O-Linked N-Acetylglucosamine Modification of Insulin Receptor Substrate-1 Occurs in Close Proximity to Multiple SH2 Domain Binding Motifs
2009; Elsevier BV; Volume: 8; Issue: 12 Linguagem: Inglês
10.1074/mcp.m900207-mcp200
ISSN1535-9484
AutoresAmanda Klein, Mary N. Berkaw, Maria G. Buse, Lauren E. Ball,
Tópico(s)Metabolism, Diabetes, and Cancer
ResumoInsulin 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 Ser914, Ser1009, Ser1036, and Ser1041. Human IRS-1 was O-GlcNAc-modified at Ser984 or Ser985, at Ser1011, and possibly at multiple sites within residues 1025–1045. O-GlcNAc 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 (PTPN11). Immunoblot analysis using an antibody generated against human IRS-1 Ser1011 GlcNAc 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 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. 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 Ser914, Ser1009, Ser1036, and Ser1041. Human IRS-1 was O-GlcNAc-modified at Ser984 or Ser985, at Ser1011, and possibly at multiple sites within residues 1025–1045. O-GlcNAc 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 (PTPN11). Immunoblot analysis using an antibody generated against human IRS-1 Ser1011 GlcNAc 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 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. Insulin receptor substrate-1 (IRS-1) 1The abbreviations used are:IRS-1insulin receptor substrate-1ETDelectron transfer dissociationSH2Src homology 2SHP2SH2 domain-containing tyrosine phosphatase 2PUGNAcO-(2-acetamido-2-deoxy-d-glucopyranosylidene) amino-N-phenylcarbamateHRPhorseradish peroxidasePI3Kphosphatidylinositol 3-kinaseMAPKmitogen-activated protein kinaseERKextracellular signal-regulated kinase. is a highly phosphorylated adaptor protein critical to insulin and IGF-1 receptor signaling. Many of the metabolic and mitogenic effects elicited by insulin and IGF-1 are mediated and modulated by posttranslational modifications of IRS-1, and tight regulation at the posttranslational level is crucial for maintaining insulin sensitivity and controlling growth factor-induced proliferation. Following hormonal stimulation, IRS-1 is phosphorylated by the receptor tyrosine kinases creating SH2 domain docking sites for downstream binding partners including the p85 regulatory subunits of phosphatidylinositol 3-kinase, Grb2, and the tyrosine phosphatase SHP2 (PTPN11) (1White M.F. IRS proteins and the common path to diabetes.Am. J. Physiol. Endocrinol. Metab. 2002; 283: E413-E422Crossref PubMed Scopus (758) Google Scholar). Binding of p85 phosphatidylinositol 3-kinase and Grb2 activate the PI3K/Akt and Ras-MAPK pathways, respectively, whereas binding of SHP2 results in tyrosine dephosphorylation and signal attenuation (2Myers Jr., M.G. Mendez R. Shi P. Pierce J.H. Rhoads R. White M.F. The COOH-terminal tyrosine phosphorylation sites on IRS-1 bind SHP-2 and negatively regulate insulin signaling.J. Biol. Chem. 1998; 273: 26908-26914Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). Positive and negative feedback regulation by Ser/Thr kinases, such as Akt (3Paz K. Liu Y.F. Shorer H. Hemi R. LeRoith D. Quan M. Kanety H. Seger R. Zick Y. Phosphorylation of insulin receptor substrate-1 (IRS-1) by protein kinase B positively regulates IRS-1 function.J. Biol. Chem. 1999; 274: 28816-28822Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar), c-Jun N-terminal kinase (JNK) (4Miller B.S. Shankavaram U.T. Horney M.J. Gore A.C. Kurtz D.T. Rosenzweig S.A. Activation of cJun NH2-terminal kinase/stress-activated protein kinase by insulin.Biochemistry. 1996; 35: 8769-8775Crossref PubMed Scopus (58) Google Scholar), S6K (5Harrington L.S. Findlay G.M. Gray A. Tolkacheva T. Wigfield S. Rebholz H. Barnett J. Leslie N.R. Cheng S. Shepherd P.R. Gout I. Downes C.P. Lamb R.F. The TSC1–2 tumor suppressor controls insulin-PI3K signaling via regulation of IRS proteins.J. Cell Biol. 2004; 166: 213-223Crossref PubMed Scopus (932) Google Scholar), and ERK (6Bouzakri K. 