RESUMO
Myeloid-derived suppressor cells (MDSCs) have a wide spectrum of immunosuppressive activity; control of these cells is a new target for improving clinical outcomes in cancer patients. MDSCs originate from unusual differentiation of neutrophils or monocytes induced by inflammatory cytokines, including granulocyte-colony stimulating factor (G-CSF) and granulocyte-macrophage (GM)-CSF. However, MDSCs are difficult to detect in neutrophil or monocyte populations because they are not uniform cells, resembling both neutrophils and monocytes; thus, they exist in a heterogeneous population. In this study, we investigated GPI-80, a known regulator of Mac-1 (CD11b/CD18) and associated closely with neutrophil maturation, to clarify this unusual differentiation. First, we demonstrated that the mean fluorescence intensity (MFI) of GPI-80 and coefficient of variation (CV) of GPI-80 were increased by treatment with G-CSF and GM-CSF, respectively, using a human promyelocytic leukaemia (HL60) cell differentiation model. To confirm the value of GPI-80 as a marker of unusual differentiation, we measured GPI-80 expression and MDSC functions using peripheral blood cells from metastatic renal cell carcinoma patients. The GPI-80 CV was augmented significantly in the CD16hi neutrophil cell population, and GPI-80 MFI was increased significantly in the CD33hi monocyte cell population. Furthermore, the GPI-80 CV in the CD16hi population was correlated inversely with the proliferative ability of T cells and the GPI-80 MFI of the CD33hi population was correlated with reactive oxygen species production. These results led us to propose that the pattern of GPI-80 expression in these populations is a simple and useful marker for unusual differentiation, which is related to MDSC functions.
Assuntos
Amidoidrolases/genética , Moléculas de Adesão Celular/genética , Diferenciação Celular/genética , Expressão Gênica , Células Mieloides/citologia , Células Mieloides/metabolismo , Adulto , Idoso , Biomarcadores , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/metabolismo , Carcinoma de Células Renais/patologia , Adesão Celular/imunologia , Linhagem Celular Tumoral , Citocinas/metabolismo , Feminino , Proteínas Ligadas por GPI/genética , Células HL-60 , Humanos , Masculino , Pessoa de Meia-Idade , Células Mieloides/imunologia , Metástase Neoplásica , Estadiamento de Neoplasias , Neutrófilos/imunologia , Neutrófilos/metabolismo , Fagocitose/imunologia , Espécies Reativas de Oxigênio/metabolismoRESUMO
A large-scale transformation procedure handling an adequate number of stable transformants with highly efficient positive-negative selection is a necessary prerequisite to successful gene targeting by homologous recombination, as the integration of a transgene by somatic homologous recombination in higher plants has been reported to be 10(-3) to 10(-5) compared with random integration by non-homologous end joining. We established an efficient and large-scale Agrobacterium-mediated rice transformation protocol that generated around 10(3) stable transformants routinely from 150 seeds and a strong positive-negative selection procedure that resulted in survivors at 10(-2) using the gene for diphtheria toxin A fragment as a negative marker. The established transformation procedure provides a basis for efficient gene targeting in rice.
Assuntos
Oryza/genética , Plantas Geneticamente Modificadas/genética , Rhizobium/genética , Transformação Genética , Toxina Diftérica/genética , Marcação de Genes , Marcadores Genéticos , Vetores Genéticos , Fragmentos de Peptídeos/genética , Recombinação GenéticaRESUMO
Transforming growth factor-beta1 (TGF-beta1) is a pluripotent cytokine that controls peripheral T cell tolerance mainly in mucosal immunity. It is secreted by regulatory T cells (Tr /Th3) but also by other immununologically active cells. Smad anchor for receptor activation (SARA) and hepatic growth factor-regulated tyrosine kinase substrate (Hgs) are involved in TGF-beta1 signaling. Both molecules are known to present Smad2 and Smad3 to the TGF-beta receptor complex. The role of SARA and Hgs in TGF-beta1 susceptibility of human CD4+ T cells is unclear. We demonstrate here that TGF-beta1 up-regulates SARA mRNA expression in CD4+ T cells similar to that of Smad7. However, the increase in SARA expression was lower (6.1+/-0.3-fold vs. 25+/-4.1-fold) compared with Smad7 and delayed, with a maximum at 12 h compared with 2 h. Th1 and Th2 cell subsets expressed the same levels of SARA and Hgs. Compared with resting cells, significantly lower levels of the two molecules were found in antigen/allergen- or anti-CD3/CD28-stimulated cells. Down-regulation of SARA and Hgs mRNA in preactivated CD4+ T cells was accompanied by a twofold increase in a TGF-beta1 responsive reporter gene assay. Overexpression of SARA and Hgs in T cells yielded a dose-dependent decrease in cotransfected reporter gene expression, indicating an inhibitory function of both molecules. Thus, SARA and Hgs are regulators of TGF-beta1 susceptibility in T cells and integrate regulatory signals into the influence of TGF-beta1-mediated suppression of human T cells.
