RESUMO
B cell antigen receptor (BCR) signaling is positively and negatively regulated by various cell surface receptors such as CD19 and CD45. Functional analysis of these receptors has been performed using gene targeting technology, which is a valid approach to elucidate their functions. However, this type of analysis is restricted when multiple molecules are evaluated simultaneously. From a different perspective, synthetic biology provides a high degree of freedom for analyzing various molecules. Here we developed a system to reconstruct BCR signaling using the J558L myeloma cell line in combination with the protein-based Ca(2+) indicator YC3.60. BCR-reconstituted J558L cells harboring YC3.60 (J558Lµv11 cells) permitted monitoring of Ca(2+) mobilization. Reconstituting CD19 in J558Lµv11 cells resulted in detectable BCR-induced Ca(2+) mobilization but with kinetics different from that of CD45-expressing cells. Furthermore, we evaluated the validity of the J558L system by proteomic analysis of tyrosine-phosphorylated proteins after antigen stimulation. Identification of more than 100 BCR-induced tyrosine-phosphorylated proteins in J558Lµv11 cells revealed a similarity to that observed in B cells, and a novel member, non-receptor protein tyrosine kinase Fer, was found. Thus, this reconstruction system using J558L cells appeared to be valid for comprehensively investigating BCR signaling.
Assuntos
Mieloma Múltiplo/patologia , Receptores de Antígenos de Linfócitos B/metabolismo , Transdução de Sinais , Sequência de Aminoácidos , Animais , Antígenos CD19/metabolismo , Sinalização do Cálcio , Linhagem Celular Tumoral , Regulação para Baixo , Cinética , Antígenos Comuns de Leucócito/metabolismo , Camundongos , Dados de Sequência Molecular , Fosfatidilinositol 3-Quinases/metabolismo , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Fosforilação , Tirosina/metabolismo , Quinases da Família src/metabolismoRESUMO
Src homology region 2 domain-containing phosphatase 1 (SHP-1), a cytoplasmic protein tyrosine phosphatase, plays an important role for the regulation of signaling from various hematopoietic cell receptors. Although SHP-1 is shown to be a negative signal modulator in mast cells, its precise molecular mechanisms are not well defined. To elucidate how SHP-1 regulates mast cell signaling, we established bone marrow-derived mast cells from SHP-1-deficient motheaten and wild-type mice and analyzed downstream signals induced by cross-linking of high affinity IgE receptor, Fc epsilonRI. Upon Fc epsilonRI ligation, motheaten-derived bone marrow-derived mast cells showed enhanced tyrosine phosphorylation of Src homology region 2 domain-containing leukocyte protein of 76 kDa (SLP-76) and linker for activation of T cells, activation of mitogen-activated protein kinases and gene transcription and production of cytokine. Because the activity of Syk, responsible for the phosphorylation of SLP-76 and linker for activation of T cells, is comparable irrespective of SHP-1, both molecules might be substrates of SHP-1 in mast cells. Interestingly, the absence of SHP-1 expression disrupted the association between SLP-76 and phospholipase Cgamma, which resulted in the decreased phospholipase Cgamma phosphorylation, calcium mobilization, and degranulation. Collectively, these results suggest that SHP-1 regulates Fc epsilonRI-induced downstream signaling events both negatively and positively by functioning as a protein tyrosine phosphatase and as an adaptor protein contributing to the formation of signaling complex, respectively.
