgE receptors (FcɛRI) in mast cells. The present study provides genetic, biochemical, and pharmacological evidence that, on FcɛRI stimulation, Syk regulates Btk, and Btk selectively regulates the membrane translocation and enzymatic activity of PKCβI among the conventional PKC isoforms (α, βI, and βII) expressed in mast cells. Syk/Btk-mediated PKCβI regulation is involved in transcriptional activation of the IL-2 and tumor necrosis factor α genes through the JNK pathway induced by FcɛRI stimulation. Accordingly, FcɛRI-induced production of these cytokines is inhibited by specific inhibitors of Btk and Syk, as well as broad-specificity inhibitors of PKC and a selective inhibitor of PKCβ. Specific regulation of PKCβI by Btk is consistent with the selective association of Btk with PKCβI. Components of this signaling pathway may represent an attractive set of potential targets of pharmaceutical interference for the treatment of allergic and other immunologic diseases.
... The importance of Lck and Fyn has been extensively studied in T cell development and differentiation (van Leeuwen and Samelson 1999). Nevertheless, much has to be understood about regulating these proximal events of TCR signaling and the sole involvement of Src kinases in them. ...
... This review will focus on major advancements in TCR signaling in the past year. The reader is referred here and at various points to more detailed reviews on previous advances in the field in general123 and also specific areas that are covered. ...
Article
The past several years have seen the beginning of a shift in the way that TCR signal transduction is studied. Although many investigators continue to identify new molecules, particularly adaptor proteins, others have attempted to look at signaling events in a larger cellular context. Thus the identification of distinct formations of signaling molecules at junctions between T cells and antigen-presenting cells, the role of the cytoskeleton and the partitioning of molecules into specialized lipid subdomains have been the subjects of many publications. Such concepts are helping to assemble a blueprint of how the myriad adaptors and kinases fit together to effect T cell activation.
... Die Aktivierung der T-Zellen erfolgt durch die Bindung des T-Zell-Rezeptors an den Komplex aus MHC-Protein und Peptid. Dadurch kommt es zur Auslösung einer Signalkaskade in der T-Zelle und letztlich zur Proliferation(Gascoigne & Zal 2004;van Leeuwen & Samelson 1999).Dendritische Zellen hingegen befinden sich im unreifen Zustand im Gewebe, zumBeispiel die Langerhanszellen in der Haut. Kommt es lokal zu einer Vermehrung pathogener Erreger, so treten im Gewebe verstärkt Gefahrensignale wie bakterielles LPS oder virale Einzelstrang-DNA auf, die zur Aktivierung der dendritischen Zellen über die Toll-Like-Rezeptoren (TLR) führen(Matzinger 1994;Janeway, Jr. & Medzhitov 2002). ...
... Phosphorylated ITAM generate docking sites for 70-kD ζ -chain-associated protein kinase (ZAP70). Lck also phosphorylates ZAP70, which propagates signaling events such as intracellular calcium influx and the MAPK kinase known as Ras-MAPK or extracellular signal-regulated kinase (ERK) (van Leeuwen & Samelson, 1999). Both of these events are necessary for T-cell activation (Smith-Garvin et al, 2009;Courtney et al, 2018;Gaud et al, 2018). ...
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Special AT-rich sequence binding protein-1 (SATB1) is localized to the nucleus and remodels chromatin structure in T cells. SATB1-deficient CD4 T cells cannot respond to TCR stimulation; however, the cause of this unresponsiveness is to be clarified. Here, we demonstrate that SATB1 is indispensable to proper mitochondrial functioning and necessary for the activation of signal cascades via the TCR in CD4 T cells. Naïve SATB1-deficient CD4 T cells contain fewer mitochondria than WT T cells, as the former do not express mitochondrial transcription factor A (TFAM). Impaired mitochondrial function in SATB1-deficient T cells subverts mitochondrial ROS production and SHP-1 inactivation by constitutive oxidization. Ectopic TFAM expression increases mitochondrial mass and mitochondrial ROS production and rescues defects in the antigen-specific response in the SATB1-deficient T cells. Thus, SATB1 is vital for maintaining mitochondrial mass and function by regulating TFAM expression, which is necessary for TCR signaling.
... The biochemical events that occur subsequent to CD3/TCR ligation have been extensively analyzed with regard to transcriptional regulation of the IL-2 gene (7,8). Activation of src family kinases results in tyrosine phosphorylation of immunoreceptor tyrosine-based activation motifs on CD3 subunits of the TCR complex, as well as tyrosine phosphorylation of the Syk family tyrosine kinase ZAP-70 (9 -12). ...
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Stimulation of the CD3/TCR results within minutes in an increase in T cell adhesion mediated by b1 integrins. The biochemical pathways that control CD3-mediated increases in b1 integrin-mediated adhesion remain poorly characterized. In this study, the role of the tyrosine kinase ZAP-70 in the regulation of b1 integrin activity by the CD3/TCR was investigated. CD3 stimulation did not increase b1 integrin-mediated adhesion of the ZAP-70-deficient Jurkat T cell line, P116, to the b1 integrin ligand fibronectin. Reintroduction of wild-type ZAP-70, but not a kinase-inactive variant, K369R, corrected the adhesive defect observed in P116 T cells. In addition, the kinase-inactive ZAP-70 mutant inhibited CD3-induced adhesion of primary human T cell blasts. Interest- ingly, a ZAP-70 mutant with a tyrosine to phenylalanine substitution at position 319 (Y319F) restored the adhesive defect in P116 T cells, even though Y319F ZAP-70 failed to fully reconstitute CD3-initiated NF-AT-dependent transcription and tyrosine phos- phorylation of the LAT adapter protein. Finally, expression of mutants of LAT and the SLP-76 adapter protein that modulate CD3-mediated activation of an NF-AT reporter gene failed to block CD3-induced increases in b1 integrin-mediated adhesion. These observations support a model in which the tyrosine kinase activity of ZAP-70 kinase is critical for regulation of b1 integrin activity by CD3/TCR. However, the signaling events downstream of ZAP-70 that regulate CD3/TCR-mediated activation of b1 integrin function exhibit key differences when compared with the signaling pathways that regulate transcriptional events initiated by CD3/TCR stimulation. The Journal of Immunology, 2000, 165: 4941- 4949.
... In contrast, properly activated T cells are able to eliminate pathogen-infected cells as well as cancer cells, while avoiding damage to the healthy tissues of the host organism. The speed, sensitivity and specificity of this process is remarkable and conveyed by the activation of downstream pathways that regulate the expression and function of a plethora of immune modulatory genes/proteins (11). These include an upregulation of inhibitory molecules, a decrease of effector functions and a reduced proliferation that are required to shutdown the immune response after removal of the "unhealthy cells" (12). ...
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Despite the broad application of different immunotherapeutic strategies for the treatment of solid as well as hematopoietic cancers, the efficacy of these therapies is still limited, with only a minority of patients having a long-term benefit resulting in an improved survival rate. In order to increase the response rates of patients to the currently available immunotherapies, a better understanding of the molecular mechanisms underlying the intrinsic and/or extrinsic resistance to treatment is required. There exist increasing evidences that activation of different oncogenic pathways as well as inactivation of tumor suppressor genes (TSG) in tumor cells inhibit the immune cell recognition and influegnce the composition of the tumor microenvironment (TME), thus leading to an impaired anti-tumoral immune response. A deeper understanding of the link between the tumor milieu and genomic alterations of TSGs and oncogenes is indispensable for the optimization of immunotherapies and to predict the patients’ response to these treatments. This review summarizes the role of different cancer-related, oncogene- and TSG-controlled pathways in the context of anti-tumoral immunity and response to different immunotherapies.
... Presentation of antigen to a memory T cell by an antigen-presenting cell triggers signal transduction through the T-cell receptor (CD3) and co-stimulatory molecules such as CD28, which results in T-cell activation and clonal expansion (Acuto and Michel, 2003). Programs are thus initiated (van Leeuwen and Samelson, 1999; van Oers, 1999 ) that lead to T-cell proliferation , increase in cell size (blastogenesis) and the induction of effector molecules (activation), which include cytokines and cell surface receptors (Mosmann et al, 1986; Jain et al, 1995). We previously identified a G 0 -G 1 commitment point (CP) in primary human T cells that controls entry into the cell cycle from quiescence (Lea et al, 2003), which provided proof of principle that entry into the cell cycle and cellular growth cycle can be uncoupled from early expression of T-cell effector (activation) functions, such as production of IL-2, g-IFN and cell surface CD69, CD44 and CD62L. ...
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Regulating the transition of cells such as T lymphocytes from quiescence (G(0)) into an activated, proliferating state involves initiation of cellular programs resulting in entry into the cell cycle (proliferation), the growth cycle (blastogenesis, cell size) and effector (functional) activation. We show the first proteomic analysis of protein interaction networks activated during entry into the first cell cycle from G(0). We also provide proof of principle that blastogenesis and proliferation programs are separable in primary human T cells. We employed a proteomic profiling method to identify large-scale changes in chromatin/nuclear matrix-bound and unbound proteins in human T lymphocytes during the transition from G(0) into the first cell cycle and mapped them to form functionally annotated, dynamic protein interaction networks. Inhibiting the induction of two proteins involved in two of the most significantly upregulated cellular processes, ribosome biogenesis (eIF6) and hnRNA splicing (SF3B2/SF3B4), showed, respectively, that human T cells can enter the cell cycle without growing in size, or increase in size without entering the cell cycle.