Assuntos
Proteínas de Transporte/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular , Fosfoproteínas/fisiologia , Serina Endopeptidases , Linfócitos T/efeitos dos fármacos , Fator de Crescimento Transformador beta/farmacologia , Alérgenos/farmacologia , Anticorpos/farmacologia , Antígenos CD28/imunologia , Complexo CD3/imunologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/metabolismo , Proteínas de Transporte/genética , Linhagem Celular , Complexos Endossomais de Distribuição Requeridos para Transporte , Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas de Fluorescência Verde , Humanos , Interferon gama/farmacologia , Interleucina-2/farmacologia , Células Jurkat , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Ativação Linfocitária/efeitos dos fármacos , Fosfoproteínas/genética , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Linfócitos T/metabolismo , Células Th1/efeitos dos fármacos , Células Th1/metabolismo , Células Th2/efeitos dos fármacos , Células Th2/metabolismo , Transfecção , Fator de Crescimento Transformador beta1RESUMO
AMSH, a molecule that associates with STAM1, is involved in the in vitro cell growth signaling mediated by interleukin 2 and granulocyte-macrophage colony-stimulating factor. To investigate the in vivo functional role of AMSH, we have generated AMSH-deficient mice by gene targeting. The AMSH-deficient mice were morphologically indistinguishable from their littermates at birth, and histopathological examinations revealed normal morphogenesis in all tissues tested. However, all the AMSH-deficient mice exhibited postnatal growth retardation and died between postnatal day 19 (P19) and P23. Examination of brain sections at P6 demonstrated significant loss of neurons and apoptotic cells in the CA1 subfield of the hippocampus. Brain atrophy developed by P16 and was accompanied by complete loss of the CA1 neurons in the hippocampus and marked atrophy of the cerebral cortex. Furthermore, AMSH-deficient hippocampal neuronal cells were unable to survive in vitro, even in the presence of several stimulatory cytokines, while AMSH-deficient cerebellar neurons, thymocytes, and embryonic fibroblasts survived normally. Taken together, these observations indicate that AMSH is an essential molecule for the survival of neuronal cells in early postnatal mice.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteínas de Transporte/química , Córtex Cerebral/citologia , Córtex Cerebral/metabolismo , Endopeptidases , Hipocampo/citologia , Hipocampo/metabolismo , Neurônios/metabolismo , Animais , Apoptose , Northern Blotting , Encéfalo/metabolismo , Proteínas de Transporte/biossíntese , Divisão Celular , Sobrevivência Celular , Células Cultivadas , Complexos Endossomais de Distribuição Requeridos para Transporte , Citometria de Fluxo , Marcação de Genes , Hibridização In Situ , Marcação In Situ das Extremidades Cortadas , Metaloproteases , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Modelos Genéticos , Fenótipo , Fosfoproteínas/metabolismo , Estrutura Terciária de Proteína , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais , Fatores de Tempo , Ubiquitina Tiolesterase , Domínios de Homologia de srcRESUMO