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Purification and characterization of a uridine diphospho-N-acetylglucosamine:polypeptide beta-N-acetylglucosaminyltransferase.J. Biol. Chem. 1992; 267: 9005-9013Abstract Full Text PDF PubMed Google Scholar, 14Gao Y. Wells L. Comer F.I. Parker G.J. Hart G.W. Dynamic O-glycosylation of nuclear and cytosolic proteins: cloning and characterization of a neutral, cytosolic beta-N-acetylglucosaminidase from human brain.J. Biol. Chem. 2001; 276: 9838-9845Abstract Full Text Full Text PDF PubMed Scopus (525) Google Scholar). Recent studies suggest that the activity of O-GlcNAc-transferase is regulated by insulin (15Whelan S.A. Lane M.D. Hart G.W. Regulation of the O-linked beta-N-acetylglucosamine transferase by insulin signaling.J. Biol. Chem. 2008; 283: 21411-21417Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar) and that localization of O-GlcNAc-transferase to the membrane is driven by direct association with phosphatidylinositide 3-phosphate (16Yang X. Ongusaha P.P. Miles P.D. Havstad J.C. Zhang F. So W.V. Kudlow J.E. Michell R.H. Olefsky J.M. Field S.J. Evans R.M. Phosphoinositide signalling links O-GlcNAc transferase to insulin resistance.Nature. 2008; 451: 964-969Crossref PubMed Scopus (451) Google Scholar). The abundance of O-GlcNAc modification on many proteins in the insulin signaling pathway increases with sustained high glucose and chronic insulin stimulation, and elevated O-GlcNAc modification of IRS-1 correlates with the development of insulin resistance in multiple cell types including 3T3-L1 adipocytes (17Vosseller K. Wells L. Lane M.D. Hart G.W. Elevated nucleocytoplasmic glycosylation by O-GlcNAc results in insulin resistance associated with defects in Akt activation in 3T3-L1 adipocytes.Proc. Natl. Acad. Sci. U.S.A. 2002; 99: 5313-5318Crossref PubMed Scopus (393) Google Scholar, 18Park S.Y. Ryu J. Lee W. O-GlcNAc modification on IRS-1 and Akt2 by PUGNAc inhibits their phosphorylation and induces insulin resistance in rat primary adipocytes.Exp. Mol. Med. 2005; 37: 220-229Crossref PubMed Scopus (123) Google Scholar), MIN6 pancreatic beta cells (19D'Alessandris C. Andreozzi F. Federici M. Cardellini M. Brunetti A. Ranalli M. Del Guerra S. Lauro D. Del Prato S. Marchetti P. Lauro R. Sesti G. Increased O-glycosylation of insulin signaling proteins results in their impaired activation and enhanced susceptibility to apoptosis in pancreatic beta-cells.FASEB J. 2004; 18: 959-961Crossref PubMed Scopus (72) Google Scholar), Fao rat hepatoma cells (16Yang X. Ongusaha P.P. Miles P.D. Havstad J.C. Zhang F. So W.V. Kudlow J.E. Michell R.H. Olefsky J.M. Field S.J. Evans R.M. Phosphoinositide signalling links O-GlcNAc transferase to insulin resistance.Nature. 2008; 451: 964-969Crossref PubMed Scopus (451) Google Scholar), human aortic endothelial cells (20Federici M. Menghini R. Mauriello A. Hribal M.L. Ferrelli F. Lauro D. Sbraccia P. Spagnoli L.G. Sesti G. Lauro R. Insulin-dependent activation of endothelial nitric oxide synthase is impaired by O-linked glycosylation modification of signaling proteins in human coronary endothelial cells.Circulation. 2002; 106: 466-472Crossref PubMed Scopus (294) Google Scholar), and skeletal muscle (21Patti M.E. Virkamäki A. Landaker E.J. Kahn C.R. Yki-Järvinen H. Activation of the hexosamine pathway by glucosamine in vivo induces insulin resistance of early postreceptor insulin signaling events in skeletal muscle.Diabetes. 1999; 48: 1562-1571Crossref PubMed Scopus (230) Google Scholar). The impact of O-GlcNAcylation on insulin signaling and diabetic complications was reviewed recently (22Copeland R.J. Bullen J.W. Hart G.W. Cross-talk between GlcNAcylation and phosphorylation: roles in insulin resistance and glucose toxicity.Am. J. Physiol. Endocrinol. Metab. 