RESUMO
The Jak family of protein-tyrosine kinases are crucial for the signaling of a large number of different polypeptide ligands, including the interferons, many cytokines, erythropoietin, and growth factors. Through their interaction with receptors, the Jaks initiate a signaling cascade resulting in the activation of gene transcription and ultimately a cellular response to various ligands. In addition to their role in cellular signaling, alteration of Jak activity has been implicated in several disease states. In identifying Jak2-interacting proteins with the yeast two-hybrid system, we cloned the human homologue of the Drosophila melanogaster tumor suppressor gene lethal () tumorous imaginal discs, which encodes the protein Tid56. Drosophila Tid56 and its human homologue hTid-1 represent members of the DnaJ family of molecular chaperones. The TID1 gene encodes two splice variants hTid-1(S) and hTid-1(L). We confirmed the interaction between Jak2 and hTid-1(S) or hTid-1(L) by immunoprecipitation from COS-1 cells expressing these proteins. The interaction between endogenous hTid-1 and Jak2 was shown in HEp2 cells. We further showed that hTid-1 interacts with the human interferon-gamma (Hu-IFN-gamma) receptor subunit IFN-gamma R2. In addition, using a chimeric construct where the extracellular domain of IFN-gamma R2 was fused to the kinase domain of Jak2, we showed that hTid-1 binds more efficiently to the chimera with an active kinase domain than to a similar construct with an inactive kinase domain. Additionally, the data demonstrate that hTid-1 isoforms as well as Jak2 interact with Hsp70/Hsc70 in vivo, and the interaction between Hsp70/Hsc70 and hTid-1 is reduced after IFN-gamma treatment. Furthermore, both hTid-1(S) and hTid-1(L) can modulate IFN-gamma-mediated transcriptional activity.
Assuntos
Proteínas de Choque Térmico/metabolismo , Interferon gama/metabolismo , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas , Transdução de Sinais/fisiologia , Animais , Células COS , Fracionamento Celular , Genes Reporter , Proteínas de Choque Térmico HSP40 , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico/genética , Humanos , Janus Quinase 2 , Modelos Biológicos , Dados de Sequência Molecular , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/metabolismo , Transcrição Gênica , Células Tumorais Cultivadas , Técnicas do Sistema de Duplo-HíbridoRESUMO
The yeast protein Hsl7p is a homologue of Janus kinase binding protein 1, JBP1, a newly characterized protein methyltransferase. In this report, Hsl7p also is shown to be a methyltransferase. It can be crosslinked to [(3)H]S-adenosylmethionine and exhibits in vitro protein methylation activity. Calf histones H2A and H4 and bovine myelin basic protein were methylated by Hsl7p, whereas histones H1, H2B, and H3 and bovine cytochrome c were not. We demonstrated that JBP1 can complement Saccharomyces cerevisiae with a disrupted HSL7 gene as judged by a reduction of the elongated bud phenotype, and a point mutation in the JBP1 S-adenosylmethionine consensus binding sequence eliminated all complementation by JBP1. Therefore, we conclude the yeast protein Hsl7p is a sequence and functional homologue of JBP1. These data provide evidence for an intricate link between protein methylation and macroscopic changes in yeast morphology.
Assuntos
Proteínas Quinases/metabolismo , Proteínas Metiltransferases/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/enzimologia , Animais , Bovinos , Grupo dos Citocromos c/metabolismo , Galactose/metabolismo , Teste de Complementação Genética , Histonas/metabolismo , Humanos , Metilação , Metiltransferases/metabolismo , Mutagênese , Proteína Básica da Mielina/metabolismo , Fenótipo , Plasmídeos/metabolismo , Mutação Puntual , Testes de Precipitina , Proteínas Quinases/química , Proteínas Quinases/genética , Proteínas Metiltransferases/química , Proteínas Metiltransferases/genética , Estrutura Terciária de Proteína , Proteína-Arginina N-Metiltransferases , S-Adenosilmetionina/metabolismo , Raios UltravioletaRESUMO
To expand our understanding of the role of Jak2 in cellular signaling, we used the yeast two-hybrid system to identify Jak2-interacting proteins. One of the clones identified represents a human homologue of the Schizosaccaromyces pombe Shk1 kinase-binding protein 1, Skb1, and the protein encoded by the Saccharomyces cerevisiae HSL7 (histone synthetic lethal 7) gene. Since no functional motifs or biochemical activities for this protein or its homologues had been reported, we sought to determine a biochemical function for this human protein. We demonstrate that this protein is a protein methyltransferase. This protein, designated JBP1 (Jak-binding protein 1), and its homologues contain motifs conserved among protein methyltransferases. JBP1 can be cross-linked to radiolabeled S-adenosylmethionine (AdoMet) and methylates histones (H2A and H4) and myelin basic protein. Mutants containing substitutions within a conserved region likely to be involved in AdoMet binding exhibit little or no activity. We mapped the JBP1 gene to chromosome 14q11.2-21. In addition, JBP1 co-immunoprecipitates with several other proteins, which serve as methyl group acceptors and which may represent physiological targets of this methyltransferase. Messenger RNA for JBP1 is widely expressed in human tissues. We have also identified and sequenced a homologue of JBP1 in Drosophila melanogaster. This report provides a clue to the biochemical function for this conserved protein and suggests that protein methyltransferases may have a role in cellular signaling.
