RESUMEN
T cell development and activation are usually accompanied by expansion and production of numerous proteins that require active translation. The eukaryotic translation initiation factor 4E (eIF4E) binds to the 5' cap structure of mRNA and is critical for cap-dependent translational initiation. It has been hypothesized that MAPK-interacting kinase 1 and 2 (Mnk1/2) promote cap-dependent translation by phosphorylating eIF4E at serine 209 (S209). Pharmacologic studies using inhibitors have suggested that Mnk1/2 have important roles in T cells. However, genetic evidence supporting such conclusions is lacking. Moreover, the signaling pathways that regulate Mnk1/2 in T cells remain unclear. We demonstrate that TCR engagement activates Mnk1/2 in primary T cells. Such activation is dependent on Ras-Erk1/2 signaling and is inhibited by diacylglycerol kinases α and ζ. Mnk1/2 double deficiency in mice abolishes TCR-induced eIF4E S209 phosphorylation, indicating their absolute requirement for eIF4E S209 phosphorylation. However, Mnk1/2 double deficiency does not affect the development of conventional αß T cells, regulatory T cells, or NKT cells. Furthermore, T cell activation, in vivo primary and memory CD8 T cell responses to microbial infection, and NKT cell cytokine production were not obviously altered by Mnk1/2 deficiency. Although Mnk1/2 deficiency causes decreased IL-17 and IFN-γ production by CD4 T cells following immunization of mice with myelin oligodendrocyte glycoprotein peptide in complete Freund's adjuvant, correlating with milder experimental autoimmune encephalomyelitis scores, it does not affect Th cell differentiation in vitro. Together, these data suggest that Mnk1/2 has a minimal role in T cell development and activation but may regulate non-T cell lineages to control Th1 and Th17 differentiation in vivo.
Asunto(s)
Encefalomielitis Autoinmune Experimental/enzimología , Activación de Linfocitos/fisiología , Linfopoyesis/fisiología , Proteínas Serina-Treonina Quinasas/fisiología , Subgrupos de Linfocitos T/inmunología , Traslado Adoptivo , Compuestos de Anilina/farmacología , Animales , Células Cultivadas/efectos de los fármacos , Células Cultivadas/inmunología , Encefalomielitis Autoinmune Experimental/inmunología , Factor 4E Eucariótico de Iniciación/metabolismo , Femenino , Interferón gamma/biosíntesis , Interferón gamma/genética , Interleucina-17/biosíntesis , Interleucina-17/genética , Listeriosis/inmunología , Activación de Linfocitos/efectos de los fármacos , Coriomeningitis Linfocítica/inmunología , Linfopoyesis/efectos de los fármacos , Sistema de Señalización de MAP Quinasas , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Glicoproteína Mielina-Oligodendrócito/inmunología , Células T Asesinas Naturales/inmunología , Ovalbúmina/inmunología , Fragmentos de Péptidos/inmunología , Fosforilación/efectos de los fármacos , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/deficiencia , Proteínas Serina-Treonina Quinasas/genética , Purinas/farmacología , Caperuzas de ARNRESUMEN
As an approach to elucidating dopamine transporter (DAT) phosphorylation characteristics, we examined in vitro phosphorylation of a recombinant rat DAT N-terminal peptide (NDAT) using purified protein kinases. We found that NDAT becomes phosphorylated at single distinct sites by protein kinase A (Ser-7) and calcium-calmodulin-dependent protein kinase II (Ser-13) and at multiple sites (Ser-4, Ser-7, and Ser-13) by protein kinase C (PKC), implicating these residues as potential sites of DAT phosphorylation by these kinases. Mapping of rat striatal DAT phosphopeptides by two-dimensional thin layer chromatography revealed basal and PKC-stimulated phosphorylation of the same peptide fragments and comigration of PKC-stimulated phosphopeptide fragments with NDAT Ser-7 phosphopeptide markers. We further confirmed by site-directed mutagenesis and mass spectrometry that Ser-7 is a site for PKC-stimulated phosphorylation in heterologously expressed rat and human DATs. Mutation of Ser-7 and nearby residues strongly reduced the affinity of rat DAT for the cocaine analog (-)-2ß-carbomethoxy-3ß-(4-fluorophenyl) tropane (CFT), whereas in rat striatal tissue, conditions that promote DAT phosphorylation caused increased CFT affinity. Ser-7 mutation also affected zinc modulation of CFT binding, with Ala and Asp substitutions inducing opposing effects. These results identify Ser-7 as a major site for basal and PKC-stimulated phosphorylation of native and expressed DAT and suggest that Ser-7 phosphorylation modulates transporter conformational equilibria, shifting the transporter between high and low affinity cocaine binding states.
