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1.
Immunity ; 56(12): 2682-2698.e9, 2023 Dec 12.
Artigo em Inglês | MEDLINE | ID: mdl-38091950

RESUMO

T cell responses are inhibited by acidic environments. T cell receptor (TCR)-induced protein phosphorylation is negatively regulated by dephosphorylation and/or ubiquitination, but the mechanisms underlying sensitivity to acidic environments are not fully understood. Here, we found that TCR stimulation induced a molecular complex of Cbl-b, an E3-ubiquitin ligase, with STS1, a pH-sensitive unconventional phosphatase. The induced interaction depended upon a proline motif in Cbl-b interacting with the STS1 SH3 domain. STS1 dephosphorylated Cbl-b interacting phosphoproteins. The deficiency of STS1 or Cbl-b diminished the sensitivity of T cell responses to the inhibitory effects of acid in an autocrine or paracrine manner in vitro or in vivo. Moreover, the deficiency of STS1 or Cbl-b promoted T cell proliferative and differentiation activities in vivo and inhibited tumor growth, prolonged survival, and improved T cell fitness in tumor models. Thus, a TCR-induced STS1-Cbl-b complex senses intra- or extra-cellular acidity and regulates T cell responses, presenting a potential therapeutic target for improving anti-tumor immunity.


Assuntos
Transdução de Sinais , Linfócitos T , Ubiquitina-Proteína Ligases/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Concentração de Íons de Hidrogênio
2.
Nat Immunol ; 24(1): 174-185, 2023 01.
Artigo em Inglês | MEDLINE | ID: mdl-36564464

RESUMO

The kinase LCK and CD4/CD8 co-receptors are crucial components of the T cell antigen receptor (TCR) signaling machinery, leading to key T cell fate decisions. Despite decades of research, the roles of CD4-LCK and CD8-LCK interactions in TCR triggering in vivo remain unknown. In this study, we created animal models expressing endogenous levels of modified LCK to resolve whether and how co-receptor-bound LCK drives TCR signaling. We demonstrated that the role of LCK depends on the co-receptor to which it is bound. The CD8-bound LCK is largely dispensable for antiviral and antitumor activity of cytotoxic T cells in mice; however, it facilitates CD8+ T cell responses to suboptimal antigens in a kinase-dependent manner. By contrast, the CD4-bound LCK is required for efficient development and function of helper T cells via a kinase-independent stabilization of surface CD4. Overall, our findings reveal the role of co-receptor-bound LCK in T cell biology, show that CD4- and CD8-bound LCK drive T cell development and effector immune responses using qualitatively different mechanisms and identify the co-receptor-LCK interactions as promising targets for immunomodulation.


Assuntos
Proteína Tirosina Quinase p56(lck) Linfócito-Específica , Linfócitos T Citotóxicos , Camundongos , Animais , Linfócitos T Citotóxicos/metabolismo , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/metabolismo , Antígenos CD4 , Transdução de Sinais , Receptores de Antígenos de Linfócitos T/metabolismo , Antígenos CD8/metabolismo
3.
Sci Signal ; 14(668)2021 02 02.
Artigo em Inglês | MEDLINE | ID: mdl-33531381

RESUMO

The cytoplasmic kinase ZAP70 is critical for T cell antigen receptor (TCR) signaling. The R360P mutation in ZAP70 is responsible for an early-onset familial autoimmune syndrome. The structural location and biochemical signaling effects of the R360P mutation are consistent with weakening of the autoinhibitory conformation of ZAP70. Mice with a ZAP70 R360P mutation and polyclonal TCR repertoires exhibited relatively normal T cell development but showed evidence of increased signaling. In addition, the R360P mutation resulted in enhanced follicular helper T cell expansion after LCMV infection. To eliminate the possibility of a TCR repertoire shift, the OTI transgenic TCR was introduced into R360P mice, which resulted in enhanced T cell responses to weaker stimuli, including weak agonists and a self-peptide. These observations suggest that disruption of ZAP70 autoinhibition by the R360P mutation enables increased mature T cell sensitivity to self-antigens that would normally be ignored by wild-type T cells, a mechanism that may contribute to the break of tolerance in human patients with R360P mutation.


