RESUMEN

Receptor-mediated activation of phospholipase C (PLC) leads to the hydrolysis of membrane inositol phospholipids, generating diacylglycerol (DAG) and water-soluble inositol phosphates. This signaling mechanism is used by antigen receptors on T and B cells that have been implicated as mediators of receptor-induced influx of extracellular Ca(2+). This unit provides protocols that describe the resolution of InsP by Dowex anion-exchange chromatography. This technique provides a reliable means of separating inositol monophosphate, inositol bisphosphate, and inositol trisphosphate, but does not resolve isomers of these. An Alternate Protocol describes the separation of inositol phosphates by anion-exchange HPLC. A protocol for resolution of inositol phospholipids by thin-layer chromatography (TLC) is also provided.

Asunto(s)

Activación de Linfocitos/inmunología , Linfocitos/inmunología , Fosfatidilinositoles/metabolismo , Cromatografía Líquida de Alta Presión/métodos , Cromatografía por Intercambio Iónico/métodos , Cromatografía en Capa Delgada/métodos , Humanos , Hidrólisis , Fosfatos de Inositol/análisis , Linfocitos/química , Fosfatidilinositoles/análisis , Receptores de Antígenos de Linfocitos T/inmunología , Linfocitos T/inmunología , Fosfolipasas de Tipo C/química

Asunto(s)

Immunoblotting/instrumentación , Immunoblotting/métodos , Animales , Humanos , Membranas Artificiales , Sensibilidad y Especificidad

RESUMEN

CTLA-4 is an important inhibitor of T cell activation. We used Jurkat cells expressing mutants of murine CTLA-4 to study the structural requirements for inhibitory signaling. We find that signals for the inhibition of IL-2 secretion are delivered efficiently by a CTLA-4 mutant in which both cytoplasmic tyrosines have been replaced by phenylalanines. A CTLA-4 mutant that lacks the carboxyl-terminal half of the intracellular domain also retains the ability to inhibit, but deletion of an additional 11 aa completely abrogates that capability. We conclude that delivery of an inhibitory signal requires the membrane-proximal region of the CTLA-4 cytoplasmic domain and does not depend upon the tyrosine phosphorylation of CTLA-4.

Asunto(s)

Antígenos de Diferenciación/fisiología , Inmunoconjugados , Inmunosupresores/farmacología , Transducción de Señal/inmunología , Tirosina/fisiología , Abatacept , Secuencias de Aminoácidos/genética , Secuencias de Aminoácidos/inmunología , Animales , Anticuerpos Monoclonales/metabolismo , Antígenos CD , Antígenos de Diferenciación/biosíntesis , Antígenos de Diferenciación/genética , Antígenos de Diferenciación/inmunología , Antígenos CD28/fisiología , Complejo CD3/fisiología , Antígeno CTLA-4 , Citoplasma/genética , Humanos , Interleucina-2/antagonistas & inhibidores , Interleucina-2/biosíntesis , Células Jurkat , Activación de Linfocitos , Ratones , Eliminación de Secuencia , Transducción de Señal/genética , Linfocitos T/metabolismo , Tirosina/genética

RESUMEN

The cytoplasmic domain of CD28 contains four tyrosine residues. Because signal transduction by CD28 appears to involve its tyrosine phosphorylation, we determined sites of CD28 tyrosine phosphorylation using mutants of mouse CD28 that retained tyrosine at one position, with the remaining three positions mutated to phenylalanine. When expressed in Jurkat cells and stimulated by mAb, only the mutants with tyrosine at position 170 or 188 were tyrosine phosphorylated. Phosphorylation of Tyr170 recruits phosphatidylinositol 3-kinase to CD28. Tyr188 has not been associated with any specific signaling event, but we found that ligation of CD28 by the natural ligand B7.2 also induced phosphorylation of Tyr188, suggesting that this event is of physiological importance. Consistent with that possibility, mutation of Tyr188 to phenylalanine severely impaired the ability of mouse CD28 to deliver a costimulus for the expression of CD69 and the production of IL-2. The functional consequences of the mutation of Tyr188 were unique; mutation of the other three tyrosines, individually or in combination, did not impair costimulation. Therefore, of the four CD28 tyrosine residues only Tyr188 is required for signaling in Jurkat cells, suggesting that its phosphorylation is a key event in the costimulation of T cells.

