RESUMO
Although IgE binding to mast cells is thought to be a passive presensitization step, we demonstrate herein that monomeric IgE (mIgE) in the absence of antigen (Ag) stimulates multiple phosphorylation events in normal murine bone marrow-derived mast cells (BMMCs). While mIgE does not induce degranulation or leukotriene synthesis, it leads to a more potent production of cytokines than IgE + Ag. Moreover, mIgE prevents the apoptosis of cytokine-deprived BMMCs, likely by maintaining Bcl-X(L) levels and producing autocrine-acting cytokines. The addition of Ag does not increase this IgE-induced survival. Since IgE concentrations as low as 0.1 microg/ml enhance BMMC survival, elevated plasma IgE levels in humans with atopic disorders may contribute to the elevated mast cell numbers seen in these individuals.
Assuntos
Citocinas/biossíntese , Imunoglobulina E/imunologia , Mastócitos/imunologia , Proteínas Serina-Treonina Quinases , Transdução de Sinais/imunologia , Animais , Apoptose/imunologia , Sobrevivência Celular , Células Cultivadas , Citocinas/genética , Imunoglobulina E/farmacologia , Mastócitos/efeitos dos fármacos , Microdomínios da Membrana/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-akt , Receptores de IgE/imunologia , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
Antigen stimulation of mast cells via FcepsilonRI, the high-affinity receptor for IgE, triggers a signaling cascade that requires Ca(2+) mobilization for exocytosis of secretory granules during an allergic response. This study investigates critical signaling components by using mutant RBL mast cells that are defective in antigen-stimulated phospholipase Cgamma (PLCgamma) activation, as well as other signaling activities downstream of stimulated tyrosine phosphorylation. We show that the expression of activated versions of the Cdc42 or Rac1 GTPase restores antigen-stimulated Ca(2+) mobilization necessary for degranulation in these mutant cells. Wild-type Cdc42 and Rac1, as well as activated Cdc42 containing effector domain mutations, all fail to restore antigen-stimulated signaling leading to exocytosis. Expression of oncogenic Dbl, a guanine nucleotide exchange factor for Cdc42 and Rac1, partially restores sustained Ca(2+) mobilization and degranulation, suggesting that activation of endogenous Cdc42 and/or Rac1 is impaired in the mutant cells. Overexpression of PLCgamma1 with either activated Cdc42 or Rac1 synergistically stimulates degranulation, consistent with a critical defect in PLCgamma activation in these cells. Thus, our results point to activation of Cdc42 and/or Rac1 playing an essential role in antigen stimulation of early events that culminate in mast cell degranulation.
Assuntos
Sinalização do Cálcio/fisiologia , Cálcio/metabolismo , Degranulação Celular/fisiologia , Mastócitos/fisiologia , Receptores de IgE/fisiologia , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteínas rac de Ligação ao GTP/metabolismo , Animais , Linhagem Celular , Ativação Enzimática , Isoenzimas/genética , Isoenzimas/metabolismo , Fosfolipase C gama , Ratos , Proteínas Recombinantes/metabolismo , Transfecção , Fosfolipases Tipo C/genética , Fosfolipases Tipo C/metabolismoRESUMO
Aggregation of the high-affinity IgE receptor induces the tyrosine phosphorylation of subunits of the receptor and the subsequent association with the receptor of the cytosolic protein tyrosine kinase Syk. The current experiments examined the functional importance of membrane association of Syk and the role of the SH2 domain in receptor-mediated signal transduction. Wild-type Syk and chimeric Syk molecules with the c-Src myristylation sequence at the amino-terminus were expressed in a Syk-negative mast cell line. Chimeric Syk with the myristylation sequence was membrane associated, and a small fraction was constitutively colocalized with FcepsilonRI, Lyn, and LAT (linker for T-cell activation) in the glycolipid-enriched microdomains or rafts. However, even under these conditions, the tyrosine phosphorylation of Syk and the downstream propagation of signals required FcepsilonRI aggregation. This chimeric Syk was less active than wild-type Syk in FcepsilonRI-mediated signal transduction. In contrast, a truncated membrane-associated form of Syk that lacked the SH2 domains was not tyrosine phosphorylated by receptor aggregation and failed to transduce intracellular signals. These findings suggest that SH2 domain-mediated membrane translocation of Syk is essential for the FcepsilonRI-mediated activation of Syk for downstream signaling events leading to histamine release. Furthermore, the localization of Syk in glycolipid-enriched microdomains by itself is not enough to generate or enhance signaling events.
