RESUMO
In this study experiments were conducted to elucidate the physical/functional relationship between CD45 and casein kinase 2 (CK2). Immunoprecipitation experiments demonstrated that CK2 associates with CD45 and that this interaction is inducible upon Ag receptor cross-linking in B and T cell lines as well as murine thymocytes and splenic B cells. However, yeast two-hybrid analysis failed to demonstrate a physical interaction between the individual CK2 alpha, alpha', or beta subunits and CD45. In contrast, a yeast three-hybrid assay in which either CK2 alpha and beta or alpha' and beta subunits were coexpressed with the cytoplasmic domain of CD45, demonstrated that both CK2 subunits are necessary for the interaction with CD45. Experiments using the yeast three-hybrid assay also revealed that a 19-aa acidic insert in domain II of CD45 mediates the physical interaction between CK2 and CD45. Structure/function experiments in which wild-type or mutant CD45RA and CD45RO isoforms were expressed in CD45-deficient Jurkat cells revealed that the 19-aa insert is important for optimal CD45 function. The ability of both CD45RA and CD45RO to reconstitute CD3-mediated signaling based on measurement of calcium mobilization and mitogen-activated protein kinase activation was significantly decreased by deletion of the 19-aa insert. Mutation of four serine residues within the 19-aa insert to alanine affected CD45 function to a similar extent compared with that of the deletion mutants. These findings support the hypothesis that a physical interaction between the CD45 cytoplasmic domain and CK2 is important for post-translational modification of CD45, which, in turn, regulates its catalytic function.
Assuntos
Antígenos Comuns de Leucócito/metabolismo , Fragmentos de Peptídeos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Linfócitos B/enzimologia , Linfócitos B/imunologia , Sítios de Ligação/genética , Sítios de Ligação/imunologia , Caseína Quinase II , Holoenzimas/metabolismo , Holoenzimas/fisiologia , Humanos , Concentração de Íons de Hidrogênio , Células Jurkat , Antígenos Comuns de Leucócito/genética , Antígenos Comuns de Leucócito/fisiologia , Camundongos , Mutagênese Insercional , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/fisiologia , Fosforilação , Estrutura Terciária de Proteína/genética , Proteínas Tirosina Fosfatases/genética , Proteínas Tirosina Fosfatases/fisiologia , Proteínas Recombinantes de Fusão/síntese química , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/fisiologia , Deleção de Sequência , Serina/genética , Baço/citologia , Baço/enzimologia , Baço/imunologia , Relação Estrutura-Atividade , Linfócitos T/enzimologia , Linfócitos T/imunologia , Timo/citologia , Timo/enzimologia , Timo/imunologia , Células Tumorais CultivadasRESUMO
The transmembrane protein tyrosine phosphatase CD45 is expressed throughout B cell development and differentiation, with the exception of terminally differentiated plasma cells on which its expression is down regulated. Numerous studies using CD45-deficient B cell lines and CD45-deficient mice have clearly demonstrated that CD45 plays an important role in modulating the signal that is transduced via the B cell antigen receptor by regulating the phosphorylation state of Src family kinases. Spatial and temporal controls enable CD45 to promote B cell antigen receptor signal transduction by constitutively maintaining Src family kinases in a partially active state, such that the B cell is able to effectively respond to an antigenic challenge. Moreover, CD45 is required for optimal activation of Ca2+-dependent and MAP kinase-dependent signal transduction pathways in the B cell. The net result is that CD45 affects the B cell response by controlling the relative threshold of sensitivity to a given antigenic stimulus. Thus, CD45 expression and function is required for normal B cell development, tolerance induction, and responsiveness to antigen.
