RESUMO
The serine/threonine kinase glycogen synthase kinase-3 (GSK3) plays an important role in balancing pro- and anti-inflammatory cytokines. We have examined the role of GSK3 in production of IL-10 by subsets of CD4(+) T helper cells. Treatment of naive murine CD4(+) T cells with GSK3 inhibitors did not affect their production of IL-10. However, treatment of Th1 and Th2 cells with GSK3 inhibitors dramatically increased production of IL-10. GSK3 inhibition also led to upregulation of IL-10 among Th1, Th2, and Th17 subsets isolated from human blood. The encephalitogenic potential of GSK3 inhibitor treated murine Th1 cells was significantly reduced in adoptive transfer experiments by an IL-10-dependent mechanism. Analysis of the murine IL-10 promoter in response to inhibition of GSK3 in Th1 cells showed modification to a transcriptionally active state indicated by changes in histone H3 acetylation and methylation. Additionally, GSK3 inhibition increased expression of the transcription factors c-Maf, Nfil3, and GATA3, correlating with the increase in IL-10. These findings are important in the context of autoimmune disease since they show that it is possible to reprogram disease-causing cells through GSK3 inhibition.
Assuntos
Quinase 3 da Glicogênio Sintase/metabolismo , Interleucina-10/biossíntese , Células Th1/imunologia , Células Th17/imunologia , Células Th2/imunologia , Acetilação , Transferência Adotiva , Animais , Fatores de Transcrição de Zíper de Leucina Básica/biossíntese , Células Cultivadas , Células Dendríticas/imunologia , Encefalomielite Autoimune Experimental/imunologia , Fator de Transcrição GATA3/biossíntese , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Histonas/metabolismo , Humanos , Inflamação/imunologia , Interleucina-10/genética , Metilação , Camundongos , Camundongos Knockout , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-maf/biossíntese , Células Th1/transplanteRESUMO
PIKfyve (FYVE domain-containing phosphatidylinositol 3-phosphate 5-kinase), the lipid kinase that phosphorylates PtdIns3P to PtdIns(3,5)P2, has been implicated in insulin-stimulated glucose uptake. We investigated whether PIKfyve could also be involved in contraction/AMPK (AMP-activated protein kinase)-stimulated glucose uptake in skeletal muscle. Incubation of rat epitrochlearis muscles with YM201636, a selective PIKfyve inhibitor, reduced contraction- and AICAriboside (5-amino-4-imidazolecarboxamide riboside)-stimulated glucose uptake. Consistently, PIKfyve knockdown in C2C12 myotubes reduced AICAriboside-stimulated glucose transport. Furthermore, muscle contraction increased PtdIns(3,5)P2 levels and PIKfyve phosphorylation. AMPK phosphorylated PIKfyve at Ser307 both in vitro and in intact cells. Following subcellular fractionation, PIKfyve recovery in a crude intracellular membrane fraction was increased in contracting versus resting muscles. Also in opossum kidney cells, wild-type, but not S307A mutant, PIKfyve was recruited to endosomal vesicles in response to AMPK activation. We propose that PIKfyve activity is required for the stimulation of skeletal muscle glucose uptake by contraction/AMPK activation. PIKfyve is a new AMPK substrate whose phosphorylation at Ser307 could promote PIKfyve translocation to endosomes for PtdIns(3,5)P2 synthesis to facilitate GLUT4 (glucose transporter 4) translocation.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Glucose/metabolismo , Contração Muscular/fisiologia , Músculo Esquelético/enzimologia , Músculo Esquelético/metabolismo , Fosfatidilinositol 3-Quinase/metabolismo , Proteínas Quinases Ativadas por AMP/genética , Aminoimidazol Carboxamida/análogos & derivados , Aminoimidazol Carboxamida/metabolismo , Animais , Linhagem Celular , Transportador de Glucose Tipo 4/metabolismo , Humanos , Insulina/metabolismo , Masculino , Gambás , Fosfatidilinositol 3-Quinase/genética , Fosfatos de Fosfatidilinositol/metabolismo , Fosforilação , Ratos , Ratos WistarRESUMO
Inward rectifier potassium channels of the Kir2 subfamily are important determinants of the electrical activity of brain and muscle cells. Genetic mutations in Kir2.1 associate with Andersen-Tawil syndrome (ATS), a familial disorder leading to stress-triggered periodic paralysis and ventricular arrhythmia. To identify the molecular mechanisms of this stress trigger, we analyze Kir channel function and localization electrophysiologically and by time-resolved confocal microscopy. Furthermore, we employ a mathematical model of muscular membrane potential. We identify a novel corticoid signaling pathway that, when activated by glucocorticoids, leads to enrichment of Kir2 channels in the plasma membranes of mammalian cell lines and isolated cardiac and skeletal muscle cells. We further demonstrate that activation of this pathway can either partly restore (40% of cases) or further impair (20% of cases) the function of mutant ATS channels, depending on the particular Kir2.1 mutation. This means that glucocorticoid treatment might either alleviate or deteriorate symptoms of ATS depending on the patient's individual Kir2.1 genotype. Thus, our findings provide a possible explanation for the contradictory effects of glucocorticoid treatment on symptoms in patients with ATS and may open new pathways for the design of personalized medicines in ATS therapy.