Assuntos
Doenças Autoimunes/imunologia , Linfócitos T CD8-Positivos/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Proteína-Tirosina Quinase ZAP-70/imunologia , Animais , Linfócitos T CD8-Positivos/citologia , Células HEK293 , Humanos , Tolerância Imunológica , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mutação
4.
Proc Natl Acad Sci U S A ; 116(22): 10798-10803, 2019 05 28.
Artigo em Inglês | MEDLINE | ID: mdl-31076553

RESUMO

The transformation of molecular binding events into cellular decisions is the basis of most biological signal transduction. A fundamental challenge faced by these systems is that reliance on protein-ligand chemical affinities alone generally results in poor sensitivity to ligand concentration, endangering the system to error. Here, we examine the lipid-binding pleckstrin homology and Tec homology (PH-TH) module of Bruton's tyrosine kinase (Btk). Using fluorescence correlation spectroscopy (FCS) and membrane-binding kinetic measurements, we identify a phosphatidylinositol (3-5)-trisphosphate (PIP3) sensing mechanism that achieves switch-like sensitivity to PIP3 levels, surpassing the intrinsic affinity discrimination of PIP3:PH binding. This mechanism employs multiple PIP3 binding as well as dimerization of Btk on the membrane surface. Studies in live cells confirm that mutations at the dimer interface and peripheral site produce effects comparable to that of the kinase-dead Btk in vivo. These results demonstrate how a single protein module can institute an allosteric counting mechanism to achieve high-precision discrimination of ligand concentration. Furthermore, this activation mechanism distinguishes Btk from other Tec family member kinases, Tec and Itk, which we show are not capable of dimerization through their PH-TH modules. This suggests that Btk plays a critical role in the stringency of the B cell response, whereas T cells rely on other mechanisms to achieve stringency.


Assuntos
Tirosina Quinase da Agamaglobulinemia/química , Tirosina Quinase da Agamaglobulinemia/metabolismo , Transdução de Sinais/fisiologia , Animais , Linfócitos B/metabolismo , Linhagem Celular , Galinhas , Camundongos , Modelos Moleculares , Mutação , Fosfatos de Fosfatidilinositol/metabolismo , Fosforilação , Conformação Proteica , Domínios Proteicos/fisiologia , Multimerização Proteica
5.
Mol Cell ; 67(3): 498-511.e6, 2017 Aug 03.
Artigo em Inglês | MEDLINE | ID: mdl-28735895

RESUMO

The Src Family kinase Lck sets a critical threshold for T cell activation because it phosphorylates the TCR complex and the Zap70 kinase. How a T cell controls the abundance of active Lck molecules remains poorly understood. We have identified an unappreciated role for a phosphosite, Y192, within the Lck SH2 domain that profoundly affects the amount of active Lck in cells. Notably, mutation of Y192 blocks critical TCR-proximal signaling events and impairs thymocyte development in retrogenic mice. We determined that these defects are caused by hyperphosphorylation of the inhibitory C-terminal tail of Lck. Our findings reveal that modification of Y192 inhibits the ability of CD45 to associate with Lck in cells and dephosphorylate the C-terminal tail of Lck, which prevents its adoption of an active open conformation. These results suggest a negative feedback loop that responds to signaling events that tune active Lck amounts and TCR sensitivity.