Assuntos
Linfócitos B/enzimologia , Linfócitos B/imunologia , Antígenos Comuns de Leucócito/metabolismo , Proteínas Tirosina Fosfatases/metabolismo , Animais , Linfócitos B/citologia , Diferenciação Celular , Precursores Enzimáticos/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Ativação Linfocitária , Camundongos , Camundongos Knockout , Modelos Imunológicos , Proteínas Tirosina Quinases/metabolismo , Receptores de Antígenos de Linfócitos B/metabolismo , Transdução de Sinais , Quinase Syk , Quinases da Família src/metabolismoRESUMO
The major histocompatability class II heterodimer (class II) is expressed on the surface of both resting and activated B cells. Although it is clear that class II expression is required for Ag presentation to CD4(+) T cells, substantial evidence suggests that class II serves as a signal transducing receptor that regulates B cell function. In ex vivo B cells primed by Ag receptor (BCR) cross-linking and incubation with IL-4, or B cell lines such as K46-17 micromlambda, class II ligation leads to the activation of protein tyrosine kinases, including Lyn and Syk and subsequent phospholipase Cgamma-dependent mobilization of Ca(2+). In this study, experiments demonstrated reciprocal desensitization of class II and BCR signaling upon cross-linking of either receptor, suggesting that the two receptors transduce signals via common processes and/or effector proteins. Because class II and BCR signal transduction pathways exhibit functional similarities, additional studies were conducted to evaluate whether class II signaling is regulated by BCR coreceptors. Upon cross-linking of class II, the BCR coreceptors CD19 and CD22 were inducibly phosphorylated on tyrosine residues. Phosphorylation of CD22 was associated with increased recruitment and binding of the protein tyrosine phosphatase SHP-1. Similarly, tyrosine phosphorylation of CD19 resulted in recruitment and binding of Vav and phosphatidylinositol 3-kinase. Finally, co-cross-linking studies demonstrated that signaling via class II was either attenuated (CD22/SHP-1) or enhanced (CD19/Vav and phosphatidylinositol 3-kinase), depending on the coreceptor that was brought into close proximity. Collectively, these results suggest that CD19 and CD22 modulate class II signaling in a manner similar to that for the BCR.
Assuntos
Antígenos CD19/fisiologia , Antígenos CD/fisiologia , Antígenos de Diferenciação de Linfócitos B/fisiologia , Linfócitos B/metabolismo , Moléculas de Adesão Celular , Antígenos de Histocompatibilidade Classe II/fisiologia , Lectinas , Receptores de Antígenos de Linfócitos B/fisiologia , Transdução de Sinais/imunologia , Animais , Antígenos CD/metabolismo , Antígenos CD19/metabolismo , Antígenos de Diferenciação de Linfócitos B/metabolismo , Linfócitos B/imunologia , Células Cultivadas , Citoplasma/metabolismo , Antígenos de Histocompatibilidade Classe II/imunologia , Antígenos de Histocompatibilidade Classe II/metabolismo , Ligantes , Camundongos , Camundongos Endogâmicos C57BL , Fosforilação , Fosfotirosina/metabolismo , Receptores de Antígenos de Linfócitos B/imunologia , Receptores de Antígenos de Linfócitos B/metabolismo , Lectina 2 Semelhante a Ig de Ligação ao Ácido Siálico , Células Tumorais CultivadasAssuntos
Moléculas de Adesão Celular , Lectinas , Proteínas Tirosina Fosfatases/fisiologia , Proteínas Tirosina Quinases/fisiologia , Receptores de Antígenos de Linfócitos B/fisiologia , Transdução de Sinais/fisiologia , Proteínas Adaptadoras de Transdução de Sinal , Tirosina Quinase da Agamaglobulinemia , Animais , Antígenos/metabolismo , Antígenos CD/metabolismo , Antígenos CD/fisiologia , Antígenos de Diferenciação de Linfócitos B/metabolismo , Linfócitos B/metabolismo , Antígenos CD79 , Cálcio/metabolismo , Proteínas de Transporte/metabolismo , Ativação Enzimática , Precursores Enzimáticos/metabolismo , Regulação da Expressão Gênica , Humanos , Imunoglobulina M/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas Oncogênicas/metabolismo , Fosfoproteínas/metabolismo , Fosforilação , Proteína Tirosina Fosfatase não Receptora Tipo 11 , Proteína Tirosina Fosfatase não Receptora Tipo 6 , Proteínas Tirosina Fosfatases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Proteínas/metabolismo , Proteínas Proto-Oncogênicas c-vav , Receptores de Antígenos de Linfócitos B/imunologia , Receptores de Antígenos de Linfócitos B/metabolismo , Receptores de IgG/metabolismo , Lectina 2 Semelhante a Ig de Ligação ao Ácido Siálico , Transdução de Sinais/imunologia , Quinase Syk , Fosfolipases Tipo C/metabolismo , Quinases da Família src/metabolismoRESUMO