2008; 295: E17-E28Crossref PubMed Scopus (193) Google Scholar, 23Buse M.G. Hexosamines, insulin resistance, and the complications of diabetes: current status.Am. J. Physiol. Endocrinol. Metab. 2006; 290: E1-E8Crossref PubMed Scopus (372) Google Scholar). The direct effect of O-GlcNAc modification on signaling via IRS-1 is not known because conditions that mimic those in the uncontrolled diabetic patient may also result in phosphorylation of IRS-1 at inhibitory sites (16Yang X. Ongusaha P.P. Miles P.D. Havstad J.C. Zhang F. So W.V. Kudlow J.E. Michell R.H. Olefsky J.M. Field S.J. Evans R.M. Phosphoinositide signalling links O-GlcNAc transferase to insulin resistance.Nature. 2008; 451: 964-969Crossref PubMed Scopus (451) Google Scholar, 24Andreozzi F. D'Alessandris C. Federici M. Laratta E. Del Guerra S. Del Prato S. Marchetti P. Lauro R. Perticone F. Sesti G. Pandolfi A. De Filippis E.A. Pellegrini G. Menghini R. Lauro D. Cardellini M. Romano M. Consoli A. Activation of the hexosamine pathway leads to phosphorylation of insulin receptor substrate-1 on Ser307 and Ser612 and impairs the phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin insulin biosynthetic pathway in RIN pancreatic beta-cells.Endocrinology. 2004; 145: 2845-2857Crossref PubMed Scopus (58) Google Scholar) and O-GlcNAc modification of other proteins in the insulin signaling pathway, such as the insulin receptor, Akt (18Park S.Y. Ryu J. Lee W. O-GlcNAc modification on IRS-1 and Akt2 by PUGNAc inhibits their phosphorylation and induces insulin resistance in rat primary adipocytes.Exp. Mol. Med. 2005; 37: 220-229Crossref PubMed Scopus (123) Google Scholar), FoxO (25Housley M.P. Rodgers J.T. Udeshi N.D. Kelly T.J. Shabanowitz J. Hunt D.F. Puigserver P. Hart G.W. O-GlcNAc regulates FoxO activation in response to glucose.J. Biol. Chem. 2008; 283: 16283-16292Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar), AMP-activated protein kinase (26Luo B. Parker G.J. Cooksey R.C. Soesanto Y. Evans M. Jones D. McClain D.A. Chronic hexosamine flux stimulates fatty acid oxidation by activating AMP-activated protein kinase in adipocytes.J. Biol. Chem. 2007; 282: 7172-7180Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar), and β-catenin (17Vosseller K. Wells L. Lane M.D. Hart G.W. Elevated nucleocytoplasmic glycosylation by O-GlcNAc results in insulin resistance associated with defects in Akt activation in 3T3-L1 adipocytes.Proc. Natl. Acad. Sci. U.S.A. 2002; 99: 5313-5318Crossref PubMed Scopus (393) Google Scholar). To elucidate site-specific effects of O-GlcNAc modification on IRS-1-mediated signal transduction, we identified the sites of O-GlcNAc modification of rat and human IRS-1 by tandem mass spectrometry. To facilitate detection of the O-GlcNAc-modified peptides and assign the sites of modification, CID coupled with neutral loss-triggered MS3 and electron transfer dissociation (ETD) (27Dearth R.K. Cui X. Kim H.J. Kuiatse I. Lawrence N.A. Zhang X. Divisova J. Britton O.L. Mohsin S. Allred D.C. Hadsell D.L. Lee A.V. Mammary tumorigenesis and metastasis caused by overexpression of insulin receptor substrate 1 (IRS-1) or IRS-2.Mol. Cell. Biol. 2006; 26: 9302-9314Crossref PubMed Scopus (138) Google Scholar) tandem spectrometric approaches were used. Fragmentation of O-GlcNAc-modified peptides by ETD did not destroy the labile O-linkage (28Mikesh L.M. Ueberheide B. Chi A. Coon J.J. Syka J.E. Shabanowitz J. Hunt D.F. The utility of ETD mass spectrometry in proteomic analysis.Biochim. Biophys. Acta. 2006; 1764: 1811-1822Crossref PubMed Scopus (466) Google Scholar) permitting direct detection of these peptides by the database searching algorithm ProteinProspector2 (29Chalkley R.J. Baker P.R. Huang L. Hansen K.C. Allen N.P. Rexach M. Burlingame A.L. Comprehensive analysis of a multidimensional liquid chromatography mass spectrometry dataset acquired on a quadrupole selecting, quadrupole collision cell, time-of-flight mass spectrometer: II. new developments in Protein Prospector allow for reliable and comprehensive automatic analysis of large datasets.Mol. Cell. Proteomics. 