RESUMO
Reactive oxygen species (ROS) regulate growth factor receptor signalling at least in part by inhibiting oxidation-sensitive phosphatases. An emerging concept is that ROS act locally to affect signal transduction in different subcellular compartments and that ROS levels are regulated by antioxidant proteins at the same local level. Here, we show that the ER-resident antioxidant peroxiredoxin 4 (Prdx4) interacts with the cytoplasmic domain of the granulocyte colony-stimulating factor receptor (G-CSFR). This interaction occurs when the activated G-CSFR resides in early endosomes. Prdx4 inhibits G-CSF-induced signalling and proliferation in myeloid progenitors, depending on its redox-active cysteine core. Protein tyrosine phosphatase 1b (Ptp1b) appears to be a major downstream effector controlling these responses. Conversely, Ptp1b might keep Prdx4 active by reducing its phosphorylation. These findings unveil a new signal transduction regulatory circuitry involving redox-controlled processes in the ER and activated cytokine receptors in endosomes.
Assuntos
Regulação para Baixo , Retículo Endoplasmático/metabolismo , Endossomos/metabolismo , Fator Estimulador de Colônias de Granulócitos/metabolismo , Peroxirredoxinas/metabolismo , Transdução de Sinais , Animais , Linhagem Celular , Proliferação de Células , Retículo Endoplasmático/enzimologia , Retículo Endoplasmático/genética , Endossomos/enzimologia , Endossomos/genética , Fator Estimulador de Colônias de Granulócitos/genética , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Humanos , Camundongos , Peroxirredoxinas/genética , Fosforilação , Ligação Proteica , Estrutura Terciária de Proteína , Proteína Tirosina Fosfatase não Receptora Tipo 1/genética , Proteína Tirosina Fosfatase não Receptora Tipo 1/metabolismo , Espécies Reativas de Oxigênio , Receptores de Fator Estimulador de Colônias de Granulócitos/genética , Receptores de Fator Estimulador de Colônias de Granulócitos/metabolismoRESUMO
Ubiquitination of cytokine receptors controls intracellular receptor routing and signal duration, but the underlying molecular determinants are unclear. The suppressor of cytokine signaling protein SOCS3 drives lysosomal degradation of the granulocyte colony-stimulating factor receptor (G-CSFR), depending on SOCS3-mediated ubiquitination of a specific lysine located in a conserved juxtamembrane motif. Here, we show that, despite ubiquitination of other lysines, positioning of a lysine within the membrane-proximal region is indispensable for this process. Neither reallocation of the motif nor fusion of ubiquitin to the C-terminus of the G-CSFR could drive lysosomal routing. However, within this region, the lysine could be shifted 12 amino acids toward the C-terminus without losing its function, arguing against the existence of a linear sorting motif and demonstrating that positioning of the lysine relative to the SOCS3 docking site is flexible. G-CSFR ubiquitination peaked after endocytosis, was inhibited by methyl-beta-cyclodextrin as well as hyperosmotic sucrose and severely reduced in internalization-defective G-CSFR mutants, indicating that ubiquitination mainly occurs at endosomes. Apart from elucidating structural and spatio-temporal aspects of SOCS3-mediated ubiquitination, these findings have implications for the abnormal signaling function of G-CSFR mutants found in severe congenital neutropenia, a hematopoietic disorder with a high leukemia risk.
Assuntos
Lisossomos/metabolismo , Transporte Proteico/fisiologia , Receptores de Fator Estimulador de Colônias de Granulócitos/metabolismo , Transdução de Sinais/fisiologia , Sequência de Aminoácidos , Animais , Análise Mutacional de DNA , Endocitose/fisiologia , Células HeLa , Humanos , Lisina/metabolismo , Dados de Sequência Molecular , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de Sequência , Proteína 3 Supressora da Sinalização de Citocinas , Proteínas Supressoras da Sinalização de Citocina/metabolismo , UbiquitinaçãoRESUMO
The proliferation of neutrophil granulocyte lineage is driven largely by granulocyte colony-stimulating factor (G-CSF) acting via the G-CSF receptors. In this study, we show that mice lacking cyclin D3, a component of the core cell cycle machinery, are refractory to stimulation by the G-CSF. Consequently, cyclin D3-null mice display deficient maturation of granulocytes in the bone marrow and have reduced levels of neutrophil granulocytes in their peripheral blood. The mutant mice are unable to mount a normal response to bacterial challenge and succumb to microbial infections. In contrast, the expansion of hematopoietic stem cells and lineage-committed myeloid progenitors proceeds relatively normally in mice lacking cyclin D3, revealing that the requirement for cyclin D3 function operates at later stages of neutrophil development. Importantly, we verified that this requirement is specific to cyclin D3, as mice lacking other G(1) cyclins (D1, D2, E1, or E2) display normal granulocyte counts. Our analyses revealed that in the bone marrow cells of wild-type mice, activation of the G-CSF receptor leads to upregulation of cyclin D3. Collectively, these results demonstrate that cyclin D3 is an essential cell cycle recipient of G-CSF signaling, and they provide a molecular link of how G-CSF-dependent signaling triggers cell proliferation.
