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Sites of Posttranslational O‐GlcNAc Modification of Rat and Human Insulin Receptor Substrate‐1 (IRS‐1)
Author(s) -
Ball Lauren Elizabeth,
Berkaw Mary Neale
Publication year - 2008
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.22.2_supplement.302
Subject(s) - chemistry , iodoacetamide , insulin receptor , glycosylation , phosphorylation , receptor , biochemistry , effector , irs1 , insulin , enzyme , cysteine , biology , insulin resistance , endocrinology
The purpose of this study is to interrogate the functional relevance of O‐GlcNAc modification of IRS‐1 on insulin/IGF‐1 receptor signaling. IRS‐1 is a highly phosphorylated adaptor molecule that transduces signals from insulin and IGF‐1 receptors to downstream effectors. The interactions of IRS‐1 with binding partners are modulated by many S/T kinases. These interactions may be further modulated by O‐GlcNAc glycosylation of S/T. To determine the sites of O‐GlcNAc modification, rat and human IRS‐1 cDNAs were expressed in HEK293cells treated overnight with an O‐GlcNAcase inhibitor, PUGNAc. The His‐tagged protein was enriched by Ni/NTA affinity chromatography, gel purified, reduced and alkylated with iodoacetamide, and proteolytically digested. Peptides were analyzed by C18‐nLC‐MS/MS with an LTQ ion‐trap mass spectrometer (Thermo). O‐GlcNAc modified peptides were identified by pseudo‐neutral loss MS3 and electron transfer dissociation (ETD) aided in determination of the sites of modification. Within rat IRS‐1, O‐GlcNAc modification was identified at residues S902, S914, S1009, S1041, and as we previously reported at S1036. Initial studies of human IRS‐1 revealed O‐GlcNAcylation of 3 peptides, 1029–1074, 1099–1112, and 999–1016. Ongoing studies are testing the effect of mutagenesis of the O‐GlcNAcylated residues on insulin stimulated interactions of IRS‐1 with downstream effectors.