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1.
Nat Immunol ; 16(9): 950-60, 2015 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-26214742

RESUMEN

The modification of proteins by ubiquitin has a major role in cells of the immune system and is counteracted by various deubiquitinating enzymes (DUBs) with poorly defined functions. Here we identified the ubiquitin-specific protease USP8 as a regulatory component of the T cell antigen receptor (TCR) signalosome that interacted with the adaptor Gads and the regulatory molecule 14-3-3ß. Caspase-dependent processing of USP8 occurred after stimulation of the TCR. T cell-specific deletion of USP8 in mice revealed that USP8 was essential for thymocyte maturation and upregulation of the gene encoding the cytokine receptor IL-7Rα mediated by the transcription factor Foxo1. Mice with T cell-specific USP8 deficiency developed colitis that was promoted by disturbed T cell homeostasis, a predominance of CD8(+) γδ T cells in the intestine and impaired regulatory T cell function. Collectively, our data reveal an unexpected role for USP8 as an immunomodulatory DUB in T cells.


Asunto(s)
Linfocitos T CD8-positivos/inmunología , Diferenciación Celular/inmunología , Endopeptidasas/inmunología , Complejos de Clasificación Endosomal Requeridos para el Transporte/inmunología , Timocitos/inmunología , Ubiquitina Tiolesterasa/inmunología , Proteínas Adaptadoras Transductoras de Señales/inmunología , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Linfocitos T CD8-positivos/metabolismo , Diferenciación Celular/genética , Colitis/genética , Colitis/inmunología , Endopeptidasas/genética , Complejos de Clasificación Endosomal Requeridos para el Transporte/genética , Proteína Forkhead Box O1 , Factores de Transcripción Forkhead/inmunología , Factores de Transcripción Forkhead/metabolismo , Homeostasis , Humanos , Células Jurkat , Ratones , Receptores de Antígenos de Linfocitos T/inmunología , Receptores de Antígenos de Linfocitos T/metabolismo , Receptores de Interleucina-7/inmunología , Receptores de Interleucina-7/metabolismo , Linfocitos T/inmunología , Linfocitos T/metabolismo , Timocitos/metabolismo , Ubiquitina Tiolesterasa/genética
2.
J Allergy Clin Immunol ; 152(3): 807-813.e7, 2023 09.
Artículo en Inglés | MEDLINE | ID: mdl-37211057

RESUMEN

BACKGROUND: Inborn errors affecting components of the T-cell receptor signaling cascade cause combined immunodeficiency with various degrees of severity. Recently, homozygous variants in LCP2 were reported to cause pediatric onset of severe combined immunodeficiency with neutrophil, platelet, and T- and B-cell defects. OBJECTIVE: We sought to unravel the genetic cause of combined immunodeficiency and early-onset immune dysregulation in a 26-year-old man who presented with specific antibody deficiency, autoimmunity, and inflammatory bowel disease since early childhood. METHODS: The patient was subjected to whole-exome sequencing of genomic DNA and examination of blood neutrophils, platelets, and T and B cells. Expression levels of the Src homology domain 2-containing leukocyte protein of 76 kDa (SLP76) and tonic and ligand-induced PI3K signaling were evaluated by flow-cytometric detection of phosphorylated ribosomal protein S6 in B and T cells. RESULTS: Compound heterozygous missense variants were identified in LCP2, affecting the proline-rich repeat domain of SLP76 (p.P190R and p.R204W). The patient's total B- and T-cell numbers were within the normal range, as was platelet function. However, neutrophil function, numbers of unswitched and class-switched memory B cells, and serum IgA were decreased. Moreover, intracellular SLP76 protein levels were reduced in the patient's B cells, CD4+ and CD8+ T cells, and natural killer cells. Tonic and ligand-induced levels of phosphorylated ribosomal protein S6 and ligand-induced phosphorylated PLCγ1 were decreased in the patient's B cells and CD4+ and CD8+ T cells. CONCLUSIONS: Biallelic variants in LCP2 impair neutrophil function and T-cell and B-cell antigen-receptor signaling and can cause combined immunodeficiency with early-onset immune dysregulation, even in the absence of platelet defects.