The B cell-restricted transmembrane glycoprotein CD22 is rapidly phosphorylated on tyrosine in response to cross-linking of the B cell antigen receptor, thereby generating phosphotyrosine motifs in the cytoplasmic domain which recruit intracellular effector proteins that contain Src homology 2 domains. By virtue of its interaction with these effector proteins CD22 modulates signal transduction through the B cell antigen receptor. To define further the molecular mechanism by which CD22 mediates its co-receptor function, phosphopeptide mapping experiments were conducted to determine which of the six tyrosine residues in the cytoplasmic domain are involved in recruitment of the stimulatory effector proteins phospholipase Cgamma (PLCgamma), phosphoinositide 3-kinase (PI3K), Grb2, and Syk. The results obtained indicate that the protein tyrosine kinase Syk interacts with multiple CD22-derived phosphopeptides in both immunoprecipitation and reverse Far Western assays. In contrast, the Grb2.Sos complex was observed to bind exclusively to the fourth phosphotyrosine motif (Y828ENV) from CD22 and does so via a direct interaction based on Far Western and reverse Far Western blotting. Although both PLCgamma and PI3K were observed to bind to multiple phosphopeptides in precipitation experiments, subsequent studies using reverse Far Western blot analysis demonstrated that only the carboxyl-terminal phosphopeptide of CD22 (Y863VTL) binds directly to either one. This finding suggests that PLCgamma and PI3K may be recruited to CD22 either through a direct interaction with Tyr863 or indirectly through an association with one or more intermediate proteins.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Antígenos CD/metabolismo , Antígenos de Diferenciação de Linfócitos B/metabolismo , Linfócitos B/metabolismo , Moléculas de Adesão Celular , Precursores Enzimáticos/metabolismo , Isoenzimas/metabolismo , Lectinas , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Fosfolipases Tipo C/metabolismo , Tirosina/metabolismo , Animais , Sítios de Ligação , Bovinos , Citoplasma/metabolismo , Proteína Adaptadora GRB2 , Fatores de Troca do Nucleotídeo Guanina , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Fosfolipase C gama , Fosforilação , Ligação Proteica , Proteínas/metabolismo , Coelhos , Ratos , Receptores de Antígenos de Linfócitos B/metabolismo , Lectina 2 Semelhante a Ig de Ligação ao Ácido Siálico , Transdução de Sinais , Quinase SykRESUMO
Cross-linking of CD45 induced capping and physical sequestration from CD22 leading to an increase in tyrosine phosphorylation of CD22 and SHP-1 recruitment. Additionally, CD22 isolated from a CD45-deficient B cell line exhibited increased basal/inducible tyrosine phosphorylation and enhanced recruitment of SHP-1 compared with CD22 isolated from CD45-positive parental cells. Subsequent experiments were performed to determine whether enhanced SHP-1 recruitment to CD22 is responsible for attenuation of receptor-mediated Ca2+ responses in CD45-deficient cells. Catalytically inactive SHP-1 expressed in CD45-deficient cells interacted with CD22 and decreased phosphatase activity in CD22 immunoprecipitates to levels that were comparable to those in CD45-positive cells. Expression of catalytically inactive SHP-1 restored intracellular mobilization of Ca2+ in response to MHC class II cross-linking, but did not affect B cell Ag receptor- or class II-mediated Ca2+ influx from the extracellular space. These results indicate that CD45 regulates tyrosine phosphorylation of CD22 and binding of SHP-1. The data further indicate that enhanced recruitment and activation of SHP-1 in CD45-deficient cells affect intracellular mobilization of Ca2+, but are not responsible for abrogation of receptor-mediated Ca2+ influx from the extracellular space.
