RESUMEN
Neurotrophins mediate neuronal growth, differentiation, and survival via tropomyosin receptor kinase (Trk) or p75 neurotrophin receptor (p75NTR ) signaling. The p75NTR is not exclusively expressed by neurons but also by certain immune cells, implying a role for neurotrophin signaling in the immune system. In this study, we investigated the effect of p75NTR on innate immune cell behavior and on neuronal morphology upon chronic Toxoplasma gondii (T. gondii) infection-induced neuroinflammation. Characterization of the immune cells in the periphery and central nervous system (CNS) revealed that innate immune cell subsets in the brain upregulated p75NTR upon infection in wild-type mice. Although cell recruitment and phagocytic capacity of p75NTRexonIV knockout (p75-/- ) mice were not impaired, the activation status of resident microglia and recruited myeloid cell subsets was altered. Importantly, recruited mononuclear cells in brains of infected p75-/- mice upregulated the production of the cytokines interleukin (IL)-10, IL-6 as well as IL-1α. Protein levels of proBDNF, known to negatively influence neuronal morphology by binding p75NTR , were highly increased upon chronic infection in the brain of wild-type and p75-/- mice. Moreover, upon infection the activated immune cells contributed to the proBDNF release. Notably, the neuroinflammation-induced changes in spine density were rescued in the p75-/- mice. In conclusion, these findings indicate that neurotrophin signaling via the p75NTR affects innate immune cell behavior, thus, influencing the structural plasticity of neurons under inflammatory conditions.
Asunto(s)
Leucocitos Mononucleares/fisiología , Neuronas/fisiología , Receptor de Factor de Crecimiento Nervioso/fisiología , Toxoplasma , Toxoplasmosis/inmunología , Animales , Femenino , Inmunidad Innata/fisiología , Inflamación/inmunología , Inflamación/patología , Leucocitos Mononucleares/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Toxoplasmosis/patologíaRESUMEN
Regulatory T (Treg) cells require T-cell receptor (TCR) signalling to exert their immunosuppressive activity, but the precise organization of the TCR signalling network compared to conventional T (Tconv) cells remains elusive. By using accurate mass spectrometry and multi-epitope ligand cartography (MELC) we characterized TCR signalling and recruitment of TCR signalling components to the immunological synapse (IS) in Treg cells and Tconv cells. With the exception of Themis which we detected in lower amounts in Treg cells, other major TCR signalling components were found equally abundant, however, their phosphorylation-status notably discriminates Treg cells from Tconv cells. Overall, this study identified 121 Treg cell-specific phosphorylations. Short-term triggering of T cell subsets via CD3 and CD28 widely harmonized these variations with the exception of eleven TCR signalling components that mainly regulate cytoskeleton dynamics and molecular transport. Accordingly, conjugation with B cells indeed caused variant cellular morphology and revealed a Treg cell-specific recruitment of TCR signalling components such as PKCθ, PLCγ1 and ZAP70 as well as B cell-derived CD86 into the IS. Together, results from this study support the existence of a Treg cell-specific IS and suggest Treg cell-specific cytoskeleton dynamics as a novel determinant for the unique functional properties of Treg cells.
Asunto(s)
Sinapsis Inmunológicas/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Transducción de Señal/inmunología , Linfocitos T Reguladores/inmunología , Animales , Células Cultivadas , Femenino , Ratones Endogámicos BALB C , Microscopía Fluorescente , Fosforilación , Proteoma/inmunología , Proteoma/metabolismo , Proteómica/métodos , Receptores de Antígenos de Linfocitos T/metabolismo , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Linfocitos T Reguladores/metabolismo , Proteína Tirosina Quinasa ZAP-70/inmunología , Proteína Tirosina Quinasa ZAP-70/metabolismoRESUMEN
Microglia cells are brain macrophages whose proper functioning is essential for maintenance and repair processes of the central nervous system (CNS). Migration and phagocytosis are critical aspects of microglial activity. By using genetically modified cell lines and knockout mice we demonstrate here that the receptor protein-tyrosine phosphatase (PTP) DEP-1 (also known as PTPRJ or CD148) acts as a positive regulator of both processes in vitro and in vivo. Notably, reduced microglial migration was detectable in brains of Ptprj-/- mice using a wounding assay. Mechanistically, density-enhanced phosphatase-1 (DEP-1) may in part function by inhibiting the activity of the Src family kinase Fyn. In the microglial cell line BV2 DEP-1 depletion by shRNA-mediated knockdown resulted in enhanced phosphorylation of the Fyn activating tyrosine (Tyr420 ) and elevated specific Fyn-kinase activity in immunoprecipitates. Moreover, Fyn mRNA and protein levels were reduced in DEP-1 deficient microglia cells. Consistent with a negative regulatory role of Fyn for microglial functions, which is inhibited by DEP-1, microglial cells from Fyn-/- mice exhibited elevated migration and phagocytosis. Enhanced microglia migration to a site of injury was also observed in Fyn-/- mice in vivo. Taken together our data revealed a previously unrecognized role of DEP-1 and suggest the existence of a potential DEP-1-Fyn axis in the regulation of microglial functions. GLIA 2017;65:416-428.