Assuntos
Membrana Celular/metabolismo , Precursores Enzimáticos/metabolismo , Proteínas Tirosina Quinases/metabolismo , Receptores de IgE/metabolismo , Transdução de Sinais/efeitos dos fármacos , Domínios de Homologia de src/fisiologia , Animais , Proteína Tirosina Quinase CSK , Membrana Celular/ultraestrutura , Glicolipídeos/metabolismo , Immunoblotting , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas de Membrana/metabolismo , Proteínas de Membrana/fisiologia , Fosforilação/efeitos dos fármacos , Ratos , Receptores de IgE/fisiologia , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/fisiologia , Quinase Syk , Transfecção , Células Tumorais Cultivadas , Quinases da Família srcRESUMO
Previous studies of mast cell maturation, structure, and function have been hampered by the lack of mast cell-specific markers. In this study, using a well-characterized mast cell-specific monoclonal antibody, MAb AA4, mast cells from rat bone marrow in various stages of maturation were isolated and characterized. The very immature mast cells, which have not been previously described, contained few granules and would not be recognized as mast cells by standard cytological methods. Pure populations of mast cells were isolated from the bone marrow using MAb AA4-conjugated magnetic beads. The same stages of maturation were observed in the isolated mast cells as were seen in the unfractionated bone marrow. All of these cells were immunopositive for the alpha-subunit of Fc epsilon RI, IgE, and c-kit, confirming their identity as mast cells. By direct counting of immunolabled cells and by flow cytometry, approximately 2.4% of the cells in the bone marrow are mast cells. Staining with toluidine blue and berberine sulfate, as well as RT-PCR of the cells, indicates that these cells are connective tissue-type mast cells. The use of immunological methods for identification of mast cell precursors should facilitate the study of these cells. (J Histochem Cytochem 49:219-228, 2001)
Assuntos
Anticorpos Monoclonais , Células da Medula Óssea/citologia , Mastócitos/citologia , Animais , Células da Medula Óssea/metabolismo , Células da Medula Óssea/ultraestrutura , Separação Celular , Feminino , Citometria de Fluxo , Imunoglobulina E/metabolismo , Masculino , Mastócitos/imunologia , Mastócitos/metabolismo , Mastócitos/ultraestrutura , Microscopia Eletrônica , Proteínas Proto-Oncogênicas c-kit/metabolismo , Ratos , Ratos Sprague-Dawley , Ratos Wistar , Receptores de IgE/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Characterization of defects in a variant subline of RBL mast cells has revealed a biochemical event proximal to IgE receptor (Fc epsilon RI)-stimulated tyrosine phosphorylation that is required for multiple functional responses. This cell line, designated B6A4C1, is deficient in both Fc epsilon RI-mediated degranulation and biosynthesis of several lipid raft components. Agents that bypass receptor-mediated Ca(2+) influx stimulate strong degranulation responses in these variant cells. Cross-linking of IgE-Fc epsilon RI on these cells stimulates robust tyrosine phosphorylation but fails to mobilize a sustained Ca(2+) response. Fc epsilon RI-mediated inositol phosphate production is not detectable in these cells, and failure of adenosine receptors to mobilize Ca(2+) suggests a general deficiency in stimulated phospholipase C activity. Antigen stimulation of phospholipases A(2) and D is also defective. Infection of B6A4C1 cells with vaccinia virus constructs expressing constitutively active Rho family members Cdc42 and Rac restores antigen-stimulated degranulation, and active Cdc42 (but not active Rac) restores ganglioside and GPI expression. The results support the hypothesis that activation of Cdc42 and/or Rac is critical for Fc epsilon RI-mediated signaling that leads to Ca(2+) mobilization and degranulation. Furthermore, they suggest that Cdc42 plays an important role in the biosynthesis and expression of certain components of lipid rafts.
Assuntos
Metabolismo dos Lipídeos , Mastócitos/fisiologia , Receptores de IgE/fisiologia , Proteínas rho de Ligação ao GTP/metabolismo , Actinas/metabolismo , Animais , Cálcio/metabolismo , Linhagem Celular , Grânulos Citoplasmáticos/genética , Grânulos Citoplasmáticos/ultraestrutura , Ativação Enzimática , Immunoblotting , Cinética , Leucemia Basofílica Aguda , Mastócitos/citologia , Fosforilação , Fosfotirosina/análise , Fosfotirosina/metabolismo , Ratos , Transdução de SinaisRESUMO
Syk is an important protein-tyrosine kinase in immunoreceptor signaling. FcepsilonRI aggregation in mast cells induces tyrosine phosphorylation and increased enzymatic activity of Syk. The two adjacent tyrosines in the Syk activation loop are thought to be important for the propagation of FcepsilonRI signaling. To evaluate the phosphorylation of these tyrosines in vivo and further understand the relationship of Syk tyrosine phosphorylation with its function, an antibody was developed specific for phosphorylated tyrosines in the activation loop of Syk. FcepsilonRI aggregation on mast cells induced the phosphorylation of both tyrosine residues of the activation loop. The kinase activity of Syk played the major role in phosphorylating its activation loop tyrosines both in vivo and in vitro. In FcepsilonRI-stimulated mast cells, the total Syk tyrosine phosphorylation paralleled the phosphorylation of its activation loop tyrosines and downstream propagation of signals for histamine release. In contrast, the cell surface binding of anti-ganglioside monoclonal antibody AA4 induced only strong general tyrosine phosphorylation of Syk and minimal histamine release and weak phosphorylation of activation loop tyrosines. These results demonstrate that phosphorylation of the activation loop tyrosines is important for mediating receptor signaling and is a better marker of Syk function than is total Syk tyrosine phosphorylation.