Assuntos
Antígenos CD/metabolismo , Antígenos de Diferenciação de Linfócitos B/metabolismo , Moléculas de Adesão Celular , Lectinas , Antígenos Comuns de Leucócito/fisiologia , Proteínas Tirosina Fosfatases/metabolismo , Tirosina/metabolismo , Animais , Sinalização do Cálcio/imunologia , Catálise , Líquido Intracelular/enzimologia , Líquido Intracelular/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Antígenos Comuns de Leucócito/genética , Antígenos Comuns de Leucócito/metabolismo , Linfoma de Células B/genética , Linfoma de Células B/imunologia , Camundongos , Fosforilação , Proteína Tirosina Fosfatase não Receptora Tipo 11 , Proteína Tirosina Fosfatase não Receptora Tipo 6 , Receptores de Antígenos de Linfócitos B/metabolismo , Proteínas Tirosina Fosfatases Contendo o Domínio SH2 , Lectina 2 Semelhante a Ig de Ligação ao Ácido Siálico , Células Tumorais Cultivadas , Domínios de Homologia de src/genética , Domínios de Homologia de src/imunologia , Quinases da Família src/metabolismoRESUMO
Major histocompatibility complex class II molecules and the B cell antigen receptor (BCR) transduce similar signals when cross-linked by ligand. Therefore, studies were conducted to determine whether the protein tyrosine phosphatase CD45 regulates signaling via these transmembrane receptors in an analogous manner. Cross-linking of either class II molecules or the BCR on CD45-positive K46-17micromlambda B lymphoma cells was observed to induce activation of the Src family protein- tyrosine kinase Lyn, tyrosine phosphorylation of Syk and phospholipase Cgamma, and the production of inositol 1,4,5-trisphosphate leading to intracellular mobilization as well as extracellular influx of Ca2+. In the absence of CD45, cross-linking of either class II molecules or the BCR failed to induce activation of Lyn. Syk was inducibly phosphorylated on tyrosine in a normal manner, whereas phospholipase Cgamma exhibited a high basal level of tyrosine phosphorylation that was not significantly increased upon stimulation. Nevertheless, phospholipase Cgamma appeared to be functional because CD45-negative cells produced elevated levels of inositol 1,4,5-trisphosphate following stimulation through class II or the BCR. Regardless of this, CD45-negative cells exhibited Ca2+ mobilization responses that were greatly diminished and transient in nature. Whereas little or no mobilization of Ca2+ was observed in response to class II cross-linking, CD45-deficient cells mobilized Ca2+ from intracellular stores but not the extracellular environment in response to BCR cross-linking. These results demonstrate that CD45 regulates both Src family kinase activation and Ca2+ mobilization associated with class II- and BCR-mediated signal transduction.
Assuntos
Linfócitos B/fisiologia , Antígenos de Histocompatibilidade Classe II/imunologia , Antígenos Comuns de Leucócito/fisiologia , Animais , Cálcio/metabolismo , Ativação Enzimática , Precursores Enzimáticos/metabolismo , Expressão Gênica , Peptídeos e Proteínas de Sinalização Intracelular , Isoenzimas/metabolismo , Linfoma de Células B , Camundongos , Fosfolipase C gama , Proteínas Tirosina Quinases/metabolismo , RNA Mensageiro/genética , Agregação de Receptores , Receptores Proteína Tirosina Quinases/metabolismo , Receptores de Antígenos de Linfócitos B/metabolismo , Transdução de Sinais , Quinase Syk , Células Tumorais Cultivadas , Fosfolipases Tipo C/metabolismo , Quinases da Família src/metabolismoRESUMO
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.