Asunto(s)
Fosfatidilinositol 3-Quinasas , Inmunodeficiencia Combinada Grave , Masculino , Niño , Humanos , Preescolar , Adulto , Fosfatidilinositol 3-Quinasas/genética , Linfocitos T CD8-positivos , Ligandos , Proteína S6 Ribosómica/genética , Transducción de Señal/genética , Inmunodeficiencia Combinada Grave/genética , Inmunodeficiencia Combinada Grave/diagnóstico , Mutación
3.
J Clin Immunol ; 43(3): 625-635, 2023 04.
Artículo en Inglés | MEDLINE | ID: mdl-36474126

RESUMEN

Increased susceptibility to develop severe forms of Epstein-Barr virus (EBV) infection in early age is a significant hallmark of an underlying primary immunodeficiency (PID). Here, we present immunologic and genetic evaluations of a 3-year-old child who was born to first-cousins parents and presented with recurrent infections, failure to thrive, and severe EBV-related infection and proliferation. A diagnosis of diffuse large B cell lymphoma was made and the immunological workup was suggestive of T cell immunodeficiency. Unfortunately, the patient succumbed to EBV-related lymphoma. Whole-exome sequencing revealed a novel homozygous mutation, c.991del.C; p. Q331Sfs*6 in the SLP76 gene. The SLP76 protein, a TCR signaling molecule, was recently linked to a human disease of the immune system. In order to examine the effect of this new SLP76 mutation on T cell signaling, a SLP76-deficient Jurkat-derived T cell line was transduced either with wild-type (WT), or with the specific SLP76 mutant, or with a mock vector. Downstream TCR signaling events, including ERK1/2 phosphorylation, CD69 expression, and Ca2 + mobilization, were reduced in cells harboring the reported mutation, linking this novel mutation to the expected immunological outcome. SLP76 deficiency should be added to the growing list of monogenetic diseases that predispose affected individuals to acquire severe and uncontrolled EBV infections and to develop substantial complications. This case further links mutations in the SLP76 gene to a significant human immunodeficiency and extends its clinical phenotype.


Asunto(s)
Infecciones por Virus de Epstein-Barr , Síndromes de Inmunodeficiencia , Linfoma , Enfermedades de Inmunodeficiencia Primaria , Preescolar , Humanos , Herpesvirus Humano 4 , Síndromes de Inmunodeficiencia/diagnóstico , Linfoma/complicaciones , Mutación , Enfermedades de Inmunodeficiencia Primaria/complicaciones , Receptores de Antígenos de Linfocitos T/genética
4.
J Immunol ; 206(10): 2322-2337, 2021 05 15.
Artículo en Inglés | MEDLINE | ID: mdl-33931484

RESUMEN

The costimulatory receptor CD28 synergizes with the TCR to promote IL-2 production, cell survival, and proliferation; yet the obligatory interdependence of TCR and CD28 signaling is not well understood. Upon TCR stimulation, Gads, a Grb2-family adaptor, bridges the interaction of two additional adaptors, LAT and SLP-76, to form a TCR-induced effector signaling complex. SLP-76 binds the Tec-family tyrosine kinase, Itk, which phosphorylates SLP-76 Y173 and PLC-γ1 Y783. In this study, we identified TCR-inducible, Itk-mediated phosphorylation of Gads Y45 in a human T cell line and in mouse primary T cells. Y45 is found within the N-terminal SH3 domain of Gads, an evolutionarily conserved domain with no known signaling function. Gads Y45 phosphorylation depended on the interaction of Gads with SLP-76 and on the dimerization-dependent binding of Gads to phospho-LAT. We provide evidence that Itk acts through SLP-76 and Gads to promote the TCR/CD28-induced activation of the RE/AP transcriptional element from the IL-2 promoter. Two Itk-related features of SLP-76, Y173 and a proline-rich Itk SH3 binding motif on SLP-76, were dispensable for activation of NFAT but selectively required for the TCR/CD28-induced increase in cytoplasmic and nuclear c-Rel and consequent RE/AP activation. We provide evidence that unphosphorylated, monomeric Gads mediates an RE/AP-directed inhibitory activity that is mitigated upon Gads dimerization and Y45 phosphorylation. This study illuminates a new, to our knowledge, regulatory module, in which TCR-induced, Itk-mediated phosphorylation sites on SLP-76 and Gads control the transcriptional response to TCR/CD28 costimulation, thus enforcing the obligatory interdependence of the TCR and CD28 signaling pathways.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/deficiencia , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Antígenos CD28/metabolismo , Fosfoproteínas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Transducción de Señal/genética , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Dimerización , Vectores Genéticos , Humanos , Interleucina-2/metabolismo , Células Jurkat , Ratones , Ratones Endogámicos BALB C , Fosfoproteínas/genética , Fosforilación/genética , Unión Proteica , Transfección
5.
EMBO J ; 30(15): 3160-72, 2011 Jul 01.
Artículo en Inglés | MEDLINE | ID: mdl-21725281