Asunto(s)
Movimiento Celular/fisiología , Regulación de la Expresión Génica/genética , Microglía/fisiología , Fagocitosis/genética , Proteínas Proto-Oncogénicas c-fyn/metabolismo , Animales , Animales Recién Nacidos , Línea Celular Transformada , Movimiento Celular/genética , Células Cultivadas , Corteza Cerebral/citología , Inmunoprecipitación , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fagocitosis/fisiología , Proteínas Proto-Oncogénicas c-fyn/genética , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Proteínas Tirosina Fosfatasas Clase 3 Similares a Receptores/genética , Proteínas Tirosina Fosfatasas Clase 3 Similares a Receptores/metabolismoRESUMEN
TCR ligation is critical for the selection, activation, and integrin expression of T lymphocytes. Here, we explored the role of the TCR adaptor protein slp-76 on iNKT-cell biology. Compared to B6 controls, slp-76(ace/ace) mice carrying a missense mutation (Thr428Ile) within the SH2-domain of slp-76 showed an increase in iNKT cells in the thymus and lymph nodes, but a decrease in iNKT cells in spleens and livers, along with reduced ADAP expression and cytokine response. A comparable reduction in iNKT cells was observed in the livers and spleens of ADAP-deficient mice. Like ADAP(-/-) iNKT cells, slp-76(ace/ace) iNKT cells were characterized by enhanced CD11b expression, correlating with an impaired induction of the TCR immediate-early gene Nur77 and a decreased adhesion to ICAM-1. Furthermore, CD11b-intrinsic effects inhibited cytokine release, concanavalin A-mediated inflammation, and iNKT-cell accumulation in the liver. Unlike B6 and ADAP(-/-) mice, the expression of the transcription factors Id3 and PLZF was reduced, whereas NP-1-expression was enhanced in slp-76(ace/ace) mice. Blockade of NP-1 decreased the recovery of iNKT cells from peripheral lymph nodes, identifying NP-1 as an iNKT-cell-specific adhesion factor. Thus, slp-76 contributes to the regulation of the tissue distribution, PLZF, and cytokine expression of iNKT cells via ADAP-dependent and -independent mechanisms.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Citocinas/biosíntesis , Mutación , Células T Asesinas Naturales/inmunología , Células T Asesinas Naturales/metabolismo , Fosfoproteínas/genética , Dominios Homologos src/genética , Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Biomarcadores , Proteína C-Reactiva/genética , Proteína C-Reactiva/metabolismo , Antígeno CD11b/genética , Antígeno CD11b/metabolismo , Eliminación de Gen , Expresión Génica , Hepatitis/etiología , Hepatitis/metabolismo , Hepatitis/patología , Proteínas Inhibidoras de la Diferenciación/genética , Proteínas Inhibidoras de la Diferenciación/metabolismo , Molécula 1 de Adhesión Intercelular/metabolismo , Factores de Transcripción de Tipo Kruppel/genética , Factores de Transcripción de Tipo Kruppel/metabolismo , Hígado/inmunología , Ganglios Linfáticos/inmunología , Antígeno-1 Asociado a Función de Linfocito/genética , Antígeno-1 Asociado a Función de Linfocito/metabolismo , Ratones , Ratones Noqueados , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Especificidad de Órganos/inmunología , Fenotipo , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Proteína de la Leucemia Promielocítica con Dedos de Zinc , Unión Proteica , Receptores de Antígenos de Linfocitos T/metabolismo , Transducción de Señal , Bazo/inmunología , Timo/inmunologíaRESUMEN
Stimulation of T cells leads to distinct changes of their adhesive and migratory properties. Signal propagation from activated receptors to integrins depends on scaffolding proteins such as the adhesion and degranulation promoting adaptor protein (ADAP)(1). Here we have comprehensively investigated the phosphotyrosine interactome of ADAP in T cells and define known and novel interaction partners of functional relevance. While most phosphosites reside in unstructured regions of the protein, thereby defining classical SH2 domain interaction sites for master regulators of T cell signaling such as SLP76, Fyn-kinase, and NCK, other binding events depend on structural context. Interaction proteomics using different ADAP constructs comprising most of the known phosphotyrosine motifs as well as the structured domains confirm that a distinct set of proteins is attracted by pY571 of ADAP, including the ζ-chain-associated protein kinase of 70 kDa (ZAP70). The interaction of ADAP and ZAP70 is inducible upon stimulation either of the T cell receptor (TCR) or by chemokine. NMR spectroscopy reveals that the N-terminal SH2 domains within a ZAP70-tandem-SH2 construct is the major site of interaction with phosphorylated ADAP-hSH3(N) and microscale thermophoresis (MST) indicates an intermediate binding affinity (Kd = 2.3 µm). Interestingly, although T cell receptor dependent events such as T cell/antigen presenting cell (APC) conjugate formation and adhesion are not affected by mutation of Y571, migration of T cells along a chemokine gradient is compromised. Thus, although most phospho-sites in ADAP are linked to T cell receptor related functions we have identified a unique phosphotyrosine that is solely required for chemokine induced T cell behavior.