Inhibitory Smads, i.e. Smad6 and Smad7, are potent antagonists of the BMP-Smad pathway by interacting with activated bone morphogenetic protein (BMP) type I receptors and thereby preventing the activation of receptor-regulated Smads, or by competing with activated R-Smads for heteromeric complex formation with Smad4. The molecular mechanisms that underlie the regulation of I-Smad activity have remained elusive. Here we report the identification of a cytoplasmic protein, previously termed associated molecule with the SH3 domain of STAM (AMSH), as a direct binding partner for Smad6. AMSH interacts with Smad6, but not with R- and Co-Smads, upon BMP receptor activation in cultured cells. Consistent with this finding, stimulation of cells with BMP induces a co-localization of Smad6 with AMSH in the cytoplasm. Ectopic expression of AMSH prolongs BMP-induced Smad1 phosphorylation, and potentiates BMP-induced activation of transcriptional reporter activity, growth arrest and apoptosis. The data strongly suggest that the molecular mechanism by which AMSH exerts its action is by inhibiting the binding of Smad6 to activated type I receptors or activated R-Smads.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteínas Morfogenéticas Ósseas/metabolismo , Proteínas de Ligação a DNA/metabolismo , Regulação para Baixo , Fosfoproteínas/metabolismo , Transdução de Sinais , Transativadores/metabolismo , Fator de Crescimento Transformador beta , Receptores de Ativinas , Animais , Apoptose , Proteína Morfogenética Óssea 7 , Células COS , Divisão Celular , Chlorocebus aethiops , Citoplasma/metabolismo , Proteínas de Ligação a DNA/genética , Complexos Endossomais de Distribuição Requeridos para Transporte , Genes Reporter , Humanos , Luciferases/genética , Camundongos , Fosfoproteínas/genética , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Smad , Proteína Smad1 , Proteína Smad4 , Proteína Smad5 , Proteína Smad6 , Proteína Smad7 , Transativadores/genética , Células Tumorais CultivadasRESUMO
Human parvovirus B19 infects specifically erythroid progenitor cells, which causes transient aplastic crises and hemolytic anemias. Here, we demonstrate that erythroblastoid UT7/Epo cells infected with B19 virus fall into growth arrest with 4N DNA, indicating G(2)/M arrest. These B19 virus-infected cells displayed accumulation of cyclin A, cyclin B1, and phosphorylated cdc2 and were accompanied by an up-regulation in the kinase activity of the cdc2-cyclin B1 complex, similar to that in cells treated with the mitotic inhibitor. However, degradation of nuclear lamina and phosphorylation of histone H3 and H1 were not seen in B19 virus-infected cells, indicating that the infected cells do not enter the M phase. Accumulation of cyclin B1 was persistently localized in the cytoplasm, but not in the nucleus, suggesting that B19 virus infection of erythroid cells raises suppression of nuclear import of cyclin B1, resulting in cell cycle arrest at the G(2) phase. The B19 virus-induced G(2)/M arrest may be the critical event in the damage of erythroid progenitor cells seen in patients with B19 virus infection.
Assuntos
Ciclinas/fisiologia , Infecções por Parvoviridae/metabolismo , Parvovirus B19 Humano/fisiologia , Proteína Quinase CDC2/fisiologia , Ciclo Celular , Linhagem Celular , Humanos , Infecções por Parvoviridae/patologia , Transdução de Sinais , Replicação ViralRESUMO
The common gamma-chain (gamma(c)) is an indispensable subunit of the functional receptor complexes for IL-4, IL-7, IL-9, and IL-15 as well as IL-2. Here we show that the gamma(c) is also shared with the IL-21R complex. Although IL-21 binds to the IL-21R expressed on gamma(c)-deficient ED40515(-) cells, IL-21 is unable to transduce any intracytoplasmic signals. However, in EDgamma-16 cells, a gamma(c)-transfected ED40515(-) cell line, IL-21 binds to the IL-21R and can activate Janus kinase (JAK)1, JAK3, STAT1, and STAT3. The chemical cross-linking study reveals the direct binding of IL-21 to the gamma(c). These data clearly demonstrate that the gamma(c) is an indispensable subunit of the functional IL-21R complex.