Assuntos
Precursores Enzimáticos/metabolismo , Precursores Enzimáticos/fisiologia , Proteínas Tirosina Quinases/metabolismo , Proteínas Tirosina Quinases/fisiologia , Tirosina/metabolismo , Animais , Relação Dose-Resposta a Droga , Ativação Enzimática , Histamina/metabolismo , Immunoblotting , Peptídeos e Proteínas de Sinalização Intracelular , Mastócitos/metabolismo , Fosforilação , Testes de Precipitina , Ratos , Transdução de Sinais , Quinase Syk , Fatores de Tempo , Transfecção , Células Tumorais Cultivadas , Tirosina/químicaRESUMO
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 (FcepsilonRI) in mast cells. The present study provides genetic, biochemical, and pharmacological evidence that, on FcepsilonRI stimulation, Syk regulates Btk, and Btk selectively regulates the membrane translocation and enzymatic activity of PKCbetaI among the conventional PKC isoforms (alpha, betaI, and betaII) expressed in mast cells. Syk/Btk-mediated PKCbetaI regulation is involved in transcriptional activation of the IL-2 and tumor necrosis factor alpha genes through the JNK pathway induced by FcepsilonRI stimulation. Accordingly, FcepsilonRI-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 PKCbeta. Specific regulation of PKCbetaI by Btk is consistent with the selective association of Btk with PKCbetaI. 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.
Assuntos
Linfócitos B/imunologia , Precursores Enzimáticos/imunologia , Isoenzimas/imunologia , Proteína Quinase C/imunologia , Proteínas Tirosina Quinases/imunologia , Transdução de Sinais/imunologia , Tirosina Quinase da Agamaglobulinemia , Animais , Células Cultivadas , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Camundongos Knockout , Proteína Quinase C beta , Quinase SykRESUMO
Vav is a hematopoietic cell-specific guanine nucleotide exchange factor (GEF) whose activation is mediated by receptor engagement. The relationship of Vav localization to its function is presently unclear. We found that Vav redistributes to the plasma membrane in response to Fcin receptor I (FcinRI) engagement. The redistribution of Vav was mediated by its Src homology 2 (SH2) domain and required Syk activity. The FcinRI and Vav were found to colocalize and were recruited to glycosphingolipid-enriched microdomains (GEMs). The scaffold protein, linker for activation of T cells (LAT), and Rac1 (a target of Vav activity) were constitutively present in GEMs. Expression of an SH2 domain-containing COOH-terminal fragment of Vav inhibited Vav phosphorylation and movement to the GEMs but had no effect on the tyrosine phosphorylation of the adaptor protein, SLP-76 (SH2 domain-containing leukocyte protein of 76 kD), and LAT. However, assembly of the multiprotein complex containing these proteins was inhibited. In addition, FcinRI-dependent activation of c-Jun NH(2)-terminal kinase 1 (JNK1) was also inhibited. Thus, Vav localization to the plasma membrane is mediated by its SH2 domain and may serve to regulate downstream effectors like JNK1.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteínas de Ciclo Celular , Proteínas de Membrana , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteínas Proto-Oncogênicas/fisiologia , Domínios de Homologia de src , Animais , Proteínas de Transporte/análise , Membrana Celular/química , Ativação Enzimática , Precursores Enzimáticos/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas Quinases JNK Ativadas por Mitógeno , Camundongos , Fosfoproteínas/análise , Proteínas Tirosina Quinases/fisiologia , Proteínas Proto-Oncogênicas/análise , Proteínas Proto-Oncogênicas/química , Proteínas Proto-Oncogênicas c-vav , Receptores de IgE/metabolismo , Quinase Syk , Proteínas rac1 de Ligação ao GTP/análiseRESUMO
The SH2-containing protein tyrosine phosphatase1 (SHP-1) is important for signaling from immune receptors. To investigate the role of SHP-1 in mast cells we overexpressed the wild-type and the phosphatase-inactive forms of SHP-1 in rat basophilic leukemia 2H3 (RBL-2H3) mast cell line. The phosphatase-inactive SHP-1 (C453S or D419A) retains its ability to bind tyrosine phosphorylated substrates and thereby competes with the endogenous wild-type enzyme. Overexpression of wild-type SHP-1 decreased the FcepsilonRI aggregation-induced tyrosine phosphorylation of the beta and gamma subunits of the receptor whereas the dominant negative SHP-1 enhanced phosphorylation. There were also similar changes in the tyrosine phosphorylation of Syk. However, receptor-induced histamine release in the cells expressing either wild-type or dominant negative SHP-1 was similar to that in the parental control cells. In contrast, compared with the parental RBL-2H3 cells, FcepsilonRI-induced c-Jun N-terminal kinase phosphorylation and the level of TNF-alpha mRNA was increased in the cells overexpressing wild-type SHP-1 whereas the dominant negative SHP-1 had the opposite effect. The substrate-trapping mutant SHP1/D419A identified pp25 and pp30 as two major potential substrates of SHP-1 in RBL-2H3 cells. Therefore, SHP-1 may play a role in allergy and inflammation by regulating mast cell cytokine production.
