RESUMEN
The inhibitory forms of killer cell Ig-like receptors (KIR) are MHC class I-binding receptors that are expressed by human NK cells and prevent their attack of normal cells. Substantial evidence indicates that the mechanism of KIR-mediated inhibition involves recruitment of the protein tyrosine phosphatase, Src homology region 2-containing protein tyrosine phosphatase (SHP)-1, to phosphorylated immunoreceptor tyrosine-based inhibitory motifs (ITIMs). However, the functional significance of parallel recruitment of a SHP-1-related phosphatase, SHP-2, to KIR ITIMs has not been addressed. In the present study, our results with mutant forms of a classical KIR, KIR3DL1, show a direct correlation between SHP-2 recruitment and functional inhibition of target cell conjugation and cytotoxicity. In addition, KIR3DL1 inhibition of target cell cytotoxicity is blocked by overexpression of a dominant-negative form of SHP-2. Finally, KIR3DL1 fused directly with the catalytic domain of SHP-2 inhibits both target cell conjugation and cytotoxicity responses. These results strongly indicate that SHP-2 catalytic activity plays a direct role in inhibitory KIR functions, and SHP-2 inhibits NK cell activation in concert with SHP-1.
Asunto(s)
Adyuvantes Inmunológicos/fisiología , Células Asesinas Naturales/enzimología , Células Asesinas Naturales/inmunología , Proteínas Tirosina Fosfatasas/fisiología , Receptores Inmunológicos/fisiología , Dominios Homologos src/inmunología , Adyuvantes Inmunológicos/biosíntesis , Adyuvantes Inmunológicos/genética , Secuencias de Aminoácidos/genética , Secuencias de Aminoácidos/inmunología , Secuencia de Aminoácidos , Animales , Dominio Catalítico/genética , Dominio Catalítico/inmunología , Línea Celular , Pruebas Inmunológicas de Citotoxicidad , Citotoxicidad Inmunológica/genética , Citotoxicidad Inmunológica/inmunología , Vectores Genéticos , Humanos , Péptidos y Proteínas de Señalización Intracelular , Células Asesinas Naturales/metabolismo , Ratones , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Proteína Fosfatasa 2 , Proteína Tirosina Fosfatasa no Receptora Tipo 11 , Proteína Tirosina Fosfatasa no Receptora Tipo 6 , Proteínas Tirosina Fosfatasas/biosíntesis , Proteínas Tirosina Fosfatasas/genética , Receptores Inmunológicos/antagonistas & inhibidores , Receptores Inmunológicos/genética , Receptores KIR , Receptores KIR3DL1 , Proteínas Tirosina Fosfatasas con Dominio SH2 , Eliminación de Secuencia , Células Tumorales Cultivadas , Tirosina/genética , Dominios Homologos src/genéticaRESUMEN
Stimulation of lymphocytes through multichain immune recognition receptors activates multiple signaling pathways. Adaptor proteins play an important role in integrating these pathways by their ability to simultaneously bind multiple signaling components. Recently, the 3BP2 adaptor protein has been shown to positively regulate the transcriptional activity of T cells. However, the mechanisms by which signaling components are involved in this regulation remain unclear, as does a potential role for 3BP2 in the regulation of other cellular functions. Here we describe a positive regulatory role for 3BP2 in NK cell-mediated cytotoxicity. We also identify p95(vav) and phospholipase C-gamma isoforms as binding partners of 3BP2. Our results show that tyrosine-183 of 3BP2 is specifically involved in this interaction and that this residue critically influences 3BP2-dependent function. Therefore, 3BP2 regulates NK cell-mediated cytotoxicity by mobilizing key downstream signaling effectors.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Proteínas Portadoras/fisiología , Proteínas de Ciclo Celular , Citotoxicidad Inmunológica , Células Asesinas Naturales/inmunología , Proteínas de la Membrana/fisiología , Adyuvantes Inmunológicos/biosíntesis , Adyuvantes Inmunológicos/metabolismo , Adyuvantes Inmunológicos/fisiología , Secuencia de Aminoácidos , Proteínas Portadoras/biosíntesis , Proteínas Portadoras/metabolismo , Células HeLa , Humanos , Isoenzimas/metabolismo , Células Jurkat , Células K562 , Células Asesinas Naturales/metabolismo , Activación de Linfocitos , Proteínas de la Membrana/biosíntesis , Proteínas de la Membrana/metabolismo , Datos de Secuencia Molecular , Fosfolipasa C gamma , Fosfoproteínas/metabolismo , Fosforilación , Pruebas de Precipitina , Isoformas de Proteínas/biosíntesis , Isoformas de Proteínas/fisiología , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-vav , Receptores de IgG/inmunología , Receptores de IgG/metabolismo , Receptores Inmunológicos/metabolismo , Receptores Inmunológicos/fisiología , Transducción de Señal/inmunología , Fosfolipasas de Tipo C/metabolismo , Tirosina/metabolismo , Tirosina/fisiología , Proteína Tirosina Quinasa ZAP-70 , Dominios Homologos src/inmunologíaRESUMEN