Asunto(s)

Antígenos CD28/metabolismo , Citoplasma/metabolismo , Activación de Linfocitos , Fragmentos de Péptidos/metabolismo , Linfocitos T/metabolismo , Tirosina/metabolismo , Animales , Anticuerpos Monoclonales/farmacología , Antígenos CD/fisiología , Antígeno B7-2 , Sitios de Unión/inmunología , Antígenos CD28/inmunología , Antígenos CD28/fisiología , Citoplasma/inmunología , Femenino , Humanos , Células Jurkat , Glicoproteínas de Membrana/fisiología , Ratones , Ratones Endogámicos BALB C , Fragmentos de Péptidos/inmunología , Fragmentos de Péptidos/fisiología , Fosforilación , Linfocitos T/inmunología , Tirosina/fisiología

RESUMEN

The accessory molecule CD28 delivers a costimulus that acts in concert with TCR signals to promote T cell activation. Activation of Jun-N-terminal kinases (JNK) requires simultaneous stimulation of the TCR and CD28 and, therefore, likely plays an important role in signal integration during costimulation. We investigated the effects of mutations in the 41-amino acid cytoplasmic domain of murine CD28 on its ability to deliver costimuli for JNK activation and IL-2 production when expressed in Jurkat T cells. Our results indicate that the costimulus for JNK activation requires the membrane-proximal 24 amino acids of the CD28 cytoplasmic domain and is not mediated by the tyrosine-based recruitment of signaling molecules, including phosphatidylinositol 3-kinase. Deletion of the carboxyl-terminal 17 amino acids does not affect the ability of CD28 to augment JNK activation but impairs its ability to enhance TCR-mediated production of IL-2, demonstrating that optimal costimulation of IL-2 production requires CD28 signals in addition to the activation of JNK.

Asunto(s)

Antígenos CD28/genética , Antígenos CD28/fisiología , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Interleucina-2/biosíntesis , Proteínas Quinasas Activadas por Mitógenos , Secuencia de Aminoácidos , Sustitución de Aminoácidos/genética , Androstadienos/farmacología , Animales , Antígenos CD28/biosíntesis , Complejo CD3/fisiología , Proteínas Quinasas Dependientes de Calcio-Calmodulina/antagonistas & inhibidores , Citoplasma/inmunología , Análisis Mutacional de ADN , Activación Enzimática/efectos de los fármacos , Activación Enzimática/genética , Activación Enzimática/inmunología , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos , Células Jurkat , Ratones , Datos de Secuencia Molecular , Fragmentos de Péptidos/inmunología , Fragmentos de Péptidos/fisiología , Transfección/inmunología , Tirosina/fisiología , Wortmanina

RESUMEN

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 Cgamma-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 Cgamma-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.

Asunto(s)

Proteínas Adaptadoras Transductoras de Señales , Desoxiuridina/análogos & derivados , Células Asesinas Naturales/inmunología , Propanolaminas/química , Linfocitos T/inmunología , Secuencia de Aminoácidos , Animales , Células Cultivadas , Clonación Molecular , Desoxiuridina/química , Proteína Adaptadora GRB2 , Humanos , Isoenzimas/metabolismo , Datos de Secuencia Molecular , Fragmentos de Péptidos/inmunología , Fosfolipasa C gamma , Fosfoproteínas/química , Proteínas/metabolismo , ARN Mensajero/metabolismo , Ratas , Proteínas Recombinantes/inmunología , Análisis de Secuencia de ADN , Timo/fisiología , Fosfolipasas de Tipo C/metabolismo , Dominios Homologos src/genética