Assuntos
Liberação de Histamina/imunologia , Mastócitos/enzimologia , Proteínas Quinases Ativadas por Mitógeno/biossíntese , Proteínas Tirosina Fosfatases/fisiologia , Fator de Necrose Tumoral alfa/biossíntese , Domínios de Homologia de src/imunologia , Animais , Antígenos/farmacologia , Regulação para Baixo/genética , Regulação para Baixo/imunologia , Precursores Enzimáticos/metabolismo , Liberação de Histamina/genética , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas Quinases JNK Ativadas por Mitógeno , Sistema de Sinalização das MAP Quinases/imunologia , Mastócitos/imunologia , Mastócitos/metabolismo , Peso Molecular , Fosfoproteínas/metabolismo , Fosforilação , Proteína Tirosina Fosfatase não Receptora Tipo 11 , Proteína Tirosina Fosfatase não Receptora Tipo 6 , Proteínas Tirosina Fosfatases/biossíntese , Proteínas Tirosina Fosfatases/genética , Proteínas Tirosina Quinases/metabolismo , Ratos , Receptores de IgE/metabolismo , Receptores de IgE/fisiologia , Proteínas Tirosina Fosfatases Contendo o Domínio SH2 , Especificidade por Substrato , Quinase Syk , Transfecção , Células Tumorais Cultivadas , Tirosina/metabolismo , Domínios de Homologia de src/genéticaRESUMO
The protein tyrosine kinase Syk plays an essential role in Fc epsilon RI-mediated histamine release in mast cells by regulating the phosphorylation of other proteins. We investigated the functional role of a putative Syk phosphorylation site, Tyr317. This tyrosine in the linker region of Syk is a possible site for binding by the negative regulator Cbl. Syk with Tyr317 mutated to Phe (Y317F) was expressed in a Syk-negative variant of the RBL-2H3 mast cells. Compared with cells expressing wild-type Syk, expression of the Y317F mutant resulted in an increase in the Fc epsilon RI-mediated tyrosine phosphorylation of phospholipase C-gamma and a dramatic enhancement of histamine release. The in vivo Fc epsilon RI-induced tyrosine phosphorylation of wild-type Syk and that of the Y317F mutant were similar. Although the Fc epsilon RI-induced tyrosine phosphorylation of total cellular proteins was enhanced in the cells expressing the Y317F Syk, the phosphorylation of some other molecules, including the receptor subunits, Vav and mitogen-activated protein kinase, was not increased. The Fc epsilon RI-induced phosphorylation of Cbl was downstream of Syk kinase activity and was unchanged by expression of the Y317F mutation. These data indicate that Tyr317 in the linker region of Syk functions to negatively regulate the signals leading to degranulation.