To examine the role of CTLA-4 in controlling Ag-specific CD8(+) T cell activation, TCR-transgenic/CTLA-4 wild-type or -deficient mice were generated in a recombination-activating gene 2-deficient background. Naive T cells from these mice responded comparably whether or not CTLA-4 was expressed. In contrast, primed T cells responded more vigorously if they lacked CTLA-4 expression. We took advantage of the difference between naive and primed T cell responses to approach the mechanism of CTLA-4 function. Single-cell analyses demonstrated that a greater fraction of CTLA-4-deficient cells responded to a fixed dose of Ag compared with CTLA-4-expressing cells, whereas the magnitude of response per cell was comparable. A shift in the dose-response curve to APCs was also observed such that fewer APCs were required to activate CTLA-4-deficient T cells to produce intracellular IFN-gamma and to proliferate. These results suggest that CTLA-4 controls the threshold of productive TCR signaling. Biochemical analysis comparing stimulated naive and primed TCR-transgenic cells revealed no obvious differences in expression of total CTLA-4, tyrosine-phosphorylated CTLA-4, and associated Src homology domain 2-containing protein tyrosine phosphatase. Thus, the biochemical mechanism explaining the differential inhibitory effect of CTLA-4 on naive and primed CD8(+) T cells remains unclear.
Asunto(s)
Antígenos de Diferenciación/genética , Linfocitos T CD8-positivos/inmunología , Inmunoconjugados , Inmunosupresores/farmacología , Activación de Linfocitos/genética , Proteínas Tirosina Fosfatasas/fisiología , Receptores de Antígenos de Linfocitos T/genética , Dominios Homologos src/inmunología , Abatacept , Adyuvantes Inmunológicos/biosíntesis , Adyuvantes Inmunológicos/deficiencia , Adyuvantes Inmunológicos/genética , Adyuvantes Inmunológicos/fisiología , Animales , Antígenos CD , Antígenos de Diferenciación/biosíntesis , Antígenos de Diferenciación/fisiología , Linfocitos T CD8-positivos/enzimología , Linfocitos T CD8-positivos/metabolismo , Antígeno CTLA-4 , Proteínas de Unión al ADN/deficiencia , Proteínas de Unión al ADN/genética , Inmunización , Inmunosupresores/metabolismo , Interfase/genética , Interfase/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Ratones Noqueados , Ratones Transgénicos , Transfección , Células Tumorales Cultivadas , Dominios Homologos src/genéticaRESUMEN
In this study, we describe human FDF03, a novel member of the Ig superfamily expressed as a monomeric 44-kDa transmembrane glycoprotein and containing a single extracellular V-set Ig-like domain. Two potential secreted isoforms were also identified. The gene encoding FDF03 mapped to chromosome 7q22. FDF03 was mostly detected in hemopoietic tissues and was expressed by monocytes, macrophages, and granulocytes, but not by lymphocytes (B, T, and NK cells), indicating an expression restricted to cells of the myelomonocytic lineage. FDF03 was also strongly expressed by monocyte-derived dendritic cells (DC) and preferentially by CD14+/CD1a- DC derived from CD34+ progenitors. Moreover, flow cytometric analysis showed FDF03 expression by CD11c+ blood and tonsil DC, but not by CD11c- DC precursors. The FDF03 cytoplasmic tail contained two immunoreceptor tyrosine-based inhibitory motif (ITIM)-like sequences. When overexpressed in pervanadate-treated U937 cells, FDF03 was tyrosine-phosphorylated and recruited Src homology-2 (SH2) domain-containing protein tyrosine phosphatase (SHP)-2 and to a lesser extent SHP-1. Like engagement of the ITIM-bearing receptor LAIR-1/p40, cross-linking of FDF03 inhibited calcium mobilization in response to CD32/FcgammaRII aggregation in transfected U937 cells, thus demonstrating that FDF03 can function as an inhibitory receptor. However, in contrast to LAIR-1/p40, cross-linking of FDF03 did not inhibit GM-CSF-induced monocyte differentiation into DC. Thus, FDF03 is a novel ITIM-bearing receptor selectively expressed by cells of myeloid origin, including DC, that may regulate functions other than that of the broadly distributed LAIR-1/p40 molecule.