RESUMEN

Optimal T cell activation requires crosslinking of the T cell receptor (TCR) concurrently with an accessory receptor, most efficiently CD28. Crosslinking of CD28 leads to increased interleukin 2 (IL2) production, inhibition of anergy and prevention of programmed cell death. Crosslinking of CD28 leads to rapid increases in tyrosine phosphorylation of specific intracellular substrates including CD28 itself. Since CD28 does not encode an intrinsic tyrosine kinase domain, CD28 must activate an intracellular tyrosine kinase(s). Indeed, crosslinking of CD28 increases the activity of the intracellular tyrosine kinases EMT/ITK and LCK. The phosphatidylinositol 3-kinase (PI3K) and GRB2 binding site in CD28 is dispensable for optimal IL2 production in Jurkat T cells. We demonstrate herein that murine Y170 (equivalent to human Y173) in CD28 is also dispensable for activation of the SRC family tyrosine kinase LCK and the TEC family tyrosine kinase EMT/ITK. In contrast, the distal three tyrosines in CD28 are required for optimal IL2 production as well as for optimal activation of the LCK and EMT/ITK tyrosine kinases. The distal three tyrosines of CD28, however, are not required for recruitment of PI3K to CD28. Furthermore, PI3K is recruited to CD28 in JCaM1 cells which lack LCK and in which EMT/ITK is not activated by ligation of CD28. Thus optimal activation of LCK or EMT/ITK is not obligatory for recruitment of PI3K to CD28 and thus is also not required for tyrosine phosphorylation of the YMNM motif in CD28. Taken together the data indicate that the distal three tyrosines in CD28 are integral to the activation of LCK and EMT/ITK and for subsequent IL2 production.

Asunto(s)

Proteínas Adaptadoras Transductoras de Señales , Antígenos CD28/fisiología , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Secuencia de Aminoácidos , Animales , Antígenos CD28/biosíntesis , Anergia Clonal , Reactivos de Enlaces Cruzados , Proteína Adaptadora GRB2 , Humanos , Interleucina-2/biosíntesis , Células Jurkat , Ratones , Péptidos/química , Péptidos/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas/metabolismo , Proteínas Recombinantes/biosíntesis , Transducción de Señal , Especificidad por Sustrato , Linfocitos T , Transfección

RESUMEN

The activation of naive CD4+ T cells requires two discrete signals: a signal delivered by the T cell receptor following recognition of antigen and an accessory signal transduced when costimulatory receptors interact with their ligands. Particularly important in the development of an immune response to foreign antigens is the T cell molecule CD28, which delivers a potent costimulus when engaged by ligands, B7-1 and B7-2, on antigen-presenting cells. It is interesting that blockade of B7 molecules, which disrupts interactions with CD28 and prevents delivery of the CD28 costimulus, also alters the immune responses to self antigens and prevents the development of clinical disease in murine models of systemic and organ-specific autoimmunity. Herein we review the roles of CD28 and its B7 ligands in the pathogenesis of autoimmunity, discuss efforts to treat animal models of autoimmunity by modifying the CD28 signal, and consider the mechanisms by which manipulation of the CD28 signal alters the course of experimental autoimmune disease.

Asunto(s)

Antígenos de Diferenciación/farmacología , Enfermedades Autoinmunes/fisiopatología , Antígeno B7-1/fisiología , Antígenos CD28/fisiología , Inmunoconjugados , Linfocitos T/inmunología , Abatacept , Animales , Antígenos CD , Antígeno CTLA-4 , Inmunosupresores/farmacología , Ligandos , Ratones , Ratones Noqueados , Transducción de Señal

RESUMEN

Mutagenesis studies have demonstrated the requirement for the CD28-responsive element (CD28RE) within the interleukin-2 (IL-2) promoter for transcriptional upregulation by CD28. Here, we demonstrate that CD28 responsiveness is conferred by a composite element containing both the CD28RE and the NF-IL-2B AP-1 sites (RE/AP). Mutations at either site within the RE/AP composite element abolish activity. The RE/AP composite element is a site for signal integration within the IL-2 promoter, since its activation is dependent on at least two separate signalling pathways being activated, through the T-cell receptor, CD28, and/or phorbol myristate acetate. Activation is maximal when all three signals occur simultaneously. By using a panel of CD28 cytoplasmic domain mutants, it was found that the transcriptional activation of the RE/AP composite element correlates exactly with the pattern of IL-2 secretion induced by these mutants upon stimulation. Similar to the upregulation of IL-2 secretion, the transcriptional upregulation of the RE/AP composite element by CD28 is FK506 insensitive. The pattern of activation of the RE/AP composite element is different from that observed for either an NFAT or consensus AP-1 site, implying that RE/AP represents a unique element. Using gel shift analysis, we demonstrate that stimulation by CD28 induces the association of the NF-kappaB family member c-Rel to the CD28RE within the RE/AP composite element. The transcriptional upregulation of IL-2 by CD28 appears, therefore, to be mediated through the RE/AP composite element, involving the association of c-Rel with the CD28RE.