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Quimiocina CXCL12/farmacología , Linfocitos T/efectos de los fármacos , Proteína Tirosina Quinasa ZAP-70/genética , Proteínas Adaptadoras Transductoras de Señales/inmunología , Sitios de Unión , Adhesión Celular/efectos de los fármacos , Degranulación de la Célula/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Quimiocina CXCL12/genética , Quimiocina CXCL12/inmunología , Expresión Génica , Humanos , Células Jurkat , Cinética , Modelos Moleculares , Fosforilación/efectos de los fármacos , Cultivo Primario de Células , Unión Proteica , Mapeo de Interacción de Proteínas , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Linfocitos T/citología , Linfocitos T/inmunología , Tirosina/metabolismo , Proteína Tirosina Quinasa ZAP-70/inmunologíaRESUMEN
INTRODUCTION: The Adhesion and Degranulation promoting Adaptor Protein (ADAP) is phosphorylated upon T cell activation and acts as a scaffold for the formation of a signaling complex that integrates molecular interactions between T cell or chemokine receptors, the actin cytoskeleton, and integrin-mediated cellular adhesion and migration. AREAS COVERED: This article reviews current knowledge of the functions of the adapter protein ADAP in T cell signaling with a focus on the role of individual phosphotyrosine (pY) motifs for SH2 domain mediated interactions. The data presented was obtained from literature searches (PubMed) as well as the authors own research on the topic. Expert commentary: ADAP can be regarded as a paradigmatic example of how tyrosine phosphorylation sites serve as dynamic interaction hubs. Molecular crowding at unstructured and redundant sites (pY595, pY651) is contrasted by more specific interactions enabled by the three-dimensional environment of a particular phosphotyrosine motif (pY571).
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Transducción de Señal , Linfocitos T/metabolismo , Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Adaptadoras Transductoras de Señales/fisiología , Animales , Humanos , Fosforilación , Tirosina/química , Tirosina/metabolismoRESUMEN
Adaptor proteins mediate protein-protein interactions in signal transduction cascades. These signaling molecules are organized in multimolecular complexes that translate information from cell surface receptors into cellular responses. The cytosolic adhesion- and degranulation-promoting adaptor protein (ADAP) is expressed in T cells, natural killer cells, myeloid cells, and platelets. Here we summarize the data about the function of ADAP in these cells with respect to their contribution to the pathogenesis of experimental autoimmune encephalomyelitis. We discuss possible mechanisms of strongly attenuated experimental autoimmune encephalomyelitis in ADAP-deficient mice.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Encefalomielitis Autoinmune Experimental/inmunología , Esclerosis Múltiple/inmunología , Complejos Multiproteicos/metabolismo , Linfocitos T/inmunología , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Adhesión Celular/genética , Degranulación de la Célula/genética , Modelos Animales de Enfermedad , Humanos , Inmunomodulación , Ratones , Ratones Noqueados , Transducción de Señal/genéticaRESUMEN
Integrins have been implicated in various processes of nervous system development, including proliferation, migration, and differentiation of neuronal cells. In this study, we show that the serine/threonine kinase Ndr2 controls integrin-dependent dendritic and axonal growth in mouse hippocampal neurons. We further demonstrate that Ndr2 is able to induce phosphorylation at the activity- and trafficking-relevant site Thr(788/789) of ß1-integrin to stimulate the PKC- and CaMKII-dependent activation of ß1-integrins, as well as their exocytosis. Accordingly, Ndr2 associates with integrin-positive early and recycling endosomes in primary hippocampal neurons and the surface expression of activated ß1-integrins is reduced on dendrites of Ndr2-deficient neurons. The role of Ndr2 in dendritic differentiation is also evident in vivo, because Ndr2-null mutant mice show arbor-specific alterations of dendritic complexity in the hippocampus. This indicates a role of Ndr2 in the fine regulation of dendritic growth; in fact, treatment of primary neurons with Semaphorin 3A rescues Ndr2 knock-down-induced dendritic growth deficits but fails to enhance growth beyond control level. Correspondingly, Ndr2-null mutant mice show a Semaphorin 3A(-/-)-like phenotype of premature dendritic branching in the hippocampus. The results of this study show that Ndr2-mediated integrin trafficking and activation are crucial for neurite growth and guidance signals during neuronal development.