Assuntos
Degranulação Celular/imunologia , Regulação Enzimológica da Expressão Gênica/imunologia , Mastócitos/imunologia , Proteína Tirosina Fosfatase não Receptora Tipo 6/imunologia , Receptores de IgE/imunologia , Transdução de Sinais/imunologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/imunologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Cálcio/imunologia , Cálcio/metabolismo , Degranulação Celular/genética , MAP Quinases Reguladas por Sinal Extracelular , Regulação Enzimológica da Expressão Gênica/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/imunologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Ativação Linfocitária/genética , Ativação Linfocitária/imunologia , Mastócitos/enzimologia , Camundongos , Camundongos Mutantes , Fosfolipase C gama/genética , Fosfolipase C gama/imunologia , Fosfoproteínas/genética , Fosfoproteínas/imunologia , Fosfoproteínas/metabolismo , Fosforilação , Proteína Tirosina Fosfatase não Receptora Tipo 6/genética , Proteína Tirosina Fosfatase não Receptora Tipo 6/metabolismo , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/imunologia , Proteínas Tirosina Quinases/metabolismo , Receptores de IgE/genética , Receptores de IgE/metabolismo , Transdução de Sinais/genética , Quinase Syk , Linfócitos T/enzimologia , Linfócitos T/imunologiaRESUMO
From the time of Thucydides in the 5th century BC, it has been known that specific recognition of pathogens and memory formation are critical components of immune functions. In contrast to the immune system of jawed vertebrates, such as humans and mice, plants lack a circulatory system with mobile immune cells and a repertoire of clonally distributed antigen receptors with almost unlimited specificities. However, without these systems and mechanisms, plants can live and survive in the same hostile environment faced by other organisms. In fact, they achieve specific pathogen recognition and elimination, with limited self-reactivity, and generate immunological memory, sometimes with transgenerational characteristics. Thus, the plant immune system satisfies minimal conditions for constituting an immune system, namely, the recognition of signals in the milieu, integration of that information, subsequent efficient reaction based on the integrated information, and memorization of the experience. In the previous report, this set of elements was proposed as an example of minimal cognitive functions. In this essay, I will first review current understanding of plant immunity and then discuss the unique features of cognitive activities, including recognition of signals from external as well as internal environments, autoimmunity, and memory formation. In doing so, I hope to reach a deeper understanding of the significance of immunity omnipresent in the realm of living organisms.
RESUMO
SHP-1 plays an important role for the regulation of signaling from various hematopoietic cell receptors. In this study, we examined IL-3-induced cell proliferation and IL-3 depletion-induced apoptosis in bone marrow-derived mast cells (BMMC) established from motheaten (me) that lack SHP-1 expression, viable motheaten (me(v)) expressing phosphatase-deficient SHP-1, and wild-type (WT) mice. When BMMC were stimulated with IL-3, increased ERK activation was evident in resting state and sustained in me-BMMC relative to WT-BMMC. ERK is known to be involved in the regulation of cell proliferation and apoptosis in some cells. In accordance with sustained ERK activation, apoptosis was decreased in me- and me(v)-BMMC compared with WT-BMMC. In contrast to the predicted role of ERK as a pro-survival molecule, IL-3-induced cell proliferation was much lower in me- and me(v)-BMMC than WT-BMMC. Stimulation with lower concentration of IL-3 or addition of PD98059, a MEK inhibitor, to the culture resulted in the suppression of decreased apoptosis and cell proliferation in me- and me(v)-BMMC. Collectively, these results suggest that SHP-1 positively regulates IL-3-dependent mast cell proliferation and apoptosis by inhibiting ERK activity through its phosphatase activity. Furthermore, our results indicate that ERK would act as a negative regulator for cell proliferation and induce apoptosis when its activity is highly increased.
Assuntos
MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Interleucina-3/farmacologia , Mastócitos/citologia , Mastócitos/enzimologia , Proteína Tirosina Fosfatase não Receptora Tipo 6/deficiência , Animais , Biocatálise/efeitos dos fármacos , Células da Medula Óssea/citologia , Morte Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , Flavonoides/farmacologia , Mastócitos/efeitos dos fármacos , Camundongos , Proteína Tirosina Fosfatase não Receptora Tipo 6/metabolismoAssuntos
Sistema Imunitário/fisiologia , Antígenos Comuns de Leucócito/fisiologia , Proteínas Tirosina Fosfatases , Animais , Fenômenos Fisiológicos Celulares , Dimerização , Regulação Enzimológica da Expressão Gênica/genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Antígenos Comuns de Leucócito/química , Síndrome de Noonan/genética , Fosforilação , Proteína Tirosina Fosfatase não Receptora Tipo 11 , Proteína Tirosina Fosfatase não Receptora Tipo 12 , Proteínas Tirosina Fosfatases/química , Proteínas Tirosina Fosfatases/genética , Proteínas Tirosina Fosfatases/fisiologia , Proteínas Tirosina Fosfatases Contendo o Domínio SH2 , Domínios de Homologia de src/fisiologiaRESUMO
Src homology region 2-domain-containing phosphatase-1 (SHP-1) plays an important role in the regulation of signaling from various receptors in hematopoietic cells. In mast cells, SHP-1 has been shown to negatively regulate the initial signaling triggered by high-affinity receptor for IgE (FcepsilonRI) and positively regulate downstream outputs. To clarify the molecular mechanisms of SHP-1 in mast cells, we determined substrates for SHP-1 by using the substrate-trapping approach. When phosphatase-inactive SHP-1 was over-expressed in rat basophilic leukemia (RBL)-2H3 cells, tyrosine phosphorylation of a 68-kDa protein was enhanced before and after FcepsilonRI aggregation. Immunoprecipitation and western blot analyses revealed that this protein is SHP-1, either endogenous or ectopically expressed. FcepsilonRI-induced activation of Lyn and Syk was comparable between cells expressing wild-type (wt) and phosphatase-inactive SHP-1. In vitro phosphatase assay and combined transfection, immunoprecipitation and immunoblot analyses showed that tyrosine 536 of SHP-1 was potent phosphorylation site and that SHP-1 could dephosphorylate this site that had been phosphorylated by Lyn. Furthermore, the phosphatase activity of SHP-1 immunoprecipitated from cells expressing a phosphatase-inactive SHP-1 was increased compared with that from vector-transfected or wt SHP-1-expressing cells. Finally, expression of phosphatase-inactive SHP-1 resulted in decreased activation of mitogen-activated protein kinases and suppressed transcription of cytokine genes, whereas wt SHP-1 enhanced these processes. Taken collectively, these results suggest that SHP-1 may be a physiological substrate of SHP-1 in RBL-2H3 cells and that dephosphorylation of SHP-1 leads to a decrease in its catalytic activity and an enhancement of downstream signaling. A negative autoregulatory circuit of SHP-1 may contribute to mast cell regulation.