Assuntos
Proliferação de Células , Ciclinas/metabolismo , Fator Estimulador de Colônias de Granulócitos/metabolismo , Neutrófilos/fisiologia , Animais , Infecções Bacterianas , Células da Medula Óssea/citologia , Células da Medula Óssea/fisiologia , Ciclo Celular/fisiologia , Linhagem da Célula , Ciclina D3 , Ciclinas/genética , Suscetibilidade a Doenças/metabolismo , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/fisiologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neutropenia , Neutrófilos/citologia , Receptores de Fator Estimulador de Colônias de Granulócitos/metabolismo , Transdução de Sinais/fisiologiaRESUMO
Most severe congenital neutropenia (SCN) cases possess constitutive neutrophil elastase mutations; a smaller cohort has acquired mutations truncating the granulocyte colony-stimulating factor receptor (G-CSF-R). We have described a case with constitutive extracellular G-CSF-R mutation hyporesponsive to ligand. Here we report two independent acquired G-CSF-R truncation mutations and a novel constitutive neutrophil elastase mutation in this patient. Co-expression of a truncated receptor chain restored STAT5 signalling responses of the extracellular G-CSF-R mutant, while constitutively-active STAT5 enhanced its proliferative capacity. These data add to our knowledge of SCN and further highlight the importance of STAT5 in mediating proliferative responses to G-CSF.
Assuntos
Elastase de Leucócito/genética , Mutação/genética , Neutropenia/congênito , Receptores de Fator Estimulador de Colônias de Granulócitos/genética , Criança , Análise Mutacional de DNA , Humanos , Neutropenia/enzimologia , Neutropenia/genética , Reação em Cadeia da Polimerase , Receptores de Fator Estimulador de Colônias de Granulócitos/metabolismo , Fator de Transcrição STAT5/genética , Serina Endopeptidases/genéticaRESUMO
Granulocyte-colony stimulating factor (G-CSF) induces proliferation of myeloid progenitor cells and controls their differentiation into mature neutrophils. Signal transducer and activator of transcription (STAT) proteins STAT3 and STAT5 are activated by G-CSF and play distinct roles in neutrophil development. Suppressor of cytokine signaling (SOCS) proteins are induced by STATs and inhibit signaling through various negative-feedback mechanisms. SOCS proteins can compete with docking of signaling substrates to receptors, interfere with Janus tyrosine kinase activity, and target proteins for proteasomal degradation. The latter process is mediated through the conserved C-terminal SOCS box. We determined the role of various SOCS proteins in controlling G-CSF responses and investigated the involvement of the SOCS box therein. We show that SOCS1 and SOCS3, but not CIS and SOCS2, inhibited G-CSF-induced STAT activation in human embryo kidney 293 cells. In myeloid 32D cells, SOCS1 and SOCS3 are induced by G-CSF. However, relative to interleukin-3-containing cultures, during G-CSF-induced neutrophilic differentiation, SOCS3 expression was further elevated, while SOCS1 levels remained constant. SOCS box deletion mutants of SOCS1 and SOCS3 were severely hampered in their abilities to inhibit STAT activation and to efficiently suppress colony formation by primary myeloid progenitors in response to G-CSF. These data demonstrate the importance of the SOCS box for the inhibitory effects of SOCS proteins on G-CSF signaling and show that among the different SOCS family members, SOCS3 is the major negative regulator of G-CSF responses during neutrophilic differentiation.
