O-Linked N-Acetylglucosamine Modification of Insulin Receptor Substrate-1 Occurs in Close Proximity to Multiple SH2 Domain Binding Motifs

Amanda L Klein,Mary N Berkaw,Maria G Buse,Lauren E Ball

doi:10.1074/mcp.m900207-mcp200

Amanda L Klein, Mary N Berkaw + Show 2 more

Open Access

https://doi.org/10.1074/mcp.m900207-mcp200

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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-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.

Highlights

Insulin receptor substrate-1 (IRS-1) is a highly phosphorylated adaptor protein critical to insulin and IGF-1 receptor signaling
To identify the sites of O-GlcNAc modification of human and rat IRS-1, the proteins were transiently expressed in HEK293 cells grown in the presence of the O-GlcNAcase inhibitor PUGNAc
The fragmentation pattern of the O-GlcNAcylated tryptic peptide 1027–1074 of rat IRS-1 by electron transfer dissociation (ETD) is consistent with the co-elution of a mixture of monoGlcNAc-modified peptides in which only the site of modification at Ser1036 could be resolved (Fig. 1A)

Summary

EXPERIMENTAL PROCEDURES

Generation of Wild Type and Mutant Rat and Human IRS-1 cDNAs—His- and S-tagged rat IRS-1, amino acid residues 6 –1235, in pTriEX vector [31] served as a template for site-directed substitution of O-GlcNAc-modified serine residues with alanine. To characterize the specificity of the antibody for the site of O-GlcNAc modification, wild type IRS-1 or a mutant of rat IRS-1 in which the site of O-GlcNAc modification was substituted with alanine (S1009A) was transiently expressed in HEK293 cells in the presence of the O-GlcNAcase inhibitor PUGNAc. Nickel-purified proteins were separated by gel electrophoresis and transferred to nitrocellulose, and the membranes were probed with a 1:3000 dilution of rabbit sera followed by a fluorescently labeled secondary IgG. To further confirm that the antibody recognized the monosaccharide and not the naked peptide backbone, human IRS-1 isolated from PUGNAc-treated HEK293 cells was incubated in the presence or absence of ␤-Nacetyl-hexosaminidase (New England Biolabs). Estimates of the relative abundance of differentially modified peptides of rat IRS-1 isolated from cells treated with the O-GlcNAcase inhibitor PUGNAc were determined from the chromatographic peak areas of the extracted ion chromatograms corresponding to the modified and unmodified peptides. The membrane was washed thoroughly, and peptide binding to the protein array was visualized by enhanced chemiluminescence

RESULTS

Site assignment

GGPQGPGGMSAFTR ATMAAASSSSAASASPTGPQGAAELAAHSSLL

DISCUSSION