... In addition, myristoylation-dependent localization of Lck to the CD4 receptor is very integral to the activation of T cells since un-myristoylated Lck is cytosolic and unable to facilitate TCR signaling cascades ( Figure 3B) (15). In the presence of NMT activity, TCR ligation activates myristoylated Lck that in turn phosphorylates tyrosine containing immunoreceptor tyrosinebased activation motifs (ITAMs) (69). On phosphorylation of ITAMs, ZAP-70 is recruited that activates various signaling molecules leading to T cell activation ( Figure 3A). ...
... Once phosphorylated, ITAMS become recruitment sites for ZAP-70, activated Zap-70 is then recruited to phosphorylate the multiple transmembrane molecules. These signalling molecules activate multiple secondary signalling pathways including Ca 2+ signalling, Ras/MAP kinase, and protein kinase C (PKC) pathways that ultimately activate several transcription factors including NFAT, AP-1 and NF-κB that are essential for full IL-2 gene expression and T cell activation [12][13][14]. We observed that ESAT-6 curtailed TCR triggered calcium mobilization in pulmonary TB patients and PPD+ve healthy individuals both but the inhibition is more in patients as compared to healthy individuals. ...
Article
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Background Mycobacterium tuberculosis (M. tuberculosis) modulates host immune response, mainly T cell responses for its own survival leading to disease or latent infection. The molecules and mechanisms utilized to accomplish immune subversion by M. tuberculosis are not fully understood. Understanding the molecular mechanism of T cell response to M. tuberculosis is important for development of efficacious vaccine against TB. Methods Here, we investigated effect of M. tuberculosis antigens Ag85A and ESAT-6 on T cell signalling events in CD3/CD28 induced Peripheral blood mononuclear cells (PBMCs) of PPD+ve healthy individuals and pulmonary TB patients. We studied CD3 induced intracellular calcium mobilization in PBMCs of healthy individuals and TB patients by spectrofluorimetry, CD3 and CD28 induced activation of mitogen activated protein kinases (MAPKs) in PBMCs of healthy individuals and TB patients by western blotting and binding of transcription factors NFAT and NFκB by Electrophorectic mobility shift assay (EMSA). Results We observed CD3 triggered modulations in free intracellular calcium concentrations in PPD+ve healthy individuals and pulmonary TB patients after the treatment of M. tuberculosis antigens. As regards the downstream signalling events, phosphorylation of MAPKs, Extracellular signal-regulated kinase 1 and 2 (ERK1/2) and p38 was curtailed by M. tuberculosis antigens in TB patients whereas, in PPD+ve healthy individuals only ERK1/2 phosphorylation was inhibited. Besides, the terminal signalling events like binding of transcription factors NFAT and NFκB was also altered by M. tuberculosis antigens. Altogether, our results suggest that M. tuberculosis antigens, specifically ESAT-6, interfere with TCR/CD28-induced upstream as well as downstream signalling events which might be responsible for defective IL-2 production which further contributed in T-cell unresponsiveness, implicated in the progression of disease. Conclusion To the best of our knowledge, this is the first study to investigate effect of Ag85A and ESAT-6 on TCR- and TCR/CD28- induced upstream and downstream signalling events of T-cell activation in TB patients. This study showed the effect of secretory antigens of M. tuberculosis in the modulation of T cell signalling pathways. This inflection is accomplished by altering the proximal and distal events of signalling cascade which could be involved in T-cell dysfunctioning during the progression of the disease. Electronic supplementary material The online version of this article (doi:10.1186/s12865-015-0128-6) contains supplementary material, which is available to authorized users.
... The presentation of a peptide linked to the major histocompatibility complex on an antigen-presenting cell to a T-cell receptor leads to crosslinking of the receptor and the transmission of intracellular signals via its associated CD3 complex. This causes tyrosine phosphorylation of the immunoreceptor tyrosine-based activation motifs (ITAMs) at the cytoplasmic tails of the CD3 complex, which (via a cascade of protein phosphorylation) leads to the transcriptional activation of genes that encode cytokines and induce cellular proliferation and differentiation (643). ...
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Maintenance of periodontal health or transition to a periodontal lesion reflects the continuous and ongoing battle between the vast microbial ecology in the oral cavity and the array of resident and emigrating inflammatory/immune cells in the periodontium. This war clearly signifies many ‘battlefronts’ representing the interface of the mucosal-surface cells with the dynamic biofilms composed of commensal and potential pathogenic species, as well as more recent knowledge demonstrating active invasion of cells and tissues of the periodontium leading to skirmishes in connective tissue, the locality of bone and even in the local vasculature. Research in the discipline has uncovered a concerted effort of the microbiome, using an array of survival strategies, to interact with other bacteria and host cells. These strategies aid in colonization by ‘ambushing, infiltrating and outflanking’ host cells and molecules, responding to local environmental changes (including booby traps for host biomolecules), communicating within and between genera and species that provide MASINT (Measurement and Signature Intelligence) to enhance sustained survival, sabotage the host inflammatory and immune responses and by potentially adopting a ‘Fabian strategy’ with a war of attrition and resulting disease manifestations. Additionally, much has been learned regarding the ever-increasing complexity of the host-response armamentarium at both cellular and molecular levels that is addressed in this review. Knowledge regarding how these systems fully interact requires both new laboratory and clinical tools, as well as sophisticated modeling of the networks that help maintain homeostasis and are dysregulated in disease. Finally, the triggers resulting in a ‘coup de main’ by the microbiome (exacerbation of disease) and the characteristics of susceptible hosts that can result in ‘pyrrhic victories’ with collateral damage to host tissues, the hallmark of periodontitis, remains unclear. While much has been learned, substantial gaps in our understanding of the ‘parameters of this war’ remain elusive toward fulfilling the Sun Tzu adage: ‘If you know the enemy and know yourself, you need not fear the result of a hundred battles.’
... Following TCR encounter of antigenic peptide bound to MHC-I in association with accessory molecules, such as CD8, CD28, and lymphocyte function-associated antigen-1 (LFA-1), the T cell is activated (9)(10)(11), and its functional antiviral activity may be commensurate to the ability of the TCR to initiate and sustain intracellular signal transduction (12,13). T-cell stimulation involves TCR-induced activation of cellular kinases that phosphorylate multiple downstream protein targets (14)(15)(16) that induce the translocation of transcription factors into the nucleus to activate a network of genes whose level of expression is determined by the initial strength and duration of the TCR triggering (17). ...
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Importance: The greater ex vivo antiviral inhibitory activity of CD8(+) T cells from elite controllers as compared to those from HIV-1 progessors supports the crucial role of effective HIV-specific CD8(+) T cells in controlling HIV-1 replication. The contribution of TCR clonotype to inhibitory potency was investigated by delineating the responsiveness of effective and ineffective CD8(+) T cell clones recognizing the identical HLA-B*2705-restricted HIV-1 Gag-derived peptide, KK10 (KRWIILGLNK). KK10-stimulated "effective" CD8(+) T cell clones displayed significantly more rapid TCR signal propagation, initial lytic granule release and sustained cytokine and chemokine secretion compared to "ineffective" CD8(+) T cell clones. However, TCRs cloned from an effective and one of two ineffective clones conferred primary CD8(+) T cells with the equivalent potent capacity to inhibit HIV-1 infection. Taken together, these data suggest that other factors aside from intrinsic TCR:peptide-MHC complex reactivity can contribute to the potent antiviral capacity of some HIV-specific CD8(+) T cell clones.
... Diese Ketten besitzen einen variablen Teil, der der Antigenerkennung und Antigenbindung dient, und einen konstanten Teil, der mit seinem Ende in der Zellmembran verankert ist. Mit der α-und β-Kette assoziiert ist ein Homodimer aus ζ-Ketten Raum hineinragen [36,62]. ...
... In addition, myristoylation-dependent localization of Lck to the CD4 receptor is very integral to the activation of T cells since un-myristoylated Lck is cytosolic and unable to facilitate TCR signaling cascades ( Figure 3B) (15). In the presence of NMT activity, TCR ligation activates myristoylated Lck that in turn phosphorylates tyrosine containing immunoreceptor tyrosinebased activation motifs (ITAMs) (69). On phosphorylation of ITAMs, ZAP-70 is recruited that activates various signaling molecules leading to T cell activation ( Figure 3A). ...
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Protein N-myristoylation is a cotranslational lipidic modification specific to the alpha-amino group of an N-terminal glycine residue of many eukaryotic and viral proteins. The ubiquitous eukaryotic enzyme, N-myristoyltransferase, catalyzes the myristoylation process. Precisely, attachment of a myristoyl group increases specific protein–protein interactions leading to subcellular localization of myristoylated proteins with its signaling partners. The birth of the field of myristoylation, a little over three decades ago, has led to the understanding of the significance of protein myristoylation in regulating cellular signaling pathways in several biological processes especially in carcinogenesis and more recently immune function. This review discusses myristoylation as a prerequisite step in initiating many immune cell signaling cascades. In particular, we discuss the hitherto unappreciated implication of myristoylation during myelopoiesis, innate immune response, lymphopoiesis for T cells, and the formation of the immunological synapse. Furthermore, we discuss the role of myristoylation in inducing the virological synapse during human immunodeficiency virus infection as well as its clinical implication. This review aims to summarize existing knowledge in the field and to highlight gaps in our understanding of the role of myristoylation in immune function so as to further investigate into the dynamics of myristoylation-dependent immune regulation.
... This signaling axis has been demonstrated to be active and targetable in MM [81] and may lead to new therapeutic strategies in combination with proteasome inhibitors and immunomodulatory drugs. The signaling pathways that regulate mTOR and AMPK for the induction of autophagy have been well described in the T cell literature as downstream of the T cell receptor and CD28 signaling, which is the prototypic T cell costimulatory molecule [82]. Through both direct signaling from Ca++ downstream of PLCγ and through nutrient sensing, AMPK can be activated and can facilitate the induction of autophagy. ...