Assuntos
Adjuvantes Imunológicos/fisiologia , Proteínas Tirosina Quinases/fisiologia , Receptores de Antígenos de Linfócitos B/fisiologia , Transdução de Sinais/imunologia , Domínios de Homologia de src/imunologia , Animais , Proteína Tirosina Quinase CSK , Linfoma de Células B , Camundongos , Fosforilação , Proteínas Tirosina Quinases/biossíntese , Proteínas Tirosina Quinases/deficiência , Proteínas Tirosina Quinases/metabolismo , Receptores de Antígenos de Linfócitos B/metabolismo , Transdução de Sinais/efeitos dos fármacos , Células Tumorais Cultivadas , Tirosina/metabolismo , Quinases da Família src/metabolismoRESUMO
Recently, we and others have demonstrated that negative signaling in B cells selectively induces the tyrosine phosphorylation of a novel inositol polyphosphate phosphatase, p145SHIP. In this study, we present data indicating that p145SHIP binds directly a phosphorylated motif, immunoreceptor tyrosine-based inhibition motif (ITIM), present in the cytoplasmic domain of Fc gammaRIIB1. Using recombinant SH2 domains, we show that binding is mediated via the Src homology region 2 (SH2)-containing inositol phosphatase (SHIP) SH2 domain. SHIP also bound to a phosphopeptide derived from CD22, raising the possibility that SHIP contributes to negative signaling by this receptor as well as Fc gammaRIIB1. The association of SHIP with the ITIM phosphopeptide was activation independent, while coassociation with Shc was activation dependent. Furthermore, experiments with Fc gammaRIIB1-deficient B cells demonstrated a genetic requirement for expression of Fc gammaRIIB1 in the induction of SHIP phosphorylation and its interaction with Shc. Based on these results, we propose a model of negative signaling in which co-cross-linking of surface immunoglobulin and Fc gammaRIIB1 results in sequential tyrosine phosphorylation of the ITIM, recruitment and phosphorylation of p145SHIP, and subsequent binding of Shc.
Assuntos
Antígenos CD/metabolismo , Moléculas de Adesão Celular , Lectinas , Monoéster Fosfórico Hidrolases/metabolismo , Receptores de IgG/metabolismo , Animais , Antígenos de Diferenciação de Linfócitos B/metabolismo , Linfócitos B/metabolismo , Linhagem Celular , Cinética , Camundongos , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases , Fosfopeptídeos/metabolismo , Fosforilação , Ligação Proteica , Proteínas Recombinantes de Fusão , Lectina 2 Semelhante a Ig de Ligação ao Ácido Siálico , Tirosina/metabolismo , Domínios de Homologia de srcAssuntos
Antígenos Comuns de Leucócito/fisiologia , Transdução de Sinais/fisiologia , Animais , Antígenos/imunologia , Complexo CD3/fisiologia , Adesão Celular/fisiologia , Diferenciação Celular/fisiologia , Regulação Enzimológica da Expressão Gênica , Humanos , Isoenzimas/química , Isoenzimas/fisiologia , Antígenos Comuns de Leucócito/química , Ativação Linfocitária/fisiologia , Subpopulações de Linfócitos/citologia , Subpopulações de Linfócitos/enzimologia , Subpopulações de Linfócitos/imunologia , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Fosfoproteínas/metabolismo , Fosforilação , Especificidade por Substrato , Quinases da Família src/fisiologiaRESUMO
Autoantibodies to the thyrotropin receptor (TSHR) have been shown to mediate the hyperthyroidism associated with Graves' disease (GD). A number of hypotheses have been proposed which link an infectious agent to the mechanism(s) involved in the induction of GD. Several studies have suggested that the development of GD may be linked to infection with the enteric pathogen Yersinia enterocolitica. We have recently identified two low molecular weight (5.5 and 8 kDa) envelope proteins of Y. enterocolitica that are cross-reactive with the extracellular domain of human TSHR (ETSHR). In this study, we have purified these ETSHR-crossreactive Yersinia proteins (TSHR-CRP) and have further characterized their immunoreactivity. Both the 5.5 and 8 kDa TSHR-CRPs were shown to be mitogenic for mouse spleen cells. This mitogenic activity was specific for B cells and was not due to lipopolysaccharide (LPS) contamination. TSHR-CRPs were mitogenic for LPS-non-responsive spleen cells obtained from C3H/Hej mice, and polymyxin B did not inhibit the mitogenic activity of the TSHR-CRPs. TSHR-CRPs also induced high levels of IL-6 production in B cells and induced production and secretion of significant levels of IgG and IgM. Finally, culture supernatants from TSHR-CRP-stimulated spleen cells were shown by Western blot analysis to contain antibodies that recognized the ETSHR These results identify for the first time two envelope proteins of Yersinia that have mitogenic activity and therefore could represent important proteins involved in the pathogenesis of Yersinia infections. Because these mitogenic proteins also contain epitopes crossreactive with the TSHR, they are potentially important for advancing our understanding of the role molecular mimicry plays in the induction of autoimmunity to the TSHR.