Assuntos
Proteínas de Transporte/metabolismo , Fator Estimulador de Colônias de Granulócitos/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas do Leite , Proteínas Repressoras/metabolismo , Transdução de Sinais/fisiologia , Fatores de Transcrição/metabolismo , Western Blotting , Medula Óssea/fisiologia , Proteínas de Transporte/genética , Diferenciação Celular/fisiologia , Linhagem Celular , Proteínas de Ligação a DNA/efeitos dos fármacos , Proteínas de Ligação a DNA/metabolismo , Ativação Enzimática/efeitos dos fármacos , Fator Estimulador de Colônias de Granulócitos/farmacologia , Células-Tronco Hematopoéticas/citologia , Humanos , Proteínas Repressoras/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Transcrição STAT3 , Fator de Transcrição STAT5 , Proteína 1 Supressora da Sinalização de Citocina , Proteína 3 Supressora da Sinalização de Citocinas , Proteínas Supressoras da Sinalização de Citocina , Transativadores/efeitos dos fármacos , Transativadores/metabolismo , Fatores de Transcrição/genética , TransfecçãoRESUMO
Thioredoxin-interacting protein (TXNIP) is involved in reactive oxygen species-induced stress responses. In a screen for novel disease genes in murine leukemia virus (MLV)-induced mouse leukemias, we identified Txnip as a frequent target for proviral integration. Ectopic TXNIP expression inhibited the proliferation of myeloid progenitor cells. TXNIP transcript and protein levels were significantly elevated in human AML blasts of certain patients, particularly those harboring translocation t(8;21). Nucleotide sequencing revealed no abnormalities in the TXNIP coding region in AML. These findings suggest that deregulated TXNIP expression contributes to MLV-induced murine leukemia as well as human AML.
Assuntos
Proteínas de Transporte/genética , Regulação da Expressão Gênica , Leucemia Experimental/genética , Leucemia Mieloide Aguda/genética , Leucemia/genética , Tiorredoxinas/genética , Integração Viral/genética , Animais , Crise Blástica/genética , Crise Blástica/patologia , Linhagem Celular , Primers do DNA , Genes Reporter , Humanos , Vírus da Leucemia Murina/genética , Leucemia Mieloide Aguda/patologia , Camundongos , Reação em Cadeia da Polimerase , Regiões Promotoras GenéticasRESUMO
The hematopoietic system provides an attractive model for studying growth factor-controlled expansion and differentiation of cells in relation to receptor routing and its consequences for signal transduction. Suppressor of cytokine signaling (SOCS) proteins regulate receptor signaling partly via their ubiquitin ligase (E3)-recruiting SOCS box domain. Whether SOCS proteins affect signaling through modulating intracellular trafficking of receptors is unknown. Here, we show that a juxtamembrane lysine residue (K632) of the granulocyte colony-stimulating factor receptor (G-CSFR) plays a key role in receptor routing and demonstrate that the effects of SOCS3 on G-CSF signaling to a major extent depend on this lysine. Mutation of K632 causes accumulation of G-CSFR in early endosomes and leads to sustained activation of signal transducer and activator of transcription 5 and ERK, but not protein kinase B. Myeloid progenitors expressing G-CSFR mutants lacking K632 show a perturbed proliferation/differentiation balance in response to G-CSF. This is the first demonstration of SOCS-mediated ubiquitination and routing of a cytokine receptor and its impact on maintaining an appropriate signaling output.
