Phosphotyrosine profiling to identify novel components of interferon and interleukin 6-family cytokine signalling.
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.
Is'harc
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ICRF, 44-Lincoln's Inn Fields, London, WC2A 3PX, UK. h.isharc@icrf.icnet.uk
Watling
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Kerr
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D015153:Blotting, Western; D002460:Cell Line; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D015180:Electrophoresis, Gel, Two-Dimensional; D006801:Humans; D007372:Interferons / Q000378:metabolism*; D015850:Interleukin-6 / Q000378:metabolism*; D019000:Phosphotyrosine / Q000378:metabolism*; D011233:Precipitin Tests; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015398:Signal Transduction
D015850:Interleukin-6; D011993:Recombinant Fusion Proteins; D019000:Phosphotyrosine; D007372:Interferons