Assuntos
Antígenos Comuns de Leucócito/metabolismo , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/metabolismo , Timócitos/enzimologia , Domínios de Homologia de src , Animais , Ativação Enzimática , Genótipo , Células HEK293 , Humanos , Células Jurkat , Antígenos Comuns de Leucócito/química , Antígenos Comuns de Leucócito/genética , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/química , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/deficiência , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/genética , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Moleculares , Mutação , Fenótipo , Fosforilação , Ligação Proteica , Proteínas Proto-Oncogênicas c-fyn/genética , Proteínas Proto-Oncogênicas c-fyn/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais , Timócitos/imunologia , Fatores de Tempo , Transfecção
6.
Elife ; 52016 10 04.
Artigo em Inglês | MEDLINE | ID: mdl-27700984

RESUMO

The sequence of events that initiates T cell signaling is dictated by the specificities and order of activation of the tyrosine kinases that signal downstream of the T cell receptor. Using a platform that combines exhaustive point-mutagenesis of peptide substrates, bacterial surface-display, cell sorting, and deep sequencing, we have defined the specificities of the first two kinases in this pathway, Lck and ZAP-70, for the T cell receptor ζ chain and the scaffold proteins LAT and SLP-76. We find that ZAP-70 selects its substrates by utilizing an electrostatic mechanism that excludes substrates with positively-charged residues and favors LAT and SLP-76 phosphosites that are surrounded by negatively-charged residues. This mechanism prevents ZAP-70 from phosphorylating its own activation loop, thereby enforcing its strict dependence on Lck for activation. The sequence features in ZAP-70, LAT, and SLP-76 that underlie electrostatic selectivity likely contribute to the specific response of T cells to foreign antigens.


Assuntos
Proteína Tirosina Quinase p56(lck) Linfócito-Específica/química , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais , Eletricidade Estática , Proteína-Tirosina Quinase ZAP-70/química , Proteína-Tirosina Quinase ZAP-70/metabolismo , Células HEK293 , Humanos , Especificidade por Substrato
7.
J Exp Med ; 213(2): 155-65, 2016 Feb 08.
Artigo em Inglês | MEDLINE | ID: mdl-26783323

RESUMO

A brother and sister developed a previously undescribed constellation of autoimmune manifestations within their first year of life, with uncontrollable bullous pemphigoid, colitis, and proteinuria. The boy had hemophilia due to a factor VIII autoantibody and nephrotic syndrome. Both children required allogeneic hematopoietic cell transplantation (HCT), which resolved their autoimmunity. The early onset, severity, and distinctive findings suggested a single gene disorder underlying the phenotype. Whole-exome sequencing performed on five family members revealed the affected siblings to be compound heterozygous for two unique missense mutations in the 70-kD T cell receptor ζ-chain associated protein (ZAP-70). Healthy relatives were heterozygous mutation carriers. Although pre-HCT patient T cells were not available, mutation effects were determined using transfected cell lines and peripheral blood from carriers and controls. Mutation R192W in the C-SH2 domain exhibited reduced binding to phosphorylated ζ-chain, whereas mutation R360P in the N lobe of the catalytic domain disrupted an autoinhibitory mechanism, producing a weakly hyperactive ZAP-70 protein. Although human ZAP-70 deficiency can have dysregulated T cells, and autoreactive mouse thymocytes with weak Zap-70 signaling can escape tolerance, our patients' combination of hypomorphic and activating mutations suggested a new disease mechanism and produced previously undescribed human ZAP-70-associated autoimmune disease.


Assuntos
Doenças Autoimunes/enzimologia , Doenças Autoimunes/genética , Proteínas Mutantes/genética , Mutação de Sentido Incorreto , Proteína-Tirosina Quinase ZAP-70/genética , Sequência de Aminoácidos , Animais , Doenças Autoimunes/imunologia , Sequência de Bases , Linhagem Celular , Pré-Escolar , Feminino , Transplante de Células-Tronco Hematopoéticas , Hemofilia A/enzimologia , Hemofilia A/genética , Hemofilia A/imunologia , Heterozigoto , Humanos , Lactente , Masculino , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Linhagem , Penfigoide Bolhoso/enzimologia , Penfigoide Bolhoso/genética , Penfigoide Bolhoso/patologia , Fenótipo , Conformação Proteica , Receptores de Antígenos de Linfócitos T/metabolismo , Imunodeficiência Combinada Severa/enzimologia , Imunodeficiência Combinada Severa/genética , Imunodeficiência Combinada Severa/imunologia , Irmãos , Síndrome , Linfócitos T/enzimologia , Linfócitos T/imunologia , Transplante Homólogo , Proteína-Tirosina Quinase ZAP-70/química , Proteína-Tirosina Quinase ZAP-70/deficiência , Proteína-Tirosina Quinase ZAP-70/imunologia , Proteína-Tirosina Quinase ZAP-70/metabolismo
8.
Sci Signal ; 8(377): ra49, 2015 May 19.
Artigo em Inglês | MEDLINE | ID: mdl-25990959