RESUMEN

Cooperatively assembled signalling complexes, nucleated by adaptor proteins, integrate information from surface receptors to determine cellular outcomes. In T and mast cells, antigen receptor signalling is nucleated by three adaptors: SLP-76, Gads and LAT. Three well-characterized SLP-76 tyrosine phosphorylation sites recruit key components, including a Tec-family tyrosine kinase, Itk. We identified a fourth, evolutionarily conserved SLP-76 phosphorylation site, Y173, which was phosphorylated upon T-cell receptor stimulation in primary murine and Jurkat T cells. Y173 was required for antigen receptor-induced phosphorylation of phospholipase C-γ1 (PLC-γ1) in both T and mast cells, and for consequent downstream events, including activation of the IL-2 promoter in T cells, and degranulation and IL-6 production in mast cells. In intact cells, Y173 phosphorylation depended on three, ZAP-70-targeted tyrosines at the N-terminus of SLP-76 that recruit and activate Itk, a kinase that selectively phosphorylated Y173 in vitro. These data suggest a sequential mechanism whereby ZAP-70-dependent priming of SLP-76 at three N-terminal sites triggers reciprocal regulatory interactions between Itk and SLP-76, which are ultimately required to couple active Itk to its substrate, PLC-γ1.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Activación de Linfocitos , Mastocitos/inmunología , Fosfoproteínas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Transducción de Señal , Linfocitos T/inmunología , Proteína Tirosina Quinasa ZAP-70/metabolismo , Animales , Células Cultivadas , Humanos , Interleucina-2/metabolismo , Interleucina-6/metabolismo , Ratones , Fosfolipasa C gamma/metabolismo , Fosforilación , Tirosina/metabolismo
6.
J Immunol ; 183(9): 5756-67, 2009 Nov 01.
Artículo en Inglés | MEDLINE | ID: mdl-19812192

RESUMEN

Engagement of the TCR or of chemokine receptors such as CXCR4 induces adhesion and migration of T cells via so-called inside-out signaling pathways. The molecular processes underlying inside-out signaling events are as yet not completely understood. In this study, we show that TCR- and CXCR4-mediated activation of integrins critically depends on the membrane recruitment of the adhesion- and degranulation-promoting adapter protein (ADAP)/Src kinase-associated phosphoprotein of 55 kDa (SKAP55)/Rap1-interacting adapter protein (RIAM)/Rap1 module. We further demonstrate that the Src homology 2 domain containing leukocyte-specific phosphoprotein of 76 kDa (SLP76) is crucial for TCR-mediated inside-out signaling and T cell/APC interaction. Besides facilitating membrane recruitment of ADAP, SKAP55, and RIAM, SLP76 regulates TCR-mediated inside-out signaling by controlling the activation of Rap1 as well as Rac-mediated actin polymerization. Surprisingly, however, SLP76 is not mandatory for CXCR4-mediated inside-out signaling. Indeed, both CXCR4-induced T cell adhesion and migration are not affected by loss of SLP76. Moreover, after CXCR4 stimulation, the ADAP/SKAP55/RIAM/Rap1 module is recruited to the plasma membrane independently of SLP76. Collectively, our data indicate a differential requirement for SLP76 in TCR- vs CXCR4-mediated inside-out signaling pathways regulating T cell adhesion and migration.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/fisiología , Quimiotaxis de Leucocito/inmunología , Activación de Linfocitos/inmunología , Antígeno-1 Asociado a Función de Linfocito/fisiología , Fosfoproteínas/fisiología , Receptores CXCR4/fisiología , Transducción de Señal/inmunología , Subgrupos de Linfocitos T/inmunología , Receptores Toll-Like/fisiología , Proteínas Adaptadoras Transductoras de Señales/deficiencia , Proteínas Adaptadoras Transductoras de Señales/genética , Adhesión Celular/genética , Adhesión Celular/inmunología , Células Cultivadas , Quimiocina CXCL12/fisiología , Quimiotaxis de Leucocito/genética , Humanos , Células Jurkat , Activación de Linfocitos/genética , Fosfoproteínas/deficiencia , Fosfoproteínas/genética , Transducción de Señal/genética , Subgrupos de Linfocitos T/citología , Subgrupos de Linfocitos T/metabolismo
7.
J Exp Med ; 218(3)2021 03 01.
Artículo en Inglés | MEDLINE | ID: mdl-33231617

