Roles of the Tetratricopeptide Repeat Domain in O-GlcNAc Transferase Targeting and Protein Substrate Specificity

Sai Prasad N Iyer, Gerald Warren Hart

Research output: Contribution to journalArticle

Abstract

The abundant and dynamic post-translational modification of nuclear and cytosolic proteins by β-O-linked N-acetylglucosamine (O-GlcNAc) is catalyzed by O-Glc-NAc-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 μM. 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.

Original languageEnglish (US)
Pages (from-to)24608-24616
Number of pages9
JournalJournal of Biological Chemistry
Volume278
Issue number27
DOIs
StatePublished - Jul 4 2003

ASJC Scopus subject areas

  • Biochemistry

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