Assuntos
Precursores Enzimáticos/genética , Precursores Enzimáticos/metabolismo , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Mutação Puntual , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , Tirosina/genética , Animais , Precursores Enzimáticos/fisiologia , Liberação de Histamina , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Isoenzimas/metabolismo , Sistema de Sinalização das MAP Quinases/genética , Proteína Oncogênica v-cbl , Fragmentos de Peptídeos/fisiologia , Fenilalanina/genética , Fosfolipase C gama , Fosforilação , Proteínas Tirosina Quinases/fisiologia , Ratos , Receptores de IgE/fisiologia , Proteínas Oncogênicas de Retroviridae/metabolismo , Quinase Syk , Transfecção , Células Tumorais Cultivadas , Fosfolipases Tipo C/metabolismo , Tirosina/metabolismo , Tirosina/fisiologiaRESUMO
The ZAP70/Syk family of protein tyrosine kinases plays an important role in Ag receptor signaling. Structural similarity of Syk and ZAP70 suggests their functional overlap. Previously, it was observed that expression of either ZAP70 or Syk reconstitutes Ag receptor signaling in Syk-negative B cells. However, in CD45-deficient T cells, Syk, but not ZAP70, restores T cell receptor-signaling pathway. To study the function of Syk, ZAP70, and CD45 in mast cells, a Syk/CD45 double-deficient variant of RBL-2H3 cells was characterized. After transfection, stable cell lines were isolated that expressed ZAP70, Syk, CD45, ZAP70 plus CD45, and Syk plus CD45. IgE stimulation did not induce degranulation in parental double-deficient cells, nor in the cells expressing only CD45. ZAP70 expression did not restore Fc epsilon RI signaling unless CD45 was coexpressed in the cells. However, Syk alone restored the IgE signal transduction pathway. The coexpression of CD45 with Syk had no significant effects on the responses to FcepsilonRI-aggregation. There was much better binding of Syk than ZAP70 to the phosphorylated Fc epsilon RI gamma-ITAM. Furthermore, unlike Syk, ZAP70 required CD45 to display receptor-induced increase in kinase activity. Therefore, in mast cells, ZAP70, but not Syk, requires CD45 for Ag receptor-induced signaling.
Assuntos
Precursores Enzimáticos/fisiologia , Antígenos Comuns de Leucócito/fisiologia , Mastócitos/enzimologia , Mastócitos/imunologia , Proteínas Tirosina Quinases/fisiologia , Receptores de IgE/fisiologia , Transdução de Sinais/imunologia , Animais , Degranulação Celular/genética , Degranulação Celular/imunologia , Precursores Enzimáticos/deficiência , Precursores Enzimáticos/genética , Precursores Enzimáticos/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Leucemia Basofílica Aguda , Antígenos Comuns de Leucócito/biossíntese , Antígenos Comuns de Leucócito/genética , Ativação Linfocitária , Mastócitos/fisiologia , Fosforilação , Ligação Proteica/imunologia , Proteínas Tirosina Quinases/deficiência , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , Ratos , Receptores de IgE/metabolismo , Receptores Imunológicos/metabolismo , Transdução de Sinais/genética , Quinase Syk , Transfecção , Células Tumorais Cultivadas , Tirosina/metabolismo , Proteína-Tirosina Quinase ZAP-70RESUMO
The protein tyrosine kinase Syk plays a pivotal role in mediating the high-affinity IgE receptor (Fc epsilonRI)-induced degranulation of mast cells. To examine the mechanism of Syk regulation, the two tyrosine residues at 519 and 520 in the putative activation loop of rat Syk were mutated to phenylalanine either singly or in combination. The various mutants were expressed in a Syk-negative variant of the RBL-2H3 (rat basophilic leukemia 2H3) mast cell line. In these transfected cell lines, mutant Syk did show increased tyrosine phosphorylation in vivo and increased enzymatic activity in vitro after Fc epsilonRI aggregation. There were conformational changes detected by an Ab when the wild-type and mutant Syk were either tyrosine phosphorylated or bound to tyrosine-phosphorylated immunoreceptor tyrosine-based activation motif peptides. However, these mutant Syk were incapable of transducing Fc epsilonRI signaling. In cells in which the expression level of mutant Syk was similar to that of the wild-type Syk, Fc epsilonRI cross-linking induced no increase in cellular protein tyrosine phosphorylation, no increase in tyrosine phosphorylation of phospholipase C-gamma2 and mitogen-activated protein kinase, and no histamine release. Overexpression of Y519F or Y520F Syk mutants partially reconstituted the signaling pathways. These results indicate that these tyrosines in the putative activation loop are not essential for the enzymatic activity of Syk or for the conformational changes induced by binding of tyrosine-phosphorylated immunoreceptor tyrosine-based activation motif peptides. However, these tyrosines are necessary for Syk-mediated propagation of Fc epsilonRI signaling.