Assuntos
Proteínas do Leite , Receptores de Interleucina-7/fisiologia , Receptores de Interleucina/fisiologia , Linhagem Celular , Proteínas de Ligação a DNA/metabolismo , Ativação Enzimática/genética , Ativação Enzimática/imunologia , Humanos , Subunidade gama Comum de Receptores de Interleucina , Subunidade alfa de Receptor de Interleucina-21 , Interleucinas/metabolismo , Interleucinas/fisiologia , Janus Quinase 1 , Janus Quinase 3 , Ligação Proteica/genética , Ligação Proteica/imunologia , Proteínas Tirosina Quinases/metabolismo , Receptores de Interleucina-21 , Receptores de Interleucina-7/deficiência , Receptores de Interleucina-7/genética , Fator de Transcrição STAT1 , Fator de Transcrição STAT3 , Fator de Transcrição STAT5 , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Transativadores/metabolismoRESUMO
Gads, a hematopoietic-lineage-specific Grb2 family member, is involved in the signaling mediated by the TCR through its interactions with SLP-76 and LAT. Here, we generated transgenic mice expressing Grf40-dSH2, an SH2-deleted dominant-negative form of Gads, which is driven by the lck proximal promoter. The total number of thymocytes was profoundly reduced in the transgenic mice, whereas in the double-negative (CD4(-)CD8(-)) thymocyte subset, in particular the CD25(+)CD44(-) pre-T cell population, it was significantly increased. However, CD5 expression, which is mediated by pre-TCR stimulation, was significantly suppressed on the CD4(-)CD8(-) thymocytes of the transgenic mice. Furthermore, the SLP-76-dependent signaling was markedly suppressed as well. These data suggest that Gads plays an important role in the pre-TCR as well as TCR signaling in thymocytes.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteínas de Transporte/genética , Regulação da Expressão Gênica no Desenvolvimento , Genes Dominantes , Inibidores do Crescimento/genética , Timo/crescimento & desenvolvimento , Animais , Proteínas de Transporte/metabolismo , Proteínas de Transporte/fisiologia , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Linhagem da Célula/genética , Linhagem da Célula/imunologia , Células Cultivadas , Feminino , Regulação da Expressão Gênica no Desenvolvimento/genética , Genes Dominantes/imunologia , Inibidores do Crescimento/fisiologia , Humanos , Ativação Linfocitária/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fosfoproteínas/genética , Fosfoproteínas/fisiologia , Deleção de Sequência/genética , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Células-Tronco/citologia , Células-Tronco/imunologia , Linfócitos T/citologia , Linfócitos T/imunologia , Timo/citologia , Timo/embriologiaRESUMO
Smad proteins are effector molecules that transmit signals from the receptors for the transforming growth factor beta (TGF-beta) superfamily to the nucleus; of the Smad proteins, Smad2 and Smad4 are essential components for mouse early embryogenesis. We demonstrated that Hgs, a FYVE domain protein, binds to Smad2 in its C-terminal half and cooperates with another FYVE domain protein, the Smad anchor for receptor activation (SARA), to stimulate activin receptor-mediated signaling through efficient recruitment of Smad2 to the receptor. Furthermore, a LacZ knock-in allele of the C-terminal half-deletion mutant of mouse Hgs was created by gene targeting. The introduced mutation causes an embryonic lethality between embryonic days 8.5 and 10.5. Mutant cells showed significantly decreased responses to stimulation with activin and TGF-beta. These findings suggest that the two FYVE domain proteins, Hgs and SARA, are prerequisites for receptor-mediated activation of Smad2.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Ligação a DNA/metabolismo , Fosfoproteínas/metabolismo , Receptores de Fatores de Crescimento/metabolismo , Transdução de Sinais , Transativadores/metabolismo , Receptores de Ativinas , Ativinas , Animais , Proteínas de Transporte/genética , Diferenciação Celular , Linhagem Celular , Quimera/genética , Quimera/imunologia , Quimera/metabolismo , Proteínas de Ligação a DNA/genética , Embrião de Mamíferos/anatomia & histologia , Embrião de Mamíferos/fisiologia , Complexos Endossomais de Distribuição Requeridos para Transporte , Marcação de Genes , Genes Reporter/efeitos dos fármacos , Inibinas/farmacologia , Substâncias Macromoleculares , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos/anatomia & histologia , Camundongos Transgênicos/genética , Camundongos Transgênicos/metabolismo , Fosfoproteínas/genética , Fosforilação , Testes de Precipitina , Proteína Smad2 , Proteína Smad3 , Transativadores/genética , Fator de Crescimento Transformador beta/farmacologiaRESUMO
We here cloned a cDNA encoding STAM2, a new member of the STAM family, which contains an SH3 domain and ITAM. STAM2 like STAM1 is associated with Jak2 and Jak3, and involved in the signaling for DNA synthesis and c-myc induction mediated by IL-2 and GM-CSF. Co-expression of the SH3 deletion mutants of STAM1 and STAM2 induces an additive effect on suppressing DNA synthesis upon stimulation with IL-2 and GM-CSF, suggesting that STAM1 and STAM2 exhibit compensatory effects on the signaling pathways downstream of Jak2 and Jak3 upon stimulation with GM-SCF and IL-2, respectively.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Família Multigênica/genética , Fosfoproteínas/química , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas , Animais , Sítios de Ligação , Linhagem Celular , Clonagem Molecular , DNA/biossíntese , Complexos Endossomais de Distribuição Requeridos para Transporte , Perfilação da Expressão Gênica , Genes Dominantes/genética , Genes myc/genética , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Substâncias de Crescimento/farmacologia , Humanos , Interleucina-2/farmacologia , Janus Quinase 2 , Janus Quinase 3 , Dados de Sequência Molecular , Fosfoproteínas/genética , Fosforilação/efeitos dos fármacos , Fosfotirosina/metabolismo , Ligação Proteica , Deleção de Sequência/genética , Transdução de Sinais/efeitos dos fármacos , Especificidade por Substrato , Ativação Transcricional/efeitos dos fármacos , Domínios de Homologia de srcRESUMO
