Abstract
In addition to a role in response to insulin and insulin-like growth factors, insulin receptor substrate 1 (IRS-1) is phosphorylated in response to IL-4, the interferons (IFNs) and oncostatin M (OSM). Here mutant cell lines lacking JAK1, JAK2, or Tyk2 were used to determine the role(s) of the Janus kinase (JAK) family of protein-tyrosine kinases in IRS-1 phophorylation. 32D cells, which do not express IRS proteins, were analyzed for any requirement for these proteins in response to the IFNs. For the mutant human fibrosarcoma cell lines, phosphorylation of IRS-1 through the insulin-like growth factor receptor is independent of JAK1, JAK2, or Tyk2. In contrast, phosphorylation of IRS-1 mediated by the Type I IFNs, IL-4, and OSM is JAK-dependent. For the alphabeta-IFNs, activation of IRS-1 is dependent on JAK1 and Tyk2, consistent with the interdependence of these kinases in the IFN-alphabeta response. Neither IRS-1 nor IRS-2 was detectably activated by IFN-gamma. Consistent with this, activation of neither IRS proteins appears to be an absolute requirement for an antiproliferative or an antiviral response to the IFNs. For IL-4 and OSM phosphorylation of IRS-1 in the human fibrosarcoma cells is largely dependent on JAK1 but can also be mediated through Tyk2 or JAK2. Activation of phosphatidylinositol 3'-kinase in response to IL-4 and OSM, at least, was also JAK-dependent. The JAKs are, therefore, required not only for STAT activation but also for the activation, through a variety of different types of cytokine receptor, of an additional signaling pathway(s) through IRS-1 and phosphatidylinositol 3'-kinase.
Highlights
Receptors that have an intrinsic tyrosine kinase domain recruit and activate a variety of signal transducers
Insulin receptor substrate 1 (IRS-1)1 is a cytosolic protein of ϳ180 kDa in mass that is tyrosyl-phosphorylated at multiple sites through activated insulin and insulin-like growth factor 1 (IGF-1) receptors [1, 2]
It is independent of the Janus kinase (JAK) and is thought to reflect activation of the intrinsic IGF-1 receptor (IGFR) kinase through an autocrine loop
Summary
Antibodies—Rabbit polyclonal antisera were raised against the JH2 domain of JAK1 [44], the JH1 and JH6 domains of JAK2,3 amino acids 289 – 450 of TYK2 [45], and the last 12 residues in the C terminus of IRS-1 [46]. U4A cells stably transfected with JAK1 (U4/J1) or JAK2 (U4/J2) were maintained in both G418 and Hygromycin at the above concentrations [50]. 32D cells expressing transfected IRS proteins were as described [51, 52] and maintained in medium containing 5–10 mM histidinol (Sigma). Cells were lysed in ice-cold lysis buffer (1% Triton X-100 (v/v), 10% glycerol (v/v), 50 mM Hepes, 150 mM NaCl, 1 mM EDTA, pH 8, 200 M sodium orthovanadate, 0.5 mM phenylmethylsulfonyl fluoride, 10 mM sodium pyrophosphate, 100 mM sodium fluoride 1.5 mM magnesium chloride, 10 g/ml aprotinin, 10 g/ml leupeptin). The association of PI 3Ј-kinase activity with immunoprecipitation of IRS-1 was assessed by Friedman’s nonparametric analysis of variance [61], and the effect of IFN on growth of murine 32D cells was assessed by standard analysis of variance
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