RESUMO
Cytokine receptors consist of multiple subunits, which are often shared between different receptors, resulting in the functional redundancy sometimes observed between cytokines. The interleukin 5 (IL-5) receptor consists of an IL-5-specific alpha-subunit (IL-5Ralpha) and a signal-transducing beta-subunit (betac) shared with the IL-3 and granulocyte-macrophage colony-stimulating factor (GM-CSF) receptors. In this study, we sought to find a role for the cytoplasmic domain of IL-5Ralpha. We show that syntenin, a protein containing PSD-95/Discs large/zO-1 (PDZ) domains, associates with the cytoplasmic tail of the IL-5Ralpha. Syntenin was found to directly associate with the transcription factor Sox4. Association of syntenin with IL-5Ralpha was required for IL-5-mediated activation of Sox4. These studies identify a mechanism of transcriptional activation by cytokine-specific receptor subunits.
Assuntos
Linfócitos B/metabolismo , Proteínas de Transporte/metabolismo , Proteínas de Grupo de Alta Mobilidade/metabolismo , Interleucina-5/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas de Membrana , Receptores de Interleucina/metabolismo , Transativadores/metabolismo , Ativação Transcricional , Animais , Linfócitos B/imunologia , Células COS , Proteínas de Transporte/química , Linhagem Celular , Genes Reporter , Humanos , Camundongos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosforilação , Mutação Puntual , Estrutura Terciária de Proteína , Receptores de Interleucina/química , Receptores de Interleucina/genética , Receptores de Interleucina-5 , Proteínas Recombinantes de Fusão/metabolismo , Fatores de Transcrição SOXC , Deleção de Sequência , Transdução de Sinais , Sinteninas , Transfecção , Técnicas do Sistema de Duplo-HíbridoRESUMO
Cytokines mediate the transduction of proliferative, differentiation and survival signals in the hematopoietic system. Although the cytokine family is large and diverse, many different cytokines display broadly overlapping functions. This can be explained by the fact that cytokine receptors often share multiple subunits. Specificity in signal transduction can however be achieved through several mechanisms. This review focuses on how signal specificity can be achieved within the IL-3, IL-5 and GM-CSF receptor family. This is discussed in terms of receptor expression, recent advances in our understanding of intracellular signalling components, and analysis of null mutant knock-out mice.
Assuntos
Citocinas/metabolismo , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/metabolismo , Receptores de Interleucina-3/metabolismo , Receptores de Interleucina/metabolismo , Transdução de Sinais , Animais , Humanos , Camundongos , Camundongos Knockout , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/química , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/genética , Receptores de Interleucina/química , Receptores de Interleucina/genética , Receptores de Interleucina-3/química , Receptores de Interleucina-3/genética , Receptores de Interleucina-5 , TransgenesRESUMO
Granulocyte macrophage colony stimulating factor (GM-CSF), interleukin-3 (IL-3) and interleukin-5 (IL-5 belong to a family of cytokines that regulate proliferation, differentiation and function of haematopoietic cells. Their receptor consists of a ligand specific alpha-chain and a signal transducing beta-chain (betac). While, the role of phosphotyrosine residues in the betac as mediators of downstream signalling cascades has been established, little is known about non-phosphotyrosine mediated events. To identify proteins interacting with betac, we screened a yeast two-hybrid library with the intracellular domain of betac. We found that RACK1, a molecule associating with activated PKC, PLCgamma and Src kinases, associated with the membrane proximal region of betac in both yeast two-hybrid, immunoprecipitation and GST-pull-down assays. The association of RACK1 was constitutive, demonstrating no alteration upon cellular stimulation. Furthermore, upon stimulation of cells with IL-5 or PMA, a complex of betac and PKCbeta was found. Together, these findings suggest a novel role for RACK1 as a possible adapter molecule associating with the intracellular domain of cytokine receptors.
Assuntos
Isoenzimas/metabolismo , Peptídeos/metabolismo , Proteína Quinase C/metabolismo , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/metabolismo , Receptores de Interleucina-3/metabolismo , Receptores de Interleucina/metabolismo , Animais , Sequência de Bases , Células COS , DNA , Ligação Proteica , Proteína Quinase C beta , Receptores de Quinase C Ativada , Receptores de Interleucina-5RESUMO
Cytosolic phospholipase A(2) (cPLA(2)) plays a critical role in various neutrophil functions including the generation of leukotrienes and platelet-activating factor release. Enzyme activity is regulated both by translocation to the membrane in a Ca(2+)-dependent manner and serine phosphorylation by members of the mitogen-activated protein kinase (MAPK) family. In this report, we have investigated the role of granulocyte/macrophage colony-stimulating factor (GM-CSF)-mediated signalling pathways in the regulation of cPLA(2). GM-CSF-induced cPLA(2) phosphorylation was not affected by pharmacological inhibition of p38 MAPK, phosphatidylinositol 3-kinase or Src. However, inhibition of extracellular signal-regulated kinase (ERK) MAPK activation resulted in a partial inhibition of cPLA(2) phosphorylation, revealed in a slower onset of phosphorylation. A cell line stably transfected with the GM-CSF receptor was used to further analyze GM-CSF-mediated cPLA(2) phosphorylation. Mutation of tyrosine residues 577 and 612 resulted in a delayed cPLA(2) phosphorylation similar to the pharmacological ERK inhibition. Furthermore, inhibition of p38 MAPK in cells bearing the double mutant betac577/612 completely abrogated GM-CSF-induced cPLA(2) phosphorylation. We conclude that GM-CSF can mediate cPLA(2) phosphorylation through the redundant activation of both p38 and ERK MAP kinases.