Asunto(s)
Células Dendríticas/metabolismo , Granulocitos/metabolismo , Inmunoglobulinas/química , Glicoproteínas de Membrana/biosíntesis , Monocitos/metabolismo , Receptores Inmunológicos/biosíntesis , Homología de Secuencia de Aminoácido , Adulto , Empalme Alternativo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Señalización del Calcio/inmunología , Diferenciación Celular/inmunología , Células Cultivadas , Cromosomas Humanos Par 7 , Clonación Molecular , ADN Complementario/aislamiento & purificación , Células Dendríticas/inmunología , Granulocitos/inmunología , Humanos , Inmunoglobulinas/genética , Integrina alfaXbeta2/biosíntesis , Péptidos y Proteínas de Señalización Intracelular , Receptores de Lipopolisacáridos/biosíntesis , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/inmunología , Glicoproteínas de Membrana/metabolismo , Ratones , Datos de Secuencia Molecular , Monocitos/citología , Monocitos/inmunología , Familia de Multigenes/inmunología , Especificidad de Órganos/genética , Especificidad de Órganos/inmunología , Fosforilación , Proteína Tirosina Fosfatasa no Receptora Tipo 11 , Proteína Tirosina Fosfatasa no Receptora Tipo 6 , Proteínas Tirosina Fosfatasas/metabolismo , ARN Mensajero/biosíntesis , Receptores de IgG/antagonistas & inhibidores , Receptores de IgG/fisiología , Receptores Inmunológicos/genética , Receptores Inmunológicos/inmunología , Receptores Inmunológicos/metabolismo , Proteínas Tirosina Fosfatasas con Dominio SH2 , Solubilidad , Células U937 , Dominios Homologos src/inmunologíaRESUMEN
Thapsigargin, which elevates cytosolic calcium levels by inhibiting the sarcoplasmic/endoplasmic reticulum calcium-dependent ATPase, was tested for its ability to degranulate bone marrow-derived mast cells (BMMCs) from src homology 2-containing inositol phosphatase +/+ (SHIP+/+) and SHIP-/- mice. As was found previously with steel factor, thapsigargin stimulated far more degranulation in SHIP-/- than in SHIP+/+ BMMCs, and this was blocked with the phosphatidylinositol-3 (PI-3) kinase inhibitors, LY294002 and wortmannin. In contrast to steel factor, however, this heightened degranulation of SHIP-/- BMMCs was not due to a greater calcium influx into these cells, nor was the thapsigargin-induced calcium influx inhibited by LY294002, suggesting that the heightened thapsigargin-induced degranulation of SHIP-/- BMMCs was due to a PI-3 kinase-regulated step distinct from that regulating calcium entry. An investigation of thapsigargin-stimulated pathways in both cell types revealed that MAPK was heavily but equally phosphorylated. Interestingly, the protein kinase C inhibitor, bisindolylmaleimide (compound 3), totally blocked thapsigargin-induced degranulation in both SHIP+/+ and SHIP-/- BMMCs. As well, thapsigargin stimulated a PI-3 kinase-dependent, transient activation of protein kinase B, and this activation was far greater in SHIP-/- than in SHIP+/+ BMMCs. Consistent with this, thapsigargin was found to be a potent survival factor, following cytokine withdrawal, for both cell types and was more potent with SHIP-/- cells. These studies have both identified an additional PI-3 kinase-dependent step within the mast cell degranulation process, possibly involving 3-phosphoinositide-dependent protein kinase-1 and a diacylglycerol-independent protein kinase C isoform, and shown that the tumor-promoting activity of thapsigargin may be due to its activation of protein kinase B and subsequent promotion of cell survival.