Asunto(s)

Antígenos CD28/fisiología , Interleucina-2/genética , Factor de Transcripción AP-1/fisiología , Transcripción Genética , Regulación hacia Arriba , Secuencia de Aminoácidos , Secuencia de Bases , Sitios de Unión , Línea Celular , Núcleo Celular/metabolismo , Proteínas de Unión al ADN/fisiología , Humanos , Sustancias Macromoleculares , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-rel

RESUMEN

Perturbation of several distinct T cell molecules, including the CD3/TCR complex, CD7, and CD28, activates phosphatidylinositol 3-kinase (PI3-K), but a clear consensus on the role of PI3-K in T cell activation has yet to emerge. We report here that CD3 mAb-induced IL-2 production by CD4+ T cells from DO11.10 TCR-alphabeta-transgenic mice is refractory to the potent PI3-K inhibitor, wortmannin, demonstrating that activation under these conditions is independent of PI3-K. In marked contrast, wortmannin substantially inhibits IL-2 production elicited by Ag (OVA(323-339) peptide) presented by appropriate APCs (syngeneic B7+ B cell blasts) and blocks Ag-induced differentiation of naive CD4+ DO11.10 T cells into IL-4-producing cells. Wortmannin inhibits Ag-induced conjugate formation between T cells and B7+ B cell blasts. Because T cell activation by Ag requires stable interactions with APCs, this inhibitory effect on conjugate formation may underlie the ability of wortmannin to block Ag-induced IL-2 production and differentiation.

Asunto(s)

Androstadienos/farmacología , Complejo CD3/fisiología , Linfocitos T CD4-Positivos/inmunología , Inhibidores Enzimáticos/farmacología , Activación de Linfocitos/efectos de los fármacos , Fosfotransferasas (Aceptor de Grupo Alcohol)/antagonistas & inhibidores , Receptores de Antígenos de Linfocitos T/fisiología , Animales , Antígeno B7-1/fisiología , Diferenciación Celular/efectos de los fármacos , Interleucina-2/biosíntesis , Interleucina-4/biosíntesis , Cooperación Linfocítica , Ratones , Ratones Transgénicos , Ovalbúmina/inmunología , Péptidos/inmunología , Fosfatidilinositol 3-Quinasas , Transducción de Señal , Wortmanina

RESUMEN

Although under certain conditions an association with phosphatidylinositol 3'-kinase (PI3-K) appears to be critical for CD28 signaling, mutation of the PI3-K binding site (Tyr 170) does not alter the costimulatory ability of murine CD28 (mCD28) in Jurkat T cells. To define the structural requirements for this PI3-K-independent signaling, we expressed a series of mCD28 mutants in Jurkat. Mutation to Phe of all four cytoplasmic Tyr residues together (ALL F mutant) greatly reduced the ability of mCD28 to augment IL-2 production. Isolated re-constitution of Tyr 188, but not 170, 185, or 197, restored the ability of ALL F mCD28 to deliver a costimulus. Thus, a signal based upon Tyr 188 can deliver a costimulus for the enhancement of IL-2 production by Jurkat cells.