Asunto(s)
Cocaína/química , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/química , Serina/química , Animales , Sitios de Unión , Cromatografía en Capa Delgada/métodos , Cocaína/análogos & derivados , Cuerpo Estriado/efectos de los fármacos , Cuerpo Estriado/metabolismo , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/metabolismo , Inhibidores de Captación de Dopamina/química , Células HEK293 , Humanos , Cinética , Masculino , Espectrometría de Masas/métodos , Mutagénesis Sitio-Dirigida , Mutación , Fosforilación , Unión Proteica , Conformación Proteica , Ratas , Ratas Sprague-DawleyRESUMEN
Regulatory T cells (Treg) are crucial for self-tolerance. It has been an enigma that Treg exhibit an anergic phenotype reflected by hypoproliferation in vitro after TCR stimulation but undergo vigorous proliferation in vivo. We report in this study that murine Treg are prone to death but hyperproliferative in vitro and in vivo, which is different from conventional CD4(+)Foxp3(-) T cells (Tcon). During in vitro culture, most Treg die with or without TCR stimulation, correlated with constitutive activation of the intrinsic death pathway. However, a small portion of the Treg population is more sensitive to TCR stimulation, particularly weak stimulation, proliferates more vigorously than CD4(+) Tcon, and is resistant to activation-induced cell death. Treg proliferation is enhanced by IL-2 but is less dependent on CD28-mediated costimulation than that of Tcon. We demonstrate further that the surviving and proliferative Treg are ICOS(+) whereas the death-prone Treg are ICOS(-). Moreover, ICOS(+) Treg contain much stronger suppressive activity than that of ICOS(-) Treg. Our data indicate that massive death contributes to the anergic phenotype of Treg in vitro and suggest modulation of Treg survival as a therapeutic strategy for treatment of autoimmune diseases and cancer.
Asunto(s)
Proteína Coestimuladora de Linfocitos T Inducibles/biosíntesis , Subgrupos de Linfocitos T/metabolismo , Linfocitos T Reguladores/metabolismo , Animales , Antígenos CD28/inmunología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Proliferación Celular , Factores de Transcripción Forkhead/análisis , Interleucina-2/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Fenotipo , Receptores de Antígenos de Linfocitos T/inmunología , Transducción de Señal , Subgrupos de Linfocitos T/inmunología , Linfocitos T Reguladores/inmunología , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
The engagement of TCR induces T-cell activation, which initiates multiple characteristic changes such as increase in cell size, cell division, and the production of cytokines and other effector molecules. The mammalian target of rapamycin (mTOR) regulates protein synthesis, transcription, cell survival, and autophagy. Critical roles of mTOR in T-cell activation and effector/memory differentiation have been revealed using chemical inhibitors or by genetic ablation of mTOR in T cells. However, the connection between mTOR signaling and other signaling cascades downstream of TCR is unclear. We demonstrate that diacylglycerol (DAG) and TCR engagement activate signaling in both mTOR complexes 1 and 2 through the activation of the Ras-mitogen-activated protein kinase/extracellular signal-regulated kinase 1/2 (Mek1/2)-extracellular signal-regulated kinase 1/2 (Erk1/2)-activator protein 1 (AP-1), known collectively as the Ras-Mek1/2-Erk1/2-AP-1 pathway. Deficiency of RasGRP1 or inhibition of Mek1/2 activity drastically decreases TCR-induced mTOR activation, whereas constitutively active Ras or Mek1 promotes mTOR activation. Although constitutively active Akt promotes TCR-induced mTOR activation, such activation is attenuated by Mek1/2 inhibition. We demonstrated further that DAG kinases (DGKs) α and ζ, which terminate DAG-mediated signaling, synergistically inhibit TCR-induced mTOR activation by inhibiting the Ras-Mek1/2-Erk/12 pathway. These observations provide novel insights into the regulation of mTOR activation.