Asunto(s)
Integrina beta1/metabolismo , Neuritas/metabolismo , Proteínas/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Animales , Procesos de Crecimiento Celular , Células Cultivadas , Endosomas/metabolismo , Eliminación de Gen , Células HEK293 , Hipocampo/citología , Hipocampo/metabolismo , Humanos , Ratones , Ratones Endogámicos C57BL , Células 3T3 NIH , Neuritas/efectos de los fármacos , Neuritas/fisiología , Células PC12 , Fosforilación , Unión Proteica , Transporte de Proteínas , Proteínas/genética , Ratas , Semaforina-3A/farmacologíaRESUMEN
The adhesion- and degranulation-promoting adaptor protein (ADAP), expressed in T cells, myeloid cells, and platelets, is known to regulate receptor-mediated inside-out signaling leading to integrin activation and adhesion. In this study, we demonstrate that, upon induction of active experimental autoimmune encephalomyelitis (EAE) by immunization with the myelin oligodendrocyte glycoprotein35-55 peptide, ADAP-deficient mice developed a significantly milder clinical course of EAE and showed markedly less inflammatory infiltrates in the CNS than wild-type mice. Moreover, ADAP-deficient recipients failed to induce EAE after adoptive transfer of myelin oligodendrocyte glycoprotein-specific TCR-transgenic T cells (2D2 T cells). In addition, ex vivo fully activated 2D2 T cells induced significantly less severe EAE in ADAP-deficient recipients. The ameliorated disease in the absence of ADAP was not due to expansion or deletion of a particular T cell subset but rather because of a strong reduction of all inflammatory leukocyte populations invading the CNS. Monitoring the adoptively transferred 2D2 T cells over time demonstrated that they accumulated within the lymph nodes of ADAP-deficient hosts. Importantly, transfer of complete wild-type bone marrow or even bone marrow of 2D2 TCR-transgenic mice was unable to reconstitute EAE in the ADAP-deficient animals, indicating that the milder EAE was dependent on (a) radio-resistant nonhematopoietic cell population(s). Two-photon microscopy of lymph node explants revealed that adoptively transferred lymphocytes accumulated at lymphatic vessels in the lymph nodes of ADAP-deficient mice. Thus, our data identify a T cell-independent mechanism of EAE modulation in ADAP-deficient mice.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/deficiencia , Encefalomielitis Autoinmune Experimental/inmunología , Linfocitos T/inmunología , Traslado Adoptivo , Animales , Trasplante de Médula Ósea , Inflamación/inmunología , Ganglios Linfáticos/citología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Glicoproteína Mielina-Oligodendrócito/inmunología , Fragmentos de Péptidos/inmunología , Transducción de Señal/inmunologíaRESUMEN
Integrins in effector T cells are highly expressed and important for trafficking of these cells and for their effector functions. However, how integrins are regulated in effector T cells remains poorly characterized. Here, we have investigated effector T cell leukocyte function-associated antigen-1 (LFA-1) regulation in primary murine effector T cells. These cells have high LFA-1 integrin expression and display high spontaneous binding to intercellular adhesion molecule-1 (ICAM-1) ligand under static conditions. In addition, these cells are able to migrate spontaneously on ICAM-1. Atomic force microscopy measurements showed that the force required for unbinding of integrin-ligand interactions increases over time (0.5-20-s contact time). The maximum unbinding force for this interaction was â¼140 piconewtons at 0.5-s contact time, increasing to 580 piconewtons at 20-s contact time. Also, the total work required to disrupt the interaction increased over the 20-s contact time, indicating LFA-1-mediated adhesion strengthening in primary effector T cells over a very quick time frame. Effector T cells adhered spontaneously to ICAM-1 under conditions of shear flow, in the absence of chemokine stimulation, and this binding was independent of protein kinase B/Akt and protein kinase C kinase activity, but dependent on calcium/calmodulin signaling and an intact actin cytoskeleton. These results indicate that effector T cell integrins are highly expressed and spontaneously adhesive in the absence of inside-out integrin signaling but that LFA-1-mediated firm adhesion under conditions of shear flow requires downstream integrin signaling, which is dependent on calcium/calmodulin and the actin cytoskeleton.