RESUMEN

The T cell receptor (TCR) signaling pathway is an ensemble of numerous proteins that are crucial for an adequate immune response. Disruption of any protein involved in this pathway leads to severe immunodeficiency and unfavorable clinical outcomes. Here, we describe an infant with severe immunodeficiency who was found to have novel biallelic mutations in SLP76. SLP76 is a key protein involved in TCR signaling and in other hematopoietic pathways. Previous studies of this protein were performed using Jurkat-derived human leukemic T cell lines and SLP76-deficient mice. Our current study links this gene, for the first time, to a human immunodeficiency characterized by early-onset life-threatening infections, combined T and B cell immunodeficiency, severe neutrophil defects, and impaired platelet aggregation. Hereby, we characterized aspects of the patient's immune phenotype, modeled them with an SLP76-deficient Jurkat-derived T cell line, and rescued some consequences using ectopic expression of wild-type SLP76. Understanding human diseases due to SLP76 deficiency is helpful in explaining the mixed T cell and neutrophil defects, providing a guide for exploring human SLP76 biology.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/deficiencia , Plaquetas/patología , Neutrófilos/patología , Fosfoproteínas/deficiencia , Inmunodeficiencia Combinada Grave/metabolismo , Inmunodeficiencia Combinada Grave/patología , Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Plaquetas/metabolismo , Resultado Fatal , Humanos , Lactante , Recién Nacido , Células Jurkat , Mutación/genética , Neutrófilos/metabolismo , Fenotipo , Fosfoproteínas/química , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Receptores de Antígenos de Linfocitos B/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Inmunodeficiencia Combinada Grave/inmunología , Transducción de Señal
8.
Front Immunol ; 10: 1704, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-31402911

RESUMEN

Antigen receptor signaling pathways are organized by adaptor proteins. Three adaptors, LAT, Gads, and SLP-76, form a heterotrimeric complex that mediates signaling by the T cell antigen receptor (TCR) and by the mast cell high affinity receptor for IgE (FcεRI). In both pathways, antigen recognition triggers tyrosine phosphorylation of LAT and SLP-76. The recruitment of SLP-76 to phospho-LAT is bridged by Gads, a Grb2 family adaptor composed of two SH3 domains flanking a central SH2 domain and an unstructured linker region. The LAT-Gads-SLP-76 complex is further incorporated into larger microclusters that mediate antigen receptor signaling. Gads is positively regulated by dimerization, which promotes its cooperative binding to LAT. Negative regulation occurs via phosphorylation or caspase-mediated cleavage of the linker region of Gads. FcεRI-mediated mast cell activation is profoundly impaired in LAT- Gads- or SLP-76-deficient mice. Unexpectedly, the thymic developmental phenotype of Gads-deficient mice is much milder than the phenotype of LAT- or SLP-76-deficient mice. This distinction suggests that Gads is not absolutely required for TCR signaling, but may modulate its sensitivity, or regulate a particular branch of the TCR signaling pathway; indeed, the phenotypic similarity of Gads- and Itk-deficient mice suggests a functional connection between Gads and Itk. Additional Gads binding partners include costimulatory proteins such as CD28 and CD6, adaptors such as Shc, ubiquitin regulatory proteins such as USP8 and AMSH, and kinases such as HPK1 and BCR-ABL, but the functional implications of these interactions are not yet fully understood. No interacting proteins or function have been ascribed to the evolutionarily conserved N-terminal SH3 of Gads. Here we explore the biochemical and functional properties of Gads, and its role in regulating allergy, T cell development and T-cell mediated immunity.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteína Adaptadora GRB2/metabolismo , Hipersensibilidad/inmunología , Hipersensibilidad/metabolismo , Inmunidad Celular , Transducción de Señal , Animales , Biomarcadores , Susceptibilidad a Enfermedades , Humanos , Inmunomodulación , Activación de Linfocitos/inmunología , Modelos Biológicos , Complejos Multiproteicos , Unión Proteica , Receptores de Antígenos de Linfocitos T/metabolismo
9.
Biochem J ; 404(3): 487-97, 2007 Jun 15.
Artículo en Inglés | MEDLINE | ID: mdl-17355222

RESUMEN

The Paks (p21-activated kinases) Pak1, Pak2 and Pak3 are among the most studied effectors of the Rho-family GTPases, Rac, Cdc42 (cell division cycle 42) and Chp (Cdc42 homologous protein). Pak kinases influence a variety of cellular functions, but the process of Pak down-regulation, following activation, is poorly understood. In the present study, we describe for the first time a negative-inhibitory loop generated by the small Rho-GTPases Cdc42 and Chp, resulting in Pak1 inhibition. Upon overexpression of Chp, we unexpectedly observed a T-cell migration phenotype consistent with Paks inhibition. In line with this observation, overexpression of either Chp or Cdc42 caused a marked reduction in the level of Pak1 protein in a number of different cell lines. Chp-induced degradation was accompanied by ubiquitination of Pak1, and was dependent on the proteasome. The susceptibility of Pak1 to Chp-induced degradation depended on its p21-binding domain, kinase activity and a number of Pak1 autophosphorylation sites, whereas the PIX- (Pak-interacting exchange factor) and Nck-binding sites were not required. Together, these results implicate Chp-induced kinase autophosphorylation in the degradation of Pak1. The N-terminal domain of Chp was found to be required for Chp-induced degradation, although not for Pak1 activation, suggesting that Chp provides a second function, distinct from kinase activation, to trigger Pak degradation. Collectively, our results demonstrate a novel mechanism of signal termination mediated by the Rho-family GTPases Chp and Cdc42, which results in ubiquitin-mediated degradation of one of their direct effectors, Pak1.