Many cytokines have dual functions of promoting or inhibiting cell proliferation; however, the molecular mechanism of the dual functions of cytokines is not well understood. Under normal conditions, interleukin (IL)-3 is required for Ba/F3 cell proliferation, whereas interferon (IFN)-gamma inhibits Ba/F3 cell proliferation. It is known that Stat1 play a major role in inhibition of cell growth in response to IFN-gamma. We have examined the possibility of whether IFN-gamma can act as a growth-promoting cytokine if the Stat1 function is selectively blocked. We have established variant Ba/F3 cell lines in which Stat1 function is inhibited by a dominant-negative Stat1 mutant. Intriguingly, once Stat1 function is inhibited, IFN-gamma can replace IL-3 acting as an essential growth factor for cell proliferation. To understand the molecular mechanism of regulation of cell proliferation by the cytokines, the signaling pathways and gene induction by IL-3 and IFN-gamma are further studied. Although IL-3 activates mitogenic-activated protein kinase and Akt kinase, IFN-gamma does not. Interestingly, both IL-3 and IFN-gamma induce expression of the c-Myc gene that is not dependent on the Stat1 activity. Expression of a dominant-negative mutant Myc can block IFN-gamma-mediated Ba/F3 cell proliferation, suggesting that c-Myc gene induction is required for IFN-gamma-mediated cell proliferation. These findings suggest that IFN-gamma intrinsically and simultaneously induces specific and conflicting signaling pathways and transcriptional programs that contribute to the potential dual effects of IFN-gamma in promoting or inhibiting cell proliferation.
Assuntos
Divisão Celular/efeitos dos fármacos , Proteínas de Ligação a DNA/fisiologia , Interferon gama/farmacologia , Transativadores/fisiologia , Substituição de Aminoácidos , Animais , Linfócitos B/efeitos dos fármacos , Linfócitos B/imunologia , Células da Medula Óssea , Linhagem Celular , DNA/biossíntese , Proteínas de Ligação a DNA/genética , Humanos , Interleucina-3/farmacologia , Camundongos , Mutagênese Sítio-Dirigida , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Fator de Transcrição STAT1 , Transativadores/genética , TransfecçãoRESUMO
STAM containing an SH3 (Src homology 3) domain and an immunoreceptor tyrosine-based activation motif was previously revealed to be implicated in signaling pathways immediately downstream of Jak2 and Jak3 tyrosine kinases associated with cytokine receptors. We molecularly cloned a novel molecule interacting with the SH3 domain of STAM, which was named AMSH (associated molecule with the SH3 domain of STAM). AMSH contains a putative bipartite nuclear localization signal and a homologous region of a c-Jun activation domain-binding protein 1 (JAB1) subdomain in addition to a binding site for the SH3 domain of STAM. AMSH mutant deleted of the C-terminal half conferred dominant negative effects on signaling for DNA synthesis and c-myc induction mediated by interleukin 2 and granulocyte macrophage-colony-stimulating factor. These results suggest that AMSH plays a critical role in the cytokine-mediated intracellular signal transduction downstream of the Jak2/Jak3.STAM complex.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteínas de Transporte/metabolismo , Proteínas Quinases Ativadas por Mitógeno , Fosfoproteínas/metabolismo , Transdução de Sinais , Domínios de Homologia de src , Sequência de Aminoácidos , Complexo do Signalossomo COP9 , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Proteínas de Transporte/genética , Clonagem Molecular , Sequência Consenso , Replicação do DNA , Proteínas de Ligação a DNA/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Hibridização in Situ Fluorescente , Interleucina-2/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas Quinases JNK Ativadas por Mitógeno , Dados de Sequência Molecular , Peptídeo Hidrolases , Proteínas Proto-Oncogênicas c-myc/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Our previous study demonstrated that IL-2 suppressed growth of human T cell lines, in which the suppression was observed with members among HTLV-I-infected T cell lines independent of IL-2 for growth. In this study, we examined the molecular mechanism of IL-2-induced growth suppression with two HTLV-I-infected T cell lines; TL-OmI expressing endogenously three subunits, i.e. alpha, beta and gamma chains, of the IL-2 receptor, and an MT-1 transfectant expressing the endogenous alpha and gamma chains and exogenous beta chain. Our analysis revealed that IL-2 induced apoptosis in both T cell lines. Experiments with inhibitors for the proteases responsible for apoptosis signals showed that caspase 1 (IL-1 beta-converting enzyme) was not involved in apoptosis induced by IL-2. Other MT-1 sublines introduced with mutant beta chains demonstrated that IL-2-induced apoptosis required signals from both the serine-rich (S) region and acidic (A) region of the IL-2 receptor beta chain, which are essential but not critical for IL-2-mediated cell growth respectively. Collectively, IL-2 functions not only on growth promotion and prevention of apoptosis but also on induction of apoptosis, which may be implicated in physiological regulation of immune reactions by controlling growth and activation of T cells.