Assuntos
Leucemia Basofílica Aguda/imunologia , Proteína Tirosina Fosfatase não Receptora Tipo 6/imunologia , Animais , Linhagem Celular Tumoral , Citocinas/genética , Citocinas/imunologia , Regulação para Baixo/imunologia , Perfilação da Expressão Gênica , Peptídeos e Proteínas de Sinalização Intracelular/imunologia , Mastócitos/imunologia , Proteínas Quinases Ativadas por Mitógeno/imunologia , Fosforilação , Proteína Tirosina Fosfatase não Receptora Tipo 6/genética , Proteínas Tirosina Quinases/imunologia , Ratos , Receptores de IgE/imunologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Transdução de Sinais/imunologia , Quinase Syk , Transcrição Gênica , Quinases da Família src/imunologiaRESUMO
Despite the important role in the development and activation of T cells, NK cells, mast cells, and macrophages, the expression and function of SLP-76 in B cells have been largely unknown. Here we demonstrate that SLP-76 is expressed in all mouse B cell lines tested and in normal splenic B cells, and serves as an SHP-1 substrate. Dephosphorylation of SLP-76 by SHP-1 inhibits its association with Nck, down-regulating c-Jun N-terminal kinase (JNK) activation and exerting a positive effect on apoptosis. Knockdown of SLP-76 in WEHI-231 cells by small interfering RNA attenuated JNK activation, but showed little effects on extracellular signal-regulated kinase (ERK) or p38 activation. Although WEHI-231 does not express linker for activation of T cells (LAT), SLP-76 localizes in membrane fraction, which increases following B cell receptor (BCR) cross-linking. Further analyses revealed that SLP-76 complexed with Gads is associated with tyrosine-phosphorylated CD22 through the SH2 domains of SLP-76 and Gads. Given that SHP-1 binds to CD22 upon BCR ligation, our findings suggest that dephosphorylation of SLP-76 recruited to CD22 by SHP-1 inhibits BCR-induced JNK activation, dictating apoptosis.
Assuntos
Antígenos CD/metabolismo , Antígenos de Diferenciação de Linfócitos B/metabolismo , Linfócitos B/metabolismo , Moléculas de Adesão Celular/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Lectinas/metabolismo , Ativação Linfocitária/fisiologia , Fosfoproteínas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Proteínas Oncogênicas/metabolismo , Fosforilação , Proteína Tirosina Fosfatase não Receptora Tipo 11 , Proteínas Tirosina Fosfatases , Receptores de Antígenos de Linfócitos B/metabolismo , Lectina 2 Semelhante a Ig de Ligação ao Ácido Siálico , Transdução de Sinais/fisiologia , Tirosina/metabolismoRESUMO
CD72 is a 45 kDa B cell-specific type II transmembrane protein of the C-type lectin superfamily. It was originally defined as a receptor-like molecule that regulates B cell activation and differentiation; however, its precise function remains unclear since more recent functional analyses, including a gene targeting study, suggest that CD72 may serve as a negative or a positive regulator of B cell signaling. In the present study, we analyzed the cell-autonomous function of CD72 in B cell receptor (BCR) signaling using CD72-deficient cells generated from mature BAL-17 cells. We found that BCR-mediated phosphorylation of CD19, Btk, Vav and phospholipase Cgamma2 and association of CD19 with phosphatidylinositol-3 kinase were impaired in CD72-deficient cells. Inositol trisphosphate synthesis was normally induced initially but ablated at 1 min of stimulation in CD72-deficient cells. In the event, Ca(2+) release from intracellular stores remained intact, though influx of extracellular Ca(2+) was severely impaired in CD72-deficient cells. Furthermore, BCR-evoked activation of mitogen-activated protein kinases (MAPKs), extracellular signal-regulated kinase and c-Jun NH(2)-terminal kinase, and growth inhibition in BAL-17 cells were blocked in the absence of CD72. Significantly, these effects were largely reversed by re-expression of CD72. Thus, CD72 appears to exert a positive effect on BCR signaling pathways leading to Ca(2+) influx and MAPK activation, which in turn may determine the fate of BAL-17 cells.