Asunto(s)
Degranulación de la Célula/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Mastocitos/efectos de los fármacos , Mastocitos/enzimología , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas , Tapsigargina/farmacología , Adyuvantes Inmunológicos/deficiencia , Adyuvantes Inmunológicos/genética , Adyuvantes Inmunológicos/fisiología , Animales , Células de la Médula Ósea/efectos de los fármacos , Células de la Médula Ósea/enzimología , Células de la Médula Ósea/inmunología , Degranulación de la Célula/inmunología , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/inmunología , Células Cultivadas , Sinergismo Farmacológico , Activación Enzimática/efectos de los fármacos , Activación Enzimática/inmunología , Mastocitos/inmunología , Ratones , Ratones Noqueados , Fosfatidilinositol 3-Quinasas/fisiología , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatasas , Inhibidores de las Quinasa Fosfoinosítidos-3 , Monoéster Fosfórico Hidrolasas/deficiencia , Monoéster Fosfórico Hidrolasas/genética , Proteína Quinasa C/fisiología , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-akt , Dominios Homologos src/inmunologíaRESUMEN
Lnk was originally cloned from a rat lymph node cDNA library and shown to participate in T cell signaling. Human Lnk (hLnk) was cloned by screening a Jurkat cell cDNA library. hLnk has a calculated molecular mass of 63 kDa, and its deduced amino acid sequence indicates the presence of an N-terminal proline-rich region, a pleckstrin homology domain, and a Src homology 2 domain. When expressed in COS cells, hLnk migrates with an apparent molecular mass of 75 kDa. Confocal fluorescence microscope analysis indicates that in COS cells transfected with an expression vector encoding a chimeric Lnk-green fluorescent protein, hLnk is found at the juxtanuclear compartment and also appears to be localized at the plasma membrane. Lnk is tyrosine-phosphorylated by p56lck. Following phosphorylation, p56lck binds to tyrosine-phosphorylated hLnk through its Src homology 2 domain. In COS cells cotransfected with hLnk, p56lck, and CD8-zeta, hLnk associated with tyrosine-phosphorylated TCR zeta-chain through its Src homology 2 domain. The overexpression of Lnk in Jurkat cells led to an inhibition of anti-CD3 mediated NF-AT-Luc activation. Our study reveals a potentially new mechanism of T cell-negative regulation.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Proteínas Adaptadoras del Transporte Vesicular , Proteínas Sanguíneas/química , Activación de Linfocitos/inmunología , Proteínas Nucleares , Fosfoproteínas/química , Proteínas/química , Proteínas/genética , Homología de Secuencia de Aminoácido , Linfocitos T/inmunología , Dominios Homologos src/inmunología , Secuencia de Aminoácidos , Animales , Células COS , Proteínas Portadoras/química , ADN Complementario/aislamiento & purificación , Proteínas de Unión al ADN/antagonistas & inhibidores , Precursores Enzimáticos/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular , Células Jurkat , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/metabolismo , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/fisiología , Datos de Secuencia Molecular , Factores de Transcripción NFATC , Fosforilación , Unión Proteica/inmunología , Proteínas Tirosina Quinasas/metabolismo , Proteínas/metabolismo , Proteínas/fisiología , ARN Mensajero/biosíntesis , Ratas , Receptores de Antígenos de Linfocitos T/antagonistas & inhibidores , Receptores de Antígenos de Linfocitos T/metabolismo , Receptores de Antígenos de Linfocitos T/fisiología , Transducción de Señal/inmunología , Quinasa Syk , Factores de Transcripción/antagonistas & inhibidores , Transfección , Tirosina/metabolismoRESUMEN