Asunto(s)
Diacilglicerol Quinasa/metabolismo , Activación de Linfocitos/fisiología , Sistema de Señalización de MAP Quinasas/inmunología , Linfocitos T/enzimología , Serina-Treonina Quinasas TOR/metabolismo , Animales , Línea Celular , Diacilglicerol Quinasa/genética , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Memoria Inmunológica/fisiología , MAP Quinasa Quinasa 1/metabolismo , MAP Quinasa Quinasa 2/metabolismo , Ratones , Ratones Mutantes , Fosfatidilinositol 3-Quinasas/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Serina-Treonina Quinasas TOR/genética , Timo/citología , Timo/inmunología , Timo/metabolismo , Factor de Transcripción AP-1/metabolismo , Proteínas ras/metabolismoRESUMEN
Type I NKT cells, or invariant NKT (iNKT) cells, express a semi-invariant TCR characterized by its unique Vα14-Jα18 usage (iVα14TCR). Upon interaction with glycolipid/CD1d complexes, the iVα14TCRs transduce signals that are essential for iNKT selection and maturation. However, it remains unclear how these signals are regulated and how important such regulations are during iNKT development. Diacylglycerol (DAG) is an essential second messenger downstream of the TCR that activates the protein kinase C-IκB kinase (IKK)α/ß-NF-κB pathway, known to be crucial for iNKT development, as well as the RasGRP1-Ras-Erk1/2 pathway in T cells. DAG kinases play an important role in controlling intracellular DAG concentration and thereby negatively regulate DAG signaling. In this article, we report that simultaneous absence of DAG kinase α and ζ causes severe defects in iNKT development, coincident with enhanced IKK-NF-κB and Ras-Erk1/2 activation. Moreover, constitutive IKKß and Ras activities also result in iNKT developmental defects. Thus, DAG-mediated signaling is not only essential but also needs to be tightly regulated for proper iNKT cell development.
Asunto(s)
Diferenciación Celular/inmunología , Diacilglicerol Quinasa/inmunología , Células T Asesinas Naturales/inmunología , Transducción de Señal/inmunología , Animales , Western Blotting , Diferenciación Celular/genética , Separación Celular , Diacilglicerol Quinasa/genética , Diacilglicerol Quinasa/metabolismo , Diglicéridos/inmunología , Diglicéridos/metabolismo , Citometría de Flujo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Células T Asesinas Naturales/citología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/genéticaRESUMEN
The invariant NKT (iNKT) cell lineage contains CD4(+) and CD4(-) subsets. The mechanisms that control such subset differentiation and iNKT cell maturation in general have not been fully understood. RasGRP1, a guanine nucleotide exchange factor for TCR-induced activation of the Ras-ERK1/2 pathway, is critical for conventional αß T cell development but dispensable for generating regulatory T cells. Its role in iNKT cells has been unknown. In this study, we report severe decreases of iNKT cells in RasGRP1(-/-) mice through cell intrinsic mechanisms. In the remaining iNKT cells in RasGRP1(-/-) mice, there is a selective absence of the CD4(+) subset. Furthermore, RasGRP1(-/-) iNKT cells are defective in TCR-induced proliferation in vitro. These observations establish that RasGRP1 is not only important for early iNKT cell development but also for the generation/maintenance of the CD4(+) iNKT cells. Our data provide genetic evidence that the CD4(+) and CD4(-) iNKT cells are distinct sublineages with differential signaling requirements for their development.