Assuntos
Precursores Enzimáticos/imunologia , Mastócitos/imunologia , Proteínas Tirosina Quinases/imunologia , Receptores de IgE/imunologia , Transdução de Sinais/imunologia , Animais , Sítios de Ligação/genética , Sítios de Ligação/imunologia , Degranulação Celular/imunologia , Precursores Enzimáticos/genética , Peptídeos e Proteínas de Sinalização Intracelular , Mutação , Fosforilação , Proteínas Tirosina Quinases/genética , Ratos , Transdução de Sinais/genética , Quinase SykRESUMO
Accumulating data indicate that the 'linker' region of Syk, which lies between its tandem Src homology 2 (SH2) domains and kinase region, provides a critical function for the biological activity of Syk. This importance has been ascribed to the presence of tyrosine phosphorylation sites capable of mediating the recruitment of cellular effectors. We and others previously identified an alternatively spliced variant of Syk, termed SykB, which lacks a 23 amino acid sequence in the linker domain. As this 'linker insert' is also not present in the closely related enzyme Zap-70, it seems plausible that Syk possesses this unique sequence for functional reasons. To understand its role better, we have compared the abilities of Syk and SykB to participate in immunoreceptor-triggered signal transduction. The results of our experiments revealed that, unlike Syk, SykB was inefficient at coupling stimulation of FcepsilonRI on basophils or the antigen receptor on T cells to the early and late events of cellular activation. Further studies showed that the functional defect in SykB was not caused by the absence of crucial tyrosine phosphorylation sites, or by a reduced intrinsic kinase activity. Rather, it correlated with the reduced ability of SykB to bind phosphorylated immunoreceptor tyrosine-based activation motifs (ITAMs) in vitro and in vivo. In combination, these results demonstrated that the unique insert in the linker domain of Syk is crucial for its capacity to participate in immunoreceptor signalling. Furthermore, they provided evidence that the linker region can regulate the ability of Syk to bind ITAMs, thus identifying a novel function for this domain.
Assuntos
Precursores Enzimáticos/química , Precursores Enzimáticos/metabolismo , Proteínas Tirosina Quinases/química , Proteínas Tirosina Quinases/metabolismo , Receptores de IgE/fisiologia , Transdução de Sinais , Linfócitos T/imunologia , Sequência de Aminoácidos , Animais , Sítios de Ligação , Linhagem Celular , Precursores Enzimáticos/biossíntese , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Dados de Sequência Molecular , Mutagênese Insercional , Fosforilação , Proteínas Tirosina Quinases/biossíntese , Ratos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Suínos , Quinase Syk , Transfecção , Células Tumorais Cultivadas , Domínios de Homologia de srcRESUMO
Tyrosine phosphorylation of proteins is critical for the Fc epsilon RI-induced signal transduction that leads to the release of inflammatory mediators from mast cells. Here we report the isolation of a monoclonal antibody, mAb BD2, to a 72 kDa protein that becomes rapidly tyrosine phosphorylated after Fc epsilon RI aggregation. By immunoprecipitation, immunoblotting and/or protease digestion this 72 kDa protein was different from the previously identified 68-76 kDa tyrosine phosphorylated proteins Btk, paxillin, SLP-76 or Syk. The phosphorylation of this 72 kDa protein was detectable within 15 sec after receptor aggregation and was independent of Ca2+ influx or the activation of protein kinase C. By in vitro kinase reaction, the 72 kDa protein did not autophosphorylate, which suggests that it is not a kinase, but is associated with a 140 kDa protein that was strongly phosphorylated. Studies in Syk deficient and Syk transfected variants of the RBL-2H3 cells demonstrated that the tyrosine phosphorylation of this 72 kDa protein was downstream of Syk. These data indicate that the 72 kDa protein precipitated by mAb BD2 is a novel phosphoprotein involved in Fc epsilon RI signaling.
Assuntos
Precursores Enzimáticos/metabolismo , Proteínas Tirosina Quinases/metabolismo , Agregação de Receptores , Receptores de IgE/metabolismo , Tirosina/metabolismo , Animais , Anticorpos Monoclonais/isolamento & purificação , Anticorpos Monoclonais/metabolismo , Especificidade de Anticorpos , Complexo Antígeno-Anticorpo/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Leucemia Basofílica Aguda , Camundongos , Camundongos Endogâmicos BALB C , Peso Molecular , Fosfoproteínas/imunologia , Fosfoproteínas/metabolismo , Fosforilação , Ratos , Receptores de IgE/fisiologia , Transdução de Sinais , Quinase Syk , Células Tumorais CultivadasRESUMO
Aggregation of the high affinity IgE receptor (Fc epsilonRI), a member of the immune receptor family, results in the activation of protein tyrosine kinases and downstream signaling pathways. The two cytoplasmic Src homology 2 domain-containing protein tyrosine phosphatases, SHP-1 (also called SH-PTP1, PTP1C or HCP) and SHP-2 (also known as SH-PTP2, PTP1D, PTP2C, or Syp), are expressed in the RBL-2H3 rat mast cell line. Here we report that aggregation of Fc epsilonRI induced the tyrosine phosphorylation of both SHP-1 and SHP-2. This phosphorylation was independent of the presence of the protein tyrosine kinase Syk. Both SHP-1 and SHP-2 associated with Fc epsilonRI. Whereas SHP-1 was constitutively associated with the receptor, SHP-2 coprecipitated with Fc epsilonRI only after receptor aggregation. Fusion proteins containing either the full-length or the Src homology 2 domains of SHP-2 directly bound to the tyrosine-phosphorylated beta, but not the gamma, subunit of Fc epsilonRI. In the reciprocal experiments, synthetic phosphorylated peptides based on the immunoreceptor tyrosine-based activation motif of the beta, but not the gamma, subunit precipitated SHP-2. In contrast, neither fusion proteins nor synthetic peptides detected interaction between SHP-1 and Fc epsilonRI. In vitro, both SHP-1 and SHP-2 dephosphorylated tyrosine-phosphorylated beta and gamma subunits of Fc epsilonRI. Therefore, SHP-1 and SHP-2 associate with Fc epsilonRI by different mechanisms and can regulate the extent of the tyrosine phosphorylation of the receptor subunits. Thus, unlike other immune cells in which inhibitory molecules are recruited by accessory proteins, Fc epsilonRI bind molecules that both activate and inhibit signal transduction.