Asunto(s)
Proteínas de Unión al GTP/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal/fisiología , Proteínas de Unión al GTP rho/metabolismo , Animales , Línea Celular , Movimiento Celular/fisiología , Proteínas de Unión al GTP/genética , Regulación de la Expresión Génica , Humanos , Proteínas de Neoplasias/genética , Fosforilación , Complejo de la Endopetidasa Proteasomal/metabolismo , Unión Proteica , Proteínas Serina-Treonina Quinasas/genética , Linfocitos T/citología , Linfocitos T/fisiología , Ubiquitina/metabolismo , Quinasas p21 Activadas , Proteínas de Unión al GTP rho/genética
10.
Biochem J ; 397(1): 213-22, 2006 Jul 01.
Artículo en Inglés | MEDLINE | ID: mdl-16515536

RESUMEN

Pak (p21-activated kinase) serine/threonine kinases have been shown to mediate directional sensing of chemokine gradients. We hypothesized that Pak may also mediate chemokine-induced shape changes, to facilitate leucocyte chemotaxis through restrictive barriers, such as the extracellular matrix. A potent inhibitor, Pak(i), was characterized and used to probe the role of Pak-family kinases in SDF-1alpha (stromal-cell derived factor-1alpha/CXCL12)-induced chemotaxis in a T cell model. Pak(i) potently inhibited SDF-1alpha-induced Pak activation by a bivalent mechanism, as indicated by its complete inactivation upon point mutation of two binding sites, but partial inactivation upon mutation of either site alone. Importantly, Pak(i) was not toxic to cells over the time frame of our experiments, since it did not substantially affect cell surface expression of CXCR4 (CXC chemokine receptor 4) or integrins, cell cycle progression, or a number of ligand-induced responses. Pak(i) produced dose-dependent inhibition of SDF-1alpha-induced migration through rigid filters bearing small pores; but unexpectedly, did not substantially affect the magnitude or kinetics of chemotaxis through filters bearing larger pores. SDF-1alpha-induced Pak activation was partly dependent on PIX (Pak-interactive exchange factor); correspondingly, an allele of beta-PIX that cannot bind Pak inhibited SDF-1alpha-induced chemotaxis through small, but not large pores. By contrast, other key players in chemotaxis: G(i), PI3K (phosphoinositide 3-kinase), and the Rho-family G-proteins, Rac and Cdc42 (cell division cycle 42), were required for SDF-1alpha-induced migration regardless of the barrier pore-size. These studies have revealed a distinct branch of the SDF-1alpha signalling pathway, in which the Rac/Cdc42 effector, Pak, and its partner, PIX, specifically regulate the cellular events required for chemokine-induced migration through restrictive barriers.


Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Quimiotaxis , Factores de Intercambio de Guanina Nucleótido/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Linfocitos T/fisiología , Forma de la Célula , Quimiocina CXCL12 , Quimiocinas CXC/fisiología , Citoesqueleto , Humanos , Células Jurkat , Factores de Intercambio de Guanina Nucleótido Rho , Transfección , Quinasas p21 Activadas
11.
Sci Signal ; 10(498)2017 Sep 26.
Artículo en Inglés | MEDLINE | ID: mdl-28951535

RESUMEN

The accurate assembly of signalosomes centered on the adaptor protein LAT (linker of activated T cells) is required for antigen receptor signaling in T cells and mast cells. During signalosome assembly, members of the growth factor receptor-bound protein 2 (Grb2) family of cytosolic adaptor proteins bind cooperatively to LAT through interactions with its phosphorylated tyrosine (pTyr) residues. We demonstrated the Src homology 2 (SH2) domain-mediated dimerization of the Grb2 family member, Grb2-related adaptor downstream of Shc (Gads). Gads dimerization was mediated by an SH2 domain interface, which is distinct from the pTyr binding pocket and which promoted cooperative, preferential binding of paired Gads to LAT. This SH2 domain-intrinsic mechanism of cooperativity, which we quantified by mathematical modeling, enabled Gads to discriminate between dually and singly phosphorylated LAT molecules. Mutational inactivation of the dimerization interface reduced cooperativity and abrogated Gads signaling in T cells and mast cells. The dimerization-dependent, cooperative binding of Gads to LAT may increase antigen receptor sensitivity by reducing signalosome formation at incompletely phosphorylated LAT molecules, thereby prioritizing the formation of complete signalosomes.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteína Adaptadora GRB2/metabolismo , Proteínas de la Membrana/metabolismo , Multimerización de Proteína , Receptores de Antígenos de Linfocitos T/metabolismo , Linfocitos T/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Proteína Adaptadora GRB2/genética , Humanos , Células Jurkat , Mastocitos/metabolismo , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos BALB C , Modelos Biológicos , Complejos Multiproteicos/metabolismo , Mutación , Fosforilación , Cultivo Primario de Células , Tirosina/metabolismo , Dominios Homologos src/fisiología
12.
Cell Signal ; 27(1): 125-34, 2015 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-25452106