Asunto(s)

Antígenos CD28/fisiología , Interleucina-2/biosíntesis , Activación de Linfocitos , Linfocitos T/fisiología , Secuencia de Aminoácidos , Humanos , Datos de Secuencia Molecular , Fosfatidilinositol 3-Quinasas , Fosfotransferasas (Aceptor de Grupo Alcohol)/fisiología , Fosfotirosina/química , Mutación Puntual , Transducción de Señal , Relación Estructura-Actividad , Células Tumorales Cultivadas

RESUMEN

CD2, a cell surface glycoprotein expressed on T cells and natural killer cells, can couple to signaling pathways that result in T cell proliferation. An Src-like protein tyrosine kinase, p56lck, coprecipitates with CD2, and perturbation of CD2 by monoclonal antibodies results in an increase in the activity of p56lck, suggesting that an interaction with p56lck contributes to CD2-mediated signaling. Herein, we investigate the mechanism by which CD2 associates with p56lck. We demonstrate that CD2 and p56lck associate when coexpressed in nonlymphoid cells, that this association requires the cytoplasmic domain of CD2, and that the SH3 domain of p56lck mediates its interactions with CD2. Using truncation mutants of CD2, we identify two regions in the cytoplasmic domain of CD2 involved in binding p56lck. Each region contains a proline-rich sequence that, in the form of a synthetic peptide, directly binds p56lck. Thus, proline-rich sequences in the cytoplasmic domain of CD2 allow this transmembrane receptor to bind to the SH3 domain of p56lck.

Asunto(s)

Antígenos CD2/metabolismo , Receptores de Superficie Celular/metabolismo , Familia-src Quinasas/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Antígenos CD2/genética , Células Cultivadas , Interleucina-2/biosíntesis , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito , Datos de Secuencia Molecular , Mutagénesis , Fragmentos de Péptidos/metabolismo , Prolina , Unión Proteica , Conformación Proteica , Ratas , Receptores de Superficie Celular/genética , Proteínas Recombinantes/metabolismo , Eliminación de Secuencia , Transducción de Señal , Spodoptera/citología , Relación Estructura-Actividad , Linfocitos T/metabolismo , Dominios Homologos src , Familia-src Quinasas/genética

RESUMEN

CD28, a cell-surface molecule expressed by T cells, delivers costimulatory signals during the activation of T cells by Ag. Stimulation of CD28 induces its association with phosphatidylinositol 3'-kinase (PI3-K), raising the possibility that PI3-K plays a critical role in CD28 signaling. We find, however, that wortmannin, a potent inhibitor of PI3-K, does not block CD28-mediated costimulation of Jurkat (a human T cell line) or of murine CD4+ T cells. To address further the role of PI3-K in CD28-mediated signaling, we expressed mutant murine CD28 molecules in Jurkat cells. Mutation of Tyr 170 of murine CD28 to Phe abrogates the association of murine CD28 with PI3-K but does not affect the ability of murine CD28 to augment IL-2 production by Jurkat cells in response to the combination of ionomycin and PMA. Conversely, a mutant of murine CD28 that has a Tyr at position 170 but has Phe substitutions at the remaining three cytoplasmic tyrosines retains the ability to associate with PI3-K and has an impaired ability to deliver a costimulus that augments IL-2 production. CD28, therefore, can deliver costimulatory signals independently of its interaction with PI3-K, and association with PI3-K is insufficient to mediate the full effector function of CD28. Optimal signaling by CD28 requires the integrity of one or more of the carboxyl-terminal three Tyr residues.

Asunto(s)

Antígenos CD28/metabolismo , Linfocitos T CD4-Positivos/metabolismo , Interleucina-2/biosíntesis , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Transducción de Señal , Linfocitos T/metabolismo , Androstadienos/farmacología , Animales , Antígenos CD28/genética , Células Cultivadas , Femenino , Humanos , Ratones , Ratones Endogámicos C57BL , Mutación , Fosfatidilinositol 3-Quinasas , Sistemas de Mensajero Secundario , Wortmanina

Asunto(s)

Antígenos CD2 , Linfocitos T/inmunología , Animales , Humanos

RESUMEN

In hematopoietic cells, the protein tyrosine phosphatase PTP1C appears to play a central role in the termination of signalling by receptors that activate protein tyrosine kinases.

