RESUMEN
The interferons and interleukin-6 (IL-6) family cytokines exert their biological effects via Janus kinase and signal transducer and activator of transcription (JAK-STAT) signalling pathways. Our aim is to identify "novel" signalling molecules not previously implicated in JAK-mediated signalling. Phosphotyrosine profiles of either whole cell lysates, or subcellular fractions, of unstimulated and cytokine-treated cell lines have been analysed and ligand-inducible differences observed. Recombinant src homology 2 domains, biotinylated peptides corresponding to cytokine receptor intracellular domains, antiphosphotyrosine antibodies, anion exchange columns and 2-D phosphotyrosine profiling have been used to select cohorts of molecules that are tyrosine phosphorylated in response to cytokine treatment. Tyrosine phosphorylated proteins showing cytokine specificity or differential profiles in cell lines mutated in specific JAKs are being purified and identified by mass spectrometry.
Asunto(s)
Interferones/metabolismo , Interleucina-6/metabolismo , Fosfotirosina/metabolismo , Transducción de Señal , Western Blotting , Línea Celular , Cromatografía de Afinidad , Cromatografía por Intercambio Iónico , Electroforesis en Gel Bidimensional , Humanos , Pruebas de Precipitina , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
A tripartite receptor comprising the external region of the erythropoietin (Epo) receptor, the transmembrane and JAK-binding domains of the gp130 subunit of the interleukin-6 (IL-6) receptor, and a seven amino acid STAT1 recruitment motif (Y440) from the interferon (IFN)-gamma receptor, efficiently mediates an IFN-gamma-like response. An analogous completely foreign chimeric receptor in which the Y440 motif is replaced with the Y905 motif from gp130 also mediates an IFN-gamma-like response, but less efficiently. The IFNGR1 signal-transducing subunit of the IFN-gamma receptor is tyrosine phosphorylated through the chimeric receptors and the endogenous IL-6 and OSM receptors. Cross phosphorylation of IFNGR1 is not, however, required for the IFN-gamma-like response through the chimeric receptors, nor does it mediate an IFN-gamma-like response to IL-6 or OSM. The data argue strongly for modular JAK/STAT signalling and against any rigid structural organization for the "pathways" involved. They emphasize the likely high degree of overlap between the signals generated from disparate JAK-receptor complexes and show that relatively minor changes in such complexes can profoundly affect the response.
Asunto(s)
Interferón gamma/metabolismo , Proteínas Nucleares , Receptores Inmunológicos/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica , Antígenos de Histocompatibilidad Clase II/biosíntesis , Humanos , Receptor de Interferón alfa y beta , Receptores de Eritropoyetina/genética , Receptores de Eritropoyetina/metabolismo , Receptores Inmunológicos/genética , Receptores de Interferón/genética , Receptores de Interferón/metabolismo , Receptores de Interleucina-6/genética , Receptores de Interleucina-6/metabolismo , Factor de Transcripción STAT1 , Transducción de Señal , Transactivadores/biosíntesis , Transactivadores/metabolismo , Receptor de Interferón gammaRESUMEN
The terminal portion of the Janus kinases (Jaks) contains a divergent FERM (Four-point-one, Ezrin, Radixin, Moesin) homology domain comprising 19 conserved hydrophobic regions. To determine the role of this domain in governing recruitment of Jak1, but not Jak3, to the gp130 subunit of the interleukin-6 family of cytokine receptors, the interaction of three Jak1/Jak3 chimeras with gp130 was investigated. Chimeras 1, 2 and 3 (Jak1 FERM regions 1-19, 1-18 and 1-8/Jak3, respectively) were all enzymically active. Chimeras 1 and 2 interacted with the cytoplasmic domain of gp130, although less efficiently than Jak1. Only chimera 2, however, restored gp130 signalling in Jak1-negative cells. The data are consistent with recruitment of Jak1 to gp130 through the Jak1 FERM domain, but also emphasise the likely requirement for precise Jak/receptor orientation to sustain function.
Asunto(s)
Antígenos CD/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Receptor gp130 de Citocinas , Citoplasma/metabolismo , Proteínas de Unión al ADN/metabolismo , Fibrosarcoma , Humanos , Janus Quinasa 1 , Janus Quinasa 3 , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , Proteínas Tirosina Quinasas/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Factor de Transcripción STAT1 , Transducción de Señal , Transactivadores/metabolismo , Células Tumorales CultivadasRESUMEN
Janus kinase 1 (Jak1) is a cytoplasmic tyrosine kinase that noncovalently associates with a variety of cytokine receptors. Here we show that the in vitro translated N-terminal domains of Jak1 are sufficient for binding to a biotinylated peptide comprising the membrane-proximal 73 amino acids of gp130, the signal-transducing receptor chain of interleukin-6-type cytokines. By the fold recognition approach amino acid residues 36-112 of Jak1 were predicted to adopt a beta-grasp fold, and a structural model was built using ubiquitin as a template. Substitution of Tyr(107) to alanine, a residue conserved among Jaks and involved in hydrophobic core interactions of the proposed beta-grasp domain, abrogated binding of full-length Jak1 to gp130 in COS-7 transfectants. By further mutagenesis we identified the loop 4 region of the Jak1 beta-grasp domain as essential for gp130 association and gp130-mediated signal transduction. In Jak1-deficient U4C cells reconstituted with the loop 4 Jak1 mutants L80A/Y81A and Delta(Tyr(81)-Ser(84)), the interferon-gamma, interferon-alpha, and interleukin-6 responses were similarly impaired. Thus, loop 4 of the beta-grasp domain plays a role in the association of Jak1 with both class I and II cytokine receptors. Taken together the structural model and the mutagenesis data provide further insight into the interaction of Janus kinases with cytokine receptors.