RESUMEN

T cell antigen receptor (TCR) signaling depends on three interacting adaptor proteins: SLP-76, Gads, and LAT. Their mechanisms of signaling have been extensively explored, with the aid of fortuitously isolated LAT- and SLP-76-deficient T cell lines, but no such tools were available for Gads, a Grb2-family adaptor that bridges the TCR-inducible interaction between SLP-76 and LAT. TALEN-directed genome editing was applied to disrupt the first coding exon of human Gads in the Jurkat T cell line. Gads was dispensable for TCR-induced phosphorylation of SLP-76, but was a dose-dependent amplifier of TCR-induced CD69 expression. Gads conferred responsiveness to weak TCR stimuli, leading to PLC-γ1 phosphorylation and calcium flux. TALEN-derived, Gads-deficient T cell lines provide a uniquely tractable genetic platform for exploring its regulatory features, such as Gads phosphorylation at T262, which we observed by mass spectrometry. Upon mutation of this site, TCR responsiveness and sensitivity to weak TCR stimuli were increased. This study demonstrates the feasibility of TALEN-based reverse genetics in Jurkat T cells, while enriching our understanding of Gads as a regulated modulator of TCR sensitivity.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Antígenos CD/metabolismo , Antígenos de Diferenciación de Linfocitos T/metabolismo , Secuencia de Bases , Endonucleasas/metabolismo , Genoma Humano/genética , Humanos , Células Jurkat , Lectinas Tipo C/metabolismo , Datos de Secuencia Molecular , Mutación/genética , Fosfolipasa C gamma/metabolismo , Fosfoproteínas/metabolismo , Fosforilación , Fosfotreonina/metabolismo , Unión Proteica , Transducción de Señal , Transactivadores/metabolismo
13.
J Cell Biol ; 195(5): 839-53, 2011 Nov 28.
Artículo en Inglés | MEDLINE | ID: mdl-22105350

RESUMEN

Antigen recognition within immunological synapses triggers and sustains T cell activation by nucleating protein microclusters that gather T cell receptors (TCRs), kinases, and adaptors. Dissipation of these microclusters results in signal termination, but how this process is regulated is unclear. In this paper, we reveal that release of the adaptors SLP76 and GADS from signaling microclusters is induced by the serine/threonine protein kinase HPK1 and that phosphorylation of GADS plays a major role in this process. We found that HPK1 was recruited into microclusters and triggered their dissipation by inducing the phosphorylation of a threonine-containing motif of GADS, together with the previously described serine phosphorylation of SLP76. These events induced the cooperative binding of 14-3-3 proteins to SLP76-GADS complexes, leading to their uncoupling from the transmembrane adaptor LAT and consequently reducing microcluster persistence and activation-induced gene transcription. These results demonstrate that serine/threonine phosphorylation of multiple TCR-proximal effectors controls the stability of signaling microclusters, thereby determining the intensity of T cell responses.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Activación de Linfocitos , Fosfoproteínas/metabolismo , Linfocitos T/fisiología , Proteínas 14-3-3/metabolismo , Regulación hacia Abajo , Humanos , Sinapsis Inmunológicas , Células Jurkat , Fosforilación , Proteínas Serina-Treonina Quinasas/metabolismo , Receptores de Antígenos de Linfocitos T/inmunología , Transducción de Señal/inmunología
14.
Proc Natl Acad Sci U S A ; 104(16): 6638-43, 2007 Apr 17.
Artículo en Inglés | MEDLINE | ID: mdl-17420479