Assuntos
Precursores Enzimáticos/imunologia , Proteínas Tirosina Fosfatases/imunologia , Proteínas Tirosina Quinases/imunologia , Receptores de IgE/imunologia , Transdução de Sinais/imunologia , Sequência de Aminoácidos , Animais , Linhagem Celular , Precursores Enzimáticos/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Dados de Sequência Molecular , Fosforilação , Proteína Tirosina Fosfatase não Receptora Tipo 11 , Proteína Tirosina Fosfatase não Receptora Tipo 6 , Proteínas Tirosina Fosfatases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Ratos , Agregação de Receptores , Receptores de IgE/metabolismo , Quinase Syk , Tirosina/metabolismoRESUMO
The SH2 domain-containing inositol-polyphosphate 5-phosphatase, SHIP, associates with FcgammaRIIB and negatively regulates both B-cell and mast cell function. We report here that SHIP was tyrosine-phosphorylated after high affinity IgE receptor (FcepsilonRI) aggregation in rat basophilic leukemia RBL-2H3 cells. The tyrosine phosphorylation of SHIP was an early event after receptor aggregation and was present in cells deficient in the protein-tyrosine kinase Syk. Furthermore it was not secondary to the increase of intracellular calcium or the activation of protein kinase C. SHIP was precipitated by immobilized phosphorylated synthetic peptides based on the immunoreceptor tyrosine-based activation motif (ITAM) of the beta but not the gamma subunit of the high affinity IgE receptor. Tyrosine phosphorylation of SHIP and its association with the tyrosine-phosphorylated beta subunit of FcepsilonRI could play an important role in down-regulating receptor-mediated signal transduction in mast cells. Thus, whereas the activation molecule Syk associates with the gamma subunit ITAM, the beta subunit ITAM binds the negative signaling molecule SHIP. Therefore, unlike B cells where the antigen receptor and coreceptors such as FcgammaRIIB or CD22 each recruits molecules with opposite effects, the FcepsilonRI contains subunits which recruit molecules that activate and inhibit signal transduction.
Assuntos
Monoéster Fosfórico Hidrolases/metabolismo , Receptores de IgE/metabolismo , Tirosina/metabolismo , Domínios de Homologia de src , Sequência de Aminoácidos , Animais , Calcimicina/farmacologia , Cálcio/metabolismo , Carcinógenos/farmacologia , Regulação para Baixo , Precursores Enzimáticos/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Ionóforos/farmacologia , Dados de Sequência Molecular , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases , Fosforilação , Conformação Proteica , Proteínas Tirosina Quinases/metabolismo , Ratos , Quinase Syk , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
One of the earliest events after aggregation of the high affinity receptor for IgE (FcepsilonRI) on mast cells is the activation of protein tyrosine kinases resulting in tyrosine phosphorylation of numerous proteins. Using a monoclonal antibody raised against the rat basophilic leukemia RBL-2H3 cells, we identified that platelet/endothelial cell adhesion molecule 1 (PECAM-1 or CD31) was tyrosine phosphorylated in these cells. Aggregation of PECAM-1 did not induce a detectable increase in its tyrosine phosphorylation, nor did it result in degranulation. However, the minimal tyrosine phosphorylation of PECAM-1 in nonstimulated cells was dramatically increased after FcepsilonRI aggregation. This receptor-induced tyrosine phosphorylation of PECAM-1 was an early event, independent of Ca2+ influx or of the activation of protein kinase C and of cell adhesion. PECAM-1 is an adhesion molecule that is required for the transmigration of leukocytes across the endothelium into sites of inflammation. Therefore tyrosine phosphorylation of PECAM-1 may modulate its interaction with other molecules, thereby regulating the migration of basophils into inflammatory sites.