Assuntos
Sinalização do Cálcio/imunologia , Processos de Crescimento Celular/imunologia , Lectinas Tipo C/imunologia , Proteínas Quinases Ativadas por Mitógeno/imunologia , Receptores de Antígenos de Linfócitos B/deficiência , Receptores de Antígenos de Linfócitos B/imunologia , Animais , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Camundongos , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-vavRESUMO
CD45 is a key protein tyrosine phosphatase regulating Src-family protein tyrosine kinases (Src-PTKs) in lymphocytes; precisely how it exerts its effect remains controversial, however. We previously demonstrated that CD45 negatively regulates Lyn in the WEHI-231 B-cell line. Here we show that negative regulation by CD45 is physiologically significant in B cells and that some CD45 is constitutively associated with glycolipid-enriched microdomains (GEMs), where it inhibits Src-PTKs by dephosphorylating both the negative and the positive regulatory sites. Upon B-cell receptor (BCR) ligation, however, CD45 dissociates from GEMs within 30 seconds, inducing phosphorylation of 2 regulatory sites and activation of Src-PTKs, but subsequently reassociates with the GEMs within 15 minutes. Disruption of GEMs with methyl-beta-cyclodextrin results in abrogation of BCR-induced apoptosis in WEHI-231 cells, suggesting GEMs are critical to signals leading to the fate determination. We propose that the primary function of CD45 is inhibition of Src-PTKs and that the level of Src-PTK activation and the B-cell fate are determined in part by dynamic behavior of CD45 with respect to GEMs.
Assuntos
Linfócitos B/enzimologia , Antígenos Comuns de Leucócito/genética , Antígenos Comuns de Leucócito/metabolismo , Quinases da Família src/metabolismo , Animais , Apoptose/imunologia , Linfócitos B/citologia , Linhagem Celular , Microdomínios da Membrana , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosfoproteínas/metabolismo , Receptores de Antígenos de Linfócitos B/metabolismo , Baço/citologia , Baço/imunologiaRESUMO
Src homology region 2 domain-containing phosphatase 1 (SHP-1) is a key mediator in lymphocyte differentiation, proliferation, and activation. We previously showed that B cell linker protein (BLNK) is a physiological substrate of SHP-1 and that B cell receptor (BCR)-induced activation of c-Jun NH(2)-terminal kinase (JNK) is significantly enhanced in cells expressing a form of SHP-1 lacking phosphatase activity (SHP-1-C/S). In this study, we confirmed that SHP-1 also exerts negative regulatory effects on JNK activation in splenic B cells. To further clarify the role of SHP-1 in B cells, we examined how dephosphorylation of BLNK by SHP-1 affects downstream signaling events. When a BLNK mutant (BLNK Delta N) lacking the NH(2)-terminal region, which contains four tyrosine residues, was introduced in SHP-1-C/S-expressing WEHI-231 cells, the enhanced JNK activation was inhibited. Among candidate proteins likely to regulate JNK activation through BLNK, Nck adaptor protein was found to associate with tyrosine-phosphorylated BLNK and this association was more pronounced in SHP-1-C/S-expressing cells. Furthermore, expression of dominant-negative forms of Nck inhibited BCR-induced JNK activation. Finally, BCR-induced apoptosis was suppressed in SHP-1-C/S-expressing cells and coexpression of Nck SH2 mutants or a dominant-negative form of SEK1 reversed this phenotype. Collectively, these results suggest that SHP-1 acts on BLNK, modulating its association with Nck, which in turn negatively regulates JNK activation but exerts a positive effect on apoptosis.