RESUMO

T cell activation by antigens binding to the T cell receptor (TCR) must be properly regulated to ensure normal T cell development and effective immune responses to pathogens and transformed cells while avoiding autoimmunity. The Src family kinase Lck and the Syk family kinase ZAP-70 (ζ chain-associated protein kinase of 70 kD) are sequentially activated in response to TCR engagement and serve as critical components of the TCR signaling machinery that leads to T cell activation. We performed a mass spectrometry-based phosphoproteomic study comparing the quantitative differences in the temporal dynamics of phosphorylation in stimulated and unstimulated T cells with or without inhibition of ZAP-70 catalytic activity. The data indicated that the kinase activity of ZAP-70 stimulates negative feedback pathways that target Lck and thereby modulate the phosphorylation patterns of the immunoreceptor tyrosine-based activation motifs (ITAMs) of the CD3 and ζ chain components of the TCR and of signaling molecules downstream of Lck, including ZAP-70. We developed a computational model that provides a mechanistic explanation for the experimental findings on ITAM phosphorylation in wild-type cells, ZAP-70-deficient cells, and cells with inhibited ZAP-70 catalytic activity. This model incorporated negative feedback regulation of Lck activity by the kinase activity of ZAP-70 and predicted the order in which tyrosines in the ITAMs of TCR ζ chains must be phosphorylated to be consistent with the experimental data.


Assuntos
Retroalimentação Fisiológica/fisiologia , Imunidade Celular/imunologia , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/metabolismo , Modelos Imunológicos , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais/imunologia , Proteína-Tirosina Quinase ZAP-70/metabolismo , Catálise , Humanos , Células Jurkat , Espectrometria de Massas , Fosfopeptídeos/genética , Fosfopeptídeos/metabolismo , Fosforilação , Proteômica/métodos , Receptores de Antígenos de Linfócitos T/imunologia
9.
Genome Res ; 23(9): 1522-40, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23804400

RESUMO

DNA methylation plays key roles in diverse biological processes such as X chromosome inactivation, transposable element repression, genomic imprinting, and tissue-specific gene expression. Sequencing-based DNA methylation profiling provides an unprecedented opportunity to map and compare complete DNA methylomes. This includes one of the most widely applied technologies for measuring DNA methylation: methylated DNA immunoprecipitation followed by sequencing (MeDIP-seq), coupled with a complementary method, methylation-sensitive restriction enzyme sequencing (MRE-seq). A computational approach that integrates data from these two different but complementary assays and predicts methylation differences between samples has been unavailable. Here, we present a novel integrative statistical framework M&M (for integration of MeDIP-seq and MRE-seq) that dynamically scales, normalizes, and combines MeDIP-seq and MRE-seq data to detect differentially methylated regions. Using sample-matched whole-genome bisulfite sequencing (WGBS) as a gold standard, we demonstrate superior accuracy and reproducibility of M&M compared to existing analytical methods for MeDIP-seq data alone. M&M leverages the complementary nature of MeDIP-seq and MRE-seq data to allow rapid comparative analysis between whole methylomes at a fraction of the cost of WGBS. Comprehensive analysis of nineteen human DNA methylomes with M&M reveals distinct DNA methylation patterns among different tissue types, cell types, and individuals, potentially underscoring divergent epigenetic regulation at different scales of phenotypic diversity. We find that differential DNA methylation at enhancer elements, with concurrent changes in histone modifications and transcription factor binding, is common at the cell, tissue, and individual levels, whereas promoter methylation is more prominent in reinforcing fundamental tissue identities.