Assuntos
Síndrome de Andersen/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Síndrome de Andersen/tratamento farmacológico , Síndrome de Andersen/genética , Animais , Feminino , Glucocorticoides/uso terapêutico , Cobaias , Células HEK293 , Células HeLa , Humanos , Proteínas Imediatamente Precoces/metabolismo , Técnicas In Vitro , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Miócitos Cardíacos/metabolismo , Oócitos/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização/química , Canais de Potássio Corretores do Fluxo de Internalização/genética , Proteínas Serina-Treonina Quinases/metabolismo , Estrutura Terciária de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transdução de Sinais , Estresse Fisiológico , Xenopus laevisRESUMO
cAMP-specific PDE (phosphodiesterase) 4 isoforms underpin compartmentalized cAMP signalling in mammalian cells through targeting to specific signalling complexes. Their importance is apparent as PDE4 selective inhibitors exert profound anti-inflammatory effects and act as cognitive enhancers. The p38 MAPK (mitogen-activated protein kinase) signalling cascade is a key signal transduction pathway involved in the control of cellular immune, inflammatory and stress responses. In the present study, we show that PDE4A5 is phosphorylated at Ser147, within the regulatory UCR1 (ultraconserved region 1) domain conserved among PDE4 long isoforms, by MK2 (MAPK-activated protein kinase 2, also called MAPKAPK2). Phosphorylation by MK2, although not altering PDE4A5 activity, markedly attenuates PDE4A5 activation through phosphorylation by protein kinase A. This modification confers the amplification of intracellular cAMP accumulation in response to adenylate cyclase activation by attenuating a major desensitization system to cAMP. Such reprogramming of cAMP accumulation is recapitulated in wild-type primary macrophages, but not MK2/3-null macrophages. Phosphorylation by MK2 also triggers a conformational change in PDE4A5 that attenuates PDE4A5 interaction with proteins whose binding involves UCR2, such as DISC1 (disrupted in schizophrenia 1) and AIP (aryl hydrocarbon receptor-interacting protein), but not the UCR2-independent interacting scaffold protein ß-arrestin. Long PDE4 isoforms thus provide a novel node for cross-talk between the cAMP and p38 MAPK signalling systems at the level of MK2.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Células COS , Chlorocebus aethiops , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Camundongos , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Especificidade por SubstratoRESUMO
We identify a compartmentalized signaling system that identifies a functional role for the GTP exchange factor, exchange protein activated by cAMP (EPAC) coupled to Rap2 in the nucleus. In this system, cAMP regulates the nuclear/cytoplasmic trafficking of DNA-dependent protein kinase (DNA-PK), a critical kinase that acts to repair double-stranded breaks (DSBs) in damaged DNA and to phosphorylate the cell survival kinase, PKB/Akt. Intersecting regulatory inputs for cAMP employ EPAC to transduce positive effects, namely the Rap2-dependent nuclear exit and activation of DNA-PK, whereas protein kinase A (PKA) provides the negative input by antagonizing these actions. We identify this as a compartmentalized regulatory system where modulation of cAMP input into the stimulatory, EPAC and inhibitory, PKA intersecting arms is provided by spatially discrete, cAMP degradation systems. The distribution of DNA-PK between nuclear and cytoplasmic compartments can thus potentially be influenced by relative inputs of cAMP signaling through the EPAC and PKA pathways. Through this signaling system EPAC activation can thereby impact on the Ser-473 phosphorylation status of PKB/Akt and the repair of etoposide-induced DSBs.
Assuntos
Núcleo Celular/enzimologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Sequência de Aminoácidos , Quebras de DNA de Cadeia Dupla , Ativação Enzimática , Células HeLa , Humanos , Espaço Intracelular/metabolismo , Dados de Sequência Molecular , Peptídeos/química , Diester Fosfórico Hidrolases/metabolismo , Fosforilação , Fosfosserina/metabolismo , Transporte Proteico , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Proteínas rap de Ligação ao GTP/metabolismoRESUMO
Immunotherapy with antigen-processing independent T cell epitopes (apitopes) targeting autoreactive CD4+ T cells has translated to the clinic and been shown to modulate progression of both Graves' disease and multiple sclerosis. The model apitope (Ac1-9[4Y]) renders antigen-specific T cells anergic while repeated administration induces both Tr1 and Foxp3+ regulatory cells. Here we address why CD4+ T cell epitopes should be designed as apitopes to induce tolerance and define the antigen presenting cells that they target in vivo. Furthermore, we reveal the impact of treatment with apitopes on CD4+ T cell signaling, the generation of IL-10-secreting regulatory cells and the systemic migration of these cells. Taken together these findings reveal how apitopes induce tolerance and thereby mediate antigen-specific immunotherapy of autoimmune diseases.