Asunto(s)
Actinas/metabolismo , Señalización del Calcio/fisiología , Calmodulina/metabolismo , Antígeno-1 Asociado a Función de Linfocito/metabolismo , Linfocitos T/metabolismo , Actinas/genética , Actinas/inmunología , Animales , Calmodulina/genética , Calmodulina/inmunología , Adhesión Celular/fisiología , Células Cultivadas , Citoesqueleto/genética , Citoesqueleto/inmunología , Citoesqueleto/metabolismo , Molécula 1 de Adhesión Intercelular/genética , Molécula 1 de Adhesión Intercelular/inmunología , Molécula 1 de Adhesión Intercelular/metabolismo , Antígeno-1 Asociado a Función de Linfocito/genética , Antígeno-1 Asociado a Función de Linfocito/inmunología , Ratones , Ratones Noqueados , Microscopía de Fuerza Atómica , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/inmunología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Resistencia al Corte , Linfocitos T/inmunología , Linfocitos T/ultraestructuraRESUMEN
The ß2-integrin lymphocyte function-associated antigen-1 (LFA-1) plays a crucial role within the immune system. It regulates the interaction between T cells and antigen-presenting cells and facilitates T-cell adhesion to the endothelium, a process that is important for lymphocyte extravasation and homing. Signals mediated via the T-cell receptor and the chemokine receptor CCR7 activate LFA-1 through processes known as inside-out signaling. The molecular mechanisms underlying inside-out signaling are not completely understood. Here, we have assessed the role of the ADAP/SKAP55 module for CCR7-mediated signaling. We show that loss of the module delays homing and reduces intranodal T-cell motility in vivo. This is probably because of a defect in CCR7-mediated adhesion that affects both affinity and avidity regulation of LFA-1. Further analysis of how the ADAP/SKAP55 module regulates CCR7-induced integrin activation revealed that 2 independent pools of the module are expressed in T cells. One pool interacts with a RAPL/Mst1 complex, whereas the other pool is linked to a RIAM/Mst1/Kindlin-3 complex. Importantly, both the RAPL/Mst1 and the RIAM/Mst1/Kindlin-3 complexes require ADAP/SKAP55 for binding to LFA-1 upon CCR7 stimulation. Hence, 2 independent ADAP/SKAP55 modules are essential components of the signaling machinery that regulates affinity and avidity of LFA-1 in response to CCR7.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/fisiología , Regulación de la Expresión Génica , Antígeno-1 Asociado a Función de Linfocito/metabolismo , Fosfoproteínas/metabolismo , Receptores CCR7/metabolismo , Linfocitos T/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Células Presentadoras de Antígenos/inmunología , Células Presentadoras de Antígenos/metabolismo , Apoptosis , Proteínas Reguladoras de la Apoptosis , Western Blotting , Adhesión Celular , Movimiento Celular , Proliferación Celular , Citometría de Flujo , Factor de Crecimiento de Hepatocito/metabolismo , Humanos , Inmunoprecipitación , Proteínas de la Membrana/metabolismo , Ratones , Ratones Noqueados , Proteínas de Unión al GTP Monoméricas/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Complejo Shelterina , Transducción de Señal , Linfocitos T/inmunología , Talina/metabolismo , Proteínas de Unión a Telómeros/metabolismoRESUMEN
The adapter protein Src homology 2 (SH2) domain-containing leukocyte protein of 76 kDa (SLP-76) is critical for multiple aspects of T cell development and function. Through its protein-binding domains, SLP-76 serves as a platform for the assembly of multiple enzymes and adapter proteins that function together to activate second messengers required for TCR signal propagation. The N terminus of SLP-76, which contains three tyrosines that serve as docking sites for SH2 domain-containing proteins, and the central proline-rich region of SLP-76 have been well studied and are known to be important for both thymocyte selection and activation of peripheral T cells. Less is known about the function of the C-terminal SH2 domain of SLP-76. This region inducibly associates with ADAP and HPK1. Combining regulated deletion of endogenous SLP-76 with transgenic expression of a SLP-76 SH2 domain mutant, we demonstrate that the SLP-76 SH2 domain is required for peripheral T cell activation and positive selection of thymocytes, a function not previously attributed to this region. This domain is also important for T cell proliferation, IL-2 production, and phosphorylation of protein kinase D and IκB. ADAP-deficient T cells display similar, but in some cases less severe, defects despite phosphorylation of a negative regulatory site on SLP-76 by HPK1, a function that is lost in SLP-76 SH2 domain mutant T cells.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/fisiología , Diferenciación Celular/inmunología , Activación de Linfocitos/inmunología , Fosfoproteínas/fisiología , Linfocitos T/citología , Linfocitos T/inmunología , Dominios Homologos src/inmunología , Proteínas Adaptadoras Transductoras de Señales/deficiencia , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Diferenciación Celular/genética , Humanos , Células Jurkat , Activación de Linfocitos/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Fosfoproteínas/deficiencia , Fosfoproteínas/genética , Transducción de Señal/genética , Transducción de Señal/inmunología , Dominios Homologos src/genéticaRESUMEN