The bursa of Fabricius is a gut-associated lymphoid organ that is essential for the generation of a diversified B cell repertoire in the chicken. We describe here a novel gene preferentially expressed in bursal B cells. The gene encodes an 85-kDa protein, designated BASH (B cell adaptor containing SH2 domain), that contains N-terminal acidic domains with SH2 domain-binding phosphotyrosine-based motifs, a proline-rich domain, and a C-terminal SH2 domain. BASH shows a substantial sequence similarity to SLP-76, an adaptor protein functioning in TCR-signal transduction. BASH becomes tyrosine-phosphorylated with the B cell Ag receptor (BCR) cross-link or by coexpression with Syk and Lyn and associates with signaling molecules including Syk and a putative chicken Shc homologue. Overexpression of BASH results in suppression of the NF-AT activation induced by BCR-cross-linking. These findings suggest that BASH is involved in BCR-mediated signal transduction and could play a critical role in B cell development in the bursa.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Proteínas Adaptadoras del Transporte Vesicular , Linfocitos B/metabolismo , Bolsa de Fabricio/metabolismo , Proteínas Portadoras , Proteínas Nucleares , Fosfoproteínas/biosíntesis , Transducción de Señal/inmunología , Dominios Homologos src/inmunología , Secuencia de Aminoácidos , Animales , Linfocitos B/inmunología , Bolsa de Fabricio/citología , Bolsa de Fabricio/inmunología , Línea Celular , Pollos , ADN Complementario/aislamiento & purificación , Proteínas de Unión al ADN/metabolismo , Precursores Enzimáticos/metabolismo , Precursores Enzimáticos/fisiología , Humanos , Inmunoglobulina M/metabolismo , Péptidos y Proteínas de Señalización Intracelular , Datos de Secuencia Molecular , Factores de Transcripción NFATC , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Fosforilación , Proteínas Tirosina Quinasas/metabolismo , Proteínas Tirosina Quinasas/fisiología , Proteínas/metabolismo , ARN Mensajero/biosíntesis , Receptores de Antígenos de Linfocitos B/metabolismo , Receptores de Antígenos de Linfocitos B/fisiología , Proteínas Adaptadoras de la Señalización Shc , Proteína Transformadora 1 que Contiene Dominios de Homología 2 de Src , Quinasa Syk , Factores de Transcripción/metabolismo , Tirosina/metabolismo , Familia-src Quinasas/fisiologíaRESUMEN
Signal transduction mediated by the B cell Ag receptor involves the activation of multiple protein tyrosine kinases that are members of the Src family (i.e., Fyn, Lyn, Blk, Lck). To determine whether members of the Src family possess common physical and/or enzymatic properties that enable them to potentiate signal transduction via the B cell Ag receptor, we expressed the protein tyrosine kinase Src in the B lymphoma cell line K46-17 mu m lambda. Based on coprecipitation analysis and two-color immunofluorescence, this heterologous Src family kinase was observed to physically associate with the B cell Ag receptor. Additional experiments demonstrated that B cell Ag receptor cross-linking results in increased tyrosine phosphorylation and activation of Src. Several parameters of B cell activation, including tyrosine phosphorylation of intracellular substrates, calcium mobilization, and transcription factor activation, were potentiated in cells that expressed Src when compared with control cells. To determine whether potentiation of Ag receptor-mediated signaling by Src was dependent on its catalytic activity, a kinase-deficient form of Src was expressed in K46-17 mu m lambda cells. Transfectants expressing kinase-deficient Src exhibited an enhanced responsiveness to stimulation through the B cell Ag receptor that was comparable with transfectants expressing wild-type Src. Additionally, kinase-deficient Src was observed to associate with the endogenous kinase Lyn in an activation-dependent manner. These findings indicate that members of the Src family may potentiate Ag receptor-mediated signaling via a kinase-independent mechanism(s) that involves amplification of kinase recruitment to the Ag receptor activation complex.