title: Differential effects of IRS1 phosphorylated on Ser307 or Ser632 in the induction of insulin resistance by oxidative stress.
abstract: OBJECTIVE
  Induction of stress kinases leading to serine hyperphosphorylation of IRS1 may link oxidative stress to insulin resistance. The aim of this study was to investigate the roles of the phosphorylated serine residues Ser307 and Ser632, two sites implicated in the inhibition of IRS1 function in insulin signalling.
  
  
  METHODS
  Fao hepatoma cells were exposed to an H(2)O(2)-generating system, and antibodies against the two phosphorylated serine residues were used for immunoprecipitation, immunoblot and immunofluorescence analyses.
  
  
  RESULTS
  Exposure to approximately 50 mumol/l H(2)O(2) for 2 h resulted in IRS1 phosphorylation on both Ser307 and Ser632, concomitant with activation of inhibitor kappa kinase beta (IKKbeta) and c-Jun kinase (JNK). Immunoprecipitation studies revealed that the maximum overlap between phospho (p) Ser307-IRS1 and pSer632-IRS1 was 20%, and confocal microscopy suggested distinct localisations of IRS1 molecules phosphorylated on either site. Although pSer307-IRS1 showed decreased insulin-induced tyrosine phosphorylation and interaction with phosphatidylinositol 3-kinase (PI3K) in response to insulin, pSer632-IRS1 molecules were normally tyrosine-phosphorylated and exhibited typical associated PI3K activity. Salicylic acid and SP600125 partially inhibited IKKbeta and JNK, respectively, which indicated distinct roles for these two kinases in the phosphorylation of IRS1 at the two serine sites. Protection against oxidation-mediated impairment in insulin-induced phosphorylation of protein kinase B/Akt and in glycogen synthesis was achieved only by combining salicylic acid and SP600125.
  
  
  CONCLUSIONS
  These results suggest that pSer307-IRS1 and pSer632-IRS1 may define two minimally overlapping pools of IRS1 in response to oxidative stress, contributing differentially to insulin resistance. A combination of stress kinase inhibitors is required to protect against insulin resistance and IRS1 hyperphosphorylation induced by oxidative stress.
authors.affiliation: Department of Clinical Biochemistry, Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer-Sheva, 84103, Israel.
authors.forename: A
authors.initials: A
authors.lastname: Bloch-Damti
authors.forename: R
authors.initials: R
authors.lastname: Potashnik
authors.forename: P
authors.initials: P
authors.lastname: Gual
authors.forename: Y
authors.initials: Y
authors.lastname: Le Marchand-Brustel
authors.forename: J F
authors.initials: JF
authors.lastname: Tanti
authors.forename: A
authors.initials: A
authors.lastname: Rudich
authors.forename: N
authors.initials: N
authors.lastname: Bashan
mesh_terms: D000818:Animals; D006528:Carcinoma, Hepatocellular; D045744:Cell Line, Tumor; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006861:Hydrogen Peroxide / Q000494:pharmacology; D055504:Insulin Receptor Substrate Proteins; D007333:Insulin Resistance / Q000502:physiology*; D008113:Liver Neoplasms; D018384:Oxidative Stress / Q000502:physiology*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt / Q000037:antagonists & inhibitors / Q000187:drug effects / Q000378:metabolism; D051381:Rats
chemical_list: D004791:Enzyme Inhibitors; D006861:Hydrogen Peroxide; D055504:Insulin Receptor Substrate Proteins; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010750:Phosphoproteins; D010768:Phosphoserine; D051057:Proto-Oncogene Proteins c-akt; C526296:Irs1 protein, rat