RESUMEN
Integrin engagement within the immune synapse enhances T cell activation, but our understanding of this process is incomplete. In response to T cell receptor (TCR) ligation, SLP-76 (LCP2), ADAP (FYB1) and SKAP55 (SKAP1) are recruited into microclusters and activate integrins via the effectors talin-1 and kindlin-3 (FERMT3). We postulated that integrins influence the centripetal transport and signaling of SLP-76 microclusters via these linkages. We show that contractile myosin filaments surround and are co-transported with SLP-76 microclusters, and that TCR ligand density governs the centripetal movement of both structures. Centripetal transport requires formin activity, actomyosin contraction, microtubule integrity and dynein motor function. Although immobilized VLA-4 (α4ß1 integrin) and LFA-1 (αLß2 integrin) ligands arrest the centripetal movement of SLP-76 microclusters and myosin filaments, VLA-4 acts distally, while LFA-1 acts in the lamellum. Integrin ß2, kindlin-3 and zyxin are required for complete centripetal transport, while integrin ß1 and talin-1 are not. CD69 upregulation is similarly dependent on integrin ß2, kindlin-3 and zyxin, but not talin-1. These findings highlight the integration of cytoskeletal systems within the immune synapse and reveal extracellular ligand-independent roles for LFA-1 and kindlin-3. This article has an associated First Person interview with the first author of the paper.
Asunto(s)
Dineínas , Antígeno-1 Asociado a Función de Linfocito , Adhesión Celular , Dineínas/genética , Humanos , Antígeno-1 Asociado a Función de Linfocito/metabolismo , Proteínas de la Membrana/metabolismo , Miosinas , Receptores de Antígenos de Linfocitos T/metabolismoRESUMEN
Vav family guanine nucleotide exchange factors (GEFs) are essential regulators of immune function. Despite their structural similarity, Vav1 promotes and Vav2 opposes T cell receptor (TCR)-induced Ca2+ entry. By using a Vav1-deficient Jurkat T cell line, we find that Vav1 facilitates Ca2+ entry via non-catalytic scaffolding functions that are encoded by the catalytic core of Vav1 and flanking linker regions. We implicate, in this scaffolding function, a previously undescribed polybasic motif that is strictly conserved in Vav1 and absent from Vav2 in tetrapods. Conversely, the catalytic activity of Vav2 contributes to the suppression of TCR-mediated Ca2+ entry. By performing an in vivo 'GEF trapping' assay in intact cells, we demonstrate that Cdc42 interacts with the catalytic surface of Vav2 but not Vav1, and that Vav1 discriminates Cdc42 from Rac1 via F56 (W56 in Rac1). Finally, the Cdc42-specific inhibitor ZCL278 and the shRNA-mediated suppression of Cdc42 each prevent the inhibition of TCR-induced Ca2+ entry by Vav2. These findings define stark differences in the functions of Vav1 and Vav2, and provide an explanation for the differential usage of these Vav isoforms by immune subpopulations.