Asunto(s)
Diferenciación Celular/inmunología , Factores de Intercambio de Guanina Nucleótido/fisiología , Células T Asesinas Naturales/inmunología , Animales , Antígenos CD4/biosíntesis , Antígenos CD4/genética , Diferenciación Celular/genética , Linaje de la Célula/genética , Linaje de la Célula/inmunología , Factores de Intercambio de Guanina Nucleótido/deficiencia , Factores de Intercambio de Guanina Nucleótido/genética , Linfopenia/genética , Linfopenia/inmunología , Linfopenia/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Células T Asesinas Naturales/citología , Células T Asesinas Naturales/metabolismo , Receptores de Antígenos de Linfocitos T alfa-beta/deficiencia , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Receptores de Antígenos de Linfocitos T gamma-delta/deficiencia , Receptores de Antígenos de Linfocitos T gamma-delta/genética , Transducción de Señal/genética , Transducción de Señal/inmunologíaRESUMEN
The mammalian target of rapamycin (mTOR) is a key regulator of cell growth and metabolism. It associates with multiple proteins and forms two distinct signaling complexes, mTORC1 and mTORC2. Accumulating evidence has revealed critical roles for intact mTOR signaling during T-cell activation and responses to microbial infection. However, the importance of mTOR regulation in T cells has yet to be explored. The TSC1/TSC2 complex has been shown to inhibit mTORC1 signaling in cell line models. We show here that deletion of TSC1 in the murine T-cell lineage results in a dramatic reduction of the peripheral T-cell pool, correlating with increased cell death. While mTORC1 is constitutively activated, mTORC2 signaling, reflected by Akt phosphorylation and activity, is decreased in TSC1-deficient T cells. Furthermore, TSC1-deficient T cells contain elevated reactive oxygen species (ROS) and exhibit decreased mitochondrial content and membrane potential, which is correlated with the activation of the intrinsic death pathway. Overall, our results demonstrate that TSC1 differentially regulates mTORC1 and mTORC2 activity, promotes T-cell survival, and is critical for normal mitochondrial homeostasis in T cells.
Asunto(s)
Homeostasis/inmunología , Mitocondrias/inmunología , Transducción de Señal/inmunología , Linfocitos T/metabolismo , Proteínas Supresoras de Tumor/inmunología , Animales , Apoptosis/inmunología , Línea Celular , Separación Celular , Supervivencia Celular/inmunología , Citometría de Flujo , Immunoblotting , Diana Mecanicista del Complejo 1 de la Rapamicina , Ratones , Ratones Noqueados , Microscopía Fluorescente , Mitocondrias/metabolismo , Complejos Multiproteicos , Proteínas/inmunología , Proteínas/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Linfocitos T/citología , Serina-Treonina Quinasas TOR , Transactivadores/inmunología , Transactivadores/metabolismo , Factores de Transcripción , Proteína 1 del Complejo de la Esclerosis Tuberosa , Proteínas Supresoras de Tumor/metabolismoRESUMEN
CpG oligodeoxynucleotides are potent toll-like receptor (TLR) 9 agonists and have shown promise as anticancer agents in preclinical studies and clinical trials. Binding of CpG to TLR9 initiates a cascade of innate and adaptive immune responses, beginning with activation of dendritic cells and resulting in a range of secondary effects that include the secretion of pro-inflammatory cytokines, activation of natural killer cells, and expansion of T cell populations. Recent literature suggests that local delivery of CpG in tumors results in superior antitumor effects as compared to systemic delivery. In this study, we utilized PRINT (particle replication in nonwetting templates) nanoparticles as a vehicle to deliver CpG into murine lungs through orotracheal instillations. In two murine orthotopic metastasis models of non-small-cell lung cancer-344SQ (lung adenocarcinoma) and KAL-LN2E1 (lung squamous carcinoma), local delivery of PRINT-CpG into the lungs effectively promoted substantial tumor regression and also limited systemic toxicities associated with soluble CpG. Furthermore, cured mice were completely resistant to tumor rechallenge. Additionally, nanodelivery showed extended retention of CpG within the lungs as well as prolonged elevation of antitumor cytokines in the lungs, but no elevated levels of proinflammatory cytokines in the serum. These results demonstrate that PRINT-CpG is a potent nanoplatform for local treatment of lung cancer that has collateral therapeutic effects on systemic disease and an encouraging toxicity profile and may have the potential to treat lung metastasis of other cancer types.