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Multiple myeloma (MM) is a hematological malignancy of terminally differentiated bone marrow (BM) resident B lymphocytes known as plasma cells (PC). PC that reside in the bone marrow include a distinct population of long-lived plasma cells (LLPC) that have the capacity to live for very long periods of time (decades in the human population). LLPC biology is critical for understanding MM disease induction and progression because MM shares many of the same extrinsic and intrinsic survival programs as LLPC. Extrinsic survival signals required for LLPC survival include soluble factors and cellular partners in the bone marrow microenvironment. Intrinsic programs that enhance cellular fidelity are also required for LLPC survival including increased autophagy, metabolic fitness, the unfolded protein response (UPR), and enhanced responsiveness to endoplasmic reticulum (ER) stress. Targeting LLPC cell survival mechanisms have led to standard of care treatments for MM including proteasome inhibition (Bortezomib), steroids (Dexamethasone), and immunomodulatory drugs (Lenalidomide). MM patients that relapse often do so by circumventing LLPC survival pathways targeted by treatment. Understanding the mechanisms by which LLPC are able to survive can allow us insight into the treatment of MM, which allows for the enhancement of therapeutic strategies in MM both at diagnosis and upon patient relapse.
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T cells play a central role in cellular-mediated immunity and must become activated to participate as effector cells in the immune response. The activation process is highly intricate and involves stimulation of a number of downstream signaling pathways enabling T cells to proliferate and produce cytokines that are vital for proper effector function. This increase in protein production and protein folding activity adds to the normal physiological strain on cellular machinery. One cellular compartment that has generated a mechanism to mitigate the stress induced by increased protein production is the endoplasmic reticulum (ER). In general, an increase in cellular production of proteins that overwhelms a cell’s protein folding capability can alter ER homeostasis and lead to ER stress. To counteract this stress, an adaptive cellular mechanism known as the ER stress response (ERSR) is initiated. The ERSR allows a cell to cope with normal physiological stress within the ER caused by increased protein translation. In this dissertation, we show that in vitro and in vivo T cell activation involving T cell receptor (TCR) ligation in the presence of costimulation initiates the physiological ERSR. Interestingly, the ERSR was also activated in T cells exposed only to TCR ligation, a treatment known to induce the ‘non-responsive’ states of anergy and tolerance. We further identified a key component of the downstream TCR signaling pathway, protein kinase C (PKC), as an initiator of physiological ERSR signaling, thus revealing a previously unknown role for this serine/threonine protein kinase in T cells. Therefore, induction of the physiological ERSR through PKC signaling may be an important ‘preparatory’ mechanism initiated during the early activation phase of T cells. If ER stress is persistent and ER homeostasis is not reestablished, physiological ER stress becomes pathological and initiates cellular death pathways through ER stress-induced apoptotic signaling. We further present data demonstrating that absence of functional Gimap5, a putative GTPase implicated to play a role in TCR signaling and maintenance of overall T cell homeostasis, leads to pathological ER stress and apoptosis. Using the BioBreeding diabetes-prone (BBDP) rat, a model for type 1 diabetes (T1D), we link pathological ER stress and ER stress-induced apoptotic signaling to the observed T cell lymphopenic phenotype of the animal. By depleting the ER stress apoptotic factor CHOP with siRNA, we were able to protect Gimap5-/- BBDP rat T cells from ER stress-induced death. These findings indicate a direct relationship between Gimap5 and maintenance of ER homeostasis for T cell survival. Overall, our findings suggest that the ERSR is activated by physiological and pathological conditions that disrupt T cell homeostasis. TCR signaling that leads to PKC activation initiates a physiological ERSR, perhaps in preparation for a T cell response to antigen. In addition, we also describe an example of pathological ERSR induction in T cells. Namely, we report that the absence of functional Gimap5 protein in T cells causes CHOP-dependent ER stress-induced apoptosis, perhaps initiated by deregulation of TCR signaling. This indicates a dual role for TCR signaling and regulation in the initiation of both the physiological and pathological ERSR. Future research that provides insights into the molecular mechanisms that govern ERSR induction in TCR signaling and regulation may lead to development of therapeutic modalities for treatment of immune-mediated diseases such as T1D.
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SIT (SHP2-interacting transmembrane adaptor protein) is a recently identified transmembrane adaptor protein, which is expressed in lymphocytes. Its structural properties, in particular the presence of five potential tyrosine phosphorylation sites, suggest involvement of SIT in TCR-mediated recruitment of SH2 domain-containing intracellular signaling molecules to the plasma membrane. Indeed, it has recently been demonstrated that SIT inducibly interacts with the SH2-containing protein tyrosine phosphatase 2 (SHP2) via an immunoreceptor tyrosine-based inhibition motif (ITIM). Moreover, SIT is capable to inhibit TCR-mediated signals proximal of activation of protein kinase C. However, inhibition of T cell activation by SIT occurs independently of SHP2 binding. The present study was performed to further characterize the molecular interaction between SIT and intracellular effector molecules and to identify the protein(s) mediating its inhibitory function. We demonstrate that SIT not only interacts with SHP2 but also with the adaptor protein Grb2 via two consensus YxN motifs. However, mutation of both Grb2-binding sites also does not influence the inhibitory function of SIT. In contrast, mutation of the tyrosine-based signaling motif Y168 ASV completely abrogates the ability of SIT to inhibit T cell activation. Co-precipitation experiments revealed that the tyrosine kinase p50csk could represent the negative regulatory effector molecule that binds to this motif.
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Recent therapeutic strategies to combat organ allograft rejection have focused on T-cell signaling pathways and the molecules that comprise them. While some pharmaceuticals have shown promise in blocking acute rejection, chronic graft destruction and permanent allograft acceptance in the absence of continuous immune suppression are problematic. Recent data now suggest T-cells hold the key to generation of transplantation tolerance and alleviation of long-term drug intervention. These new targets are spread across the T cell receptor, costimulatory, and T-cell growth factor signaling pathways. Development of mice deficient in T-cell genes and identification of new drug classes has accelerated our understanding of how these effector molecules contribute to T- cell activation, anergy, apoptosis, and survival. These observations have provided valuable insight into reasons for unsuccessful tolerogenic strategies while inspiring the design of new ones. This review is focused on the most recent developments in T-cell signaling cascades and their potential role for the induction of transplantation tolerance.
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The thymus is central to the establishment of a functioning immune system. Here is the place where T cells mature from hematopoietic progenitors, driven by mutual interactions of stromal cells and the developing thymocytes. As a result, different types of T cells are generated, all of which have been carefully selected for the ability to act in host defense towards non-self and against the potential to mount pathogenic self-reactive autoimmune responses. In this review we summarize our present knowlege on the lineage decisions taking place during this development, the selection processes responsible for shaping the T cell antigen-receptor repertoire, the interactions with the stromal components and the signal transduction pathways which transform the interactions with the thymic microenvironment into cellular responses of survival, proliferation, differentiation and, importantly, also of cell death.
Thesis
Les staphylocoques à coagulase négative (SCN) sont généralement considérés comme des pathogènes opportunistes à faible virulence. Cependant, des études antérieures ont rapporté une pathogénicité de certaines souches similaire à celle observée chez S. aureus ce qui laisse supposer l’expression de facteurs de virulence. Cette thèse vise à contribuer à l’importance des SCN dans les infections primitives sévères. Nous avons évalué le potentiel pathogène de souches cliniques de SCN au Bénin. Pour atteindre cet objectif, des SCN associés à diverses infections cliniques sévères ont été collectés sur une période de 10 mois au Centre National Hospitalier et Universitaire Hubert Koutoukou Maga à Cotonou. Ces souches sont identifiées d’abord par la galerie API® Staph, puis par la spectrométrie de masse MALDI-TOF et analysées pour leur susceptibilité aux antibiotiques et leur capacité à produire des facteurs de virulence. Cette partie de l’étude a montré que les espèces les plus impliquées dans les infections à SCN au Bénin sont : S. haemolyticus et S. epidermidis suivi d’autres espèces comme S. cohnii, S. sciuri, S. arlettae, S. capitis. Nous avons aussi apporté la preuve de la multi-résistance des souches aux antibiotiques, ainsi que de la présence d’au moins un, voire plusieurs facteurs de virulence tels que la protéase, l’estérase, l’hémolysine, la leucotoxine et l’entérotoxine staphylococcique C chez 44% des souches testées particulièrement dans les souches hospitalières isolées d’hémocultures. Ensuite, nous avons caractérisé un nouveau facteur de virulence identifié chez deux souches de S. epidermidis : l’entérotoxine staphylococcique C nommée SECepi qui a été dosée à environ 100 µg/mL dans les surnageants de culture bactérienne. Le gène secepi est constitué de 801 pb correspondant à 266 acides aminés. Sur la base des résultats de la comparaison d'homologie entre la chaîne peptidique de SECepi et les séquences déjà connues, nous avons constaté que SECepi est proche de SEC3 de la souche de S. aureus Mu3, avec trois substitutions d'acides aminés dans le peptide signal et neuf substitutions d'acides aminés dans la protéine mature. Cependant, plusieurs résidus qui sont impliqués dans la formation du complexe trimoléculaire CMH-SEC-TCR sont conservés dans SECepi. L’analyse de la protéine recombinante (rSECepi) révèle une parenté antigénique et une forte homologie structurale prédite avec SECaureus. De plus, cette toxine présente les activités biologiques caractéristiques d'un superantigène (SAg) incluant la stimulation de la mitogénicité et de la production concomitante de fortes doses de cytokines pro-inflammatoires et suppressives chez des lymphocytes T humains activés. Par ailleurs, SECepi résiste assez bien au chauffage à 100°C et à la digestion par les enzymes gastro-intestinales telles que la pepsine et la trypsine. Ces résultats fournissent la preuve que SECepi peut agir comme un superantigène chez l'hôte humain bien que le type sauvage comporte plusieurs mutations chez S. epidermidis. L’étude du dossier médical de l’un des patients a montré que l’entérotoxine produite par la souche de S. epidermidis a bien pu être à l’origine d’éléments de gravité du tableau clinique présenté par ce dernier à son admission en hospitalisation. Enfin, l’analyse génomique des deux souches toxinogènes de S. epidermidis, ainsi que leur aptitude à former du biofilm, confirment les possibilités variées d’échanges génétiques entre cette espèce et S. aureus. Cette thèse souligne l'importance de la surveillance des infections à SCN chez l’homme parce que certaines souches, à l’instar de S. aureus, produisent des facteurs de virulence pouvant aggraver l’état générale l’hôte.