Ras transmits manifold signals from the TCR at various crossroads in the life of a T cell. For example, selection programs in the thymus or the acquisition of a state of hypo-responsiveness known as anergy are just some of the T cell features known to be controlled by TCR-sparked signals that are intracellularly propagated by Ras. These findings raise the question of how Ras can transmit such a variety of signals leading to the shaping of equally many T cell traits. Because Ras proteins transit through endomembrane compartments on their way to the plasma membrane (PM), compartmentalized Ras activation at distinct subcellular sites represents a potential mechanism for signal diversification in TCR signaling. This hypothesis has been nurtured by studies in T cells engineered to overexpress Ras that reported distinct activation of Ras at the PM and Golgi. Contrary to this scenario, we report in this study that activation of endogenous Ras, imaged in live Jurkat T cells using novel affinity probes for Ras-GTP, proceeds only at the PM even upon enforced signal flux through the diacylglycerol/RasGRP1 pathway. Physiological engagement of the TCR at the immunological synapse in primary T cells caused focalized Ras-GTP accumulation also only at the PM. Analysis of palmitoylation-deficient Ras mutants, which are confined to endomembranes, confirmed that the TCR does not activate Ras in that compartment and revealed a critical function for palmitoylation in N-Ras/H-Ras activation. These findings identify the PM as the only site of TCR-driven Ras activation and document that endomembranes are not a signaling platform for Ras in T cells.
Asunto(s)
Membrana Celular/metabolismo , Lipoilación/inmunología , Receptores de Antígenos de Linfocitos T/fisiología , Factores de Intercambio de Guanina Nucleótido ras/metabolismo , Proteínas ras/metabolismo , Animales , Línea Celular Tumoral , Membrana Celular/genética , Membrana Celular/inmunología , Proteínas de Unión al ADN/genética , Retículo Endoplásmico/genética , Retículo Endoplásmico/inmunología , Colorantes Fluorescentes/metabolismo , Factores de Intercambio de Guanina Nucleótido/genética , Humanos , Células Jurkat , Lipoilación/genética , Proteína Oncogénica p21(ras)/metabolismo , Unión Proteica/genética , Unión Proteica/inmunología , Transporte de Proteínas/genética , Transporte de Proteínas/inmunología , Ratas , Receptores de Antígenos de Linfocitos T/metabolismo , Transducción de Señal/genética , Transducción de Señal/inmunología , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Factores de Intercambio de Guanina Nucleótido ras/biosíntesis , Factores de Intercambio de Guanina Nucleótido ras/genética , Proteínas ras/biosíntesisRESUMEN
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/metabolismoRESUMEN
Therapeutic angiogenesis is likely to require the administration of factors that complement each other. Activation of the receptor tyrosine kinase (RTK) Flk1 by vascular endothelial growth factor (VEGF) is crucial, but molecular interactions of other factors with VEGF and Flk1 have been studied to a limited extent. Here we report that placental growth factor (PGF, also known as PlGF) regulates inter- and intramolecular cross talk between the VEGF RTKs Flt1 and Flk1. Activation of Flt1 by PGF resulted in intermolecular transphosphorylation of Flk1, thereby amplifying VEGF-driven angiogenesis through Flk1. Even though VEGF and PGF both bind Flt1, PGF uniquely stimulated the phosphorylation of specific Flt1 tyrosine residues and the expression of distinct downstream target genes. Furthermore, the VEGF/PGF heterodimer activated intramolecular VEGF receptor cross talk through formation of Flk1/Flt1 heterodimers. The inter- and intramolecular VEGF receptor cross talk is likely to have therapeutic implications, as treatment with VEGF/PGF heterodimer or a combination of VEGF plus PGF increased ischemic myocardial angiogenesis in a mouse model that was refractory to VEGF alone.