2005; 4: 1194-1204Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). O-GlcNAc modification occurred in close proximity to multiple SH2 domain binding motifs and within a region of IRS-1 shown previously to interact with the insulin and IGF-1 receptors (30McGettrick A.J. Feener E.P. Kahn C.R. Human insulin receptor substrate-1 (IRS-1) polymorphism G972R causes IRS-1 to associate with the insulin receptor and inhibit receptor autophosphorylation.J. Biol. Chem. 2005; 280: 6441-6446Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). His- and S-tagged rat IRS-1, amino acid residues 6–1235, in pTriEX vector (31Ball L.E. Berkaw M.N. Buse M.G. Identification of the major site of O-linked beta-N-acetylglucosamine modification in the C terminus of insulin receptor substrate-1.Mol. Cell. Proteomics. 2006; 5: 313-323Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar) served as a template for site-directed substitution of O-GlcNAc-modified serine residues with alanine. Rat IRS-1 S1036A, S1041A, S1036A/S1041A, and S1009A were generated using the oligonucleotide primers 5′-gccccccctccatccgccacagctagcgcttctgcttctgttacacct, 5′-gccccccctccatcctccacagctagcgctgctgcttctgttacacct, 5′-gccccccctccatccgccacagctagcgctgctgcttctgttacacct, and 5′-ccagtggccccagtcgcatatgctgacatgcgg, respectively, using the QuikChange kit (Stratagene) according to the manufacturer's recommendations. The cDNA encoding C-terminally hemagglutinin-tagged human IRS-1 was kindly provided by Adrian Lee (Baylor University) (27Dearth R.K. Cui X. Kim H.J. Kuiatse I. Lawrence N.A. Zhang X. Divisova J. Britton O.L. Mohsin S. Allred D.C. Hadsell D.L. Lee A.V. Mammary tumorigenesis and metastasis caused by overexpression of insulin receptor substrate 1 (IRS-1) or IRS-2.Mol. Cell. Biol. 2006; 26: 9302-9314Crossref PubMed Scopus (138) Google Scholar). Initial mass spectrometric analysis of the expressed protein did not reveal the sites of O-GlcNAc modification; therefore, the human IRS-1 cDNA was cloned into the NotI and PmlI sites of pTriEX4 vector (Novagen) to generate an N-terminally His- and S-tagged protein. A stop codon was inserted at the 3′ end of the coding sequence to remove the C-terminal hemagglutinin tag from the expressed protein. Mutagenesis reactions and manipulations of the cDNA were verified by sequencing. HEK293 cells were maintained in Iscove's modified Eagle's medium (BioSource) containing 10% heat inactivated FCS with 1% antibiotic/antimycotic (Invitrogen). HEK293 cells were transiently transfected with rat or human IRS-1 cDNA using Polyfect transfection reagent according to the manufacturer's recommendations (Qiagen). To facilitate mass spectrometric detection of O-GlcNAc-modified peptides, cells grown in 5 mm glucose were incubated with an O-GlcNAcase inhibitor (50 µm O-(2-acetamido-2-deoxy-d-glucopyranosylidene) amino-N-phenylcarbamate (PUGNAc); Toronto Chemicals) for 18 h prior to lysis. For co-immunoprecipitation experiments, cells were serum-starved for 16 h in the presence of absence of PUGNAc and then stimulated with 100 nm insulin or 10 ng/ml IGF-1 (Sigma) for varying times. Cells were lysed in 300 mm NaCl, 50 mm sodium phosphate, pH 8, 10 mm imidazole, 10 mm β-mercaptoethanol, 1% Nonidet P-40, 50 µm PUGNAc, EDTA-free protease inhibitor mixture (Roche Applied Science), 10 mm sodium fluoride, 500 µm sodium vanadate, 1 mm sodium pyrophosphate. His-tagged IRS-1 was enriched by incubation with nickel-nitrilotriacetic acid affinity resin (Novagen) for 