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Class I major histocompatibility complex (class I MHC) molecules, known to be important for immune responses to antigen, are expressed also by neurons that undergo activity-dependent, long-term structural and synaptic modifications. Here, we show that in mice genetically deficient for cell surface class I MHC or for a class I MHC receptor component, CD3ζ, refinement of connections between retina and central targets during development is incomplete. In the hippocampus of adult mutants,N-methyl-d-aspartate receptor–dependent long-term potentiation (LTP) is enhanced, and long-term depression (LTD) is absent. Specific class I MHC messenger RNAs are expressed by distinct mosaics of neurons, reflecting a potential for diverse neuronal functions. These results demonstrate an important role for these molecules in the activity-dependent remodeling and plasticity of connections in the developing and mature mammalian central nervous system (CNS).
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Adaptor proteins lack catalytic activity and contain only protein-protein interaction domains. They have been shown to interact with an ever-growing number of signaling proteins and to play essential roles in many signaling pathways. SLP-76 and LAT are cell-type-specific adaptor proteins expressed in T cells, NK cells, platelets, and mast cells. In these cell types, SLP-76 and LAT are required for signaling by immunoreceptor tyrosine-based activation motif (ITAM)-containing receptors, including the T cell receptor (TCR), the pre-TCR, the high-affinity Fc receptor, and the platelet GPVI collagen receptor. In B cells, an analogous adaptor, BLNK/SLP-65, is required for signaling by the ITAM-containing B cell receptor. This review summarizes recent research on SLP-76, LAT, and BLNK.A major challenge in understanding adaptor protein function has been to sort out the many interactions mediated by adaptor proteins and to define the mechanisms by which adaptors mediate critical signaling events. In the case of LAT, SLP-76, and BLNK, the availability of tractable genetic systems, deficient in expression of each of these adaptor proteins, has facilitated in-depth investigation of their signaling functions and mechanisms of action. The picture that has emerged is one in which multiple adaptor proteins cooperate to bring about the formation of a large signaling complex, localized to specialized lipid microdomains within the cell membrane and known as GEMs. Adaptors not only recruit signaling proteins, but also play an active role in regulating the conformation and activation of many of the proteins recruited to the complex. In particular, recent research has shed light on the mechanisms by which multiple adaptor proteins cooperate to bring about the recruitment and activation of phospholipase C in response to the activation of ITAM-containing receptors.
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Farrerol, a new type of 2,3-dihydro-flavonoid, has been isolated from the leaves of Rhododendron dauricum L. In the present study, we found that farrerol exerted potent immunosuppressive effects on murine T cells both in vitro and in vivo. In vitro, farrerol markedly suppressed concanavalin A (ConA)-induced lymphocyte proliferation, Th1 and Th2 cytokine production, cluster of differentiation 4-positive (CD4(+)) T cell populations, and the ratio of CD4(+)/cluster of differentiation 8-positive (CD8(+)) T cells. Moreover, farrerol significantly inhibited the T cell-mediated delayed-type hypersensitivity (DTH) reaction in vivo. In addition, we investigated signal transduction mechanisms to determine how farrerol's effects by Western blotting. The data revealed that farrerol could downregulate the activation of the nuclear factor κB (NF-қB) and nuclear factor of activated T cells 2 (NFAT2) signal transduction pathways. These findings suggested that farrerol has potential effects on the regulation of the immune system and could be developed as a practicable immunosuppressive compound.
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Productive T cell activation depends on the assembly of a highly ordered and compartmentalized immunological synapse or supramolecular activation complex (SMAC). Reorganization of the actin cytoskeleton and clustering of specialized membrane microdomains, or lipid rafts, occur early following TCR/CD3 and costimulatory receptor ligation. Many key signaling molecules localize in lipid raft patches during T cell activation. Lipid raft reorganization is required for T cell activation, where it plays an apparently important role in stabilizing the T cell synapse. Here we review recent evidence supporting the role of lipid rafts in T cell activation. Particular emphasis is placed on the coupling of protein kinase C-θ (PKC θ), which is selectively expressed in T cells and is known to function as an essential signal for T cell activation, and lipid rafts.
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Engagement of immune receptors by antigen triggers processes that culminate in activation, cell proliferation, differentiation and effector functions. A growing body of evidence suggests that it is within specialized membrane regions called lipid rafts that the initiation and propagation of the signaling events taking place in immune cells occurs. These specialized membrane compartments are enriched in cholesterol and glycolipids and contain many lipid-modified signaling proteins such as GPI (glycosylphosphatidylinositol)- linked proteins, Src family tyrosine kinases and other adaptor proteins. By confining certain signaling molecules in membrane subdomains, lipid rafts have been proposed to function as platforms for the formation of multi-unit transduction complexes. Therefore, it has been suggested that the translocation of immune receptors into lipid rafts regulates immune cell activation. In lymphocytes it has been shown that immune receptors become lipid raft-associated upon antigen cross-linking, and signaling complexes are formed when additional components of the signaling pathways are recruited to lipid rafts. Furthermore, substantial evidence from a number of experiments shows that the integrity of lipid rafts is crucial for the initiation and maintenance of intracellular signals. Recent studies have also shown that the access and translocation of immune receptors to lipid rafts are developmentally regulated (immature versus mature cells, na�ve versus effector) and that the cholesterol content plays an important role in maintaining plasma membrane heterogeneity and influencing a variety of cellular processes including signaling, adhesion, and permeability. The aim of this review is to examine the role membrane lipids play in regulating immune activation.
Chapter
Five decades of clinical transplantation have seen a dramatic improvement in many aspects of patient outcome, largely attributable to better quality immunosuppressive agents and their more judicious use. The success of contemporary immunosuppression is underscored by a reduction in the incidence of acute rejection rates between 1988 and 1998 from 60 to 20 percent and an improvement in one-year renal allograft survival rates to around 90 percent over the past 2 decades [1],[2]. Over the same period, despite substantial evidence implicating acute rejection as a major risk factor for chronic allograft nephropathy, the early benefits of our current immunosuppressive agents have not been maintained in the long-term. Historically, the immunosuppressive arsenal has encompassed predominantly steroid-based therapy initially and subsequently, the introduction of antimetabolites, calcineurin phosphatase inhibition, as well as antilymphocyte antibodies. Most of these agents are broadly acting and non-specific in their immunosuppressive effect. Consequently, these drugs have been associated with the spectrum of risks of global over-immunosuppression, including the development of life-threatening infectious complications and the increased risk of certain malignancies. Besides risk related to excessive immunosuppression, these medications have all been implicated in the plethora of serious non-immune related adverse effects, including perturbations in metabolic pathways (diabetes mellitus, osteopenia, hyperlipidemia), renal function (renal failure, hypertension), as well as several cosmetically disfiguring manifestations (weight gain, skin friability and bruising, hirsutism and alopecia).
Chapter
The molecular machinery involved in Fc receptor-mediated phagocytosis in the different cell types of the immune system is still poorly defined. Cross-linking of FcγR results in activation of Src family kinases followed by activation of Syk family kinases. After Syk activation several phosphorylated proteins have been identified including: phospholipase C (PLC), phosphatidylinositol 3-kinase (PI 3-K), extracellular signal-regulated kinase (ERK), and GTPases of the Rho family. For phagocytosis, the involvement of PI 3-K and ERK seems to be dependent on the cell type. FcγR-mediated phagocytosis by monocytic cells (THP-1) was not blocked by wortmannin, a specific inhibitor of PI 3-K, nor by PD98059, a specific inhibitor of ERK. However, upon differentiation of THP-1 monocytes to a macrophage phenotype both wortmannin and PD98059 efficiently blocked FcγR-mediated phagocytosis. Additionally, phagocytosis by neutrophils, a more efficient phagocyte, was inhibited both by wortmannin and PD98059. Neutrophils and macrophage- differentiated monocytes presented significantly more efficient phagocytosis than monocytes, upon PMA stimulation. Taken together, these results indicate that less efficient phagocytic leukocytes, such as monocytes, do not require PI 3-K and ERK for phagocytosis. However, upon differentiation into macrophages ERK and PI 3-K are recruited as part of the phagocytic machinery.