Assuntos
Apresentação de Antígeno/imunologia , Doenças Autoimunes/imunologia , Doenças Autoimunes/terapia , Autoimunidade , Epitopos de Linfócito T/imunologia , Imunoterapia/métodos , Animais , Células Apresentadoras de Antígenos/imunologia , Células Apresentadoras de Antígenos/metabolismo , Citocinas/metabolismo , Modelos Animais de Doenças , Encefalomielite Autoimune Experimental , Tolerância Imunológica , Ativação Linfocitária , Camundongos , Camundongos Transgênicos , Peptídeos/química , Peptídeos/imunologiaRESUMO
Peripheral immune regulation is critical for the maintenance of self-tolerance. Here we have investigated signaling processes that distinguish T cells with regulatory capability from effector T cells. The murine Tg4 T cell receptor recognizes a peptide derived from the self-antigen myelin basic protein. T cells from Tg4 T cell receptor transgenic mice can be used to generate effector T cells and three types of T cells with regulatory capability, inducible regulatory T cells, T cells tolerized by repeated in vivo antigenic peptide exposure or T cells treated with the tolerogenic drug UCB9608 (a phosphatidylinositol 4 kinase IIIß inhibitor). We comparatively studied signaling in all of these T cells by activating them with the same antigen presenting cells presenting the same myelin basic protein peptide. Supramolecular signaling structures, as efficiently detected by large-scale live cell imaging, are critical mediators of T cell activation. The formation of a supramolecular signaling complex anchored by the adaptor protein linker for activation of T cells (LAT) was consistently terminated more rapidly in Tg4 T cells with regulatory capability. Such termination could be partially reversed by blocking the inhibitory receptors CTLA-4 and PD-1. Our work suggests that attenuation of proximal signaling may favor regulatory over effector function in T cells.
Assuntos
Células Apresentadoras de Antígenos/imunologia , Sinapses Imunológicas/imunologia , Ativação Linfocitária/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Camundongos Transgênicos , Receptores de Antígenos de Linfócitos T/imunologia , Transdução de Sinais/imunologia , Linfócitos T Reguladores/imunologiaRESUMO
PIKfyve is a protein and lipid kinase that plays an important role in membrane trafficking, including TGN to endosome retrograde sorting and in insulin-stimulated translocation of the GLUT4 glucose transporter from intracellular storage vesicles to the plasma membrane. We have previously demonstrated that PIKfyve is phosphorylated in response to insulin in a PI3-kinase and protein kinase B (PKB)-dependent manner. However, it has been implied that this was not due to direct phosphorylation of PIKfyve by PKB, but as a result of an insulin-induced PIKfyve autophosphorylation event. Here we demonstrate that purified PIKfyve is phosphorylated in vitro by a recombinant active PKB on two separate serine residues, S318 and S105, which flank the N-terminal FYVE domain of the protein. Only S318, however, becomes phosphorylated in intact cells stimulated with insulin. We further demonstrate that S318 is phosphorylated in response to hyperosmotic stress in a PI3-kinase- and PKB-independent manner. Importantly, the effects of insulin and sorbitol were not prevented by the presence of an ATP-competitive PIKfyve inhibitor (YM20163) or in a mutant PIKfyve lacking both lipid and protein kinase activity. Our results confirm, therefore, that PIKfyve is directly phosphorylated by PKB on a single serine residue in response to insulin and are not due to autophosphorylation of the enzyme. We further reveal that two stimuli known to promote glucose uptake in cells, both stimulate phosphorylation of PIKfyve on S318 but via distinct signal transduction pathways.
