title: Phosphorylation of serine 1137/1138 of mouse insulin receptor substrate (IRS) 2 regulates cAMP-dependent binding to 14-3-3 proteins and IRS2 protein degradation.
abstract: Insulin receptor substrate (IRS) 2 as intermediate docking platform transduces the insulin/IGF-1 (insulin like growth factor 1) signal to intracellular effector molecules that regulate glucose homeostasis, β-cell growth, and survival. Previously, IRS2 has been identified as a 14-3-3 interaction protein. 14-3-3 proteins can bind their target proteins via phosphorylated serine/threonine residues located within distinct motifs. In this study the binding of 14-3-3 to IRS2 upon stimulation with forskolin or the cAMP analog 8-(4-chlorophenylthio)-cAMP was demonstrated in HEK293 cells. Binding was reduced with PKA inhibitors H89 or Rp-8-Br-cAMPS. Phosphorylation of IRS2 on PKA consensus motifs was induced by forskolin and the PKA activator N(6)-Phe-cAMP and prevented by both PKA inhibitors. The amino acid region after position 952 on IRS2 was identified as the 14-3-3 binding region by GST-14-3-3 pulldown assays. Mass spectrometric analysis revealed serine 1137 and serine 1138 as cAMP-dependent, potential PKA phosphorylation sites. Mutation of serine 1137/1138 to alanine strongly reduced the cAMP-dependent 14-3-3 binding. Application of cycloheximide revealed that forskolin enhanced IRS2 protein stability in HEK293 cells stably expressing IRS2 as well as in primary hepatocytes. Stimulation with forskolin did not increase protein stability either in the presence of a 14-3-3 antagonist or in the double 1137/1138 alanine mutant. Thus the reduced IRS2 protein degradation was dependent on the interaction with 14-3-3 proteins and the presence of serine 1137/1138. We present serine 1137/1138 as novel cAMP-dependent phosphorylation sites on IRS2 and show their importance in 14-3-3 binding and IRS2 protein stability.
authors.affiliation: Division of Clinical Chemistry and Pathobiochemistry, Tuebingen 72076, Germany; Institute for Diabetes Research and Metabolic Diseases of the Helmholtz Center Munich at the University of Tuebingen (Paul Langerhans Institute Tuebingen), Tuebingen, Germany; German Center for Diabetes Research (DZD), Tuebingen 72076, Germany.
authors.forename: Sabine S
authors.initials: SS
authors.lastname: Neukamm
authors.affiliation: Medical Proteome Center, Institute for Ophtalmic Research, University Hospital Tuebingen, Tuebingen 72076, Germany.
authors.forename: Jennifer
authors.initials: J
authors.lastname: Ott
authors.affiliation: Medical Proteome Center, Institute for Ophtalmic Research, University Hospital Tuebingen, Tuebingen 72076, Germany.
authors.forename: Sascha
authors.initials: S
authors.lastname: Dammeier
authors.affiliation: Division of Clinical Chemistry and Pathobiochemistry, Tuebingen 72076, Germany; Institute for Diabetes Research and Metabolic Diseases of the Helmholtz Center Munich at the University of Tuebingen (Paul Langerhans Institute Tuebingen), Tuebingen, Germany; German Center for Diabetes Research (DZD), Tuebingen 72076, Germany.
authors.forename: Rainer
authors.initials: R
authors.lastname: Lehmann
authors.affiliation: Institute for Diabetes Research and Metabolic Diseases of the Helmholtz Center Munich at the University of Tuebingen (Paul Langerhans Institute Tuebingen), Tuebingen, Germany; German Center for Diabetes Research (DZD), Tuebingen 72076, Germany; Division of Endocrinology, Diabetology, Vascular Medicine, Nephrology and Clinical Chemistry, Department of Internal Medicine IV, Tuebingen 72076, Germany.
authors.forename: Hans-Ulrich
authors.initials: HU
authors.lastname: Häring
authors.affiliation: Division of Clinical Chemistry and Pathobiochemistry, Tuebingen 72076, Germany; Institute for Diabetes Research and Metabolic Diseases of the Helmholtz Center Munich at the University of Tuebingen (Paul Langerhans Institute Tuebingen), Tuebingen, Germany; German Center for Diabetes Research (DZD), Tuebingen 72076, Germany.
authors.forename: Erwin
authors.initials: E
authors.lastname: Schleicher
authors.affiliation: Division of Clinical Chemistry and Pathobiochemistry, Tuebingen 72076, Germany; Institute for Diabetes Research and Metabolic Diseases of the Helmholtz Center Munich at the University of Tuebingen (Paul Langerhans Institute Tuebingen), Tuebingen, Germany; German Center for Diabetes Research (DZD), Tuebingen 72076, Germany. Electronic address: Cora.Weigert@med.uni-tuebingen.de.
authors.forename: Cora
authors.initials: C
authors.lastname: Weigert
mesh_terms: D048948:14-3-3 Proteins / Q000235:genetics / Q000378:metabolism*; D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000235:genetics / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000235:genetics / Q000378:metabolism; D003513:Cycloheximide / Q000494:pharmacology; D057809:HEK293 Cells; D006801:Humans; D055504:Insulin Receptor Substrate Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D020125:Mutation, Missense; D010766:Phosphorylation / Q000502:physiology; D011485:Protein Binding / Q000187:drug effects / Q000502:physiology; D055550:Protein Stability / Q000187:drug effects; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D059748:Proteolysis; D012694:Serine / Q000235:genetics / Q000378:metabolism*
chemical_list: D048948:14-3-3 Proteins; D055504:Insulin Receptor Substrate Proteins; C526298:Irs2 protein, mouse; D011500:Protein Synthesis Inhibitors; D005576:Colforsin; D012694:Serine; D003513:Cycloheximide; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases
keywords: 14-3-3; Cyclic AMP (cAMP); IRS2; Insulin; Phosphorylation; Protein Degradation; Protein Kinase A (PKA)