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Objective: To investigate the effects of ginsenoside Rb 1 on activation and proliferation of murine T lymphocytes in vitro and to elucidate the mechanism of the immunosuppressive effect of ginsenoside Rb 1. Methods: Cell suspensions were prepared from murine lymph nodes. T lymphocytes were treated with different concentrations of ginsenoside Rb 1 and stimulated with polyclonal activator concanavalin (ConA). Fluorescence conjugated monoclonal antibodies and flow cytometry were used to detect the expression of CD3/CD69 and CD4/CD25. After the staining with CFDA-SE, T lymphocytes were stimulated with polyclonal activator ConA. The proliferation of T lymphocytes, after stimulated by ConA, was detected using the method of MTT. The distribution of the cell apoptosis was analyzed by staining both DIOC and PI. Results: In a dose-dependent manner, ginsenoside Rb 1 (5, 10, and 20 μmol/L) could significantly inhibit T lymphocytes activation index (P<0.01) stimulated by ConA and proliferation index (P<0.05) stimulated by ConA. Ginsenoside Rb 1 could also reduce the apoptosis of T lymphocytes stimulated by H 2O 2. Conclusion: Ginsenoside Rb 1 can effectively inhibit the activation and proliferation of murine T lymphocytes, and ginsenoside Rb 1 is a potential effective immunoinhibitory agent.
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Chronic lymphocytic leukemia (CLL) is the most common leukemia in western hemisphere. Although there is grooving evidence of genetic as well as epigenetic deregulation, still the exact pathomechanism of disease remains unsolved. CLL is heterogenic disease both in clinical and molecular biology characteristics. Molecular abnormalities are common and could be detected in more than 80% CLL patients. Cytogenetic heterogeneity could not clearly clarify prolonged survival as well as proliferation in CLL suggesting the key role of microenvironment in the pathogenesis. Nonetheless molecular aberrations influence the clinical course of disease, response to treatment and prognosis in CLL to the highest extent. Mutational status of IgVH genes divides CLL patients into two groups of different prognosis. Existence of mutations along with restricted B-cell receptor repertoire suggest that common antigen could be involved in etiopathogenesis of CLL. In CLL we could found two compartments: accumulative - in peripheral blood, and later in advanced stage off disease in spleen and liver, and proliferative - in lymph nodes and bone marrow. In actively proliferating cases the turnover of 50% CLL population takes only 3 months, we therefore could not assess any longer CLL as a disease caused simply by inhibition of apoptosis.
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The discovery of new immunosuppressive drugs such as rapamycin, cyclosporin A (CsA) and tacrolimus (FK506) has been very useful for preventing graft rejection and autoimmune disease. However, these drugs are not specific, and are associated with side-effects and toxicities. Therefore, understanding the molecular mechanisms of these drugs is important for designing specific immunosuppressants. Here, we show that in contrast to CsA and FK506, rapamycin blocks activation-induced expression of the linker for activation of T cells (LAT), a signaling molecule critical for initiating TCR signaling. Thus, whereas CsA and FK506 strongly enhanced TCR- and phorbol myristate acetate-induced LAT expression in T cells, rapamycin effectively inhibited activation-induced LAT expression. Importantly, these opposite effects were mutually antagonistic, as rapamycin acted as a potent antagonist for both CsA and FK506. Because CsA, unlike FK506 and rapamycin, does not bind to the intracellular immunophilin FK-binding protein (FKBP), the antagonism between these drugs is not simply due to competition for intracellular FKBP. Accordingly, RNA and protein stability analyses suggest inhibition by rapamycin at the translational level. Given the important role of LAT in initiating T cell activation, our data suggests that the effects of rapamycin, CsA and FK506 on T cell activation involve regulating early T cell signaling. These findings refine our understanding of the manifold effects of these immunosuppressants, thus providing insight into the drastic physiological contrasts observed between these drugs.
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In the present study, we examined the role of the recently identified glycosylphosphatidylinositol (GPI)-anchored cell surface molecule BY55, assigned as CD160, in TCR signaling. CD160 is expressed by most intestinal intraepithelial lymphocytes and by a minor subset of circulating lymphocytes including NK, TCRgd and cytotoxic effector CD8 bright+ CD28 ‐ T lymphocytes. We report that CD160, which has a broad specificity for MHC class Ia and Ib molecules, behaves as a coreceptor upon T cell activation. Anti-CD160 mAb enhance the CD3-induced proliferation of freshly isolated CD160-enriched peripheral blood lymphocytes and CD160 + T cell clones. Further, the engagement of CD160 receptors on normal clonal T lymphocyte populations lacking CD4, CD8 and CD28 molecules by MHC class I molecules results in an increased CD3-induced cell proliferation. Further, we found that CD160 co-precipitates with the protein tyrosine kinase p56 lck and tyrosine phosphorylated z chains upon TCR‐CD3 cell activation. Thus, we demonstrate that CD160 provides co-stimulatory signals leading to the expansion of a minor subset of circulating lymphocytes including double-negative CD4/CD8 T lymphocytes and CD8 bright+ cytotoxic effector T lymphocytes
Chapter
This chapter discusses how component boundaries should be formed, and develops leading practice for such implementations, to aid future model construction via the aggregation of existing components with minimal code alterations. Modules are unlikely to be rigid structures, with species belonging to different modules at different times, depending on perspective. This flexibility is not easily represented in computer code formulations, and can lead to conflicts when components from different researchers are combined. Modularity using CellML have also been discussed, illustrating concepts with the example of an inositol triphosphate (IP3) signaling cascade model broken into distinct conceptual modules. The model is composed of the GPCR module (reactions R1–R6), a module that describes the interactions of PLCβ, GαGTP, and Ca2+ (reactions R8–R13), and a third conceptual module that describes the production and degradation of IP3. The GPCR module receives an extracellular signal (via a ligand) and transduces this across the plasma membrane to the inside of the cell via GPCR receptor activation. The second module, in which the intracellular signal primes the key enzyme, PLCβ, exhibits a similar accounting property for that enzyme via reactions R8–R12. It is appropriate to take advantage of biological modularity to construct models at particular temporal and spatial scales. Some researchers do make available computer code or otherwise executable component-based implementations for their models but the recombination of these components to form new modules often requires the editing of this code to ensure that molecular species are linked appropriately for the biological system under investigation.
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Protein kinase C-θ (PKC-θ) is essential for mature T cell activation; however, the mechanism by which it is recruited to the TCR signaling machinery is unknown. Here we show that T cell stimulation by antibodies or peptide–major histocompatibility complex (MHC) induces translocation of PKC-θ to membrane lipid rafts, which localize to the immunological synapse. Raft translocation was mediated by the PKC-θ regulatory domain and required Lck but not ZAP-70. In addition, PKC-θ was associated with Lck in the rafts. An isolated PKC-θ catalytic fragment did not partition into rafts or activate the transcription factor NF-κB, although addition of a Lck-derived raft-localization sequence restored these functions. Thus, physiological T cell activation translocates PKC-θ to rafts, which localize to the T cell synapse; this PKC-θ translocation is important for its function.
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Ligand-induced down-regulation of two growth factor receptors, EGF receptor (ErbB-1) and ErbB-3, correlates with differential ability to recruit c-Cbl, whose invertebrate orthologs are negative regulators of ErbB. We report that ligand-induced degradation of internalized ErbB-1, but not ErbB-3, is mediated by transient mobilization of a minor fraction of c-Cbl into ErbB-1-containing endosomes. This recruitment depends on the receptor's tyrosine kinase activity and an intact carboxy-terminal region. The alternative fate is recycling of internalized ErbBs to the cell surface. Cbl-mediated receptor sorting involves covalent attachment of ubiquitin molecules, and subsequent lysosomal and proteasomal degradation. The oncogenic viral form of Cbl inhibits down-regulation by shunting endocytosed receptors to the recycling pathway. These results reveal an endosomal sorting machinery capable of controlling the fate, and, hence, signaling potency, of growth factor receptors.
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T cell receptor (TCR) triggering induces association of the protein tyrosine kinase ZAP-70, via its two src-homology 2 (SH2) domains, to di-phosphorylated Immunoreceptor Tyrosine-based Activation Motifs (2pY-ITAMs) present in the intracellular tail of the TCR-zeta chain. The crystal structure of the SH2 domains complexed with a 2pY-ITAM peptide suggests that the 60-amino acid-long inter-SH2 spacer helps the SH2 domains to interact with each other to create the binding site for the 2pY-ITAM. To investigate whether the inter-SH2 spacer has additional roles in the whole ZAP-70, we raised antibodies against two peptides of this region and probed ZAP-70 structure under various conditions. We show that the reactivity of antibodies directed at both sequences was dramatically augmented toward the tandem SH2 domains alone compared with that of the entire ZAP-70. This indicates that the conformation of the inter-SH2 spacer is not maintained autonomously but is controlled by sequences C-terminal to the SH2 domains, namely, the linker region and/or the kinase domain. Moreover, antibody binding to the same two determinants was also inhibited when ZAP-70 or the SH2 domains bound to the zeta chain or to a 2pY-ITAM. Together, these two observations suggest a model in which intramolecular contacts keep ZAP-70 in a closed configuration with the two SH2 domains near to each other.
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Cross-linking of the T cell antigen receptor (TCR)-CD3 complex induces rapid tyrosine phosphorylation and activation of Src (Lck and Fyn) and Syk (Syk and Zap-70) family protein tyrosine kinases (PTKs) which, in turn, phosphorylate multiple intracellular substrates. Cbl is a prominent PTK substrate suggesting a pivotal role for it in early signal transduction events. However, the regulation of Cbl function and tyrosine phosphorylation in T cells by upstream PTKs remains poorly understood. In the present study, we used genetic and biochemical approaches to demonstrate that Cbl directly interacts with Syk and Fyn via its N-terminal and C-terminal regions, respectively. Tyr-316 of Syk was required for the interaction with Cbl as well as for the maximal tyrosine phosphorylation of Cbl. However, both wild-type Syk and Y316F-mutated Syk phosphorylated equally well the C-terminal fragment of Cbl in vivo, suggesting the existence of an alternative, N terminus-independent mechanism for the Syk-induced tyrosine phosphorylation of Cbl. This mechanism appears to involve Fyn, since, in addition to its association with the C-terminal region of Cbl, Fyn also associated with Syk and enhanced the Syk-induced tyrosine phosphorylation of Cbl. These findings implicate Fyn as an adaptor protein that facilitates the interaction between Syk and Cbl, and suggest that Src and Syk family PTKs coordinately regulate the tyrosine phosphorylation of Cbl.