Assuntos
Metiltransferases , Proteínas Metiltransferases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas , Proteínas de Saccharomyces cerevisiae , Proteínas de Schizosaccharomyces pombe , Sequência de Aminoácidos , Proteínas de Transporte/genética , Mapeamento Cromossômico , Cromossomos Humanos Par 14 , Clonagem Molecular , Proteínas de Drosophila , Histonas/metabolismo , Humanos , Hibridização in Situ Fluorescente , Janus Quinase 2 , Metilação , Dados de Sequência Molecular , Mutação Puntual , Testes de Precipitina , Ligação Proteica , Proteínas Quinases/genética , Proteínas Metiltransferases/genética , Proteína-Arginina N-Metiltransferases , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Técnicas do Sistema de Duplo-HíbridoRESUMO
Interleukin-10 (IL-10) is a pleiotropic cytokine which signals through a specific cell surface receptor complex. Only one chain, that for ligand binding (IL-10Ralpha or IL-10R1), was identified previously. We report here that, although human IL-10 binds to the human IL-10R1 chain expressed in hamster cells, it does not induce signal transduction. However, the co-expression of CRFB4, a transmembrane protein of previously unknown function belonging to the class II cytokine receptor family, together with the IL-10R1 chain renders hamster cells sensitive to IL-10. The IL-10:CRFB4 complex was detected by cross-linking to labeled IL-10. In addition, the IL-10R1 chain was able to be co-immunoprecipitated with anti-CRF antibody when peripheral blood mononuclear cells were treated with IL-10. These results demonstrate that the CRFB4 chain is part of the IL-10 receptor signaling complex. Thus, the CRFB4 chain, which we designate as the IL-10R2 or IL-10Rbeta chain, serves as an accessory chain essential for the active IL-10 receptor complex and to initiate IL-10-induced signal transduction events.
Assuntos
Glicoproteínas de Membrana , Receptores de Citocinas/metabolismo , Receptores de Interleucina/metabolismo , Animais , Células COS , Cricetinae , Reagentes de Ligações Cruzadas/farmacologia , Haplorrinos , Humanos , Subunidade beta de Receptor de Interleucina-10 , Janus Quinase 1 , Monócitos/metabolismo , Proteínas Tirosina Quinases/metabolismo , Proteínas/metabolismo , Receptores de Citocinas/química , Receptores de Citocinas/genética , Receptores de Interleucina/química , Receptores de Interleucina/genética , Receptores de Interleucina-10 , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais , Succinimidas/farmacologia , TYK2 QuinaseRESUMO
Each cytokine which utilizes the Jak-Stat signal transduction pathway activates a distinct combination of members of the Jak and Stat families. Thus, either the Jaks, the Stats, or both could contribute to the specificity of ligand action. With the use of chimeric receptors involving the interferon gamma receptor (IFN-gammaR) complex as a model system, we demonstrate that Jak2 activation is not an absolute requirement for IFN-gamma signaling. Other members of the Jak family can functionally substitute for Jak2. IFN-gamma can signal through the activation of Jak family members other than Jak2 as measured by Statlalpha homodimerization and major histocompatibility complex class I antigen expression. This indicates that Jaks are interchangeable and indiscriminative in the Jak-Stat signal transduction pathway. The necessity for the activation of one particular kinase during signaling can be overcome by recruiting another kinase to the receptor complex. The results may suggest that the Jaks do not contribute to the specificity of signal transduction in the Jak-Stat pathway to the same degree as Stats.
Assuntos
Antígenos CD/fisiologia , Proteínas de Ligação a DNA/metabolismo , Interferon gama/farmacologia , Proteínas Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas , Receptores de Interferon/fisiologia , Transdução de Sinais , Animais , Antígenos CD/biossíntese , Sequência de Bases , Células CHO , Cricetinae , Primers do DNA , Ativação Enzimática , Antígeno HLA-B7/biossíntese , Humanos , Células Híbridas , Janus Quinase 2 , Modelos Biológicos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Fosforilação , Fosfotirosina/metabolismo , Reação em Cadeia da Polimerase , Receptores de Interferon/biossíntese , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais/efeitos dos fármacos , Receptor de Interferon gamaRESUMO
Interferon gamma (IFN-gamma) signals through a multimeric receptor complex consisting of two different chains: the IFN-gamma receptor binding subunit (IFN-gamma R, IFN-gamma R1), and a transmembrane accessory factor (AF-1, IFN-gamma R2) necessary for signal transduction. Using cell lines expressing different cloned components of the IFN-gamma receptor complex, we examined the function of the receptor components in signal transduction upon IFN-gamma treatment. A specific IFN-gamma R2:IFN-gamma cross-linked complex was observed in cells expressing both IFN-gamma R1 and IFN-gamma R2 indicating that IFN-gamma R2 (AF-1) interacts with IFN-gamma and is closely associated with IFN-gamma R1. We show that the intracellular domain of IFN-gamma R2 is necessary for signaling. Cells coexpressing IFN-gamma R1 and truncated IFN-gamma R2, lacking the COOH-terminal 51 amino acids (residues 286-337), or cells expressing IFN-gamma R1 alone were unresponsive to IFN-gamma treatment as measured by MHC class I antigen induction. Jak1, Jak2, and Stat1 alpha were activated, and IFN-gamma R1 was phosphorylated only in cells expressing both IFN-gamma R1 and IFN-gamma R2. Jak2 kinase was shown to associate with the intracellular domain of the IFN-gamma R2.