Assuntos
Algoritmos , Metilação de DNA , Genoma Humano , Análise de Sequência de DNA/métodos , Interpretação Estatística de Dados , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Especificidade de Órgãos
10.
Nat Genet ; 45(7): 836-41, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23708189

RESUMO

Transposable element (TE)-derived sequences comprise half of the human genome and DNA methylome and are presumed to be densely methylated and inactive. Examination of genome-wide DNA methylation status within 928 TE subfamilies in human embryonic and adult tissues identified unexpected tissue-specific and subfamily-specific hypomethylation signatures. Genes proximal to tissue-specific hypomethylated TE sequences were enriched for functions important for the relevant tissue type, and their expression correlated strongly with hypomethylation within the TEs. When hypomethylated, these TE sequences gained tissue-specific enhancer marks, including monomethylation of histone H3 at lysine 4 (H3K4me1) and occupancy by p300, and a majority exhibited enhancer activity in reporter gene assays. Many such TEs also harbored binding sites for transcription factors that are important for tissue-specific functions and showed evidence of evolutionary selection. These data suggest that sequences derived from TEs may be responsible for wiring tissue type-specific regulatory networks and may have acquired tissue-specific epigenetic regulation.


Assuntos
Metilação de DNA , Elementos de DNA Transponíveis/genética , Elementos Facilitadores Genéticos/genética , Família Multigênica/genética , Adulto , Sítios de Ligação/genética , Células Cultivadas , Mapeamento Cromossômico , Embrião de Mamíferos , Epigênese Genética , Genoma Humano , Histonas/genética , Histonas/metabolismo , Humanos , Especificidade de Órgãos/genética
11.
Mol Cell Biol ; 33(11): 2188-201, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23530057

RESUMO

Serial activation of the tyrosine kinases Lck and ZAP-70 initiates signaling downstream of the T cell receptor. We previously reported the structure of an autoinhibited ZAP-70 variant in which two regulatory tyrosine residues (315 and 319) in the SH2-kinase linker were replaced by phenylalanine. We now present a crystal structure of ZAP-70 in which Tyr 315 and Tyr 319 are not mutated, leading to the recognition of a five-residue sequence register error in the SH2-kinase linker of the original crystallographic model. The revised model identifies distinct roles for these two tyrosines. As seen in a recently reported structure of the related tyrosine kinase Syk, Tyr 315 of ZAP-70 is part of a hydrophobic interface between the regulatory apparatus and the kinase domain, and the integrity of this interface would be lost upon engagement of doubly phosphorylated peptides by the SH2 domains. Tyr 319 is not necessarily dislodged by SH2 engagement, which activates ZAP-70 only ∼5-fold in vitro. In contrast, phosphorylation by Lck activates ZAP-70 ∼100-fold. This difference is due to the ability of Tyr 319 to suppress ZAP-70 activity even when the SH2 domains are dislodged from the kinase domain, providing stringent control of ZAP-70 activity downstream of Lck.


Assuntos
Proteína-Tirosina Quinase ZAP-70/química , Proteína-Tirosina Quinase ZAP-70/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Cristalografia por Raios X , Transferência Ressonante de Energia de Fluorescência , Proteína Adaptadora GRB2/genética , Proteína Adaptadora GRB2/metabolismo , Humanos , Ligação de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/genética , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Modelos Moleculares , Fosforilação , Conformação Proteica , Estrutura Terciária de Proteína , Proteínas Tirosina Quinases/química , Proteínas Tirosina Quinases/metabolismo , Quinase Syk , Tirosina/química , Tirosina/metabolismo , Proteína-Tirosina Quinase ZAP-70/genética , Domínios de Homologia de src , Quinases da Família src/química , Quinases da Família src/metabolismo
12.
Sci Signal ; 6(256): ra1, 2013 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-23281368