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Activation of T and natural killer (NK) cells leads to the tyrosine phosphorylation of pp36 and to its association with several signaling molecules, including phospholipase Cγ-1 and Grb2. Microsequencing of peptides derived from purified rat pp36 protein led to the cloning, in rat and man, of cDNA encoding a T- and NK cell–specific protein with several putative Src homology 2 domain–binding motifs. A rabbit antiserum directed against a peptide sequence from the cloned rat molecule recognized tyrosine phosphorylated pp36 from pervanadate-treated rat thymocytes. When expressed in 293T human fibroblast cells and tyrosine-phosphorylated, pp36 associated with phospholipase Cγ-1 and Grb2. Studies with GST–Grb2 fusion proteins demonstrated that the association was specific for the Src homology 2 domain of Grb-2. Molecular cloning of the gene encoding pp36 should facilitate studies examining the role of this adaptor protein in proximal signaling events during T and NK cell activation.
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Tec family non-receptor tyrosine kinases have been implicated in signal transduction events initiated by cell surface receptors from a broad range of cell types, including an essential role in B-cell development. A unique feature of several Tec members among known tyrosine kinases is the presence of an N-terminal pleckstrin homology (PH) domain. We directly demonstrate that phosphatidylinositol-3,4,5-trisphosphate (PtdIns-3,4,5-P3) interacting with the PH domain acts as an upstream activation signal for Tec kinases, resulting in Tec kinase-dependent phospholipase Cgamma (PLCgamma) tyrosine phosphorylation and inositol trisphosphate production. In addition, we show that this pathway is blocked when an SH2-containing inositol phosphatase (SHIP)-dependent inhibitory receptor is engaged. Together, our results suggest a general mechanism whereby PtdIns-3,4,5-P3 regulates receptor-dependent calcium signals through the function of Tec kinases.
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Bruton's tyrosine kinase (Btk) is essential for B-lineage development and represents an emerging family of non-receptor tyrosine kinases implicated in signal transduction events initiated by a range of cell surface receptors. Increased dosage of Btk in normal B cells resulted in a striking enhancement of extracellular calcium influx following B-cell antigen receptor (BCR) cross-linking. Ectopic expression of Btk, or related Btk/Tec family kinases, restored deficient extracellular Ca2+ influx in a series of novel Btk-deficient human B-cell lines. Btk and phospholipase Cgamma (PLCgamma) co-expression resulted in tyrosine phosphorylation of PLCgamma and required the same Btk domains as those for Btk-dependent calcium influx. Receptor-dependent Btk activation led to enhanced peak inositol trisphosphate (IP3) generation and depletion of thapsigargin (Tg)-sensitive intracellular calcium stores. These results suggest that Btk maintains increased intracellular calcium levels by controlling a Tg-sensitive, IP3-gated calcium store(s) that regulates store-operated calcium entry. Overexpression of dominant-negative Syk dramatically reduced the initial phase calcium response, demonstrating that Btk/Tec and Syk family kinases may exert distinct effects on calcium signaling. Finally, co-cross-linking of the BCR and the inhibitory receptor, FcgammaRIIb1, completely abrogated Btk-dependent IP3 production and calcium store depletion. Together, these data demonstrate that Btk functions at a critical crossroads in the events controlling calcium signaling by regulating peak IP3 levels and calcium store depletion.
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To define the T-cell receptor signal transduction motif, we have transfected human and murine T-cell lines with a chimeric receptor consisting of the extracellular and transmembrane domains of human CD8 alpha and the membrane-proximal portion of CD3 zeta containing at its C terminus either an 18-amino acid segment (NQLYNELNLGRREEYDVL) or alanine-scanning point mutant derivatives. Crosslinking of the extracellular domain of the chimera is sufficient to initiate Ca2+ flux, interleukin 2 production, and tyrosine phosphorylation of cellular proteins including the chimera. Subsequently, the chimera becomes associated with several tyrosine-phosphorylated proteins, among them the 70-kDa protein tyrosine kinase ZAP70. Mutational data identify the T-cell activation motif as Y(X)2L(X)7Y(X)2L and show that each of the four designated residues is necessary for the above activation events. Recombinant protein containing the two tandem SH2 domains derived from ZAP70 binds to a synthetic peptide corresponding to the above 18-amino acid motif but only when both tyrosines are phosphorylated; in contrast, little or no binding is observed to monophosphorylated or nonphosphorylated analogues. These results imply that after receptor crosslinking in T cells, and by inference also in B cells and mast cells, the motif is phosphorylated on both tyrosine residues, thereafter serving as a docking site for protein tyrosine kinases containing tandem SH2 domains.
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The p56lck and p59fyn protein tyrosine kinases are important signal transmission elements in the activation of mature T lymphocytes by ligands to the T-cell antigen receptor (TCR)/CD3 complex. The lack of either kinase results in deficient early signaling events, and pharmacological agents that block tyrosine phosphorylation prevent T-cell activation altogether. After triggering of the TCR/CD3 complex, both kinases are moderately activated and begin to phosphorylate cellular substrates, but the molecular mechanisms responsible for these changes have remained unclear. We recently found that the p72syk protein tyrosine kinase is physically associated with the TCR/CD3 complex and is rapidly tyrosine phosphorylated and activated by receptor triggering also in T cells lacking p56lck. Here we examine the regulation of p72syk and its interaction with p56lck in transfected COS-1 cells. p72syk was catalytically active and heavily phosphorylated on its putative autophosphorylation site, Tyr-518/519. Mutation of these residues to phenylalanines abolished its activity in vitro and toward cellular substrates in vivo and reduced its tyrosine phosphorylation in intact cells by approximately 90%. Coexpression of lck did not alter the catalytic activity of p72syk, but the expressed p56lck was much more active in the presence of p72syk than when expressed alone. This activation was also seen as increased phosphorylation of cellular proteins. Concomitantly, p56lck was phosphorylated at Tyr-192 in its SH2 domain, and a Phe-192 mutant p56lck was no longer phosphorylated by p72syk. Phosphate was also detected in p56lck at Tyr-192 in lymphoid cells. These findings suggest that p56lck is positively regulated by the p72syk kinase.
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Engagement of the T cell antigen receptor (TCR) results in activation of several tyrosine kinases leading to tyrosine phosphorylation of protein substrates and activation of multiple biochemical pathways. TCR-mediated activation of the src-family kinases, Lck and Fyn, results in tyrosine phosphorylation of the TCR zeta and CD3 chains. The site of phosphorylation in these chains is the tyrosine-based activation motif (TAM), a 15-16 amino acid module containing two tyrosine residues. Tyrosine-phosphorylated TAMs serve as targets for binding of the zeta-associated protein (ZAP-70) tyrosine kinase via its tandem SH2 domains. This binding correlates with activation of ZAP-70, a critical event in T cell activation. To further define the structural requirements for ZAP-70 interaction with the TCR, we developed a binding assay using immobilized glutathione S-transferase fusion proteins containing the NH2- and/or COOH-terminal SH2 domains of ZAP-70, and soluble synthetic peptides with the sequence of the cytoplasmic region of the TCR zeta chain (TCR zeta cyt) or individual TCR zeta and CD3 epsilon TAM motifs. Direct binding studies demonstrated that the tandem ZAP-70 SH2 domains bind phosphorylated, but not nonphosphorylated, TCR zeta cyt. The NH2-terminal ZAP-70 SH2 domain also binds to TCR zeta cyt but with 100-fold lower affinity. No binding was observed with the COOH-terminal ZAP-70 SH2 domain. Similar studies demonstrated that the ZAP-70 tandem SH2 domain can bind a TCR zeta 3 TAM peptide in which both tyrosine residues are phosphorylated: Little or no binding was observed with peptides phosphorylated at only one tyrosine residue, or a nonphosphorylated peptide. Binding of the tandem SH2 domains to the other two TCR zeta TAM peptides and to a CD3 epsilon TAM peptide was also observed. All four doubly tyrosine phosphorylated TAM peptides cross-compete with each other for binding to the tandem SH2 domains of ZAP-70. The affinity of these peptides for the tandem SH2 construct demonstrated a hierarchy of TAM zeta 1 > or = TAM zeta 2 > TAM epsilon > or = TAM zeta 3. The results provide further evidence that the ZAP-70 interaction with the TCR requires prior phosphorylation of both tyrosine residues within a TAM motif. Binding of ZAP-70 to phospho-TAMs is notable for the high level of cooperativity between the two SH2 domains, which individually demonstrate low affinity interaction with the ligand. The cooperativity ensures higher affinity for the doubly phosphorylated ligand. Affinity differences of as much as 30-fold indicates a significant specificity of interaction of ZAP-70 SH2 domains for different phospho-TAMs.