Assuntos
Insulina/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Quinases Dependentes de 3-Fosfoinositídeo , Animais , Células CHO , Cricetinae , Cricetulus , Insulina/farmacologia , Camundongos , Pressão Osmótica , Fosfatidilinositol 3-Quinases/genética , Fosforilação/efeitos dos fármacos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Serina/genética , Serina/metabolismo , Sorbitol/farmacologiaRESUMO
Glucose-stimulated insulin secretion from pancreatic beta-cells requires the kinesin-1/Kif5B-mediated transport of insulin granules along microtubules. 5'-AMPK (5'-AMP-activated protein kinase) is a heterotrimeric serine/threonine kinase which is activated in beta-cells at low glucose concentrations, but inhibited as glucose levels increase. Active AMPK blocks glucose-stimulated insulin secretion and the recruitment of insulin granules to the cell surface, suggesting motor proteins may be targets for this kinase. While both kinesin-1/Kif5B and KLC1 (kinesin light chain-1) contain consensus AMPK phosphorylation sites (Thr(693) and Ser(520), respectively) only recombinant GST (glutathione transferase)-KLC1 was phosphorylated by purified AMPK in vitro. To test the hypothesis that phosphorylation at this site may modulate kinesin-1-mediated granule movement, we developed an approach to study the dynamics of all the resolvable granules within a cell in three dimensions. This cell-wide approach revealed that the number of longer excursions (>10 mum) increased significantly in response to elevated glucose concentration (30 versus 3 mM) in control MIN6 beta-cells. However, similar changes were seen in cells overexpressing wild-type KLC1, phosphomimetic (S517D/S520D) or non-phosphorylatable (S517A/S520A) mutants of KLC1. Thus, changes in the phosphorylation state of KLC1 at Ser(517)/Ser(520) seem unlikely to affect motor function.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Vesículas Secretórias/metabolismo , Serina/metabolismo , Animais , Glucose/metabolismo , Humanos , Células Secretoras de Insulina/citologia , Cinesinas/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Mutação , Fosforilação , Isoformas de Proteínas/metabolismoRESUMO
Disrupted-in-schizophrenia 1 (DISC1) is a genetic susceptibility factor for schizophrenia and related severe psychiatric conditions. DISC1 is a multifunctional scaffold protein that is able to interact with several proteins, including the independently identified schizophrenia risk factor phosphodiesterase-4B (PDE4B). Here we report that the 100 kDa full-length DISC1 isoform (fl-DISC1) can bind members of each of the four gene, cAMP-specific PDE4 family. Elevation of intracellular cAMP levels, so as to activate protein kinase A, caused the release of PDE4D3 and PDE4C2 isoforms from fl-DISC1 while not affecting binding of PDE4B1 and PDE4A5 isoforms. Using a peptide array strategy, we show that PDE4D3 binds fl-DISC1 through two regions found in common with PDE4B isoforms, the interaction of which is supplemented because of the presence of additional PDE4B-specific binding sites. We propose that the additional binding sites found in PDE4B1 underpin its resistance to release during cAMP elevation. We identify, for the first time, a functional distinction between the 100 kDa long DISC1 isoform and the short 71 kDa isoform. Thus, changes in the expression pattern of DISC1 and PDE4 isoforms offers a means to reprogram their interaction and to determine whether the PDE4 sequestered by DISC1 is released after cAMP elevation. The PDE4B-specific binding sites encompass point mutations in mouse Disc1 that confer phenotypes related to schizophrenia and depression and that affect binding to PDE4B. Thus, genetic variation in DISC1 and PDE4 that influence either isoform expression or docking site functioning may directly affect psychopathology.
Assuntos
3',5'-AMP Cíclico Fosfodiesterases/metabolismo , AMP Cíclico/metabolismo , Líquido Extracelular/metabolismo , Proteínas do Tecido Nervoso/metabolismo , 1-Metil-3-Isobutilxantina/farmacologia , 3',5'-AMP Cíclico Fosfodiesterases/genética , Animais , Linhagem Celular , Chlorocebus aethiops , Colforsina/farmacologia , AMP Cíclico/análogos & derivados , AMP Cíclico/farmacologia , Proteínas Quinases Dependentes de AMP Cíclico/fisiologia , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4 , Líquido Extracelular/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Humanos , Imunoprecipitação/métodos , Proteínas do Tecido Nervoso/genética , Inibidores de Fosfodiesterase/farmacologia , Fosforilação/efeitos dos fármacos , Isoformas de Proteínas/metabolismo , Transfecção/métodosRESUMO