Assuntos
Apoptose/efeitos dos fármacos , Apoptose/imunologia , Cisteína Endopeptidases/fisiologia , Interleucina-1/farmacologia , Linfócitos T/enzimologia , Linfócitos T/imunologia , Caspase 1 , Divisão Celular/efeitos dos fármacos , Divisão Celular/imunologia , Linhagem Celular , Cisteína Endopeptidases/imunologia , Inibidores do Crescimento/imunologia , Inibidores do Crescimento/farmacologia , Humanos , Transdução de Sinais/imunologia , Linfócitos T/citologiaRESUMO
Several tyrosine kinases such as Jak1, Jak3, Lck and Syk are known to participate in IL-2-mediated intracellular signal transduction. Jak1, Lck and Syk are associated with the cytoplasmic domain of the beta chain, whereas Jak3 is associated with the cytoplasmic domain of the gamma chain, which is shared among receptors for IL-2, IL-4, IL-7 and IL-15. We first demonstrated that Jak1 is associated with the alpha chains of receptors for IL-4, IL-7 and IL-15 as well as the IL-2 receptor beta chain. Furthermore, we revealed that two proline residues in the box1 region, which is conserved in the IL-2 receptor beta chain and the alpha chains of the cytokine receptors, are essentially involved in association with Jak1. The MOLT4 transfectants with the box1 mutants of the IL-2 receptor beta chain lacking Jak1 association showed IL-2 responsiveness, in terms of activations of Jak3 and Stat5 and induction of cell growth, indicating that Jak1 is dispensable for IL-2-mediated cell growth signaling, and that Jak1 activation is not required for activation of Jak3 and Stat5 in the MOLT4 transfectants.
Assuntos
Interleucina-2/fisiologia , Proteínas Tirosina Quinases/metabolismo , Transdução de Sinais , Sequência de Aminoácidos , Linhagem Celular , Sequência Conservada , Precursores Enzimáticos/metabolismo , Humanos , Interleucina-2/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular , Janus Quinase 1 , Janus Quinase 3 , Proteína Tirosina Quinase p56(lck) Linfócito-Específica , Mutagênese Sítio-Dirigida , Mutação Puntual , Receptores de Interleucina/fisiologia , Receptores de Interleucina-2/química , Receptores de Interleucina-2/fisiologia , Proteínas Recombinantes/metabolismo , Quinase Syk , Transfecção , Quinases da Família src/metabolismoRESUMO
We previously identified a putative signal transducing adaptor molecule, named STAM, that contains an Src homology 3 (SH3) domain and immunoreceptor tyrosine-based activation motif (ITAM). In this report, we demonstrate the functional significance of STAM in cytokine-mediated signal transduction. STAM is associated with Jak3 and Jak2 tyrosine kinases via its ITAM region and phosphorylated by Jak3 and Jak2 upon stimulation with IL-2 and GM-CSF, respectively. An SH3 deletion mutant of STAM confers a dominant-negative effect on DNA synthesis mediated by IL-2 and GM-CSF. Furthermore, the wild-type STAM, but not STAM mutants deleted of SH3 and ITAM, significantly enhances c-myc induction mediated by IL-2 and GM-CSF. These results strongly implicate STAM in the signaling pathways for cell growth and c-myc induction immediately downstream of the Jaks associated with the cytokine receptors.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Regulação da Expressão Gênica/imunologia , Genes myc/imunologia , Fosfoproteínas/metabolismo , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas , Transdução de Sinais/imunologia , Domínios de Homologia de src/imunologia , Animais , Divisão Celular/genética , Divisão Celular/imunologia , DNA/biossíntese , Complexos Endossomais de Distribuição Requeridos para Transporte , Regulação da Expressão Gênica/efeitos dos fármacos , Genes myc/efeitos dos fármacos , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Humanos , Interleucina-2/farmacologia , Janus Quinase 2 , Janus Quinase 3 , Camundongos , Mutação , Fosforilação , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/fisiologia , Receptores de Interleucina-2/genética , Transdução de Sinais/genética , Transfecção , Células Tumorais Cultivadas , Tirosina/metabolismo , Domínios de Homologia de src/genéticaRESUMO
