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Understanding the Mechanisms that Lead to Dynamic Stress‐Induced O‐GlcNAcylation; Identification of the Stress‐Dependent Interactome of the O‐GlcNAcase
Author(s) -
Groves Jennifer,
Maduka Austin,
O'Meally Robert,
Cole Robert,
Zachara Natasha
Publication year - 2015
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.29.1_supplement.570.4
Subject(s) - interactome , microbiology and biotechnology , stable isotope labeling by amino acids in cell culture , proteomics , biochemistry , gsk 3 , phosphorylation , chemistry , biology , gene
O‐linked‐β‐N‐acetylglucosamine (O‐GlcNAc) is a dynamic post‐translational modification of intracellular proteins thought to regulate protein function in a manner analogous to phosphorylation. The addition and removal of O‐GlcNAc is catalyzed by the O‐GlcNAc transferase (OGT) and the O‐GlcNAcase (OGA), respectively. O‐GlcNAc is induced in response to many forms of cellular stress and injury in ex vivo , in vivo , and in vitro models. Suggesting that O‐GlcNAc regulates cell survival decisions, elevation of O‐GlcNAc levels prior to or after injury is protective. However, little is known about how the cell communicates with OGT and OGA during times of stress to regulate pro‐survival signaling networks. One mechanism by which these enzymes may be regulated is via protein‐protein interactions that alter their expression, activity, and/or targeting. The goal of this study is to define the stress‐dependent interactome of OGA using the BioID technique. We have fused full‐length OGA to the N‐ or C‐terminus of a mutant E. coli biotin ligase, which will promiscuously biotinylate interacting partners in vivo . These fusion proteins express and localize properly in U2OS cells, possess hexosaminidase activity, and biotinylate proteins differentially in response to oxidative stress. Numerous stress‐dependent binding partners of OGA were identified by SILAC‐based proteomics including calcium/calmodulin‐dependent kinase II, fatty acid synthase, and filamin A. These data provide the framework for future physiological studies that will provide mechanistic insight into how OGA is regulated during stress and contributes to O‐GlcNAc‐mediated cell survival.