RAW macrophages, which express the PDE4D3 and PDE4D5 cAMP phosphodiesterase isoforms, exhibited increased PDE4 activity when challenged with H2O2 in a fashion that was negated by treatment with the cell permeant antioxidant, N-acetyl cysteine and by diphenyleneiodonium chloride, an inhibitor of NADPH oxidase. In Cos1 cells transfected to express PDE4D3, challenge with H2O2 caused a rapid increase in both the activity and phosphorylation of PDE4D3. Lysates from H2O2-treated COS cells caused the phosphorylation of purified, recombinant PDE4D3 at two sites. One was the established ERK phosphorylation site at Ser579, located at the extreme C-terminus of the catalytic unit, and the other was a novel site at Ser239, located at the extreme N-terminus of the catalytic unit. Double Ser239Ala:Ser579Ala mutation of PDE4D3 prevented its H2O2-dependent phosphorylation both in vitro and in intact COS cells. Phosphorylation of PDE4D3 at Ser579 was ablated by treating COS cells with the MEK inhibitor, PD98059, which also negated activation. The activity of the Ser239Ala:Ser579Ala double mutant, and the Ser579Ala single PDE4D3 mutant was unaffected by H2O2 challenge of COS cells, whilst the Ser239Ala mutant was inhibited. Wortmannin inhibited the H2O2-dependent phosphorylation of PDE4D3 in COS cells by around 50%, whilst it fully ablated phosphorylation at Ser239 as well as ablating activation of PDE4D3. Neither immunodepletion of p70S6 kinase nor siRNA-mediated knockdown of mTor inhibited the H2O2-dependent phosphorylation of PDE4D3 at Ser239. Activation of PDE4D3 by challenge with H2O2 was not additive with activation through protein kinase A (PKA)-mediated phosphorylation of PDE4D3. Challenge with H2O2 did not alter PKA-mediated phosphorylation of PDE4D3 at Ser54. H2O2 dependent phosphorylation of PDE4D3, at Ser239 and Ser579, did not alter the sensitivity of PDE4D3 to inhibition by the selective PDE4 inhibitor, rolipram. An unknown protein kinase acting downstream of phosphatidyl inositol 3-kinase phosphorylates PDE4D3 at Ser239. This switches the effect of phosphorylation by ERK at Ser579 from inhibition to activation. We propose that phosphorylation at Ser239 attenuates interaction between either UCR2 or the UCR1/UCR2 module and the PDE4 catalytic unit so as to re-programme the functional outcome effect of phosphorylation by ERK. We identify a novel process through which reactive oxygen species activate long PDE4 isoforms so as to reduce cAMP levels and thereby promote inflammatory responses.
Assuntos
3',5'-AMP Cíclico Fosfodiesterases/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Estresse Oxidativo , Fosfatidilinositol 3-Quinases/metabolismo , Serina/química , Transdução de Sinais , 3',5'-AMP Cíclico Fosfodiesterases/antagonistas & inibidores , Animais , Sítios de Ligação , Células COS , Linhagem Celular , Células Cultivadas , Chlorocebus aethiops , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4 , Ativação Enzimática/efeitos dos fármacos , Humanos , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/enzimologia , Macrófagos/metabolismo , Camundongos , Oxidantes/farmacologia , Inibidores de Fosfodiesterase/farmacologia , Fosforilação/efeitos dos fármacos , Serina/metabolismoRESUMO
IL-10 is an immunomodulatory cytokine with a critical role in limiting inflammation in immune-mediated pathologies. The mechanisms leading to IL-10 expression by CD4+ T cells are being elucidated, with several cytokines implicated. We explored the effect of IL-4 on the natural phenomenon of IL-10 production by a chronically stimulated antigen-specific population of differentiated Th1 cells. In vitro, IL-4 blockade inhibited while addition of exogenous IL-4 to Th1 cultures enhanced IL-10 production. In the in vivo setting of peptide immunotherapy leading to a chronically stimulated Th1 phenotype, lack of IL-4Rα inhibited the induction of IL-10. Exploring the interplay of Th1 and Th2 cells through co-culture, Th2-derived IL-4 promoted IL-10 expression by Th1 cultures, reducing their pathogenicity in vivo. Co-culture led to upregulated c-Maf expression with no decrease in the proportion of T-bet+ cells in these cultures. Addition of IL-4 also reduced the encephalitogenic capacity of Th1 cultures. These data demonstrate that IL-4 contributes to IL-10 production and that Th2 cells modulate Th1 cultures towards a self-regulatory phenotype, contributing to the cross-regulation of Th1 and Th2 cells. These findings are important in the context of Th1 driven diseases since they reveal how the Th1 phenotype and function can be modulated by IL-4.
Assuntos
Interleucina-10/metabolismo , Interleucina-4/metabolismo , Células Th1/imunologia , Células Th1/metabolismo , Animais , Biomarcadores , Citocinas/metabolismo , Imunofenotipagem , Ativação Linfocitária , Camundongos , Camundongos Knockout , Fenótipo , Receptores de Superfície Celular/genética , Fator de Transcrição STAT6/metabolismo , Células Th2/imunologia , Células Th2/metabolismoRESUMO
Notch is a critical regulator of T cell differentiation and is activated through proteolytic cleavage in response to ligand engagement. Using murine myelin-reactive CD4 T cells, we demonstrate that proximal T cell signaling modulates Notch activation by a spatiotemporally constrained mechanism. The protein kinase PKCθ is a critical mediator of signaling by the T cell antigen receptor and the principal costimulatory receptor CD28. PKCθ selectively inactivates the negative regulator of F-actin generation, Coronin 1A, at the center of the T cell interface with the antigen presenting cell (APC). This allows for effective generation of the large actin-based lamellum required for recruitment of the Notch-processing membrane metalloproteinase ADAM10. Such enhancement of Notch activation is critical for efficient T cell proliferation and Th17 differentiation. We reveal a novel mechanism that, through modulation of the cytoskeleton, controls Notch activation at the T cell:APC interface thereby linking T cell receptor and Notch signaling pathways.