We previously reported a new type of signal-transducing adaptor molecule, STAM, which was shown to be involved in cytokine-mediated intracellular signal transduction. In this study, we molecularly cloned a 110-kDa phosphotyrosine protein inducible by stimulation with interleukin 2 (IL-2). The 110-kDa molecule was found to be a human counterpart of mouse Hrs (hepatocyte growth factor-regulated tyrosine kinase substrate) and to be associated with STAM. Tyrosine phosphorylation of Hrs is induced rapidly after stimulation with IL-2 and granulocyte-macrophage colony-stimulating factor as well as hepatocyte growth factor. The mutual association sites of Hrs and STAM include highly conserved coiled-coil sequences, suggesting that their association is mediated by the coiled-coil structures. Exogenous introduction of the wild-type Hrs significantly suppressed DNA synthesis upon stimulation with IL-2 and granulocyte-macrophage colony-stimulating factor, while the Hrs mutant deleted of the STAM-binding site lost such suppressive ability. These results suggest that Hrs counteracts the STAM function which is critical for cell growth signaling mediated by the cytokines.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Fosfoproteínas/metabolismo , Proteínas Proto-Oncogênicas , Transdução de Sinais , Dedos de Zinco , Animais , Células COS , Linhagem Celular , Bandeamento Cromossômico , Clonagem Molecular , Replicação do DNA , Complexos Endossomais de Distribuição Requeridos para Transporte , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Humanos , Interleucina-2/metabolismo , Janus Quinase 2 , Janus Quinase 3 , Camundongos , Dados de Sequência Molecular , Peso Molecular , Fosfoproteínas/genética , Fosforilação , Proteínas Tirosina Quinases/metabolismo , Domínios de Homologia de srcRESUMO
We molecularly cloned a cDNA coding for a novel phosphotyrosine molecule with a 70 kDa molecular mass, named STAM (signal transducing adaptor molecule), which is tyrosine-phosphorylated rapidly after stimulation with various cytokines such as IL-2, IL-3, IL-4, IL-7, GM-CSF, EGF and PDGF. STAM contains an SH3 (Src-homology 3) domain and the ITAM (immunoreceptor tyrosine-based activation motif), suggesting that STAM acts as an adaptor molecule involved in signal transducing pathways from the cytokine receptors.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Fosfoproteínas/genética , Proteínas/genética , Transdução de Sinais , Sequência de Aminoácidos , Animais , Linhagem Celular , Mapeamento Cromossômico , Clonagem Molecular , Citocinas/farmacologia , DNA Complementar/genética , Complexos Endossomais de Distribuição Requeridos para Transporte , Humanos , Camundongos , Dados de Sequência Molecular , Peso Molecular , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Fosforilação , Proteínas/química , Proteínas/metabolismo , Receptores de Citocinas/metabolismo , Homologia de Sequência de Aminoácidos , Tirosina/metabolismo , Domínios de Homologia de srcRESUMO
The tyrosine kinases Jak1 and Jak3 are known to be associated with the beta and gamma chains of interleukin-2 receptor (IL-2R). They are activated by stimulation with IL-2, IL-4, IL-7, IL-9, or IL-15, receptors of which share the gamma chain of the IL-2R. We have obtained direct evidence of Jak1 association with the alpha chains of receptors for IL-4, IL-7 and IL-9 and with the beta chain of IL-2R, which is also common to the IL-15R. Furthermore, we have prepared mutant IL-2R beta chains with a mutation in the box 1 region, which is conserved among the IL-2R beta chain and the alpha chains of the other cytokine receptors sharing the IL-2R gamma chain. Using MOLT-4 transfectants with the mutant beta chains, we found that two conserved proline residues within the box 1 region are essentially involved in association with Jak1. The MOLT-4 transfectants with the mutant beta chains lacking Jak1 association showed IL-2 responsiveness, in terms of activation of Jak3 and Stat5 and induction of cell growth, indicating that Jak1 is dispensable for IL-2-mediated cell growth signaling and that Jak1 activation is not required for activation of Jak3 and Stat5 in the MOLT-4 transfectants.