Asunto(s)
Activación de Linfocitos , Proteínas Proto-Oncogénicas c-vav , Isoformas de Proteínas , Proteínas Proto-Oncogénicas c-vav/genética , Proteínas Proto-Oncogénicas c-vav/metabolismo , Receptores de Antígenos de Linfocitos T , Linfocitos TRESUMEN
Antigen recognition by the T cell receptor (TCR) directs the assembly of essential signaling complexes known as SLP-76 (also known as LCP2) microclusters. Here, we show that the interaction of the adhesion and degranulation-promoting adaptor protein (ADAP; also known as FYB1) with SLP-76 enables the formation of persistent microclusters and the stabilization of T cell contacts, promotes integrin-independent adhesion and enables the upregulation of CD69. By analyzing point mutants and using a novel phospho-specific antibody, we show that Y595 is essential for normal ADAP function, that virtually all tyrosine phosphorylation of ADAP is restricted to a Y595-phosphorylated (pY595) pool, and that multivalent interactions between the SLP-76 SH2 domain and its binding sites in ADAP are required to sustain ADAP phosphorylation. Although pY595 ADAP enters SLP-76 microclusters, non-phosphorylated ADAP is enriched in protrusive actin-rich structures. The pre-positioning of ADAP at the contact sites generated by these structures favors the retention of nascent SLP-76 oligomers and their assembly into persistent microclusters. Although ADAP is frequently depicted as an effector of SLP-76, our findings reveal that ADAP acts upstream of SLP-76 to convert labile, Ca2+-competent microclusters into stable adhesive junctions with enhanced signaling potential.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Células Jurkat/metabolismo , Fosfoproteínas/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Proteínas Adaptadoras Transductoras de Señales/inmunología , Adhesión Celular/fisiología , Comunicación Celular/fisiología , Citoesqueleto/inmunología , Citoesqueleto/metabolismo , Humanos , Células Jurkat/citología , Células Jurkat/inmunología , Activación de Linfocitos , Fosfoproteínas/inmunología , Fosforilación , Receptores de Antígenos de Linfocitos T/inmunología , Transducción de Señal , Dominios Homologos srcRESUMEN
Although dynamic imaging technologies have provided important insights into the underlying processes responsible for T-cell activation, the processes that link antigen recognition to downstream signaling remain poorly defined. Converging lines of inquiry indicate that T-cell receptor (TCR) microclusters are the minimal structures capable of directing effective TCR signaling. Furthermore, imaging studies have determined that these structures trigger the assembly of oligomeric signaling scaffolds that contain the adapters and effectors required for T-cell activation. Existing models of T-cell activation accurately explain the sensitivity and selectivity of antigen recognition. However, these models do not account for important properties of microclusters, including their peripheral formation, size, and movement on the actin cytoskeleton. Here we examine how lipid rafts, galectin lattices, and protein scaffolds contribute to the assembly, function, and fate of TCR microclusters within immune synapses. Finally, we propose a 'mechanical segregation' model of signal initiation in which cytoskeletal forces contribute to the lateral segregation of molecules and cytoskeletal scaffolds provide a template for microclusters assembly.
Asunto(s)
Actinas/inmunología , Activación de Linfocitos/inmunología , Receptores de Antígenos de Linfocitos T/metabolismo , Transducción de Señal/inmunología , Linfocitos T/metabolismo , Actinas/metabolismo , Animales , Citoesqueleto/metabolismoRESUMEN
Antigen recognition triggers the recruitment of the critical adaptor protein SLP-76 to small macromolecular clusters nucleated by the T-cell receptor (TCR). These structures develop rapidly, in parallel with TCR-induced increases in tyrosine phosphorylation and cytosolic calcium, and are likely to contribute to TCR-proximal signaling. Previously, we demonstrated that these SLP-76-containing clusters segregate from the TCR and move towards the center of the contact interface. Neither the function of these clusters nor the structural requirements governing their persistence have been examined extensively. Here we demonstrate that defects in cluster assembly and persistence are associated with defects in T-cell activation in the absence of Lck, ZAP-70, or LAT. Clusters persist normally in the absence of phospholipase C-gamma1, indicating that in the absence of a critical effector, these structures are insufficient to drive T-cell activation. Furthermore, we show that the critical adaptors LAT and Gads localize with SLP-76 in persistent clusters. Mutational analyses of LAT, Gads, and SLP-76 indicated that multiple domains within each of these proteins contribute to cluster persistence. These data indicate that multivalent cooperative interactions stabilize these persistent signaling clusters, which may correspond to the functional complexes predicted by kinetic proofreading models of T-cell activation.