RESUMEN

ITK (IL-2-inducible T cell kinase), a Tec family protein tyrosine kinase (PTK), is one of three PTKs required for T cell antigen receptor (TCR)-induced activation of phospholipase C-gamma1 (PLC-gamma1). Like Src and Abl family PTKs, ITK adopts an inactive, "closed" conformation, and its conversion to the active conformation is not well understood, nor have its direct substrates been identified. In a side-by-side comparison of ITK and ZAP-70 (zeta chain-associated protein kinase of 70 kDa), ITK efficiently phosphorylated Y(783) and Y(775) of PLC-gamma1, two phosphorylation sites that are critical for its activation, whereas ZAP-70 did not. SLP-76 (SH2-domain-containing leukocyte protein of 76 kDa), an adaptor required for TCR-induced activation of PLC-gamma1, was required for the phosphorylation of both PLC-gamma1 sites in intact cells. Furthermore, this event depended on the N-terminal tyrosines of SLP-76. Likewise, SLP-76, particularly its N-terminal tyrosines, was required for TCR-induced tyrosine phosphorylation and activation of ITK but was not required for the phosphorylation or activation of ZAP-70. Both ZAP-70 and ITK phosphorylated SLP-76 in vitro; thus, both PTKs are potential regulators of SLP-76, but only ITK is regulated by SLP-76. Upon TCR stimulation, a small fraction of ITK bound to SLP-76. This fraction, however, encompassed most of the catalytically active ITK. Catalytic activity was lost upon mild elution of ITK from the SLP-76-nucleated complex but was restored upon reconstitution of the complex. We propose that SLP-76 is required for ITK activation; furthermore, an ongoing physical interaction between SLP-76 and ITK is required to maintain ITK in an active conformation.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/fisiología , Fosfoproteínas/fisiología , Proteínas Tirosina Quinasas/metabolismo , Receptores de Antígenos de Linfocitos T/fisiología , Activación Enzimática/fisiología , Humanos , Células Jurkat , Conformación Proteica , Proteínas Tirosina Quinasas/química
15.
J Biol Chem ; 282(5): 2937-46, 2007 Feb 02.
Artículo en Inglés | MEDLINE | ID: mdl-17148460

RESUMEN

Phospholipase C-gamma1 (PLC-gamma1) activation depends on a heterotrimeric complex of adaptor proteins composed of LAT, Gads, and SLP-76. Upon T cell receptor stimulation, a portion of PLC-gamma1 is recruited to a detergent-resistant membrane fraction known as the glycosphingolipid-enriched membrane microdomains (GEMs), or lipid rafts, to which LAT is constitutively localized. In addition to LAT, PLC-gamma1 GEM recruitment depended on SLP-76, and, in particular, required the Gads-binding domain of SLP-76. The N-terminal tyrosine phosphorylation sites and P-I region of SLP-76 were not required for PLC-gamma1 GEM recruitment, but were required for PLC-gamma1 phosphorylation at Tyr(783). Thus, GEM recruitment can be insufficient for full activation of PLC-gamma1 in the absence of a second SLP-76-mediated event. Indeed, a GEM-targeted derivative of PLC-gamma1 depended on SLP-76 for T cell receptor-induced phosphorylation at Tyr783 and subsequent NFAT activation. On a biochemical level, SLP-76 inducibly associated with both Vav and catalytically active ITK, which efficiently phosphorylated a PLC-gamma1 fragment at Tyr783 in vitro. Both associations were disrupted upon mutation of the N-terminal tyrosine phosphorylation sites of SLP-76. The P-I region deletion disrupted Vav association and reduced SLP-76-associated kinase activity. A smaller deletion within the P-I region, which does not impair PLC-gamma1 activation, did not impair the association with Vav, but reduced SLP-76-associated kinase activity. These results provide new insight into the multiple roles of SLP-76 and the functional importance of its interactions with other signaling proteins.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/fisiología , Fosfolipasa C gamma/metabolismo , Fosfoproteínas/fisiología , Receptores de Antígenos de Linfocitos T/fisiología , Proteínas Adaptadoras Transductoras de Señales/deficiencia , Proteínas Adaptadoras Transductoras de Señales/genética , Línea Celular Tumoral , Activación Enzimática , Eliminación de Gen , Humanos , Células Jurkat , Cinética , Microdominios de Membrana/fisiología , Fosfoproteínas/deficiencia , Fosfoproteínas/genética , Transfección
16.
J Biol Chem ; 280(9): 8364-70, 2005 Mar 04.
Artículo en Inglés | MEDLINE | ID: mdl-15623534

