title: Serine 307 on insulin receptor substrate 1 is required for SOCS3 and TNF-α signaling in the rMC-1 cell line.
abstract: OBJECTIVE
  To establish the key insulin receptor substrate 1 (IRS-1) structural elements required in this insulin regulatory pathway, we investigated the effects of substituting alanine for serine 307 in IRS-1 on the ability of tumor necrosis factor-α (TNF-α) and a related mediator, suppressor of cytokine signaling 3 (SOCS3), to phosphorylate IRS-1 and regulate insulin signaling in the rat retinal Müller cell (rMC-1) cell line.
  
  
  METHODS
  rMC-1 cells were grown in normal (5 mM) or high (25 mM) glucose medium and transfected with either normal IRS-1(Ser307)plasmid or a mutated IRS-1(Ser307Ala) plasmid. Cells were also treated with recombinant TNF-α or SOCS3 to induce increased levels of these proteins.
  
  
  RESULTS
  In cells with IRS-1(Ser307Ala), TNF-α and SOCS3 failed to phosphorylate IRS-1. Likewise, resulting downstream effects, including changes in phosphorylation of insulin receptor(Tyr960), antiapoptotic Akt phosphorylation, and proapoptotic cleavage of caspase 3 were also blocked. We also report for the first time that SOCS3 and TNF-α are reciprocally stimulatory leading to a mutual enhancement of levels of both factors, thus forming a potential positive feedback loop that contributes to insulin receptor resistance.
  
  
  CONCLUSIONS
  Increases in TNF-α and SOCS3 are triggered by high glucose and through reciprocal stimulation of expression of these two factors, which in turn could be major drivers of insulin resistance and related cell death. The demonstration that a single phosphorylation site is key for these pathways suggests that drugs targeted to this site might be effective in protecting against diabetic damage to the retina.
authors.affiliation: Department of Ophthalmology, University of Tennessee Health Science Center, Memphis, TN.
authors.forename: Youde
authors.initials: Y
authors.lastname: Jiang
authors.affiliation: Department of Ophthalmology, University of Tennessee Health Science Center, Memphis, TN.
authors.forename: Subrata K
authors.initials: SK
authors.lastname: Biswas
authors.affiliation: Department of Ophthalmology, University of Tennessee Health Science Center, Memphis, TN ; Department of Anatomy/Neurobiology, University of Tennessee Health Science Center, Memphis, TN ; Department of Pharmaceutical Sciences, University of Tennessee Health Science Center, Memphis, TN.
authors.forename: Jena J
authors.initials: JJ
authors.lastname: Steinle
mesh_terms: D000409:Alanine / Q000235:genetics / Q000378:metabolism; D019943:Amino Acid Substitution; D000818:Animals; D053148:Caspase 3 / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D063928:Ependymoglial Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D025461:Feedback, Physiological; D005786:Gene Expression Regulation; D005947:Glucose / Q000378:metabolism* / Q000494:pharmacology; D055504:Insulin Receptor Substrate Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D007333:Insulin Resistance; D010766:Phosphorylation / Q000187:drug effects; D051057:Proto-Oncogene Proteins c-akt / Q000235:genetics / Q000378:metabolism; D051381:Rats; D012694:Serine / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D000071223:Suppressor of Cytokine Signaling 3 Protein; D050826:Suppressor of Cytokine Signaling Proteins / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology
chemical_list: D055504:Insulin Receptor Substrate Proteins; C526296:Irs1 protein, rat; C491410:Socs3 protein, rat; D000071223:Suppressor of Cytokine Signaling 3 Protein; D050826:Suppressor of Cytokine Signaling Proteins; D014409:Tumor Necrosis Factor-alpha; D012694:Serine; D051057:Proto-Oncogene Proteins c-akt; C505410:Casp3 protein, rat; D053148:Caspase 3; D005947:Glucose; D000409:Alanine