RESUMO

The Src and Syk families of kinases are two distinct sets of kinases that play critical roles in initiating membrane-proximal B cell receptor (BCR) signaling. However, unlike in other lymphocytes, such as T cells, the "division of labor" between Src family kinases (SFKs) and Syk in B cells is not well separated because both Syk and SFKs can phosphorylate immunoreceptor tyrosine-based activation motifs (ITAMs) present in proteins comprising the BCR. To understand why B cells require both SFKs and Syk for activation, we investigated the roles of both families of kinases in BCR signaling with computational modeling and in vitro experiments. Our computational model suggested that positive feedback enabled Syk to substantially compensate for the absence of SFKs when spatial clustering of BCRs was induced by multimeric ligands. We confirmed this prediction experimentally. In contrast, when B cells were stimulated by monomeric ligands that failed to produce BCR clustering, both Syk and SFKs were required for complete and rapid BCR activation. Our data suggest that SFKs could play a pivotal role in increasing BCR sensitivity to monomeric antigens of pathogens and in mediating a rapid response to soluble multimeric antigens of pathogens that can induce spatial BCR clustering.


Assuntos
Linfócitos B/imunologia , Retroalimentação Fisiológica/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Modelos Imunológicos , Proteínas Tirosina Quinases/metabolismo , Receptores de Antígenos de Linfócitos B/metabolismo , Transdução de Sinais/imunologia , Quinases da Família src/metabolismo , Animais , Anticorpos Monoclonais , Linfócitos B/metabolismo , Linfócitos B/fisiologia , Proteína Tirosina Quinase CSK , Clonagem Molecular , Simulação por Computador , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Método de Monte Carlo , Fosforilação , Proteínas Tirosina Quinases/genética , Células Sf9 , Spodoptera , Quinase Syk , Ultracentrifugação , Proteína-Tirosina Quinase ZAP-70/metabolismo
13.
Cold Spring Harb Perspect Biol ; 2(5): a002279, 2010 May.
Artigo em Inglês | MEDLINE | ID: mdl-20452964

RESUMO

ZAP-70 is a cytoplasmic protein tyrosine kinase that plays a critical role in the events involved in initiating T-cell responses by the antigen receptor. Here we review the structure of ZAP-70, its regulation, its role in development and in disease. We also describe a model experimental system in which ZAP-70 function can be interrupted by a small chemical inhibitor.


Assuntos
Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais , Linfócitos T/enzimologia , Proteína-Tirosina Quinase ZAP-70/metabolismo , Animais , Inibidores Enzimáticos/farmacologia , Humanos , Camundongos , Modelos Moleculares , Fosforilação , Conformação Proteica , Linfócitos T/citologia , Proteína-Tirosina Quinase ZAP-70/antagonistas & inibidores , Proteína-Tirosina Quinase ZAP-70/química
14.
Proc Natl Acad Sci U S A ; 106(49): 20699-704, 2009 Dec 08.
Artigo em Inglês | MEDLINE | ID: mdl-19920178

RESUMO

The delivery of signals from the activated T cell antigen receptor (TCR) inside the cell relies on the protein tyrosine kinase ZAP-70 (zeta-associated protein of 70 kDa). A recent crystal structure of inactive full-length ZAP-70 suggests that a central interface formed by the docking of the two SH2 domains of ZAP-70 onto the kinase domain is crucial for suppressing catalytic activity. Here we validate the significance of this autoinhibitory interface for the regulation of ZAP-70 catalytic activity and the T cell response. For this purpose, we perform in vitro catalytic activity assays and binding experiments using ZAP-70 proteins purified from insect cells to examine activation of ZAP-70. Furthermore, we use cell lines stably expressing wild-type or mutant ZAP-70 to monitor proximal events in T cell signaling, including TCR-induced phosphorylation of ZAP-70 substrates, activation of the MAP kinase pathway, and intracellular Ca(2+) levels. Taken together, our results directly correlate the stability of the autoinhibitory interface with the activation of these key events in the T cell response.


