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 biolo gical effects via Janus kinase and signal transducer and activator of trans cription (JAK-STAT) signalling pathways. Our aim is to identify "novel" sig nalling molecules not previously implicated in JAK-mediated signalling. Pho sphotyrosine profiles of either whole cell lysates, or subcellular fraction s, of unstimulated and cytokine-treated cell lines have been analysed and l igand-inducible differences observed. Recombinant src homology 2 domains, b iotinylated peptides corresponding to cytokine receptor intracellular domai ns, antiphosphotyrosine antibodies, anion exchange columns and 2-D phosphot yrosine profiling have been used to select cohorts of molecules that are ty rosine phosphorylated in response to cytokine treatment. Tyrosine phosphory lated proteins showing cytokine specificity or differential profiles in cel l lines mutated in specific JAKs are being purified and identified by mass spectrometry.