Premium
Site‐Specific Reciprocal Modification of Human IRS‐2 by O‐GlcNAcylation or Phosphorylation
Author(s) -
Ball Lauren Elizabeth,
Berkaw Mary Neale
Publication year - 2010
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.24.1_supplement.lb131
Subject(s) - phosphorylation , insulin receptor , chemistry , signal transduction , receptor , signal transducing adaptor protein , biochemistry , scaffold protein , insulin receptor substrate , immunoprecipitation , microbiology and biotechnology , irs1 , biology , insulin , insulin resistance , endocrinology , gene
Insulin receptor substrates 1 and 2 (IRS‐1/2) are homologous adaptor proteins that mediate differential metabolic effects of insulin and insulin like growth factor‐1 (IGF‐1). Tyr phosphorylation of IRS proteins by the insulin/IGF‐1 receptors creates interaction motifs for binding partners and subsequent activation of the PI3K/Akt and Grb2/MAPK pathways. IRS protein interactions are further modulated by Ser/Thr phosphorylation in response to nutrient excess and stress. To determine the impact of nutrient sensitive Ser/Thr O‐GlcNAc modification of IRS‐2 on protein interactions and the potential interplay between phosphorylation and O‐GlcNAcylation, the modifications of human IRS‐2 were characterized by tandem mass spectrometry. The protein was immunoprecipitated from HEK293 cells grown in the presence of the O‐GlcNAcase inhibitor, PUGNAc. The gel purified protein was proteolytically digested and the peptides were separated and analyzed by nRP‐LC‐IT‐MS utilizing fragmentation by ETD and CID. This analysis has revealed four novel sites of O‐GlcNAc modification which are distributed throughout the IRS‐2 molecule. Two of the O‐GlcNAcylated residues are known sites of phosphorylation and may represent sites of reciprocal regulation by a kinase and the O‐GlcNAc transferase. Similar to previously characterized human IRS‐1, IRS‐2 is O‐GlcNAc modified within a C‐terminal domain shown to interact directly with the insulin and IGF‐1 receptors. These findings will facilitate determination of the functional effects of O‐GlcNAc modification on IRS‐2 mediated insulin and IGF‐1 signaling.