Assuntos
Receptores de Antígenos de Linfócitos T/imunologia , Proteína-Tirosina Quinase ZAP-70/antagonistas & inibidores , Proteína-Tirosina Quinase ZAP-70/química , Biocatálise , Sinalização do Cálcio , Ativação Enzimática , Estabilidade Enzimática , Humanos , Espaço Intracelular/metabolismo , Células Jurkat , Sistema de Sinalização das MAP Quinases , Modelos Moleculares , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Mutação/genética , Peptídeos/metabolismo , Fosforilação , Fosfotirosina/metabolismo , Estrutura Terciária de Proteína , Relação Estrutura-Atividade , Fatores de Tempo , Proteína-Tirosina Quinase ZAP-70/metabolismo
15.
J Biol Chem ; 283(22): 15419-30, 2008 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-18378687

RESUMO

ZAP-70 is a cytoplasmic protein tyrosine kinase that is required for T cell antigen receptor (TCR) signaling. Both mice and humans deficient in ZAP-70 fail to develop functional T cells, thus demonstrating its necessity for T cell development and function. There is currently no highly specific, cell-permeable, small molecule inhibitor for ZAP-70; therefore, we generated a mutant ZAP-70 allele that retains kinase activity but is sensitive to inhibition by a mutant-specific inhibitor. We validated the chemical genetic inhibitor system in Jurkat T cell lines, where the inhibitor blocked ZAP-70-dependent TCR signaling in cells expressing the analog-sensitive allele. Interestingly, the inhibitor also ablated CD28 superagonist signaling, thereby demonstrating the utility of this system in dissecting the requirement for ZAP-70 in alternative mechanisms of T cell activation. Thus, we have developed the first specific chemical means of inhibiting ZAP-70 in cells, which serves as a valuable tool for studying the function of ZAP-70 in T cells.


Assuntos
Alelos , Antígenos CD28/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais/efeitos dos fármacos , Superantígenos/farmacologia , Proteína-Tirosina Quinase ZAP-70/antagonistas & inibidores , Animais , Humanos , Células Jurkat , Camundongos , Mutação , Receptores de Antígenos de Linfócitos T/agonistas , Transdução de Sinais/genética , Proteína-Tirosina Quinase ZAP-70/genética , Proteína-Tirosina Quinase ZAP-70/metabolismo
16.
Cell ; 129(4): 735-46, 2007 May 18.
Artigo em Inglês | MEDLINE | ID: mdl-17512407

RESUMO

ZAP-70, a cytoplasmic tyrosine kinase required for T cell antigen receptor signaling, is controlled by a regulatory segment that includes a tandem SH2 unit responsible for binding to immunoreceptor tyrosine-based activation motifs (ITAMs). The crystal structure of autoinhibited ZAP-70 reveals that the inactive kinase domain adopts a conformation similar to that of cyclin-dependent kinases and Src kinases. The autoinhibitory mechanism of ZAP-70 is, however, distinct and involves interactions between the regulatory segment and the hinge region of the kinase domain that reduce its flexibility. Two tyrosine residues in the SH2-kinase linker that activate ZAP-70 when phosphorylated are involved in aromatic-aromatic interactions that connect the linker to the kinase domain. These interactions are inconsistent with ITAM binding, suggesting that destabilization of this autoinhibited ZAP-70 conformation is the first step in kinase activation.


Assuntos
Apresentação de Antígeno/fisiologia , Linfócitos T/enzimologia , Proteína-Tirosina Quinase ZAP-70/química , Proteína-Tirosina Quinase ZAP-70/metabolismo , Motivos de Aminoácidos/fisiologia , Animais , Sítios de Ligação/fisiologia , Linhagem Celular , Cristalografia por Raios X , Ativação Enzimática/fisiologia , Repressão Enzimática/fisiologia , Humanos , Modelos Moleculares , Fosforilação , Ligação Proteica/fisiologia , Conformação Proteica , Domínios de Homologia de src/fisiologia
17.
Mol Cell Biol ; 25(12): 4924-33, 2005 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-15923611