RESUMEN

SLP-76 forms part of a hematopoietic-specific adaptor protein complex, and is absolutely required for T cell development and activation. T cell receptor (TCR)-induced activation of phospholipase C-gamma1 (PLC-gamma1) depends on three features of SLP-76: the N-terminal tyrosine phosphorylation sites, the Gads-binding site, and an intervening sequence, denoted the P-I region, which binds to the SH3 domain of PLC-gamma1 (SH3(PLC)) via a low affinity interaction. Despite extensive research, the mechanism whereby SLP-76 regulates PLC-gamma1 remains uncertain. In this study, we uncover and explore an apparent paradox: whereas the P-I region as a whole is essential for TCR-induced activation of PLC-gamma1 and nuclear factor of activated T cells (NFAT), no particular part of this region is absolutely required. To better understand the contribution of the P-I region to PLC-gamma1 activation, we mapped the PLC-gamma1-binding site within the region, and created a SLP-76 mutant that fails to bind SH3(PLC), but is fully functional, mediating TCR-induced phosphorylation of PLC-gamma1 at tyrosine 783, calcium flux, and nuclear factor of activated T cells activation. Unexpectedly, full functionality of this mutant was maintained even under less than optimal stimulation conditions, such as a low concentration of the anti-TCR antibody. Another SLP-76 mutant, in which the P-I region was scrambled to abolish any sequence-dependent protein-binding motifs, also retained significant functionality. Our results demonstrate that SLP-76 need not interact with SH3(PLC) to activate PLC-gamma1, and further suggest that the P-I region of SLP-76 serves a structural role that is sequence-independent and is not directly related to protein-protein interactions.


Asunto(s)
Fosfoproteínas/química , Fosfoproteínas/fisiología , Receptores de Antígenos de Linfocitos T/metabolismo , Fosfolipasas de Tipo C/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Alelos , Secuencias de Aminoácidos , Sitios de Unión , Calcio/metabolismo , Línea Celular , Epítopos/química , Eliminación de Gen , Genes Reporteros , Glutatión Transferasa/metabolismo , Humanos , Inmunoprecipitación , Células Jurkat , Luciferasas/metabolismo , Mutación , Fosfolipasa C gamma , Prolina/química , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/metabolismo , Relación Estructura-Actividad , Factores de Tiempo , Transfección , Dominios Homologos src
17.
J Immunol ; 171(2): 860-6, 2003 Jul 15.
Artículo en Inglés | MEDLINE | ID: mdl-12847255

RESUMEN

Engagement of the T cell with Ag on an APC results in a series of immediate signaling events emanating from the stimulation of the TCR. These events include the induced phosphorylation of a number of cellular proteins with a subsequent increase in intracellular calcium and the restructuring of the microtubule and actin cytoskeleton within the T cell. This restructuring of the cytoskeleton culminates in the polarization of the T cell's secretory apparatus toward the engaging APC, allowing the T cell to direct secretion of cytokines toward the appropriate APC. This polarization can be monitored by analyzing the position of the microtubule-organizing center (MTOC), as it moves toward the interface of the T cell and APC. The requirements for MTOC polarization were examined at a single-cell level by studying the interaction of a Jurkat cell line expressing a fluorescently labeled MTOC with Staphylococcal enterotoxin superantigen-bound Raji B cell line, which served as the APC. We found that repolarization of the MTOC substantially followed fluxes in calcium. We also used immobilized anti-TCR mAb and Jurkat signaling mutants, defective in TCR-induced calcium increases, to determine whether signaling components that are necessary for a calcium response also play a role in MTOC polarization. We found that zeta-associated protein-70 as well as its substrate adaptor proteins linker for activation of T cells and Src homology 2 domain-containing leukocyte protein-76 are required for MTOC polarization. Moreover, our studies revealed that a calcium-dependent event not requiring calcineurin or calcium/calmodulin-dependent kinase is required for TCR-induced polarization of the MTOC.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Complejo CD3 , Proteínas Portadoras/fisiología , Activación de Linfocitos , Proteínas de la Membrana , Centro Organizador de los Microtúbulos/metabolismo , Fosfoproteínas/fisiología , Proteínas Tirosina Quinasas/fisiología , Receptores de Antígenos de Linfocitos T/fisiología , Linfocitos T/inmunología , Linfocitos T/metabolismo , Anticuerpos Monoclonales/farmacología , Células Presentadoras de Antígenos/metabolismo , Bencilaminas/farmacología , Calcineurina/fisiología , Inhibidores de la Calcineurina , Calcio/metabolismo , Calcio/fisiología , Señalización del Calcio/inmunología , Proteínas Quinasas Dependientes de Calcio-Calmodulina/antagonistas & inhibidores , Proteínas Quinasas Dependientes de Calcio-Calmodulina/fisiología , Polaridad Celular/inmunología , Humanos , Células Jurkat , Centro Organizador de los Microtúbulos/enzimología , Receptores de Antígenos de Linfocitos T/antagonistas & inhibidores , Receptores de Antígenos de Linfocitos T/inmunología , Sulfonamidas/farmacología , Linfocitos T/enzimología , Tacrolimus/farmacología , Células Tumorales Cultivadas , Proteína Tirosina Quinasa ZAP-70
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