Assuntos
Lisossomos/metabolismo , Receptores de Fator Estimulador de Colônias de Granulócitos/metabolismo , Proteínas Supressoras da Sinalização de Citocina/metabolismo , Motivos de Aminoácidos , Animais , Proliferação de Células , Endocitose , Células HeLa , Humanos , Ligantes , Lisina/metabolismo , Camundongos , Modelos Biológicos , Proteínas Mutantes/metabolismo , Células Mieloides/citologia , Células Mieloides/metabolismo , Transporte Proteico , Fator de Transcrição STAT5/metabolismo , Proteína 3 Supressora da Sinalização de Citocinas , Ubiquitina/metabolismoRESUMO
Truncated granulocyte colony-stimulating factor receptors (G-CSF-Rs) are implicated in severe congenital neutropenia (SCN) and the consecutive development of acute myeloid leukemia (AML). Mice expressing G-CSF-R truncation mutants (gcsfr-d715) show defective receptor internalization, an increased signal transducer and activator of transcription 5 (STAT5)/STAT3 activation ratio, and hyperproliferative responses to G-CSF treatment. We determined whether a lack of negative feedback by suppressor of cytokine signaling (SOCS) proteins contributes to the signaling abnormalities of G-CSF-R-d715. Expression of SOCS3 transcripts in bone marrow cells from G-CSF-treated gcsfr-d715 mice was approximately 60% lower than in wild-type (WT) littermates. SOCS3 efficiently suppressed STAT3 and STAT5 activation by WT G-CSF-R in luciferase reporter assays. In contrast, while SOCS3 still inhibited STAT3 activation by G-CSF-R-d715, STAT5 activation was no longer affected. This was due mainly to loss of the SOCS3 recruitment site Tyr729, with an additional contribution of the internalization defects of G-CSF-R-d715. Because Tyr729 is also a docking site for the Src homology 2-containing protein tyrosine phosphatase-2 (SHP-2), which binds to and inactivates STAT5, we suggest a model in which reduced SOCS3 expression, combined with the loss of recruitment of both SOCS3 and SHP-2 to the activated receptor complex, determine the increased STAT5/STAT3 activation ratio and the resulting signaling abnormalities projected by truncated G-CSF-R mutants.
Assuntos
Proteínas de Ligação a DNA/fisiologia , Leucemia Mieloide Aguda/genética , Proteínas do Leite , Neutropenia/genética , Receptores de Fator Estimulador de Colônias de Granulócitos/genética , Proteínas Repressoras/fisiologia , Deleção de Sequência , Transativadores/fisiologia , Fatores de Transcrição/fisiologia , Sequência de Aminoácidos , Animais , Sítios de Ligação , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/genética , Modelos Animais de Doenças , Humanos , Camundongos , Camundongos Mutantes , Receptores de Fator Estimulador de Colônias de Granulócitos/fisiologia , Proteínas Repressoras/genética , Fator de Transcrição STAT3 , Fator de Transcrição STAT5 , Transdução de Sinais , Proteína 3 Supressora da Sinalização de Citocinas , Proteínas Supressoras da Sinalização de Citocina , Transativadores/antagonistas & inibidores , Transativadores/genética , Fatores de Transcrição/genética , Tirosina , Domínios de Homologia de srcRESUMO
Granulocyte colony-stimulating factor (G-CSF) is the major regulator of neutrophil production. Studies in cell lines have established that conserved tyrosines Tyr704, Tyr729, Tyr744, Tyr764 within the cytoplasmic domain of G-CSF receptor (G-CSF-R) contribute significantly to G-CSF-induced proliferation, differentiation, and cell survival. However, it is unclear whether these tyrosines are equally important under more physiologic conditions. Here, we investigated how individual G-CSF-R tyrosines affect G-CSF responses of primary myeloid progenitors. We generated G-CSF-R-deficient mice and transduced their bone marrow cells with tyrosine "null" mutant (m0), single tyrosine "add-back" mutants, or wild-type (WT) receptors. G-CSF-induced responses were determined in primary colony assays, serial replatings, and suspension cultures. We show that removal of all tyrosines had no major influence on primary colony growth. However, adding back Tyr764 strongly enhanced proliferative responses, which was reverted by inhibition of ERK activity. Tyr729, which we found to be associated with the suppressor of cytokine signaling, SOCS3, had a negative effect on colony formation. After repetitive replatings, the clonogenic capacities of cells expressing m0 gradually dropped compared with WT. The presence of Tyr729, but also Tyr704 and Tyr744, both involved in activation of signal transducer and activator of transcription 3 (STAT3), further reduced replating efficiencies. Conversely, Tyr764 greatly elevated the clonogenic abilities of myeloid progenitors, resulting in a more than 10(4)-fold increase of colony-forming cells over m0 after the fifth replating. These findings suggest that tyrosines in the cytoplasmic domain of G-CSF-R, although dispensable for G-CSF-induced colony growth, recruit signaling mechanisms that regulate the maintenance and outgrowth of myeloid progenitor cells.