Identification of the Amino Acids 300–600 of IRS-2 as 14-3-3 Binding Region with the Importance of IGF-1/Insulin-Regulated Phosphorylation of Ser-573

Neukamm, S.S., Toth, R., Morrice, N., Campbell, D.G., MacKintosh, C., Lehmann, R., Haering, H., Schleicher, E.D. and Weigert, C. (2012) Identification of the Amino Acids 300–600 of IRS-2 as 14-3-3 Binding Region with the Importance of IGF-1/Insulin-Regulated Phosphorylation of Ser-573. PLoS ONE, 7(8), e43296. (doi: 10.1371/journal.pone.0043296) (PMID:22912850) (PMCID:PMC3422239)

Abstract

Phosphorylation of insulin receptor substrate (IRS)-2 on tyrosine residues is a key event in IGF-1/insulin signaling and leads to activation of the PI 3-kinase and the Ras/MAPK pathway. Furthermore, phosphorylated serine/threonine residues on IRS-2 can induce 14-3-3 binding. In this study we searched IRS-2 for novel phosphorylation sites and investigated the interaction between IRS-2 and 14-3-3. Mass spectrometry identified a total of 24 serine/threonine residues on IRS-2 with 12 sites unique for IRS-2 while the other residues are conserved in IRS-1 and IRS-2. IGF-1 stimulation led to increased binding of 14-3-3 to IRS-2 in transfected HEK293 cells and this binding was prevented by inhibition of the PI 3-kinase pathway and an Akt/PKB inhibitor. Insulin-stimulated interaction between endogenous IRS-2 and 14-3-3 was observed in rat hepatoma cells and in mice liver after an acute insulin stimulus and refeeding. Using different IRS-2 fragments enabled localization of the IGF-1-dependent 14-3-3 binding region spanning amino acids 300–600. The 24 identified residues on IRS-2 included several 14-3-3 binding candidates in the region 300–600. Single alanine mutants of these candidates led to the identification of serine 573 as 14-3-3 binding site. A phospho-site specific antibody was generated to further characterize serine 573. IGF-1-dependent phosphorylation of serine 573 was reduced by inhibition of PI 3-kinase and Akt/PKB. A negative role of this phosphorylation site was implicated by the alanine mutant of serine 573 which led to enhanced phosphorylation of Akt/PKB in an IGF-1 time course experiment. To conclude, our data suggest a physiologically relevant role for IGF-1/insulin-dependent 14-3-3 binding to IRS-2 involving serine 573.

• Item Type: Articles
• Status: Published
• Refereed: Yes
• Glasgow Author(s) Enlighten ID: Morrice, Dr Nicholas
• Authors: Neukamm, S.S., Toth, R., Morrice, N., Campbell, D.G., MacKintosh, C., Lehmann, R., Haering, H., Schleicher, E.D., and Weigert, C.
• College/School: College of Medical Veterinary and Life Sciences > School of Cancer Sciences
• Journal Name: PLoS ONE
• Publisher: Public Library of Science
• ISSN: 1932-6203
• ISSN (Online): 1932-6203
• Published Online: 17 August 2012
• Copyright Holders: Copyright © 2012 The Authors
• First Published: First published in PloS ONE 7(8):e43296
• Publisher Policy: Reproduced under a Creative Commons License