Assuntos
Mastócitos/metabolismo , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Receptores de IgE/metabolismo , Animais , Adesão Celular , Linhagem Celular , Mastócitos/citologia , Fosforilação , Agregação de Receptores , Tirosina/metabolismoRESUMO
In rat basophilic leukemia 2H3 (RBL-2H3) cells, mAb AA4 binds to two derivatives of ganglioside GD1b that associate with the Src family kinase p53/56lyn and a serine kinase. Pre-incubation of cells with mAb AA4 blocks immunoglobulin E (IgE) mediated histamine release. In the present study we investigated the effect of incubation with mAb AA4 on signal transduction events. In addition to stimulation of the high affinity IgE receptor (Fc epsilonRI), cells were also activated by the calcium ionophore A23187 and the acetylcholine agonist carbachol in RBL-2H3 cells transfected with the G protein-coupled m3 muscarinic receptor. Incubation of cells with mAb AA4 in a dose-dependent manner inhibited the following Fc epsilonRI-induced signal transduction events: the increase of intracellular free calcium, phosphoinositol breakdown, tyrosine phosphorylation of proteins including the beta- of Fc epsilonRI and secretion. However, there was no inhibition of degranulation or of these biochemical events when cells were stimulated with calcium ionophore or activated via a G protein-coupled pathway. Our results demonstrate that mAb AA4 selectively blocks Fc epsilonRI-induced cell activation at a very early step upstream of receptor tyrosine phosphorylation. As mAb AA4 has previously been found to bind to gangliosides associated with Fc epsilonRI, inhibition of very early biochemical events may be due to interaction of mAb AA4 with the Fc epsilonRI induced signal transduction cascade at the receptor level.
Assuntos
Gangliosídeos/fisiologia , Imunoglobulina E/imunologia , Mastócitos/fisiologia , Receptores de IgE/fisiologia , Animais , Anticorpos Monoclonais , Cálcio/metabolismo , Citoplasma/metabolismo , Fosfatidilinositóis/metabolismo , Fosfoproteínas/metabolismo , Fosforilação , Fosfotirosina/metabolismo , Ratos , Serotonina/metabolismo , Transdução de Sinais , Células Tumorais CultivadasRESUMO
The rat basophilic leukemia RBL-2H3 mast cell line is widely used for studies of the structure and function of the high affinity IgE receptor (Fc epsilonRI). Here we report on a simple method to isolate large numbers of intact RBL-2H3 cells from tumors produced by injection of the cells into newborn rats. Collagenase treatment of rat tumors yields approximately 3.5 x 10(8) viable cells/animal. Aggregating Fc epsilonRI on these cells induced tyrosine phosphorylation of proteins including the protein tyrosine kinase Syk. This procedure should prove useful for the isolation and characterization of cellular molecules important for mast cell and basophil function.
Assuntos
Leucemia Basofílica Aguda/patologia , Animais , Adesão Celular , Linhagem Celular , Separação Celular/métodos , Precursores Enzimáticos/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Mastócitos , Fosfotirosina/metabolismo , Proteínas Tirosina Quinases/metabolismo , Ratos , Receptores de IgE/fisiologia , Transdução de Sinais , Quinase SykRESUMO
Stimulation of the IgE high affinity receptor on rat basophilic leukemia RBL-2H3 cells results in activation of protein tyrosine kinases and rapid tyrosine phosphorylation of several substrates, many of which remain unidentified. In this report, we demonstrate that the Grb2 adapter protein, when expressed as a glutathione S-transferase fusion protein, associates with four tyrosine-phosphorylated molecules (116, 76, 36, and 31 kDa) from lysates of stimulated RBL-2H3 cells. We show further that the 76-kDa protein is SLP-76, a hematopoietic cell-specific protein first identified as a Grb2-binding protein in T cells. Upon stimulation of the high affinity receptor for IgE, SLP-76 undergoes rapid tyrosine phosphorylation and associates with two additional tyrosine phosphoproteins of 62 and 130 kDa via the SH2 domain of SLP-76. Additional studies demonstrate that the SLP-76 SH2 domain also binds a protein kinase from stimulated RBL-2H3 cell lysates. Furthermore, the phosphorylation of SLP-76 requires Syk activity but is not dependent on Ca+2 mobilization. These data, together with our previous work documenting its role in T-cell activation, suggest that SLP-76 and the proteins with which it associates may play a fundamental role in coupling signaling events in multiple cell types in the immune system.