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Nanopartículas , Animales , Pulmón , Neoplasias Pulmonares/tratamiento farmacológico , Ratones , Ratones Endogámicos C57BL , Oligodesoxirribonucleótidos , Receptor Toll-Like 9RESUMEN
Phosphorylation of the dopamine transporter (DAT) on N-terminal serines and unidentified threonines occurs concomitantly with protein kinase C (PKC)- and substrate-induced alterations in transporter activity, subcellular distribution, and dopamine efflux, but the residues phosphorylated and identities of protein kinases and phosphatases involved are not known. As one approach to investigating these issues, we recombinantly expressed the N-terminal tail of rat DAT (NDAT) and examined its phosphorylation and dephosphorylation properties in vitro. We found that NDAT could be phosphorylated to significant levels by PKCalpha, PKA, PKG, and CaMKII, which catalyzed serine phosphorylation, and ERK1, JNK, and p38, which catalyzed threonine phosphorylation. We identified Thr53, present in a membrane proximal proline-directed kinase motif as the NDAT site phosphorylated in vitro by ERK1, JNK and p38, and confirmed by peptide mapping and mutagenesis that Thr53 is phosphorylated in vivo. Dephosphorylation studies showed that protein phosphatase 1 catalyzed near-complete in vitro dephosphorylation of PKCalpha-phosphorylated NDAT, similar to its in vivo and in vitro effects on native DAT. These findings demonstrate the ability of multiple enzymes to directly recognize the DAT N-terminal domain and for kinases to act at multiple distinct sites. The strong correspondence between NDAT and rDAT phosphorylation characteristics suggests the potential for the enzymes that are active on NDAT in vitro to act on DAT in vivo and indicates the usefulness of NDAT for guiding future DAT phosphorylation analyses.
Asunto(s)
Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/química , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/metabolismo , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Prolina/química , Secuencia de Aminoácidos , Animales , Cuerpo Estriado/química , Cuerpo Estriado/metabolismo , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/genética , Escherichia coli/genética , Humanos , Datos de Secuencia Molecular , Fragmentos de Péptidos/genética , Fosforilación , Estructura Terciaria de Proteína/genética , Ratas , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
Lung cancer is the leading cause of cancer-related deaths worldwide, and lung squamous carcinomas (LUSC) represent about 30% of cases. Molecular aberrations in lung adenocarcinomas have allowed for effective targeted treatments, but corresponding therapeutic advances in LUSC have not materialized. However, immune checkpoint inhibitors in sub-populations of LUSC patients have led to exciting responses. Using computational analyses of The Cancer Genome Atlas, we identified a subset of LUSC tumors characterized by dense infiltration of inflammatory monocytes (IMs) and poor survival. With novel, immunocompetent metastasis models, we demonstrated that tumor cell derived CCL2-mediated recruitment of IMs is necessary and sufficient for LUSC metastasis. Pharmacologic inhibition of IM recruitment had substantial anti-metastatic effects. Notably, we show that IMs highly express Factor XIIIA, which promotes fibrin cross-linking to create a scaffold for LUSC cell invasion and metastases. Consistently, human LUSC samples containing extensive cross-linked fibrin in the microenvironment correlated with poor survival.