1 h at room temperature followed by extensive washing with lysis buffer containing 20 mm imidazole. The protein was eluted in lysis buffer containing 250 mm imidazole and gel-purified by electrophoresis on 4–10% XT Criterion gels (Bio-Rad). For mass spectrometric analysis, the protein was stained with E-zinc (Pierce), and the IRS-1 band was excised from the gel. Gel pieces were washed twice for 10 min with 100 mm ammonium bicarbonate, dehydrated with acetonitrile, and dried under vacuum. Cysteines were reduced with dithiothreitol and alkylated with iodoacetamide for 30 min at room temperature in the dark. Gel pieces were dehydrated with acetonitrile and dried completely prior to proteolytic digestion with trypsin (Promega) at 37 °C or with Glu-C (Roche Applied Science) at 25 °C for 18 h. Peptides were extracted with one wash of 25 mm ammonium bicarbonate for 20 min and three washes of 5% formic acid, 50% acetonitrile for 20 min each, dried under vacuum, reconstituted in 0.1% TFA, and cleaned up on C18 microspin columns (Nest Group). Prior to LC/MS analysis, the samples were reconstituted with 10 µl of 2% acetonitrile, 0.2% formic acid. HEPG2 cells were grown in 10% FCS with low glucose (5 mm) Dulbecco's modified Eagle's medium. Murine MC3T3-E1 osteoblast precursor cells were grown in 10% FCS with low glucose (5 mm) minimum Eagle's medium α (Invitrogen) with 1% penicillin/streptomycin (Mediatech). For overnight incubation in the absence or presence of 50 µm PUGNAc, the medium was supplemented with 1% FCS. Wild type and mutant IRS-1 protein, isolated by nickel affinity chromatography or immunoprecipitation, was separated by SDS-PAGE and transferred to nitrocellulose. Membranes were blocked with nonfat dry milk in Tris-buffered saline for detection by ECL or Odyssey blocking buffer (Licor Biosciences, Inc.) prior to fluorescence detection. Antibodies used for immunoprecipitation and immunoblotting of endogenously expressed IRS-1 were purchased from Bethyl Laboratories (A301-158) and Santa Cruz Biotechnology (A-19), respectively. A pan-specific monoclonal anti-O-GlcNAc (CTD 110.6) IgM generated against YSPTS(O-GlcNAc)PSK (32Comer F.I. Vosseller K. Wells L. Accavitti M.A. Hart G.W. Characterization of a mouse monoclonal antibody specific for O-linked N-acetylglucosamine.Anal. Biochem. 2001; 293: 169-177Crossref PubMed Scopus (234) Google Scholar) was purchased for Western blot (Pierce) or immunoprecipitation (Covance). The presence of recombinant wild type or mutant IRS-1 was detected by probing for the S-tag with S-protein conjugated to HRP (Novagen) and visualized using West Pico enhanced chemiluminescent reagent (Pierce). Fluorescently labeled secondary antibodies, IRDye 800CW and 680, were purchased from Licor Biosciences, Inc. Digested peptides were separated by C18 reversed phase nano-LC using a 75-µm × 15-cm capillary column packed in house (YMC ODS-AQ 120A S5, Waters) with a gradient of 2–60% B in 110 min (B = 0.02% heptafluorobutyric acid or 0.2% formic acid in acetonitrile) at 180 nl/min using an Ultimate 3000 nanoflow system with Chromeleon 6.8 software (Dionex, Sunnyvale, CA). Eluted peptides were analyzed by CID or ETD MS/MS with an LTQ XL ion trap mass spectrometer equipped with ETD capability (ThermoFisher). Multiple mass spectrometric approaches were utilized to detect O-GlcNAc-modified peptides and confirm the assignments of the sites of O-GlcNAc modification. Data-dependent neutral loss-triggered MS3 acquisition facilitated the identification of candidate O-GlcNAc-modified peptides. The instrument was programmed to acquire MS/MS/MS (MS3) data on precursor ions that exhibited a neutral loss corresponding to N-acetylglucosamine (203.2, 101.6, 67.7, or 50 Da for the 1+, 2+, 3+, and 4+ charge states, respectively) upon collision-induced dissociation. A normalized collision