Is'harc, Hayaatun; (2002) JAK/STAT signalling. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

JAK-STAT signalling pathways play a critical role in transducing responses from Interferon (IFN) and cytokine receptors, but additional pathways are also engaged. Our objective was to characterise further JAK-receptor complexes, firstly through analysis of the structural basis of JAK-cytokine receptor interaction and secondly by identification and delineation of novel signals emanating from activated receptor complexes. The protein tyrosine kinase, JAKl, non-covalently associates with the intracellular domain of a subunit of many cytokine receptors, including the signal-transducing subunit of the receptor for the IL-6 family of cytokines, gp130. The N-terminal domains of JAKl translated in vitro were sufficient for binding to a biotinylated peptide encompassing the JAK-interaction motif of gp130. The region of JAKl concerned is predicted to adopt a ( -grasp fold. Mutation of key residues within the putative -grasp fold rendered JAK1 incapable of reconstituting IFN, as well as IL-6, responses in JAK1-deficient U4C cells, suggesting a similar mode of association between JAK1 and both class I and class II cytokine receptors. To identify 'novel' signalling molecules not previously implicated in JAK-mediated signalling, phosphotyrosine profiles of lysates from unstimulated and cytokine-treated cell lines were analysed. Recombinant SH2 domains, biotinylated receptor peptides, anti-phosphotyrosine antibodies, anion exchange and sizing columns and 2D gel electrophoresis were used to select molecules tyrosine phosphorylated in response to cytokine. Ligand inducible differences were observed and purification of inducibly tyrosine phosphorylated species to provide material for identification by mass spectrometry has been attempted. Substantial phosphorylation of the IFN receptor 1 (IFNGR1) subunit is observed in response to low level stimulation of the IL-6 and OSM on costation M receptors. It is also mediated to significant levels through EPOR/ erythropoietin receptor) gp130 chimeras and to a lesser extent through the IFN? receptor. As yet no absolute requirement for IFNGR1 in the responses through the endogenous IL-6, OSM or IFN receptors, or the chimeric receptors, has been detected. Similarly, cross-phosphorylation of IFNGR1 following OSM stimulation does not markedly prime or modulate the IFN response. The mechanistic basis for and biological significance of this striking phenomenon remain to be established.

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