Assuntos
Linfócitos B/imunologia , Proteínas da Membrana Bacteriana Externa/imunologia , Receptores da Tireotropina/imunologia , Yersinia enterocolitica/química , Animais , Anticorpos , Anticorpos Antibacterianos/imunologia , Formação de Anticorpos , Reações Cruzadas , Epitopos , Feminino , Glutamato Descarboxilase/imunologia , Imunoglobulina G/metabolismo , Imunoglobulina M/metabolismo , Interleucina-6/biossíntese , Ativação Linfocitária/fisiologia , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos CBA , Mitógenos/fisiologia , Baço/citologia , Baço/metabolismoAssuntos
Moléculas de Adesão Celular , Lectinas , Receptores de Antígenos de Linfócitos T/fisiologia , Transdução de Sinais/imunologia , Quinases da Família src/metabolismo , Animais , Antígenos CD/metabolismo , Antígenos de Diferenciação de Linfócitos B/metabolismo , Linhagem Celular , Humanos , Linfoma de Células B , Fosforilação , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/metabolismo , Lectina 2 Semelhante a Ig de Ligação ao Ácido Siálico , Transfecção , Células Tumorais CultivadasRESUMO
CD22 is a membrane immunoglobulin (mIg)-associated protein of B cells. CD22 is tyrosine-phosphorylated when mIg is ligated. Tyrosine-phosphorylated CD22 binds and activates SHP, a protein tyrosine phosphatase known to negatively regulate signaling through mIg. Ligation of CD22 to prevent its coaggregation with mIg lowers the threshold at which mIg activates the B cell by a factor of 100. In secondary lymphoid organs, CD22 may be sequestered away from mIg through interactions with counterreceptors on T cells. Thus, CD22 is a molecular switch for SHP that may bias mIg signaling to anatomic sites rich in T cells.
Assuntos
Antígenos CD/imunologia , Antígenos de Diferenciação de Linfócitos B/imunologia , Linfócitos B/imunologia , Moléculas de Adesão Celular , Lectinas , Ativação Linfocitária , Proteínas Tirosina Fosfatases/metabolismo , Sequência de Aminoácidos , Animais , Antígenos CD/metabolismo , Antígenos de Diferenciação de Linfócitos B/metabolismo , Células Cultivadas , Humanos , Imunoglobulina M/imunologia , 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 Recombinantes/metabolismo , Lectina 2 Semelhante a Ig de Ligação ao Ácido Siálico , Transdução de Sinais , Células Tumorais CultivadasRESUMO
Recent studies have demonstrated that the protein tyrosine phosphatase CD45 plays an integral role in regulation of B-cell function. Most notably, expression of this phosphatase is required for activation of B lymphocytes and entry into the cell cycle. Here, Louis Justement and colleagues review current information concerning the function of CD45 in the B cell. The discussion focuses on two questions that are of central importance: what are the physiological substrates for CD45 and how does reversible tyrosine phosphorylation affect their function?
Assuntos
Linfócitos B/imunologia , Antígenos Comuns de Leucócito/imunologia , Ativação Linfocitária/imunologia , Proteínas Tirosina Quinases/imunologia , AnimaisRESUMO
Signal transduction via the B cell antigen receptor complex is regulated by changes in tyrosine phosphorylation of several proteins. The equilibrium between tyrosine phosphorylation and dephosphorylation is regulated by the combined action of protein tyrosine kinase and protein tyrosine phosphatase enzymes. In particular, the protein tyrosine phosphatase, CD45, has been shown to play an essential role in signal transduction via the B cell antigen receptor. Therefore, experiments were performed to examine the intermolecular associations between CD45 and phosphotyrosine-containing proteins in the B cell to identify potential substrates for CD45. Based on coprecipitation experiments, CD45 was found to be physically associated with multiple components of the B cell antigen receptor complex including the MB-1/B29 heterodimer. Additionally, CD45 was selectively associated with the src family protein tyrosine kinase, lyn. Neither blk nor fyn were observed to interact with CD45 even though they have been implicated in antigen receptor signal transduction. This finding suggests that CD45 may preferentially regulate the phosphorylation of lyn and thus, its activity. In summary, these studies provide evidence to support the hypothesis that CD45 regulates antigen receptor-mediated signal transduction by controlling the tyrosine phosphorylation of multiple components of the antigen receptor complex.