Asunto(s)

Células Madre Hematopoyéticas/metabolismo , Proteínas Tirosina Fosfatasas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Transducción de Señal , Animales , Activación Enzimática , Células Madre Hematopoyéticas/enzimología , Péptidos y Proteínas de Señalización Intracelular , Proteína Tirosina Fosfatasa no Receptora Tipo 11 , Proteína Tirosina Fosfatasa no Receptora Tipo 6

RESUMEN

The T cell surface molecule CD28 can provide costimulatory signals that permit the full activation of T cells. Here we demonstrate that stimulation of CD28, either by B7, its natural ligand, or by the anti-CD28 monoclonal antibody 9.3, induces an association between CD28 and phosphatidylinositol 3-kinase (PI3-K) in Jurkat T cells, raising the possibility that an interaction with PI3-K contributes to CD28-mediated signaling. To examine the mechanism of the association, we synthesized tyrosine-phosphorylated oligopeptides corresponding to each of the four tyrosines in the CD28 cytoplasmic domain. When added to lysates of B7-stimulated Jurkat cells, the oligopeptide corresponding to Tyr 173 inhibits the coimmunoprecipitation of PI3-K with CD28; the other oligopeptides have no effect. Tyr 173 is contained within the sequence YMNM, a motif that is also found in the platelet-derived growth factor receptor and that, when phosphorylated, forms a high affinity binding site for the p85 subunit of PI3-K. These observations suggest that phosphorylation of Tyr 173 may mediate the interaction between CD28 and PI3-K. However, because CD28 is not known to be phosphorylated, it remains possible that CD28 interacts with PI3-K through a mechanism independent of tyrosine phosphorylation.

Asunto(s)

Antígenos CD/metabolismo , Antígenos CD28/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Animales , Antígenos CD/aislamiento & purificación , Secuencia de Bases , Antígenos CD28/aislamiento & purificación , Línea Celular , Humanos , Cinética , Sarcoma de Mastocitos , Ratones , Datos de Secuencia Molecular , Oligopéptidos/síntesis química , Oligopéptidos/farmacología , Fosfatidilinositol 3-Quinasas , Fosfopéptidos/síntesis química , Fosfopéptidos/farmacología , Fosfotransferasas (Aceptor de Grupo Alcohol)/aislamiento & purificación , Fosfotirosina , Unión Proteica , Transducción de Señal/efectos de los fármacos , Linfocitos T , Transfección , Células Tumorales Cultivadas , Tirosina/análogos & derivados

RESUMEN

Interleukin-2 (IL-2) signaling results in tyrosine phosphorylation of the 75-kDa IL-2 receptor (IL-2R) beta chain and the activation of phosphatidylinositol 3'-kinase (PI3-K). Herein, we demonstrate that the 85-kDa (p85) regulatory subunit of PI3-K physically associates with the tyrosine-phosphorylated IL-2R beta chain. A fusion protein containing both the amino- and the carboxyl-terminal src homology 2 domains of p85 precipitates an 80-kDa tyrosine-phosphorylated protein (pp80) from the lysates of IL-2-stimulated, but not unstimulated, human T lymphoblasts. Preclearing studies and immunoblotting with an antiserum to the IL-2R beta chain demonstrates that pp80 represents a portion of the IL-2R beta chain pool. A tyrosine-phosphorylated oligopeptide corresponding to tyrosine 392 of the IL-2R beta chain partially inhibits binding of the IL-2R beta chain by p85 fusion protein, raising the possibility that this residue plays a role in the interaction of PI3-K with the receptor.

Asunto(s)

Fragmentos de Péptidos/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Receptores de Interleucina-2/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Humanos , Cinética , Datos de Secuencia Molecular , Oligopéptidos/farmacología , Fosfatidilinositol 3-Quinasas , Fosforilación , Pruebas de Precipitina , Proteínas Proto-Oncogénicas pp60(c-src)/química , Receptores de Interleucina-2/química , Receptores del Factor de Crecimiento Derivado de Plaquetas/metabolismo , Transducción de Señal , Tirosina/metabolismo