energy of 35% was used for fragmentation by CID. The threshold for the selection of an ion for fragmentation was set to 500. In subsequent analyses, the instrument was programmed to acquire tandem mass spectra generated by alternating between fragmentation by CID and ETD or by ETD only on the five most abundant ions in the full mass spectrum survey scan (m/z range, 400–2000). The parameters used for ETD were as follows: 100-ms activation time; emission current, 130 µA; automatic gain control, 30,000; temperature, 170 °C; supplemental activation was turned off; isolation width, 4 Da. With the exception of LC-MS/MS analyses performed with a parent mass inclusion list, dynamic exclusion was enabled to exclude ions from MS/MS selection for 3 min after being selected three times in a 30-s window. Tandem mass spectra were searched against a database of human or rat IRS-1 using Bioworks 3.3.1 for the following variable modifications: phosphorylation at serine, threonine, or tyrosine; O-GlcNAc modification of serine and threonine (+203.2 Da); and oxidation of methionine. Carbamidomethylcysteine was included as a static modification. Because of the labile nature of O-GlcNAc glycosylation, CID spectra also were searched for variable N- or C-terminal O-GlcNAc modification (+203.2 Da) to mimic the partial to complete loss of the GlcNAc from the precursor and fragment ions upon collision-induced dissociation. Unlike the neutral loss of phosphoric acid (98 Da) from phosphoserine resulting in a serine that has lost the side chain hydroxyl group, neutral loss of the monosaccharide from O-GlcNAc serine leaves the serine intact. Thus, MS3 spectra acquired following neutral loss of N-acetylglucosamine were also searched using Bioworks. ETD MS/MS spectra were searched by Protein Prospector2 (29Chalkley R.J. Baker P.R. Huang L. Hansen K.C. Allen N.P. Rexach M. Burlingame A.L. Comprehensive analysis of a multidimensional liquid chromatography mass spectrometry dataset acquired on a quadrupole selecting, quadrupole collision cell, time-of-flight mass spectrometer: II. new developments in Protein Prospector allow for reliable and comprehensive automatic analysis of large datasets.Mol. Cell. Proteomics. 2005; 4: 1194-1204Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar) for phosphorylation or O-GlcNAc modification of serine and threonine and for unanticipated posttranslational modifications. The exact sites of modification were confirmed by manual interpretation of CID and ETD spectra. A peptide corresponding to human IRS-1 residues 1003–1019 with an O-linked-β-N-acetylglucosamine serine at 1011, Ac-CDTSPAAPVS(O-GlcNAc)YADMRTGI-amide, was synthesized and purified by Sussex Research (Ontario, Canada). An N-terminal Cys residue was included for conjugation of the peptide to keyhole limpet hemocyanin carrier protein, and a polyclonal antibody was generated in rabbit (Lampire Biological Laboratories, Pipersville, PA). 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. The protein was visualized using an Odyssey instrument (Licor Biosciences, Inc.). Removal of the site of O-GlcNAc modification at Ser1009 completely abolished antibody binding (supplemental Fig. 1). To confirm that the antibody recognized the monosaccharide and not the naked peptide backbone, a competition experiment was performed by preincubating the sera with either the unmodified or O-GlcNAc-modified peptide residues 1003–1019. Preincubation of the sera with the O-GlcNAc-modified peptide abolished antibody binding to rat IRS-1, whereas preincubation with the naked peptide did not alter antibody binding to IRS-1 (supplemental Fig. 1). To check the specificity of the antibody for N-acetylglucosamine, a competition experiment was performed by preincubating the sera with 1 m N-acetylg
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