Asunto(s)
Activación de Linfocitos/inmunología , Transducción de Señal , Linfocitos T/inmunología , Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Enterotoxinas/farmacología , Humanos , Células Jurkat , Activación de Linfocitos/efectos de los fármacos , Proteínas de la Membrana/metabolismo , Ratones , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Unión Proteica/efectos de los fármacos , Estructura Terciaria de Proteína , Transporte de Proteínas/efectos de los fármacos , Proteínas Recombinantes de Fusión/metabolismo , Transducción de Señal/efectos de los fármacos , Linfocitos T/citología , Linfocitos T/metabolismo , TermodinámicaRESUMEN
The tumor suppressor PTEN is mutated in a high percentage of human cancers, and is implicated in pathways regulating cell growth, proliferation, survival, and migration. Despite significant advances, our understanding of its mechanisms of action remains incomplete. We have used a high-throughput proteomic immunoblotting approach to identify proteins whose expression levels are modulated by PTEN. Out of over 800 proteins screened, 22 proteins showed significant changes in expression. Five proteins that exhibited two-fold or greater changes in expression level were further characterized. AKAP121 and G3BP expression was reduced, while dihydrofolate reductase (DHFR), Rap1 and RCC1 expression was elevated in response to PTEN expression in a PTEN-null T-cell leukemia line. The phosphatase activity of PTEN was required for these effects. However, direct inhibition of PI-3 Kinase could mimic PTEN in modulating expression of DHFR, G3BP, Rap1 and RCC1, but not AKAP121. Real-time PCR showed that the effects of PTEN were primarily post-transcriptional, and would not have been revealed by mRNA-based screens. We conclude from these data that PTEN can modulate the expression level of a number of different proteins. The identified proteins have the potential to serve as previously unrecognized effectors of PTEN, and suggest the existence of additional complexity in the modes by which PTEN can regulate cellular biology.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/análisis , Proteínas Portadoras/análisis , Proteínas de Ciclo Celular/análisis , Factores de Intercambio de Guanina Nucleótido/análisis , Proteínas Nucleares/análisis , Monoéster Fosfórico Hidrolasas/fisiología , Tetrahidrofolato Deshidrogenasa/análisis , Proteínas Supresoras de Tumor/fisiología , Proteínas de Anclaje a la Quinasa A , ADN Helicasas , Humanos , Células Jurkat , Fosfohidrolasa PTEN , Fosfatidilinositol 3-Quinasas/análisis , Proteínas de Unión a Poli-ADP-Ribosa , Proteómica , ARN Helicasas , Proteínas con Motivos de Reconocimiento de ARN , Transcripción GenéticaRESUMEN
Phosphoinositide 3-kinase (PI3K) is important in TCR signaling. PI3K generates phosphatidylinositol 3, 4, 5-trisphosphate (PI-3,4,5-P3), which regulates membrane localization and/or activity of multiple signaling proteins. PTEN (phosphatase and tensin homologue deleted on chromosome 10) opposes PI3K, reversing this reaction. Maintaining the balance between these two enzymes is important for normal T cell function. Here we use the PTEN-null Jurkat T cell line to address the role of PTEN in modulating proximal and distal TCR-signaling events. PTEN expression at levels that restored low basal Akt phosphorylation (an indicator of PI-3,4,5-P3 levels), but which were not themselves cytotoxic, had minimal effect on TCR-stimulated activation of phospholipase Cgamma1 and Ca2+ flux, but reduced the duration of extracellular signal-regulated kinase (Erk) activation. Distal signaling events, including nuclear factor of activated T cells (NFAT) activation, CD69 expression and IL-2 production, were all inhibited by PTEN expression. Notably, PTEN did not block TCR-stimulated PI-3,4,5-P3 accumulation. The effect of PTEN on distal TCR signaling events was strongly correlated with the loss of the constitutive Akt activation and glycogen synthase kinase-3 (GSK3) inhibition that is typical of Jurkat cells, and could be reversed by expression of activated Akt or pharmacologic inhibition of GSK3. These results suggest that PTEN acts in T cells primarily to control basal PI-3,4,5-P3 levels, rather than opposing PI3K acutely during TCR stimulation.