Assuntos
Antígenos CD , Linfócitos B/metabolismo , Antígenos Comuns de Leucócito/metabolismo , Proteínas Tirosina Quinases/metabolismo , Receptores de Antígenos de Linfócitos B/metabolismo , Tetra-Hidroisoquinolinas , Quinases da Família src , Animais , Antígenos CD79 , Imunoglobulina D/metabolismo , Cadeias Pesadas de Imunoglobulinas/metabolismo , Cadeias Leves de Imunoglobulina/metabolismo , Imunoglobulina M/metabolismo , Ativação Linfocitária , Substâncias Macromoleculares , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Noscapina/análogos & derivados , Noscapina/metabolismo , Fosfoproteínas/metabolismo , Fosfotirosina , Testes de Precipitina , Ligação Proteica , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais , Tirosina/análogos & derivados , Tirosina/metabolismoRESUMO
CD45 is a receptor-like protein tyrosine phosphatase expressed exclusively by cells of the hemopoietic lineage. Studies in vitro involving treatment of B cells with anti-CD45 mAb have demonstrated that ligand binding to CD45 alters the cell's response to various activation/differentiation stimuli. In general anti-CD45 treatment has been shown to exert an inhibitory effect on early activation events resulting in the failure of quiescent B cells to enter the cell cycle. These studies suggest that CD45 acts as an important regulatory molecule in vitro, that controls B cell function. In contrast, little is known concerning the role that CD45 plays in vivo regarding regulation of B cell activation and differentiation in response to T-dependent Ag. In our study the anti-CD45 mAb RA3.6B2, which recognizes a B cell-restricted epitope, was used to examine this question. Administration of anti-CD45 mAb in vivo was found to inhibit the proliferative response of splenocytes when stimulated with B cell-, but not T cell-specific mitogens. Immunization with the T-dependent Ag FITC-KLH in the presence of increasing amounts of anti-CD45 mAb resulted in a significant, dose-dependent inhibition of the plaque-forming cell response. Additionally, anti-CD45 mAb inhibited the production of FITC-specific serum antibodies indicating that the effect was systemic. Finally, anti-CD45 mAb appeared to exert a maximal effect at earlier time points, within 48 h of Ag administration, suggesting that B cell activation was primarily affected. These results provide evidence to support the conclusion that CD45 is an important regulatory molecule that is involved in the control of B cell activation in vivo.
Assuntos
Anticorpos Monoclonais/imunologia , Antígenos/imunologia , Linfócitos B/imunologia , Epitopos , Antígenos Comuns de Leucócito/fisiologia , Linfócitos T/fisiologia , Animais , Formação de Anticorpos , Hemocianinas/imunologia , Antígenos Comuns de Leucócito/imunologia , Contagem de Leucócitos , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , RatosRESUMO
Signal transduction via the B cell AgR complex has recently been shown to be dependent on the activation of one or more protein tyrosine kinases. Similarly, it has been found that signal transduction requires the expression of the protein tyrosine phosphatase CD45. Thus, transduction of a signal after AgR cross-linking must involve the coordinate interaction of these two enzymatic activities. It is therefore logical to hypothesize that the competence of the B cell to respond to ligands that bind the AgR may be dependent on the maintenance of an equilibrium between the tyrosine phosphorylation and dephosphorylation of specific signal transduction components. We have demonstrated in the present study that in resting B cells, the basal level of AgR complex tyrosine phosphorylation is regulated by cellular protein tyrosine phosphatases. Treatment of cells with the protein tyrosine phosphatase inhibitor, Na3VO4, resulted in rapid hyperphosphorylation of the receptor complex. Based on this observation, experiments were designed to examine the role of CD45 in regulation of AgR complex phosphorylation. Treatment of B cells with anti-CD45 mAb alone was found to have no effect on cytoskeletal association of CD45 or on its distribution within the membrane. Addition of a secondary cross-linking reagent, however, induced the association of CD45 with the cytoskeleton and caused capping. Subsequent studies demonstrated that increased tyrosine phosphorylation of the mIg-associated proteins MB-1 and B29 could be induced after incubating cells with anti-CD45 mAb and a secondary cross-linker, but not after the addition of anti-CD45 mAb alone. Changes in tyrosine phosphorylation of MB-1 and B29 were found to correlate with the cytoskeletal association of CD45. Interestingly, although cross-linking CD45 induced alterations in its association with the cytoskeleton and in its distribution within the membrane, no significant change in the level of protein tyrosine phosphatase activity could be detected under these conditions. These findings support the possibility that ligand binding to CD45 can induce biochemical and/or physical alterations in the molecule that presumably inhibit its ability to interact with specific substrates in the cell, thereby shifting the established equilibrium between tyrosine-specific phosphorylation and dephosphorylation.
