title: The TORC1-activated Proteins, p70S6K and GRB10, Regulate IL-4 Signaling and M2 Macrophage Polarization by Modulating Phosphorylation of Insulin Receptor Substrate-2.
abstract: Lung M2 macrophages are regulators of airway inflammation, associated with poor lung function in allergic asthma. Previously, we demonstrated that IL-4-induced M2 gene expression correlated with tyrosine phosphorylation of the insulin receptor substrate-2 (IRS-2) in macrophages. We hypothesized that negative regulation of IRS-2 activity after IL-4 stimulation is dependent upon serine phosphorylation of IRS-2. Herein, we describe an inverse relationship between tyrosine phosphorylation (Tyr(P)) and serine phosphorylation (Ser(P)) of IRS-2 after IL-4 stimulation. Inhibiting serine phosphatase activity increased Ser(P)-IRS-2 and decreased Tyr(P)-IRS-2 leading to reduced M2 gene expression (CD200R, CCL22, MMP12, and TGM2). We found that inhibition of p70S6K, downstream of TORC1, resulted in diminished Ser(P)-IRS-2 and prolonged Tyr(P)-IRS-2 as well. Inhibition of p70S6K increased expression of CD200R and CCL22 indicating that p70S6K negatively regulates some, but not all, human M2 genes. Knocking down GRB10, another negative regulatory protein downstream of TORC1, enhanced both Tyr(P)-IRS-2 and increased expression of all four M2 genes. Furthermore, GRB10 associated with IRS-2, NEDD4.2 (an E3-ubiquitin ligase), IL-4Rα, and γC after IL-4 stimulation. Both IL-4Rα and γC were ubiquitinated after 30 min of IL-4 treatment, suggesting that GRB10 may regulate degradation of the IL-4 receptor-signaling complex through interactions with NEDD4.2. Taken together, these data highlight two novel regulatory proteins that could be therapeutically manipulated to limit IL-4-induced IRS-2 signaling and polarization of M2 macrophages in allergic inflammation.
authors.affiliation: From Johns Hopkins University, School of Medicine, Department of Anesthesiology and Critical Care Medicine, Baltimore, Maryland 21205.
authors.forename: Kristi J
authors.initials: KJ
authors.lastname: Warren
authors.affiliation: From Johns Hopkins University, School of Medicine, Department of Anesthesiology and Critical Care Medicine, Baltimore, Maryland 21205.
authors.forename: Xi
authors.initials: X
authors.lastname: Fang
authors.affiliation: From Johns Hopkins University, School of Medicine, Department of Anesthesiology and Critical Care Medicine, Baltimore, Maryland 21205.
authors.forename: Nagaraj M
authors.initials: NM
authors.lastname: Gowda
authors.affiliation: From Johns Hopkins University, School of Medicine, Department of Anesthesiology and Critical Care Medicine, Baltimore, Maryland 21205.
authors.forename: Joshua J
authors.initials: JJ
authors.lastname: Thompson
authors.affiliation: From Johns Hopkins University, School of Medicine, Department of Anesthesiology and Critical Care Medicine, Baltimore, Maryland 21205 nheller@jhmi.edu.
authors.forename: Nicola M
authors.initials: NM
authors.lastname: Heller
mesh_terms: D056827:Endosomal Sorting Complexes Required for Transport / Q000235:genetics / Q000378:metabolism; D051383:GRB10 Adaptor Protein / Q000235:genetics / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000235:genetics; D006801:Humans; D006967:Hypersensitivity / Q000235:genetics / Q000378:metabolism; D055504:Insulin Receptor Substrate Proteins / Q000235:genetics / Q000378:metabolism*; D015847:Interleukin-4 / Q000235:genetics / Q000378:metabolism*; D008264:Macrophages / Q000378:metabolism*; D000076222:Mechanistic Target of Rapamycin Complex 1; D046912:Multiprotein Complexes / Q000235:genetics / Q000378:metabolism*; D000075702:Nedd4 Ubiquitin Protein Ligases; D019948:Receptors, Interleukin-4 / Q000235:genetics / Q000378:metabolism; D038762:Ribosomal Protein S6 Kinases, 70-kDa / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction; D058570:TOR Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D020298:U937 Cells; D044767:Ubiquitin-Protein Ligases / Q000235:genetics / Q000378:metabolism
chemical_list: D056827:Endosomal Sorting Complexes Required for Transport; C096358:GRB10 protein, human; C508598:IL4 protein, human; C526297:IRS2 protein, human; D055504:Insulin Receptor Substrate Proteins; D046912:Multiprotein Complexes; D019948:Receptors, Interleukin-4; D051383:GRB10 Adaptor Protein; D015847:Interleukin-4; D000075702:Nedd4 Ubiquitin Protein Ligases; C000618483:Nedd4 protein, human; C000618485:Nedd4L protein, human; D044767:Ubiquitin-Protein Ligases; D000076222:Mechanistic Target of Rapamycin Complex 1; D038762:Ribosomal Protein S6 Kinases, 70-kDa; D058570:TOR Serine-Threonine Kinases
keywords: GRB10; IL-4; IRS-2; S6 kinase; allergy; asthma; macrophage; mammalian target of rapamycin (mTOR); p70S6K