Abstract
The abundant and dynamic post-translational modification of nuclear and cytosolic proteins by beta-O-linked N-acetylglucosamine (O-GlcNAc) is catalyzed by O-GlcNAc-transferase (OGT). Recently, we reported the identification of a novel family of OGT-interacting proteins (OIPs) that interact strongly with the tetratricopeptide repeat (TPR) domain of OGT (Iyer, S. P., Akimoto, Y., and Hart, G. W. (2003) J. Biol. Chem. 278, 5399-5409). Members of this family are modified by O-GlcNAc and are excellent substrates of OGT. Here, we further investigated the role of the TPR domain in the O-GlcNAcylation of OIP106, one of the members of this OIP family. Using N-terminal deletions, we first identified the region of OIP106 that binds OGT, termed the OGT-interacting domain (OID). Deletion analysis indicated that TPRs 2-6 of OGT interact with the OID of OIP106. The apparent Km of OGT for the OID of OIP106 is 3.35 microm. Unlike small peptide substrates, glycosylation of the OID was dependent upon its interaction with the first 6 TPRs of OGT. Furthermore, the isolated TPR domain of OGT competitively inhibited glycosylation of the OID protein, but did not inhibit glycosylation of a 12-amino acid casein kinase II peptide substrate, providing kinetic evidence for the role of the TPR domain as a protein substrate docking site. Additionally, both the OID of OIP106 and nucleoporin p62 competed with each other for glycosylation by OGT. These studies support the model that the catalytic subunit of OGT achieves both high specificity and a remarkable diversity of substrates by complexing with a variety of targeting proteins via its TPR protein-protein interaction domains.
Highlights
Post-translational modification of proteins by enzymatic addition of single -O-linked N-acetylglucosamine (O-GlcNAc)1 moieties to the hydroxyl groups of Ser/Thr residues has been shown to be a major form of protein regulation [2,3,4]
When overexpressed in E. coli as a His-tagged fusion protein, the majority of recombinant full-length OIP106 was found to be insoluble, so we localized the region of OIP106 that was responsible for its interaction with OGT with the hope that this region would be a smaller portion of the molecule and be more amenable to overexpression in an E. coli based system
Previous work on OGT enzymology has clearly shown a requirement for its tetratricopeptide repeat (TPR) domain in modulating its oligomerization as well as specificity for its peptide substrates in vitro [24]
Summary
O-GlcNAc, -O-linked N-acetylglucosamine; OGT, O-GlcNAc-transferase (uridine diphospho-N-acetylglucosamine:polypeptide -N-acetylglucosaminyltransferase); TPR, tetratricopeptide repeat; GABAA, ␥-aminobutyric acid type A; OIP, OGT-interacting protein; OID, OGT-interacting domain; GST, glutathione S-transferase; CKII, casein kinase II; BSA, bovine serum albumin. An O-GlcNAc-specific -N-acetylglucosaminidase (N-acetyl--D-glucosaminidase) removes the sugar from O-GlcNAcylated proteins Both enzymes have been purified, characterized, and cloned (16 –21). OIP106 was shown to associate in a complex with RNA polymerase II, suggesting a role in targeting OGT to RNA polymerase II complexes for transcriptional machinery O-GlcNAcylation [1] Both proteins are modified by O-GlcNAc and are substrates for OGT [1]. A free TPR domain effectively inhibited glycosylation of the OID, but not glycosylation of small peptides, in a dose-dependent manner, providing kinetic evidence that the TPRs function as protein substrate “docking” sites Both recombinant nucleoporin p62 (a high affinity substrate) and OID proteins effectively competed with each other for glycosylation by OGT. Samples were analyzed by Western blotting with S-proteinhorseradish peroxidase and AL25
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