Assuntos
Citoesqueleto de Actina/metabolismo , Proteína Quinase C-theta/metabolismo , Receptores Notch/metabolismo , Transdução de Sinais , Linfócitos T/imunologia , Proteína ADAM10/metabolismo , Secretases da Proteína Precursora do Amiloide/metabolismo , Animais , Antígenos CD28/metabolismo , Proteínas de Membrana/metabolismo , Camundongos , Proteínas dos Microfilamentos/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismoRESUMO
We employ a novel, dominant negative approach to identify a key role for certain tethered cyclic AMP specific phosphodiesterase-4 (PDE4) isoforms in regulating cyclic AMP dependent protein kinase A (PKA) sub-populations in resting COS1 cells. A fraction of PKA is clearly active in resting COS1 cells and this activity increases when cells are treated with the selective PDE4 inhibitor, rolipram. Point mutation of a critical, conserved aspartate residue in the catalytic site of long PDE4A4, PDE4B1, PDE4C2 and PDE4D3 isoforms renders them catalytically inactive. Overexpressed in resting COS1 cells, catalytically inactive forms of PDE4C2 and PDE4D3, but not PDE4A4 and PDE4B1, are constitutively PKA phosphorylated while overexpressed active versions of all these isoforms are not. Inactive and active versions of all these isoforms are PKA phosphorylated in cells where protein kinase A is maximally activated with forskolin and IBMX. By contrast, rolipram challenge of COS1 cells selectively triggers the PKA phosphorylation of recombinant, active PDE4D3 and PDE4C2 but not recombinant, active PDE4A4 and PDE4B1. Purified, recombinant PDE4D3 and PDE4A4 show a similar dose-dependency for in vitro phosphorylation by PKA. Disruption of the tethering of PKA type-II to PKA anchor proteins (AKAPs), achieved using the peptide Ht31, prevents inactive forms of PDE4C2 and PDE4D3 being constitutively PKA phosphorylated in resting cells as does siRNA-mediated knockdown of PKA-RII, but not PKA-RI. PDE4C2 and PDE4D3 co-immunoprecipitate from COS1 cell lysates with 250 kDa and 450 kDa AKAPs that tether PKA type-II and not PKA type-I. PKA type-II co-localises with AKAP450 in the centrosomal region of COS1 cells. The perinuclear distribution of recombinant, inactive PDE4D3, but not inactive PDE4A4, overlaps with AKAP450 and PKA type-II. The distribution of PKA phosphorylated inactive PDE4D3 also overlaps with that of AKAP450 in the centrosomal region of COS1 cells. We propose that a novel role for PDE4D3 and PDE4C2 is to gate the activation of AKAP450-tethered PKA type-II localised in the perinuclear region under conditions of basal cAMP generation in resting cells.
Assuntos
3',5'-AMP Cíclico Fosfodiesterases/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Centrossomo/enzimologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , AMP Cíclico/biossíntese , 3',5'-AMP Cíclico Fosfodiesterases/antagonistas & inibidores , 3',5'-AMP Cíclico Fosfodiesterases/genética , Animais , Células COS , Chlorocebus aethiops , Proteína Quinase Tipo II Dependente de AMP Cíclico , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Proteínas Quinases Dependentes de AMP Cíclico/genética , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4 , Ativação Enzimática , Isoenzimas/genética , Isoenzimas/metabolismo , Microscopia Confocal , Mutação , Inibidores de Fosfodiesterase/farmacologia , Interferência de RNA , Rolipram/farmacologiaRESUMO
Recently, it has been shown that enzymes of the cyclic adenosine monophosphate (cAMP)-specific phosphodiesterase (PDE) family 4 can be directly phosphorylated by extracellular signal-regulated kinase 2 (ERK2). Phosphorylation of PDE4s by ERK2 is dependent on two docking domains on either side of the target serine that allow specificity and high-fidelity binding of the kinase. The functional consequence of PDE4 phosphorylation by ERK is either an increase or a decrease in PDE activity, depending on whether the PDE4 contains only one of the upstream conserved regions (UCR1) that are typical of PDE4s or both (UCR1 and UCR2). We detail some of the methods that have been crucial in elucidating these important discoveries that represent a novel point of cross talk between the cAMP signaling system and the ERK mitogen-activated protein kinase cascade.