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During antigen recognition by T cells, CD4 and the T-cell receptor (TCR)/CD3/zeta complex are thought to interact with the same major histocompatibility complex II molecule in a stable ternary complex. Evidence has suggested that the association of CD4 with TCR/CD3/zeta requires the interaction of the protein tyrosine kinase p56lck with CD4. We have taken a biochemical approach to understand the mechanism by which p56lck and, in particular, its src homology (SH) 2 domain contributes to the association of CD4 with TCR/CD3/zeta during activation. We have previously shown that the p56lck SH2 domain binds directly to tyrosine-phosphorylated ZAP-70. Here we formally demonstrate the in vivo association of p56lck with the homologous protein tyrosine kinases Syk and ZAP-70 after CD3 stimulation of Jurkat cells. A tyrosine-phosphorylated peptide containing the sequence predicted to be optimal for binding to the SH2 domain of src family kinases specifically competes for this association, indicating that tyrosine-phosphorylated ZAP-70 and Syk bind to p56lck by an SH2-mediated interaction. We also show that the same peptide is able to compete for the activation-dependent TCR/CD4 association in Jurkat cells. Moreover, ZAP-70 and CD4 cocap only after CD3 stimulation in human T lymphoblasts. We propose that the interaction of the p56lck SH2 domain with zeta-associated tyrosine-phosphorylated ZAP-70 and/or Syk enables CD4 to associate with antigen-stimulated TCR/CD3/zeta complexes.
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Tyrosine-based activation motifs (TAMs) define a conserved signaling sequence, EX2YX2L/IX7YX2L/I, that couples the T cell antigen receptor to protein tyrosine kinases and adapter molecules. The present study shows that phosphorylation of both tyrosines within the motif is required for high affinity binding of the tyrosine kinase ZAP-70 whereas phosphorylation of the single COOH-terminal tyrosine within the motif is optimal for the binding of the adapter Shc. There were also quantitative differences in the ZAP-70 and Shc association with the ζ1-TAM since nM concentrations of the doubly phosphorylated ζ1-TAM are sufficient for ZAP-70 recruitment whereas micromolar levels of singly phosphorylated TAMs are necessary for Shc binding. Shc is tyrosine phosphorylated in antigen receptor-activated T cells and can potentially form a complex with the adapter molecule Grb2 and could thus recruit the Ras guanine nucleotide exchange protein Sos into the antigen receptor complex. The present data show that Grb2 can bind to the phosphorylated TAM, but this binding is independent of Shc and there is no formation of ζ1-TAM·Shc·Grb2·Sos complexes in antigen receptor-activated cells. Accordingly, Shc function should not be considered in the context of Grb2/Sos recruitment to the T cell antigen receptor complex.
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Recently, a severe combined immunodeficiency syndrome with a deficiency of CD8+ peripheral T cells and a TCR signal transduction defect in peripheral CD4+ T cells was associated with mutations in ZAP-70. Since TCR signaling is required in developmental decisions resulting in mature CD4 (and CD8) T cells, the presence of peripheral CD4+ T cells expressing TCRs incapable of signaling in these patients is paradoxical. Here, we show that the TCRs on thymocytes, but not peripheral T cells, from a ZAP-70-deficient patient are capable of signaling. Moreover, the TCR on a thymocyte line derived from this patient can signal, and the homologous kinase Syk is present at high levels and is tyrosine phosphorylated after TCR stimulation. Thus, Syk may compensate for the loss of ZAP-70 and account for the thymic selection of at least a subset of T cells (CD4+) in ZAP-70-deficient patients.
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Stimulation of the T-cell antigen receptor (TCR) induces activation of multiple tyrosine kinases, resulting in phosphorylation of numerous intracellular substrates. One substrate is p95vav, which is expressed exclusively in hematopoietic and trophoblast cells. It contains a number of structural motifs, including Src homology 2, Src homology 3, and pleckstrin homology domains and a putative guanine nucleotide exchange domain. The role of p95vav in TCR-mediated signaling processes is unclear. Here, we show that overexpression of p95vav alone in Jurkat T cells leads to activation of the nuclear factors, including NFAT, involved in interleukin-2 expression. Furthermore, p95vav synergizes with TCR stimulation in inducing NFAT- and interleukin-2-dependent transcription. In contrast, NFAT activation by a G-protein-coupled receptor is not modulated by p95vav overexpression, suggesting that the effect is specific to the TCR signaling pathways. Although removal of the first 67 amino acids of p95vav activates its transforming potential in NIH 3T3 cells, this region appears to be required for its function in T cells. We further demonstrate that the p95vav-induced NFAT activation is not mimicked by Ras activation, though its function is dependent upon Ras and Raf. Furthermore, the activating function of p95vav is blocked by FK506, suggesting that its activity also depends on calcineurin. To further dissect p95vav involvement in TCR signaling, we analyzed various Jurkat mutants deficient in TCR signaling function or TCR expression and showed that an intact TCR signaling pathway is required for p95vav to function. However, overexpression of p95vav does not appear to influence TCR-induced protein tyrosine phosphorylation or increases in cytoplasmic free calcium. Taken together, our data suggest that p95vav plays an important role at an yet unidentified proximal position in the TCR signaling cascade.
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ZAP-70 is a 70-kDa protein tyrosine kinase, expressed exclusively in T cells and NK cells, and plays a critical role in mediating T cell activation in response to T cell receptor engagement. The strong correlation between tyrosine phosphorylation of ZAP-70 and its acquisition of increased kinase activity suggests that it is positively regulated by tyrosine phosphorylation. Previously, we identified tyrosines 492 and 493 of ZAP-70 as being sites of in vivo phosphorylation in response to T cell receptor engagement. To determine the role of phosphorylation in regulating ZAP-70 activity, we mutated each of these tyrosines individually to phenylalanine. When expressed in COS cells, Y493F-mutated ZAP-70 demonstrated normal basal kinase activity, but, unlike wild type ZAP-70, could not be activated by tyrosine phosphorylation induced by incubation with pervanadate or by co-expression of constitutively activated Lck. This suggests that Tyr-493 phosphorylation is required for the tyrosine phosphorylation-induced activation of ZAP-70. The Y492F mutation resulted in 4-fold higher basal kinase activity, which could be stimulated further by tyrosine phosphorylation. These results reveal that critical tyrosine residues in the kinase domain of ZAP-70 are important in regulation of its catalytic activity.
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Tyrosine phosphorylation of a 17-amino acid immunoreceptor tyrosine-based activation motif (ITAM), conserved in each of the signaling subunits of the T-cell antigen receptor (TCR), mediates the recruitment of ZAP-70 and syk protein-tyrosine kinases (PTKs) to the activated receptor. The interaction between the two tandemly arranged Src-homology 2 (SH2) domains of this family of PTKs and each of the phosphotyrosine-containing ITAMs was examined by real-time measurements of kinetic parameters. The association rate and equilibrium binding constants for the ZAP-70 and syk SH2 domains were determined for the CD3 epsilon ITAM. Both PTKs bound with ka and Kd values of 5 x 10(6) M-1.sec-1 and approximately 25 nM, respectively. Bindings to the other TCR ITAMs (zeta 1, zeta 2, gamma, and delta ITAMs) were comparable, although the zeta 3 ITAM bound approximately 2.5-fold less well. Studies of the affinity of a single functional SH2 domain of ZAP-70 provided evidence for the cooperative nature of binding of the dual SH2 domains. Mutation of either single SH2 domain decreased the Kd by > 100-fold. Finally, the critical features of the ITAM for syk binding were found to be similar to those required for ZAP-70 binding. These data provide insight into the mechanism by which the multisubunit TCR interacts with downstream effector molecules.
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ZAP-70 is a protein tyrosine kinase thought to play a critical role in T-cell receptor (TCR) signal transduction. During T-cell activation, ZAP-70 binds to a conserved signalling motif known as the immune receptor tyrosine activating motif (ITAM) and becomes tyrosine phosphorylated. To determine whether binding of ZAP-70 to the phosphorylated ITAM was able to activate its kinase activity, we measured the kinase activity of ZAP-70 both when it was bound and when it was unbound to phosphorylated TCR subunits. The ability of ZAP-70 to phosphorylate itself, but not exogenous substrates, was enhanced when it was bound to the tyrosine-phosphorylated TCR zeta and eta chains or to a construct that contained duplicated epsilon ITAMs. No enhanced ZAP-70 autophosphorylation was noted when it was bound to tyrosine-phosphorylated CD3 gamma or epsilon. In addition, autophosphorylation of ZAP-70 when bound to zeta or eta resulted in the generation of multiple distinct ZAP-70 phosphorylated tyrosine residues which had the capacity to bind the SH2 domains of fyn, lck, GAP, and abl. As the effect was noted only when ZAP-70 was bound to TCR subunits containing multiple ITAMs, we propose that one of the roles of the tandem ITAMs is to facilitate the autophosphorylation of ZAP-70. Tyrosine-phosphorylated ZAP-70 then mediates downstream signalling by recruiting SH2 domain-containing signalling proteins.
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ZAP-70 is a protein tyrosine kinase (PTK) required for T-cell development and T-cell antigen receptor (TCR) function. ZAP-70 is associated with the phosphorylated antigen receptor and undergoes tyrosine phosphorylation following receptor activation. We demonstrate here that tyrosine phosphorylation of ZAP-70 results in an increase in its catalytic activity and that this activation is mediated by the phosphorylation of tyrosine residue 493 by the src family of PTKs. The activity of baculoviral expressed ZAP-70 was up-regulated 10-fold when ZAP-70 was co-infected and phosphorylated by the src family PTK, lck. Mutation of Y493 alone abrogated the ability of ZAP-70 to be activated by lck. Moreover, we demonstrate that phosphorylation of Y493 and activation of ZAP-70 is required for antigen receptor-mediated induction of interleukin-2 (IL-2) secretion in lymphocytes.
