O-linked N-acetylglucosamine modification of insulin receptor substrate-1 occurs in close proximity to multiple SH2 domain binding motifs

Abstract

Insulin receptor substrate-1 (IRS-1) is a highly phosphorylated adaptor protein critical to insulin and IGF-1 receptor signaling. Ser/Thr kinases impact the metabolic and mitogenic effects elicited by insulin and IGF-1 through feedback and feed forward regulation at the level of IRS-1. Ser/Thr residues of IRS-1 are also O- GIcNAc-modified, which may influence the phosphorylation status of the protein. To facilitate the understanding of the functional effects of O-GIcNAc modification on IRS-1-mediated signaling, we identified the sites of O-GIcNAc modification of rat and human IRS-1. Tandem mass spectrometric analysis of IRS-1, exogenously expressed in HEK293 cells, revealed that the C terminus, which is rich in docking sites for SH2 domain-containing proteins, was O-GIcNAc-modified at multiple residues. Rat IRS-1 was O-GIcNAc-modified at Ser914, Ser1009, Ser1036, and Ser 1041. Human IRS-1 was O-GIcNAc-modified at Ser984 or Ser985, at Ser1011, and possibly at multiple sites within residues 1025-1045. O-GIcNAc modification at a conserved residue in rat (Ser1009) and human (Ser1011) IRS-1 is adjacent to a putative binding motif for the N-terminal SH2 domains of p85α and p85β regulatory subunits of phosphatidylinositol 3-kinase and the tyrosine phosphatase SHP2 (PTPN 11). Immunoblot analysis using an antibody generated against human IRS-1 Ser1011 GIcNAc further confirmed the site of attachment and the identity of the +203.2-Da mass shift as β-N- acetylglucosamine. The accumulation of IRS-1 Ser1011 GIcNAc in HEPG2 liver cells and MC3T3-E1 preosteoblasts upon inhibition of O-GIcNAcase indicates that O-GIcNAcylation of endogenously expressed IRS-1 is a dynamic process that occurs at normal glucose concentrations (5 mM). O-GIcNAc modification did not occur at any known or newly identified Ser/Thr phosphorylation sites and in most cases occurred simultaneously with phosphorylation of nearby residues. These findings suggest that O-GIcNAc modification represents an additional layer of posttranslational regulation that may im pact the specificity of effects elicited by insulin and IGF-1. © 2009 by The American Society for Biochemistry and Molecular Biology, Inc.