Asunto(s)
Proteínas Gestacionales/metabolismo , Receptor Cross-Talk/fisiología , Receptor 1 de Factores de Crecimiento Endotelial Vascular/metabolismo , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo , Animales , Células Cultivadas , Dimerización , Factores de Crecimiento Endotelial/metabolismo , Factores de Crecimiento Endotelial/farmacología , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/fisiología , Activación Enzimática , Perfilación de la Expresión Génica , Humanos , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Péptidos y Proteínas de Señalización Intercelular/farmacología , Linfocinas/metabolismo , Linfocinas/farmacología , Ratones , Isquemia Miocárdica/metabolismo , Isquemia Miocárdica/patología , Neovascularización Patológica/metabolismo , Neovascularización Fisiológica/efectos de los fármacos , Fosforilación , Factor de Crecimiento Placentario , Proteínas Gestacionales/farmacología , Transducción de Señal , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial Vascular , Proteínas Virales/metabolismo , Proteínas Virales/farmacologíaRESUMEN
T cells are the key players of the adaptive immune response. They coordinate the activation of other immune cells and kill malignant and virus-infected cells. For full activation T cells require at least two signals. Signal 1 is induced after recognition of MHC/peptide complexes presented on antigen presenting cells (APCs) by the clonotypic TCR (T-cell receptor)/CD3 complex whereas Signal 2 is mediated via the co-stimulatory receptor CD28, which binds to CD80/CD86 molecules that are present on APCs. These signaling events control the activation, proliferation and differentiation of T cells. In addition, triggering of the TCR/CD3 complex induces the activation of the integrin LFA-1 (leukocyte function associated antigen 1) leading to increased ligand binding (affinity regulation) and LFA-1 clustering (avidity regulation). This process is termed "inside-out signaling". Subsequently, ligand bound LFA-1 transmits a signal into the T cells ("outside-in signaling") which enhances T-cell interaction with APCs (adhesion), T-cell activation and T-cell proliferation. After triggering of signal transducing receptors, adapter proteins organize the proper processing of membrane proximal and intracellular signals as well as the activation of downstream effector molecules. Adapter proteins are molecules that lack enzymatic or transcriptional activity and are composed of protein-protein and protein-lipid interacting domains/motifs. They organize and assemble macromolecular complexes (signalosomes) in space and time. Here, we review recent findings regarding three cytosolic adapter proteins, ADAP (Adhesion and Degranulation-promoting Adapter Protein), SKAP1 and SKAP2 (Src Kinase Associated Protein 1 and 2) with respect to their role in TCR/CD3-mediated activation, proliferation and integrin regulation.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/inmunología , Complejo CD3/inmunología , Péptidos y Proteínas de Señalización Intracelular/inmunología , Activación de Linfocitos , Fosfoproteínas/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Transducción de Señal/inmunología , Linfocitos T/inmunología , Animales , Citosol/inmunología , HumanosRESUMEN
A high degree of regional, temporal and molecular specificity is evident in the regulation of GABAergic signaling in stress-responsive circuitry, hampering the use of systemic GABAergic modulators for the treatment of stress-related psychopathology. Here we investigated the effectiveness of local intervention with the GABA synthetic enzymes GAD65 and GAD67 in the dorsal dentate gyrus (dDG) vs ventral DG (vDG) to alleviate anxiety-like behavior and stress-induced symptoms in the rat. We induced shRNA-mediated knock down of either GAD65 or GAD67 with lentiviral vectors microinjected into the dDG or vDG of young adult male rats and examined anxiety behavior, learning and memory performance. Subsequently we tested whether reducing GAD65 expression in the dDG would also confer resilience against juvenile stress-induced behavioral and physiological symptoms in adulthood. While knock down of either isoform in the vDG increased anxiety levels in the open field and the elevated plus maze tests, the knock down of GAD65, but not GAD67, in the dDG conferred a significant reduction in anxiety levels. Strikingly, this manipulation also attenuated juvenile stress evoked anxiety behavior, cognitive and synaptic plasticity impairments. Local GABAergic circuitry in the DG plays an important and highly region-specific role in control of emotional behavior and stress responding. Reduction of GAD65 expression in the dDG appears to provide resilience to juvenile stress-induced emotional and cognitive deficits, opening a new direction towards addressing a significant risk factor for developing stress and trauma-related psychopathologies later in life.