RESUMO

ZAP-70, a Syk family cytoplasmic protein tyrosine kinase (PTK), is required to couple the activated T-cell antigen receptor (TCR) to downstream signaling pathways. It contains two tandem SH2 domains that bind to phosphorylated TCR subunits and a C-terminal catalytic domain. The region connecting the SH2 domains with the kinase domain, termed interdomain B, has previously been shown to have striking regulatory effects on ZAP-70 function, presumed to be due to the recruitment of key substrates. Paradoxically, deletion of interdomain B preserves ZAP-70 function. Recent structural studies of several receptor tyrosine kinases (RTKs) revealed that their juxtamembrane regions negatively regulate their catalytic activities. In EphB2 and several other RTKs, this autoinhibition depends upon interaction between the kinase domain and tyrosine residues within the juxtamembrane region. Autoinhibition is released when these tyrosines become phosphorylated following receptor stimulation. Sequence homology suggested analogous regulation for ZAP-70. Based on mutagenesis analysis of ZAP-70 interdomain B, we find that this region downregulates ZAP-70 catalytic activity in a similar manner as the juxtamembrane region of EphB2. Similar regulation was also noted for the related Syk kinase. These findings suggest that a general autoinhibitory mechanism employed by RTKs is also used by some cytoplasmic tyrosine kinases.


Assuntos
Proteínas Tirosina Quinases/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Transdução de Sinais/fisiologia , Sequência de Aminoácidos , Animais , Cálcio/metabolismo , Linhagem Celular , Ativação Enzimática , Precursores Enzimáticos/genética , Precursores Enzimáticos/metabolismo , Genes Reporter , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Dados de Sequência Molecular , Fosforilação , Proteínas Tirosina Quinases/genética , Ratos , Receptores Proteína Tirosina Quinases/genética , Receptor EphB2/genética , Receptor EphB2/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Alinhamento de Sequência , Quinase Syk , Tirosina/metabolismo , Proteína-Tirosina Quinase ZAP-70
18.
Mol Cell Biol ; 24(6): 2455-66, 2004 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-14993283

RESUMO

The Tec protein tyrosine kinase is the founding member of a family that includes Btk, Itk, Bmx, and Txk. Btk is essential for B-cell receptor signaling, because mutations in Btk are responsible for X-linked agammaglobulinemia (XLA) in humans and X-linked immunodeficiency (xid) in mice, whereas Itk is involved in T-cell receptor signaling. Tec is expressed in both T and B cells, but its role in antigen receptor signaling is not clear. In this study, we show that Tec protein is expressed at substantially lower levels in primary T and B cells relative to Itk and Btk, respectively. However, Tec is up-regulated upon T-cell activation and in Th1 and Th2 cells. In functional experiments that mimic Tec up-regulation, we find that Tec overexpression in lymphocyte cell lines is sufficient to induce phospholipase Cgamma (PLC-gamma) phosphorylation and NFAT (nuclear factor of activated T cells) activation. In contrast, overexpression of Btk, Itk, or Bmx does not induce NFAT activation. Tec-induced NFAT activation requires PLC-gamma, but not the adapters LAT, SLP-76, and BLNK, which are required for Btk and Itk to couple to PLC-gamma. Finally, we show that the unique effector function for Tec correlates with a unique subcellular localization. We hypothesize that Tec functions in activated and effector T lymphocytes to induce the expression of genes regulated by NFAT transcription factors.


Assuntos
Linfócitos/enzimologia , Proteínas Nucleares , Proteínas Tirosina Quinases/metabolismo , Tirosina Quinase da Agamaglobulinemia , Animais , Linfócitos B/enzimologia , Linhagem Celular , Galinhas , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica , Humanos , Células Jurkat , Ativação Linfocitária , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/genética , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/metabolismo , Camundongos , Fatores de Transcrição NFATC , Fosfolipase C gama , Estrutura Terciária de Proteína , Proteínas Tirosina Quinases/química , Proteínas Tirosina Quinases/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transdução de Sinais , Frações Subcelulares/enzimologia , Linfócitos T/enzimologia , Linfócitos T/imunologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transfecção , Fosfolipases Tipo C/genética , Fosfolipases Tipo C/metabolismo
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