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Stimulation of the T cell antigen receptor (TCR) leads to tyrosine phosphorylation of a number of cellular proteins, including the vav proto-oncogene product. We now report the detection of several phosphotyrosine proteins (80, 74, and 70 kDa) from TCR-stimulated T cells that bind to the Src homology 2 (SH2) domain of proto-Vav (Vav-SH2) and co-immunoprecipitate with the proto-Vav product. Their binding to Vav-SH2 differs from that observed with SH2 domains from other proteins. None of the Vav-SH2-associated phosphoproteins bind to either of the Src homology 3 (SH3) domains of proto-Vav or to mutant Vav-SH2 proteins. The association of the phosphoproteins with Vav-SH2 requires induction of tyrosine phosphorylation of cellular proteins since proteins from lysates of herbimycin A-treated TCR-activated T cells fail to associate with Vav-SH2. Among the proteins from T cells that co-immunoprecipitate with the proto-Vav product and bind to its SH2 domain, specific antibodies identified the 70-kDa tyrosine phosphoprotein as ZAP-70, a protein tyrosine kinase (PTK) involved in TCR signal transduction. Binding of this PTK to Vav-SH2 is inhibited by a ZAP-70-specific synthetic tyrosine phosphopeptide. We suggest that ZAP-70 may function as a PTK for proto-Vav.
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To further understand the interactions between Zap-70, Src family kinases, and other T-cell proteins, we have examined the regulation of Zap-70 in the antigen-specific T-cell line BI-141. By analyzing derivatives containing an activated version of either p56lck or p59fynT, it was observed that the two Src-related enzymes augmented T-cell receptor (TCR)-mediated tyrosine phosphorylation of Zap-70, as well as its association with components of the antigen receptor complex. Importantly, the accumulation of TCR.Zap-70 complexes quantitatively and temporally correlated with the induction of tyrosine phosphorylation of the CD3 and zeta chains of TCR. Using a CD4-positive variant of BI-141, we also found that the ability of Zap-70 to undergo tyrosine phosphorylation and associate with TCR was enhanced by aggregation of TCR with the CD4 co-receptor. Further studies allowed the identification of two distinct pools of tyrosine-phosphorylated Zap-70 in activated T-cells. While one population was associated with TCR, the other was co-immunoprecipitated with a 120-kDa tyrosine-phosphorylated protein of unknown identity. In addition to supporting the notion that Src-related enzymes regulate the recruitment of Zap-70 in TCR signaling, these data added further complexity to previous models of regulation of Zap-70. Furthermore, they suggested that p120 may be an effector and/or a regulator of Zap-70 in activated T-lymphocytes.
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We have developed a rapid and sensitive two capillary-column chromatography and mass spectrometry-based method for the determination of protein phosphorylation sites following recovery of individual phosphopeptides from two-dimensional phosphopeptide maps. With a standard phosphopeptide, we demonstrate detection sensitivity of at least 250 fmol for this system. We applied this technique to the analysis of in vitro sites of tyrosine phosphorylation induced on the T cell-specific protein tyrosine kinase ZAP-70 in the absence and presence of p56lck. We show that ZAP-70 has a primary autophosphorylation site at Tyr-292, with a secondary site at Tyr-126. We also show additional phosphorylation at Tyr-69, Tyr-178, Tyr-492, and Tyr-493 upon the addition of the protein tyrosine kinase, p56lck. By comparative two-dimensional phosphopeptide mapping, we show that ZAP-70 isolated from Jurkat T cells also autophosphorylates at Tyr-292 and Tyr-126. Similar analysis of 32P-labeled Jurkat cells stimulated with anti-T cell receptor antibodies reveals Tyr-492 and Tyr-493 as the principal sites of T cell antigen receptor-induced tyrosine phosphorylation, with additional phosphorylation at the Tyr-292, but not the Tyr-126 autophosphorylation site. The high degree of sensitivity achieved with this technology should greatly facilitate the direct biochemical determination of inducible protein phosphorylation events, an experimental strategy that until now has been both time consuming and difficult.
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A homozygous mutation in the kinase domain of ZAP-70, a T cell receptor-associated protein tyrosine kinase, produced a distinctive form of human severe combined immunodeficiency. Manifestations of this disorder included profound immunodeficiency, absence of peripheral CD8+ T cells, and abundant peripheral CD4+ T cells that were refractory to T cell receptor-mediated activation. These findings demonstrate that ZAP-70 is essential for human T cell function and suggest that CD4+ and CD8+ T cells depend on different intracellular signaling pathways to support their development or survival.
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Despite extensive study, several of the major components involved in T cell receptor–mediated signaling remain unidentified. Here we report the cloning of the cDNA for a highly tyrosine-phosphorylated 36–38 kDa protein, previously characterized by its association with Grb2, phospholipase C-γ1, and the p85 subunit of phosphoinositide 3-kinase. Deduced amino acid sequence identifies a novel integral membrane protein containing multiple potential tyrosine phosphorylation sites. We show that this protein is phosphorylated by ZAP-70/Syk protein tyrosine kinases leading to recruitment of multiple signaling molecules. Its function is demonstrated by inhibition of T cell activation following overexpression of a mutant form lacking critical tyrosine residues. Therefore, we propose to name the molecule LAT—linker for activation of T cells.
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Syk family kinases are essential for lymphocyte development and activation. Therefore the identification of their direct effectors is of critical importance. Here, we report that Syk interacts in the yeast two-hybrid system with Vav, a proto-oncogene product exclusively expressed in hematopoietic cells. This interaction was direct, required the catalytic activity of Syk, the SH2 domain of Vav, and tyrosine residues in the linker domain of Syk. Vav also associated with Syk and Zap in antigen receptor-stimulated B or T cells, respectively. Functionally, Vav was phosphorylated by Syk family kinases both in vivo and in vitro. Furthermore, Syk and Vav cooperated to activate NF-AT synergistically. These results indicate that the interaction between Syk family kinases and Vav plays an important role in coupling immune recognition receptors to signaling pathways involved in lymphokine production.
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The linker molecule LAT is a critical substrate of the tyrosine kinases activated upon TCR engagement. Phosphorylated LAT binds Grb2, PLC-γ1, and other signaling molecules. We demonstrate that human LAT is palmitoylated and that palmitoylated LAT predominantly localizes into glycolipid-enriched microdomains (GEMs). Although the LAT transmembrane domain is sufficient for membrane localization, palmitoylation at C26 and C29 is essential for efficient partitioning into GEMs. LAT palmitoylation is necessary for its tyrosine phosphorylation. After T cell activation, most tyrosine-phosphorylated LAT molecules and a fraction of PLC-γ1 and other signaling molecules are present in GEMs. LAT is central to T cell activation and is a novel linker molecule shown to require targeting to membrane microdomains for signaling.
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The development and function of αβT lymphocytes depend on signals derived from pre-T and αβT cell receptors (preTCR and αβTCR) (reviewed in refs 1, 2). The engagement of these receptors leads to the activation of Lck and Fyn, which are protein tyrosine kinases (PTKs) of the Src family. It remains unclear to what extent the activation of Src-family PTKs can direct the differentiation steps triggered by preTCR and αβTCR. Here we show that the inactivation of the negative regulator of Src-family PTKs, carboxy- terminal Src kinase (Csk), in immature thymocytes abrogates the requirement for preTCR, αβTCR and major histocompatibility complex (MHC) class II for the development of CD4+8+ double-positive and CD4+ single-positive thymocytes as well as peripheral CD4 αβT-lineage cells. These data show that Csk and its substrates are required to establish preTCR/αβTCR-mediated control over the development of αβT cells.
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CROSSLINKING of B- or T-cell antigen receptors results in the rapid tyrosine phosphorylation of a number of proteins, including Vav, a protein expressed in cells of the haematopoietic system1–3. Vav contains an array of structural motifs that include Src-homology domains SH2/SH34 and regions of homology to the guanine-nucleotide-exchange protein Dbl, pleckstrin and protein kinase C (refs 5-9). Using the RAG-complementation approach10, we have analysed in vivo differentiation and in vitro responses of B-and T-lineage cells generated by injection of embryonic stem cells homozygous for a null mutation in the vav gene into blastocysts of RAG-1- or RAG-2-deficient mice. Here we report that antigen receptor-mediated proliferative responses of B and T cells in vitro are severely reduced in the absence of Vav. We also suggest a direct link between the low proliferative response of Vav-deficient B and T cells and the reduced number of these cells in peripheral lymphoid organs of chimaeric mice.
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The T cell antigen receptor (TCR) initiates sigal transduction by activating multiple cytoplasmic protein tyrosine kinases (PTKs). Considerable progress in the field of TCR signal transduction has been made in three areas recently: first, in understanding the structure and function of the PTK ZAP-70; second, in the elucidation of the function of the substrates and pathways downstream of the PTKs; and third, in the identification of molecules that negatively regulate TCR signaling.
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The Syk cytoplasmic protein-tyrosine kinase has two amino-terminal SH2 domains and a carboxy-terminal catalytic domain. Syk, and its close relative ZAP-70, are apparently pivotal in coupling antigen- and Fc-receptors to downstream signalling events. Syk associates with activated Fc receptors, the T cell receptor complex and the B-cell antigen-receptor complex (BCR) in immature and mature B lymphocytes. On receptor activation, the tandem SH2 domains of Syk bind dual phosphotyrosine sites in the conserved ITAM motifs of receptor signalling chains, such as the immunoglobulin alpha and beta-chains of the BCR, leading to Syk activation. Here we have investigated Syk function in vivo by generating a mouse strain with a targeted mutation in the syk gene. Homozygous syk mutants suffered severe haemorrhaging as embryos and died perinatally, indicating that Syk has a critical role in maintaining vascular integrity or in wound healing during embryogenesis. Analysis of syk-/- lymphoid cells showed that the syk mutation impaired the differentiation of B-lineage cells, apparently by disrupting signalling from the pre-BCR complex and thereby preventing the clonal expansion, and further maturation, of pre-B cells.