Assuntos
3',5'-AMP Cíclico Fosfodiesterases/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Sistemas do Segundo Mensageiro/fisiologia , 3',5'-AMP Cíclico Fosfodiesterases/genética , Animais , Células COS , Domínio Catalítico/genética , Domínio Catalítico/fisiologia , Chlorocebus aethiops , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4 , Humanos , Sistema de Sinalização das MAP Quinases/genética , Fosforilação , Sistemas do Segundo Mensageiro/genéticaRESUMO
In Rat-1 cells, the dramatic decrease in the levels of both intracellular cyclic 3'5' adenosine monophosphate (cyclic AMP; cAMP) and in the activity of cAMP-activated protein kinase A (PKA) observed in mitosis was paralleled by a profound increase in cAMP hydrolyzing phosphodiesterase-4 (PDE4) activity. The decrease in PKA activity, which occurs during mitosis, was attributable to PDE4 activation as the PDE4 selective inhibitor, rolipram, but not the phosphodiesterase-3 (PDE3) inhibitor, cilostamide, specifically ablated this cell cycle-dependent effect. PDE4 inhibition caused Rat-1 cells to move from S phase into G2/M more rapidly, to transit through G2/M more quickly and to remain in G1 for a longer period. Inhibition of PDE3 elicited no observable effects on cell cycle dynamics. Selective immunopurification of each of the four PDE4 sub-families identified PDE4D as being selectively activated in mitosis. Subsequent analysis uncovered PDE4D9, an isoform whose expression can be regulated by Disrupted-In-Schizophrenia 1 (DISC1)/activating transcription factor 4 (ATF4) complex, as the sole PDE4 species activated during mitosis in Rat-1 cells. PDE4D9 becomes activated in mitosis through dual phosphorylation at Ser585 and Ser245, involving the combined action of ERK and an unidentified 'switch' kinase that has previously been shown to be activated by H2O2. Additionally, in mitosis, PDE4D9 also becomes phosphorylated at Ser67 and Ser81, through the action of MK2 (MAPKAPK2) and AMP kinase (AMPK), respectively. The multisite phosphorylation of PDE4D9 by all four of these protein kinases leads to decreased mobility (band-shift) of PDE4D9 on SDS-PAGE. PDE4D9 is predominantly concentrated in the perinuclear region of Rat-1 cells but with a fraction distributed asymmetrically at the cell margins. Our investigations demonstrate that the diminished levels of cAMP and PKA activity that characterise mitosis are due to enhanced cAMP degradation by PDE4D9. PDE4D9, was found to locate primarily not only in the perinuclear region of Rat-1 cells but also at the cell margins. We propose that the sequestration of PDE4D9 in a specific complex together with AMPK, ERK, MK2 and the H2O2-activatable 'switch' kinase allows for its selective multi-site phosphorylation, activation and regulation in mitosis.
Assuntos
Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/metabolismo , Mitose , Proteínas do Tecido Nervoso/metabolismo , Fator 4 Ativador da Transcrição/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Ativação Enzimática , Interfase , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Dados de Sequência Molecular , Fosforilação , Isoformas de Proteínas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , RatosRESUMO
Since the discovery of interleukin-10 (IL-10) in the 1980s, a large body of work has led to its recognition as a pleiotropic immunomodulatory cytokine that affects both the innate and adaptive immune systems. IL-10 is produced by a wide range of cell types, but for the purposes of this review we shall focus on IL-10 secreted by CD4(+) T cells. Here we describe the importance of IL-10 as a mediator of suppression used by both FoxP3(+) and FoxP3(-) T regulatory cells. Moreover, we discuss the molecular events leading to the induction of IL-10 secretion in T helper cell subsets, where it acts as a pivotal negative feedback mechanism. Finally we discuss how a greater understanding of this principle has allowed for the design of more efficient, antigen-specific immunotherapy strategies to exploit this natural phenomenon clinically.
RESUMO
We previously showed that the serum- and glucocorticoid-inducible kinase 3 (SGK3) increases the AMPA-type glutamate receptor GluA1 protein in the plasma membrane. The activation of AMPA receptors by NMDA-type glutamate receptors eventually leads to postsynaptic neuronal plasticity. Here, we show that SGK3 mRNA is upregulated in the hippocampus of new-born wild type Wistar rats after NMDA receptor activation. We further demonstrate in the Xenopus oocyte expression system that delivery of GluA1 protein to the plasma membrane depends on the small GTPase RAB11. This RAB-dependent GluA1 trafficking requires phosphorylation and activation of phosphoinositol-3-phosphate-5-kinase (PIKfyve) and the generation of PI(3,5)P(2). In line with this mechanism we could show PIKfyve mRNA expression in the hippocampus of wild type C57/BL6 mice and phosphorylation of PIKfyve by SGK3. Incubation of hippocampal slices with the PIKfyve inhibitor YM201636 revealed reduced CA1 basal synaptic activity. Furthermore, treatment of primary hippocampal neurons with YM201636 altered the GluA1 expression pattern towards reduced synaptic expression of GluA1. Our findings demonstrate for the first time an involvement of PIKfyve and PI(3,5)P(2) in NMDA receptor-triggered synaptic GluA1 trafficking. This new regulatory pathway of GluA1 may contribute to synaptic plasticity and memory.