Asunto(s)
Fosfatos de Fosfatidilinositol/inmunología , Monoéster Fosfórico Hidrolasas/inmunología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Receptores de Antígenos de Linfocitos T/inmunología , Proteínas Supresoras de Tumor/inmunología , Androstadienos/farmacología , Antígenos CD/inmunología , Antígenos de Diferenciación de Linfocitos T/inmunología , Calcio/inmunología , Proteínas de Unión al ADN , Glucógeno Sintasa Quinasa 3/antagonistas & inhibidores , Glucógeno Sintasa Quinasa 3/inmunología , Humanos , Interleucina-2/antagonistas & inhibidores , Interleucina-2/inmunología , Células Jurkat , Lectinas Tipo C , Microscopía Confocal , Proteína Quinasa 1 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 1 Activada por Mitógenos/inmunología , Factores de Transcripción NFATC , Proteínas Nucleares , Fosfohidrolasa PTEN , Fosfolipasa C gamma , Monoéster Fosfórico Hidrolasas/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/inmunología , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/inmunología , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-akt , Transducción de Señal/inmunología , Factores de Transcripción , Proteínas Supresoras de Tumor/metabolismo , Fosfolipasas de Tipo C/inmunología , WortmaninaRESUMEN
The balance of activities between the proto-oncogene phosphoinositide 3-kinase (PI3K) and the tumour suppressor gene PTEN has been shown to affect cellular growth and proliferation, as well as tumorigenesis. Previously, PTEN expression in the PTEN-null Jurkat T cell leukaemia line was shown to cause reduced proliferation without cell cycle arrest. Here, we further these investigations by determining the basis for this phenomenon. By BrdU pulse-chase and cell cycle arrest and release assays, we find that PTEN expression reduced proliferation by slowing progression through all phases of the cell cycle. This was associated with reduced levels of cyclins A, B1 and B2, cdk4, and cdc25A and increased p27KIP1 expression. Apoptosis played no role in the antiproliferative effect of PTEN, since only marginal increases in the rate of apoptosis were detected upon PTEN expression, and inhibitors of effector caspases did not restore proliferative capacity. Active Akt blocked the antiproliferative effects of PTEN, indicating that PTEN mediates its effects through conventional PI3K-linked signalling pathways. Similar results were obtained from a different PTEN-null leukaemia T cell line, CEM. Together, these results show that PTEN expression in leukaemic T cells leads to reduced proliferation via an apoptosis-independent mechanism involving slower passage through the cell cycle.
Asunto(s)
Ciclo Celular/fisiología , División Celular/fisiología , Leucemia de Células T/metabolismo , Monoéster Fosfórico Hidrolasas/genética , Linfocitos T/fisiología , Proteínas Supresoras de Tumor/genética , Apoptosis/genética , Apoptosis/fisiología , Humanos , Células Jurkat , Fosfohidrolasa PTEN , Monoéster Fosfórico Hidrolasas/metabolismo , Proto-Oncogenes Mas , Proteínas Supresoras de Tumor/metabolismoRESUMEN
The initiating events associated with T activation in response to stimulation of the T cell antigen receptor (TCR) and costimulatory receptors, such as CD28, are intimately associated with the enzymatically catalyzed addition of phosphate not only to key tyrosine, threonine and serine residues in proteins but also to the D3 position of the myo-inositol ring of phosphatidylinositol (PtdIns). This latter event is catalyzed by the lipid kinase phosphoinositide 3-kinase (PI3K). The consequent production of PtdIns(3,4)P2 and PtdIns(3,4,5)P3 serves both to recruit signaling proteins to the plasma membrane and to induce activating conformational changes in proteins that contain specialized domains for the binding of these phospholipids. The TCR signaling proteins that are subject to regulation by PI3K include Akt, phospholipase Cgamma1 (PLCgamma1), protein kinase C zeta (PKC-zeta), Itk, Tec and Vav, all of which play critical roles in T cell activation. As is the case for phosphorylation of protein substrates, the phosphorylation of PtdIns is under dynamic regulation, with the D3 phosphate being subject to hydrolysis by the 3-phosphatase PTEN (phosphatase and tensin homolog deleted on chromosome 10), thereby placing PTEN in direct opposition to PI3K. In this review we consider recent data concerning how PTEN may act in regulating the process of T cell activation.
