Identification of the major site of O-linked beta-N-acetylglucosamine modification in the C terminus of insulin receptor substrate-1.
Signal transduction from the insulin receptor to downstream effectors is attenuated by phosphorylation at a number of Ser/Thr residues of insulin receptor substrate-1 (IRS-1) resulting in resistance to insulin action, the hallmark of type II diabetes. Ser/Thr residues can also be reversibly glycosylated by O-linked beta-N-acetylglucosamine (O-GlcNAc) monosaccharide, a dynamic posttranslational modification that offers an alternative means of protein regulation to phosphorylation. To identify sites of O-GlcNAc modification in IRS-1, recombinant rat IRS-1 isolated from HEK293 cells was analyzed by two complementary mass spectrometric methods. Using data-dependent neutral loss MS3 mass spectrometry, MS/MS data were scanned for peptides that exhibited a neutral loss corresponding to the mass of N-acetylglucosamine upon dissociation in an ion trap. This methodology provided sequence coverage of 84% of the protein, permitted identification of a novel site of phosphorylation at Thr-1045, and facilitated the detection of an O-GlcNAc-modified peptide of IRS-1 at residues 1027-1073. The level of O-GlcNAc modification of this peptide increased when cells were grown under conditions of high glucose with or without chronic insulin stimulation or in the presence of an inhibitor of the O-GlcNAcase enzyme. To map the exact site of O-GlcNAc modification, IRS-1 peptides were chemically derivatized with dithiothreitol following beta-elimination and Michael addition prior to LC-MS/MS. This approach revealed Ser-1036 as the site of O-GlcNAc modification. Site-directed mutagenesis and Western blotting with an anti-O-GlcNAc antibody suggested that Ser-1036 is the major site of O-GlcNAc modification of IRS-1. Identification of this site will facilitate exploring the biological significance of the O-GlcNAc modification.
Ball
Lauren E
LE
Department of Endocrinology, Diabetes, and Medical Genetics, Medical University of South Carolina, Charleston, South Carolina 29425, USA.
Berkaw
Mary N
MN
Buse
Maria G
MG
D000117:Acetylglucosamine / Q000737:chemistry* / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D002853:Chromatography, Liquid; D005947:Glucose / Q000494:pharmacology; D007328:Insulin / Q000494:pharmacology; D055504:Insulin Receptor Substrate Proteins; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010449:Peptide Mapping; D010750:Phosphoproteins / Q000737:chemistry* / Q000378:metabolism*; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D012694:Serine / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013912:Threonine / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism
D007328:Insulin; D055504:Insulin Receptor Substrate Proteins; C526296:Irs1 protein, rat; D010750:Phosphoproteins; D011994:Recombinant Proteins; D013912:Threonine; D012694:Serine; D014357:Trypsin; D005947:Glucose; D000117:Acetylglucosamine