Asunto(s)
Carcinoma de Células Escamosas/inmunología , Factor XIIIa/inmunología , Fibrina/química , Neoplasias Pulmonares/inmunología , Monocitos/inmunología , Animales , Biomarcadores de Tumor/química , Biomarcadores de Tumor/inmunología , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patología , Quimiocina CCL2/genética , Quimiocina CCL2/inmunología , Factor XIIIa/genética , Femenino , Fibrina/inmunología , Regulación Neoplásica de la Expresión Génica , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Masculino , Ratones , Ratones Endogámicos DBA , Invasividad NeoplásicaRESUMEN
The dopamine transporter (DAT) is a phosphoprotein whose activity and phosphorylation state are acutely regulated by both protein kinase C (PKC) and substrate transport. DAT is a major site of action for psychostimulant and therapeutic drugs that either block transport or are transported substrates, but the effects of such drugs on DAT phosphorylation and regulation are not well understood. To examine these issues we subjected rDAT LLC-PK(1) cells to acute in vitro pretreatments with the endogenous, psychostimulant, and therapeutic compounds dopamine (DA), (-)-cocaine, 2 beta-carbomethoxy-3beta-(4-fluorophenyl)tropane (beta-CFT), GBR 12909, mazindol, and methylphenidate (MPH), in the presence or absence of the PKC activator phorbol 12 myristate 13 acetate (PMA), followed by analysis of DAT metabolic phosphorylation and transport activity. Basal phosphorylation of DAT was not affected by any of the uptake blockers tested, and PMA-stimulated phosphorylation was not affected by cocaine, beta-CFT, mazindol or MPH, but was strongly suppressed by GBR 12909. Pretreatment of cells with cocaine or MPH had no effect on subsequent DA transport activity or the extent of PMA-induced transport down-regulation, whereas GBR 12909 inhibited PMA-induced DAT internalization. These findings indicate that these DAT phosphorylation and down-regulation properties are unaffected by some classes of uptake blocking drugs, but that differential regulatory effects may be exerted by GBR compounds. Pretreatment of cells with DA had no obvious effect on basal or PMA-stimulated DAT phosphorylation but led to cocaine-blockable transport down-regulation. DA-induced down-regulation was blocked by the PKC inhibitor bisindoylmaleimide I and was not additive with down-regulation induced by PMA, consistent with PKC serving as a common step and point of integration for these DA and PMA induced processes. The results of this study provide information on the potential for endogenous and psychoactive compounds to modulate or be modulated by DAT phosphorylation-mediated regulatory mechanisms that may contribute to drug behavioral or therapeutic properties.
Asunto(s)
Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/metabolismo , Dopamina/farmacología , Psicotrópicos/farmacología , Animales , Autorradiografía/métodos , Biotinilación/métodos , Línea Celular , Inhibidores de Captación de Dopamina/farmacología , Relación Dosis-Respuesta a Droga , Interacciones Farmacológicas , Inhibidores Enzimáticos/farmacología , Inmunoprecipitación/métodos , Ésteres del Forbol/farmacología , Isótopos de Fósforo/metabolismo , Fosforilación/efectos de los fármacos , Piperazinas/farmacología , Transporte de Proteínas/efectos de los fármacos , Porcinos , Tritio/metabolismoRESUMEN
The T cell receptor (TCR) recognizes self or foreign antigens presented by major histocompatibility complex (MHC) molecules. Engagement of the TCR triggers the formation of multi-molecular signalosomes that lead to the generation of second messengers and subsequent activation of multiple distal signaling cascades, such as the Ca+2-calcineurin-NFAT, RasGRP1-Ras-Erk1/2, PKCθ-IKK-NFκB, and TSC1/2-mTOR pathways. These signaling cascades control many aspects of T cell biology. Mechanisms have been evolved to fine-tune TCR signaling to maintain T cell homeostasis and self-tolerance, and to properly mount effective responses to microbial infection. Defects or deregulation of TCR signaling has been implicated in the pathogenesis of multiple human diseases.
RESUMEN
Immune cell development and function must be tightly regulated through cell surface receptors to ensure proper responses to pathogen and tolerance to self. In T cells, the signal from the T-cell receptor is essential for T-cell maturation, homeostasis, and activation. In mast cells, the high-affinity receptor for IgE transduces signal that promotes mast cell survival and induces mast cell activation. In dendritic cells and macrophages, the toll-like receptors recognize microbial pathogens and play critical roles for both innate and adaptive immunity against pathogens. Our research explores how signaling from these receptors is transduced and regulated to better understand these immune cells. Our recent studies have revealed diacylglycerol kinases and TSC1/2-mTOR as critical signaling molecules/regulators in T cells, mast cells, dendritic cells, and macrophages.