Asunto(s)
Fosfatidilinositoles/metabolismo , Monoéster Fosfórico Hidrolasas/metabolismo , Linfocitos T/enzimología , Proteínas Supresoras de Tumor/metabolismo , Humanos , Activación de Linfocitos , Modelos Biológicos , Fosfohidrolasa PTEN , Fosfatidilinositol 3-Quinasas/metabolismo , Linfocitos T/inmunologíaRESUMEN
Phosphoinositide 3-kinases (PI3Ks) phosphorylate the D3 position of the myo -inositol ring of inositol phospholipids, producing, amongst others, phosphatidylinositol-(3,4,5)-trisphosphate. This activity is opposed by the lipid phosphatase PTEN, which catalyzes the removal of this phosphate. Stimulation of PI3Ks is elicited by engagement of receptors for antigen, cytokines and chemokines, and by co-stimulatory molecules. Kinases and other enzymes containing pleckstrin homology domains are activated by binding to these phospholipids, affecting a variety of cellular processes that control lymphocyte function, including cell survival, proliferation, chemotaxis and cytoskeletal reorganization. This review highlights the signaling pathways of these kinases and other enzymes in T cells, their biological effects, and their regulation by PTEN.
Asunto(s)
Fosfatidilinositol 3-Quinasas/metabolismo , Monoéster Fosfórico Hidrolasas/metabolismo , Proteínas Serina-Treonina Quinasas , Transducción de Señal , Linfocitos T/enzimología , Proteínas Supresoras de Tumor/metabolismo , Animales , Humanos , Células Jurkat , Ratones , Fosfohidrolasa PTEN , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-akt , Receptores de Antígenos de Linfocitos T/metabolismoRESUMEN
Eosinophils preferentially accumulate at sites of chronic allergic diseases such as bronchial asthma. Tha mechanisms by which selective eosinophil migration occurs are not fully understood. However, interactions of cell-surface adhesion molecules on the eosinophil with molecular counterligands on endothelial and epithelial cells, and on extracellular matrix proteins, are likely to be critical during the recruitment process. One possible mechanism for selective eosinophil recruitment involves the alpha4ß1 (VLA-4) integrin which is not expressed on eosinophils. Correlations have been found between infiltration of eosinophils and endothelial expression of VCAM-1, the ligand for VLA-4, in the lungs of asthmatic individuals as well as in late rections in the lungs, nose and skin. Epithelial and endothelial cells respond to the Th2-type cytokines IL-4 and IL-13 with selective de novo expression of VCAM-1, consistent with the possible role of VCAM-1/VLA-4 interactions in eosinophil influx during allergic inflammation. Both ß1 and ß2 integrins on eosinophils exist in a state of partial activation. For example, eosinophils can be maximally activated for adhesion to VCAM-1 or fibronectin after exposure to ß1 integrin-activating antibodies or divalent cations, conditions that do not necessarily affect the total cell surface expression of ß1 integrins. In contrast, cytokines like IL-5 prevent ß1 integrin activation while promoting ß2 integrin function. Furthermore, ligation of integrins can regulate the affector functions of the cell. For example, eosinophil adhesion via ß1 and/or ß2 integrins has been shown to alter a variety of functional responses including degranulation and apoptosis. Thus, integrins appear to be important in mediating eosinophil migration and activation in allergic inflammation. Strategies that interfere with these processes may prove to be useful for treatment of allergic diseases.