Assuntos
Interleucina-2/fisiologia , Proteínas do Leite , Proteínas Tirosina Quinases/fisiologia , Linfócitos T/enzimologia , Antígenos CD/fisiologia , Sequência de Bases , Divisão Celular , Células Cultivadas , Proteínas de Ligação a DNA/metabolismo , Humanos , Janus Quinase 1 , Janus Quinase 3 , Dados de Sequência Molecular , Fosfotirosina/metabolismo , Proteínas Tirosina Quinases/metabolismo , Receptores de Interleucina/fisiologia , Receptores de Interleucina-4 , Receptores de Interleucina-7 , Receptores de Interleucina-9 , Fator de Transcrição STAT5 , Transdução de Sinais , Linfócitos T/citologia , Transativadores/metabolismoRESUMO
We have examined phosphorylation mediated by cross-talk between growth signal pathways induced by IL-2 and IL-5. To analyze the phosphorylation process in the same cells, we established two sublines, T88-Mbeta1, which is a subline of a murine IL-5-dependent cell line, T88-M, by introduction of the human IL-2 receptor beta chain (IL-2Rbeta), and secondly CTLL-5Ralphabeta, which is a subline of a murine IL-2-dependent cell line, CTLL-2, by introduction of the murine IL-5 receptor alpha chain (IL-5Ralpha) and IL-5 receptor beta chain (IL-5Rbeta, betac) genes. Both T88-Mbeta1 and CTLL-5Ralphabeta expressed high-affinity receptors for IL-2 and IL-5, and proliferated in response to both factors. Tyrosine phosphorylation of IL-2Rbeta was induced by stimulation of T88-Mbeta1 with not only IL-2 but also IL-5. Anti-IL-2Rbeta-directed immune complexes from T88-Mbeta1 stimulated with IL-5 as well as with IL-2 contained an activated tyrosine kinase. However, stimulation with IL-5 but not IL-2 induced the tyrosine phosphorylation of IL-5Rbeta, betac, suggesting that IL-2 does not activate a tyrosine kinase which efficiently catalyzes the IL-5Rbeta molecule in response to IL-5. On the other hand, the detection of JAK1 and the other common set of phosphotyrosine-containing proteins after stimulation with either IL-5 or IL-2 suggests the existence of the same tyrosine phosphorylation pathways.
Assuntos
Interleucina-2/farmacologia , Interleucina-5/farmacologia , Proteínas Proto-Oncogênicas , Receptores de Interleucina-2/metabolismo , Receptores de Interleucina/metabolismo , Animais , Divisão Celular/imunologia , Linhagem Celular , Janus Quinase 1 , Janus Quinase 2 , Janus Quinase 3 , Cinética , Camundongos , Fosforilação/efeitos dos fármacos , Proteínas Tirosina Quinases/análise , Proteínas Tirosina Quinases/metabolismo , Receptores de Interleucina/biossíntese , Receptores de Interleucina-2/biossíntese , Receptores de Interleucina-5 , Linfócitos T Citotóxicos/enzimologiaRESUMO
Interleukin 2 (IL-2), a T cell-derived cytokine, targets a variety of cells to induce their growth, differentiation, and functional activation. IL-2 inserts signals into the cells through IL-2 receptors expressed on cell surfaces to induce such actions. In humans, the functional IL-2 receptor consists of the subunit complexes of the alpha, beta and gamma chains, or the beta and gamma chains. The third component, the gamma chain, of IL-2 receptor plays a pivotal role in formation of the full-fledged IL-2 receptor, together with the beta chain, the gamma chain participates in increasing the IL-2 binding affinity and intracellular signal transduction. Moreover, the cytokine receptors for at least IL-2, IL-4, IL-7, IL-9, and IL-15 utilize the same gamma chain as an essential subunit. Interestingly, mutations of the gamma chain gene cause human X-linked severe combined immunodeficiency (XSCID) characterized by a complete or profound T cell defect. Among the cytokines sharing the gamma chain, at least IL-7 is essentially involved in early T cell development in the mouse organ culture system. The molecular identification of the gamma chain brought a grasp of the structures and functions of the cytokine receptor and an in-depth understanding of the cause of human XSCID. To investigate the mechanism of XSCID and development of gene therapy for XSCID, knockout mice for the gamma chain gene were produced that showed similar but not exactly the same phenotypes as human XSCID.