title: Dynamic interplay between O-linked N-acetylglucosaminylation and glycogen synthase kinase-3-dependent phosphorylation.
abstract: O-GlcNAcylation on serine and threonine side chains of nuclear and cytoplasmic proteins is dynamically regulated in response to various environmental and biological stimuli. O-GlcNAcylation is remarkably similar to O-phosphorylation and appears to have a dynamic interplay with O-phosphate in cellular regulation. A systematic glycoproteomics analysis of the affects of inhibiting specific kinases on O-GlcNAcylation should help reveal both the global and specific dynamic relationships between these two abundant post-translational modifications. Here we report the O-GlcNAc perturbations in response to inhibition of glycogen synthase kinase-3 (GSK-3), a pivotal kinase involved in many signaling pathways. By combining immunoaffinity chromatography and SILAC (stable isotope labeling with amino acids in cell culture)-based quantitative mass spectrometry, we identified 45 potentially O-GlcNAcylated proteins. Quantitative measurements indicated that at least 10 proteins had an apparent increase of O-GlcNAcylation upon GSK-3 inhibition by lithium, whereas surprisingly 19 other proteins showed decreases. O-GlcNAcylation changes on a subset of the proteins were confirmed by follow-up experiments. By combining a new O-GlcNAc peptide enrichment method and beta-elimination followed by Michael addition with DTT, we also mapped the O-GlcNAc site (Ser-55) of vimentin, which showed an apparent increase of O-GlcNAcylation upon GSK-3 inhibition. Based on the MS data, we further investigated potential roles of O-GlcNAc on host cell factor-1, a transcription co-activator, and showed that dynamic regulation of O-GlcNAcylation on host cell factor-1 influenced its subcellular distribution. Taken together, these data indicated the complex interplay between phosphorylation and O-GlcNAcylation that occurs within signaling networks.
authors.affiliation: Department of Biological Chemistry, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205-2185, USA.
authors.forename: Zihao
authors.initials: Z
authors.lastname: Wang
authors.forename: Akhilesh
authors.initials: A
authors.lastname: Pandey
authors.forename: Gerald W
authors.initials: GW
authors.lastname: Hart
mesh_terms: D000117:Acetylglucosamine / Q000378:metabolism*; D000215:Acylation; D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D002846:Chromatography, Affinity / Q000379:methods; D004789:Enzyme Activation; D038362:Glycogen Synthase Kinase 3 / Q000037:antagonists & inhibitors / Q000378:metabolism*; D006031:Glycosylation; D051863:Host Cell Factor C1 / Q000378:metabolism; D007553:Isotope Labeling; D018021:Lithium Chloride / Q000494:pharmacology; D008969:Molecular Sequence Data; D017351:N-Acetylglucosaminyltransferases / Q000037:antagonists & inhibitors / Q000378:metabolism; D010449:Peptide Mapping; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D051863:Host Cell Factor C1; D017351:N-Acetylglucosaminyltransferases; C449880:O-GlcNAc transferase; D038362:Glycogen Synthase Kinase 3; D018021:Lithium Chloride; D000117:Acetylglucosamine