O-linked N-acetylglucosamine modification of insulin receptor substrate-1 occurs in close proximity to multiple SH2 domain binding motifs.
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-GlcNAc-modified, which may influence the phosphorylation status of the protein. To facilitate the understanding of the functional effects of O-GlcNAc modification on IRS-1-mediated signaling, we identified the sites of O-GlcNAc 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-GlcNAc-modified at multiple residues. Rat IRS-1 was O-GlcNAc-modified at Ser(914), Ser(1009), Ser(1036), and Ser(1041). Human IRS-1 was O-GlcNAc-modified at Ser(984) or Ser(985), at Ser(1011), and possibly at multiple sites within residues 1025-1045. O-GlcNAc modification at a conserved residue in rat (Ser(1009)) and human (Ser(1011)) IRS-1 is adjacent to a putative binding motif for the N-terminal SH2 domains of p85alpha and p85beta regulatory subunits of phosphatidylinositol 3-kinase and the tyrosine phosphatase SHP2 (PTPN11). Immunoblot analysis using an antibody generated against human IRS-1 Ser(1011) GlcNAc further confirmed the site of attachment and the identity of the +203.2-Da mass shift as beta-N-acetylglucosamine. The accumulation of IRS-1 Ser(1011) GlcNAc in HEPG2 liver cells and MC3T3-E1 preosteoblasts upon inhibition of O-GlcNAcase indicates that O-GlcNAcylation of endogenously expressed IRS-1 is a dynamic process that occurs at normal glucose concentrations (5 mm). O-GlcNAc 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-GlcNAc modification represents an additional layer of posttranslational regulation that may impact the specificity of effects elicited by insulin and IGF-1.
Klein
Amanda L
AL
Department of Molecular and Cellular Pharmacology, Medical University of South Carolina, Charleston, South Carolina 29425, USA.
Berkaw
Mary N
MN
Buse
Maria G
MG
Ball
Lauren E
LE
D000117:Acetylglucosamine / Q000031:analogs & derivatives* / Q000378:metabolism*; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D006801:Humans; D055504:Insulin Receptor Substrate Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D019869:Phosphatidylinositol 3-Kinases / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation; D011110:Polymorphism, Genetic; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism; D054592:Protein Tyrosine Phosphatase, Non-Receptor Type 11 / Q000737:chemistry / Q000378:metabolism; D051381:Rats; D012694:Serine / Q000378:metabolism; D018909:src Homology Domains
D055504:Insulin Receptor Substrate Proteins; D021122:Protein Subunits; D012694:Serine; D019869:Phosphatidylinositol 3-Kinases; D054592:Protein Tyrosine Phosphatase, Non-Receptor Type 11; D000117:Acetylglucosamine