Assuntos
Antígenos CD , Antígenos Comuns de Leucócito/fisiologia , Proteínas Tirosina Fosfatases/fisiologia , Receptores de Antígenos de Linfócitos B/metabolismo , Tirosina/metabolismo , Animais , Antígenos CD79 , Citoesqueleto/metabolismo , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , FosforilaçãoRESUMO
The B cell Ag receptor complex is comprised of membrane (m)IgM or mIgD noncovalently associated with one or more heterodimers, each containing one subunit of MB-1 (IgM alpha or IgD alpha) and one of B29 (Ig beta or Ig gamma). It is known that cross-linking of the B cell Ag receptor results in protein tyrosine kinase activation. Recent reports from other laboratories have demonstrated that mIg coprecipitates with multiple src family protein tyrosine kinases, including blk, lyn, and fyn. However, the mechanism by which these kinases are physically coupled to the Ag receptor has not been confirmed. It has been hypothesized that the mIg-associated proteins MB-1 and B29 provide a physical link between the Ag receptor (mIg) and one or more protein tyrosine kinases. In this study, we confirm previous findings demonstrating that the B cell Ag receptor coprecipitates with the MB-1/B29 heterodimer as well as the protein tyrosine kinases blk, lyn, and fyn under mild detergent conditions (1% digitonin). Additionally, we demonstrate that in detergent conditions (1% Nonidet P-40 (NP-40)) which disrupt the association between mIg and the MB-1/B29 heterodimer, no protein tyrosine kinase activity can be detected in association with mIg. These findings indicated that NP-40 effectively dissociates the B cell Ag receptor from ancillary signal transducing proteins. MB-1 and B29 were however, found to coprecipitate with blk, lyn, and fyn isolated from B cell lysates containing 1% NP-40. No significant difference was observed in the stoichiometry of association between the kinases and the MB-1/B29 heterodimer in the presence of 1% NP-40 when compared to 1% digitonin. It was further determined that in resting B cells, only a small fraction (approximately 1-3%) of the MB-1/B29 heterodimers appear to be complexed with protein tyrosine kinases. Finally, based on preclearing experiments, it appears that individual heterodimers may associate with a single species of protein tyrosine kinase. These data support the hypothesis that the MB-1/B29 heterodimer couples the antigen receptor to protein tyrosine kinases, thereby providing a physical link that facilitates Ag receptor-mediated regulation of kinase activity.
Assuntos
Antígenos CD , Glicoproteínas de Membrana/análise , Fosfoproteínas/análise , Proteínas Tirosina Quinases/análise , Proteínas Proto-Oncogênicas/análise , Receptores de Antígenos de Linfócitos B/análise , Animais , Antígenos CD79 , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Proteínas Proto-Oncogênicas c-fynRESUMO
CD45 is a member of a family of membrane proteins that possess phosphotyrosine phosphatase activity, and is the source of much of the tyrosine phosphatase activity in lymphocytes. In view of its enzymatic activity and high copy number, it seems likely that CD45 functions in transmembrane signal transduction by lymphocyte receptors that are coupled to activation of tyrosine kinases. The B cell antigen receptor was found to transduce a Ca(2+)-mobilizing signal only if cells expressed CD45. Also, both membrane immunoglobulin M (mIgM) and CD45 were lost from the surface of cells treated with antibody to CD45, suggesting a physical interaction between these proteins. Finally, CD45 dephosphorylated a complex of mIg-associated proteins that appears to function in signal transduction by the antigen receptor. These data indicate that CD45 occurs as a component of a complex of proteins associated with the antigen receptor, and that CD45 may regulate signal transduction by modulating the phosphorylation state of the antigen receptor subunits.