RESUMEN
Protein kinase C-theta (PKC-theta) is important for the activation of autoreactive T cells but is thought to be of minor importance for T-cell responses in infectious diseases, suggesting that PKC-theta may be a target for the treatment of T-cell-mediated autoimmune diseases. To explore the function of PKC-theta in a chronic persisting infection in which T cells are crucial for pathogen control, we infected BALB/c PKC-theta(-/-) and PKC-theta(+/+) wild-type mice with Toxoplasma gondii. The PKC-theta(-/-) mice succumbed to necrotizing Toxoplasma encephalitis due to an insufficient parasite control up to day 40, whereas the wild-type mice survived. The number of T. gondii-specific CD4 and CD8 T cells was significantly reduced in the PKC-theta(-/-) mice, resulting in the impaired production of protective cytokines (gamma interferon, tumor necrosis factor) and antiparasitic effector molecules (inducible nitric oxide, gamma interferon-induced GTPase) in the spleen and brain. In addition, Th2-cell numbers were reduced in infected the PKC-theta(-/-) mice, paralleled by the diminished GATA3 expression of PKC-theta(-/-) CD4 T cells and reduced T. gondii-specific IgG production in serum and cerebrospinal fluid. Western blot analysis of splenic CD4 and CD8 T cells revealed an impaired activation of the NF-kappaB, AP-1, and MAPK pathways in T. gondii-infected PKC-theta(-/-) mice. Adoptive transfer of wild-type CD4 plus CD8 T cells significantly protected PKC-theta(-/-) mice from death by increasing the numbers of gamma interferon-producing T. gondii-specific CD4 and CD8 T cells, illustrating a cell-autonomous, protective function of PKC-theta in T cells. These findings imply that PKC-theta inhibition drastically impairs T. gondii-specific T-cell responses with fatal consequences for intracerebral parasite control and survival.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Isoenzimas/inmunología , Proteína Quinasa C/inmunología , Toxoplasma/inmunología , Animales , Anticuerpos Antiprotozoarios/análisis , Anticuerpos Antiprotozoarios/sangre , Encéfalo/inmunología , Líquido Cefalorraquídeo/inmunología , Citocinas/inmunología , Citocinas/metabolismo , Femenino , Inmunoglobulina G/análisis , Inmunoglobulina G/sangre , Isoenzimas/deficiencia , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Proteína Quinasa C/deficiencia , Proteína Quinasa C-theta , Bazo/inmunología , Células Th2/inmunologíaRESUMEN
Lymphocyte membrane rafts contain molecules critical for immunoreceptor signaling. Here, we report identification of a new raft-associated adaptor protein LIME (Lck-interacting molecule) expressed predominantly in T lymphocytes. LIME becomes tyrosine phosphorylated after cross-linking of the CD4 or CD8 coreceptors. Phospho-LIME associates with the Src family kinase Lck and its negative regulator, Csk. Ectopic expression of LIME in Jurkat T cells results in an increase of Csk in lipid rafts, increased phosphorylation of Lck and higher Ca2+ response to CD3 stimulation. Thus, LIME appears to be involved in regulation of T cell activation by coreceptors.
Asunto(s)
Proteínas Adaptadoras del Transporte Vesicular/inmunología , Antígenos CD4/inmunología , Antígenos CD8/inmunología , Microdominios de Membrana/inmunología , Proteínas Adaptadoras del Transporte Vesicular/genética , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Secuencia de Aminoácidos , Proteína Tirosina Quinasa CSK , ADN Complementario , Bases de Datos de Proteínas , Humanos , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/metabolismo , Datos de Secuencia Molecular , Fosforilación , Proteínas Tirosina Quinasas/metabolismo , Familia-src QuinasasRESUMEN
Death-associated protein kinase (DAPK) is a serine/threonine kinase that contributes to pro-apoptotic signaling on cytokine exposure. The role of DAPK in macrophage-associated tumor cell death is currently unknown. Recently, we suggested a new function for DAPK in the induction of apoptosis during the interaction between colorectal tumor cells and tumor-associated macrophages. Using a cell-culture model with conditioned supernatants of differentiated/activated macrophages (U937) and human HCT116 colorectal tumor cells, we replicated DAPK-associated tumor cell death; this model likely reflects the in vivo tumor setting. In this study, we show that tumor necrosis factor-alpha exposure under conditions of macrophage activation induced DAPK-dependent apoptosis in the colorectal tumor cell line HCT116. Simultaneously, early phosphorylation of p38 mitogen-activated protein kinase (phospho-p38) was observed. We identified the phospho-p38 mitogen-activated protein kinase as a novel interacting protein of DAPK in tumor necrosis factor-alpha-induced apoptosis. The general relevance of this interaction was verified in two colorectal cell lines without functional p53 (ie, HCT116 p53(-/-) and HT29 mutant) and in human colon cancer and ulcerative colitis tissues. Supernatants of freshly isolated human macrophages were also able to induce DAPK and phospho-p38. Our findings highlight the mechanisms that underlie DAPK regulation in tumor cell death evoked by immune cells.