Assuntos
Receptores de AMPA/metabolismo , Transdução de Sinais , Aminopiridinas/farmacologia , Animais , Animais Recém-Nascidos , Inibidores Enzimáticos/farmacologia , Compostos Heterocíclicos com 3 Anéis/farmacologia , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Proteínas Imediatamente Precoces/genética , Proteínas Imediatamente Precoces/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Oócitos/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Fosforilação , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Receptores de N-Metil-D-Aspartato/metabolismo , Regulação para Cima , Xenopus/crescimento & desenvolvimento , Xenopus/metabolismo , Proteínas rab de Ligação ao GTP/metabolismoRESUMO
The movement of insulin granules along microtubules, driven by kinesin-1/Kif5B, is essential for glucose-stimulated insulin secretion from pancreatic ß-cells. 5ÎAMP-activated protein kinase (AMPK) is a heterotrimeric serine/threonine kinase, which is activated in ß-cells at low glucose concentrations, but inhibited as glucose levels increase. AMPK activation blocks glucose-stimulated recruitment of secretory granules to the cell surface and insulin secretion, suggesting motor proteins may be targets for this kinase. Whilst both kinesin-1/Kif5B and kinesin light chain-1 (KLC1) contain consensus AMPK phosphorylation sites only a peptide corresponding to Ser520 in mouse KLC1 and purified recombinant GST-KLC1 were phosphorylated by purified AMPK in vitro. To test the hypothesis that phosphorylation at this site may modulate kinesin-1-mediated granule movement, we developed a novel approach to study the dynamics of the granules within a cell in three-dimensions using Nokigawa spinning disc confocal microscopy. This cell-wide approach revealed that the number of longer excursions (>10 µm) increased significantly in response to elevated glucose concentration (30 vs 3 mM) in control MIN6 cells. However, similar changes were seen in cells over-expressing wild-type KLC1, phosphomimetic (S517/520D) or non-phosphorylatable (S517/520A) mutants of KLC1. Moreover, no evidence for a change in the phosphorylation state of KLC1 at Ser520 after AMPK activation was obtained using an anti-phospho Ser520-specific antibody. Thus, changes in the phosphorylation state of KLC1 at Ser517/520 are unlikely to affect motor function. In conclusion, we describe a new three-dimensional cell wide approach for the analysis of secretory granule dynamics in living ß-cells.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Insulina/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Vesículas Secretórias/metabolismo , Proteínas Quinases Ativadas por AMP/fisiologia , Sequência de Aminoácidos , Animais , Células CHO , Células Cultivadas , Cricetinae , Cricetulus , Cinesinas , Camundongos , Proteínas Associadas aos Microtúbulos/fisiologia , Modelos Biológicos , Dados de Sequência Molecular , Fosforilação/fisiologia , Processamento de Proteína Pós-Traducional/fisiologia , Ratos , Via Secretória/fisiologia , Homologia de Sequência de AminoácidosRESUMO
MicroRNAs (miRNAs) are short non-coding RNAs that have been implicated in fine-tuning gene regulation, although the precise roles of many are still unknown. Pancreatic development is characterized by the complex sequential expression of a gamut of transcription factors. We have performed miRNA expression profiling at two key stages of mouse embryonic pancreas development, e14.5 and e18.5. miR-124a2 expression was strikingly increased at e18.5 compared with e14.5, suggesting a possible role in differentiated beta-cells. Among the potential miR-124a gene targets identified by biocomputation, Foxa2 is known to play a role in beta-cell differentiation. To evaluate the impact of miR-124a2 on gene expression, we overexpressed or down-regulated miR-124a2 in MIN6 beta-cells. As predicted, miR-124a2 regulated Foxa2 gene expression, and that of its downstream target, pancreatic duodenum homeobox-1 (Pdx-1). Foxa2 has been described as a master regulator of pancreatic development and also of genes involved in glucose metabolism and insulin secretion, including the ATP-sensitive K(+) (K(ATP)) channel subunits, Kir6.2 and Sur-1. Correspondingly, miR-124a2 overexpression decreased, and anti-miR-124a2 increased Kir6.2 and Sur-1 mRNA levels. Moreover, miR-124a2 modified basal and glucose- or KCl-stimulated intracellular free Ca(2+) concentrations in single MIN6 and INS-1 (832/13) beta-cells, without affecting the secretion of insulin or co-transfected human growth hormone, consistent with an altered sensitivity of the beta-cell exocytotic machinery to Ca(2+). In conclusion, whereas the precise role of microRNA-124a2 in pancreatic development remains to be deciphered, we identify it as a regulator of a key transcriptional protein network in beta-cells responsible for modulating intracellular signaling.