Assuntos
Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosfolipases A/metabolismo , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Citosol/enzimologia , Humanos , Técnicas In Vitro , Sistema de Sinalização das MAP Quinases , Neutrófilos/enzimologia , Neutrófilos/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Transfecção , Tirosina/metabolismo , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
The small guanosine triphosphate (GTPase) p21rac is highly expressed in human neutrophils where it is thought to play a role in cytoskeletal reorganization and superoxide production. Using the p21rac binding domain of PAK (PAK-RBD) as an activation-specific probe, we have investigated agonist-stimulated activation of p21rac. Stimulation of neutrophils with the chemoattractants fMet-Leu-Phe (fMLP) or platelet-activating factor (PAF) induced an extremely rapid and transient p21rac activation, being optimal within 5 seconds. This activation correlates with the rapid changes of intracellular free Ca(2+) ([Ca(2+)](i)) stimulated by fMLP; however, changes in [Ca(2+)](i) were neither sufficient nor required for p21rac activation. Furthermore, fMLP-induced p21rac activation was not inhibited by broad tyrosine kinase inhibitors or specific inhibitors of ERK, p38 mitogen activated protein kinase, Src, or phosphatidylinositol 3-kinases. Surprisingly, the cytokines granulocyte-macrophage colony-stimulating factor (GM-CSF) and tumor necrosis factor-alpha did not cause p21rac activation or modulate fMLP-induced p21rac activation. AlF(-), a potent activator of heterotrimeric G-protein alpha-subunits, however, was found to activate p21rac. Stimulation of neutrophils with phorbol myristate acetate (PMA) strongly activated the respiratory burst, but did not induce p21rac activation, suggesting that superoxide production per se can occur independently of p21rac activation. These data suggest that in human granulocytes, G-protein coupled receptors, but not cytokine receptors, activate p21rac via a rapid, novel exchange-mechanism independently of changes in [Ca(2+)](i), tyrosine phosphorylation, or PI3K.
Assuntos
GTP Fosfo-Hidrolases/fisiologia , Proteínas de Ligação ao GTP/fisiologia , Ativação de Neutrófilo/fisiologia , Neutrófilos/fisiologia , Cálcio/fisiologia , Fatores Quimiotáticos/farmacologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Guanosina Trifosfato/fisiologia , Humanos , Ativação de Neutrófilo/efeitos dos fármacos , Fator de Ativação de Plaquetas/farmacologia , Fator de Necrose Tumoral alfa/farmacologia , Proteínas rac de Ligação ao GTPRESUMO
Although it is known that many stimuli can activate mitogen-activated protein kinases (MAPKs) and phosphatidylinositol 3-kinases (PI3K) in human neutrophils, little is known concerning either the mechanisms or function of this activation. We have utilized a selective inhibitor of MAPK kinase (MEK), PD098059, and two inhibitors of PI3K, wortmannin and LY294002, to investigate the roles of these kinases in the regulation of neutrophil effector functions. Granulocyte/macrophage colony-stimulating factor, platelet-activating factor (PAF) and N-formylmethionyl-leucyl-phenylalanine are capable of activating both p44ERK1 and p42ERK2 MAPKs and phosphotyrosine-associated PI3K in human neutrophils. The activation of extracellular signal-related protein kinases (ERKs) is correlated with the activation of p21ras by both tyrosine kinase and G-protein-coupled receptors as measured by a novel assay for GTP loading. Wortmannin and LY294002 inhibit, to various degrees, superoxide generation, neutrophil migration and PAF release. Incubation with PD098059, however, inhibits only the PAF release stimulated by serum-treated zymosan. This demonstrates that, while neither MEK nor ERK kinases are involved in the activation of respiratory burst or neutrophil migration, inhibition of PAF release suggests a potential role in the activation of cytosolic phospholipase A2. PI3K isoforms, however, seem to have a much wider role in regulating neutrophil functioning.