RESUMEN

To examine the role of CD45 in NK cell activation, we isolated three mutants and one variant of a rat NK cell line, RNK-16. Each of these lacked cell-surface expression of CD45 and did not have detectable transcripts for CD45 on Northern blot analysis. The CD45-negative cells expressed CD2, CD53, and NKR-P1, but mAb-induced perturbations of these molecules did not induce protein tyrosine phosphorylations and increases in the concentration of cytoplasmic-free calcium, as occurred in the wild-type RNK-16. Unlike the wild-type cells, the CD45-negative cells failed to lyse YAC-1 and RL-male-1 tumor targets. The cytolytic activity of the CD45-negative cells could be stimulated pharmacologically by ionomycin and PMA, which, when added to the cytotoxicity assays, induced killing of tumor targets. These studies suggest that CD45 is required for the response of RNK-16 cells to target cells and for signaling through CD2, CD53, and NKR-P1.

Asunto(s)

Citotoxicidad Inmunológica , Células Asesinas Naturales/inmunología , Antígenos Comunes de Leucocito/análisis , Animales , Antígenos CD/fisiología , Antígenos de Diferenciación de Linfocitos T/fisiología , Antígenos CD2 , Línea Celular , Mutación , Proteínas Tirosina Quinasas/fisiología , Ratas , Receptores Inmunológicos/fisiología , Tetraspanina 25

RESUMEN

The cell surface molecule CD2 has a signaling role in the activation of T lymphocytes and natural killer cells. Because perturbation of CD2 leads to the appearance of tyrosine-phosphorylated proteins, we investigated the possibility that CD2 associates with cytoplasmic protein tyrosine kinases. As determined by in vitro kinase assays and phosphoamino acid analysis, protein tyrosine kinase activity coprecipitated with CD2 from rat T lymphocytes, T lymphoblasts, thymocytes, interleukin-2-activated natural killer cells, and RNK-16 cells (a rat natural killer cell line). In each case, both p56lck and p59fyn were identified in the CD2 immunoprecipitate. In the thymus, the association between CD2 and these kinases occurred predominately in a small subset of thymocytes that had the cell surface phenotype of mature T cells, indicating that the association is a regulated event and occurs late in T-cell ontogeny. The finding that CD2 is associated with p56lck and p59fyn in detergent lysates suggests that interactions with these Src-like protein kinases play a critical role in CD2-mediated signal transduction.

Asunto(s)

Antígenos de Diferenciación de Linfocitos T/metabolismo , Células Asesinas Naturales/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Receptores Inmunológicos/metabolismo , Linfocitos T/metabolismo , Animales , Antígenos CD2 , Línea Celular , Humanos , Yodoacetamida/farmacología , Mapeo Peptídico , Pruebas de Precipitina , Proteínas Proto-Oncogénicas pp60(c-src)/metabolismo , Ratas , Ratas Endogámicas F344 , Linfocitos T/enzimología

RESUMEN

Stimulation of the T cell antigen receptor (TCR) activates a protein tyrosine kinase and leads to the tyrosine phosphorylation of phosphoinositide-specific phospholipase C-gamma 1 (PLC gamma 1). The molecular interactions involved in this phosphorylation are not known. After stimulation of the TCR on Jurkat T cells, tyrosine-phosphorylated proteins of 36, 38, 58, and 63 kD coprecipitate with PLC gamma 1. An identical pattern of proteins precipitate with TrpE fusion proteins that contain the Src homology (SH) 2 domains of PLC gamma 1, indicating that these regions of PLC gamma 1 are responsible for binding. TCR stimulation leads to an association between the SH2 domains of PLC gamma 1 and a protein tyrosine kinase, which, by peptide mapping, is identical to p56lck. These studies establish that p56lck associates with PLC gamma 1 as a result of TCR stimulation of Jurkat cells, suggesting that p56lck plays a central role in coupling the TCR to the activation of PLC gamma 1.

Asunto(s)

Proteínas Tirosina Quinasas/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Linfocitos T/metabolismo , Fosfolipasas de Tipo C/metabolismo , Línea Celular , Humanos , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito , Mapeo Peptídico , Fosforilación , Linfocitos T/enzimología , Tirosina/metabolismo