Identification and cloning of a novel family of coiled-coil domain proteins that interact with O-GlcNAc transferase

O-linked N-acetylglucosamine modification of serine/threonine residues on nuclear and cytoplasmic proteins has been shown to be a dynamic and ubiqitous form of protein regulation in eukaryotes. The enzyme that catalyzes the addition of O-GlcNAc on proteins is the O-GlcNAc transferase, and it has been shown to be essential for life, down to the single cell level. OGT is a nucleocytoplasmic enzyme that is present ubiquitously in all cell types examined. The amino terminal half of OGT consists of a protein-protein interaction domain called the tetratricopeptide repeat domain or TPR domain. We hypothesized that the TPR domain may mediate protein-protein interactions between the OGT and other putative interacting proteins, in a potentially regulatory manner. We used the yeast two-hybrid approach and isolated a novel family of highly homologous coiled-coil domain proteins that interacted with the OGT. This family consists of GABA_A receptor associated protein, GRIF-1, and O&barbelow;GT I&barbelow;nteracting P&barbelow;rotein of 106 kDa or OIP106 (KIAA1042). Both proteins interact with the TPR domain of OGT in a strong and stable manner. Additionally, both proteins are modified by O-GlcNAc, indicating that they are substrates for OGT. GRIF-1 is only expressed in excitable tissue, whereas O1P106 is ubiquitously expressed. GRIF-1 localizes to the GABA_A receptor, whereas O1P106 is a nuclear protein and co-localizes with a subset of RNA polymerase II (Pol II). Further studies showed that OIP106 was found to exist in a stable complex with Pol II and OGT. Studies performed with transiently expressed OIP106 showed that it was required for targeting OGT to Pol II complexes, and this interaction required the carboxy terminal domain of Pol II. These studies indicate that these proteins possibly regulate OGT function by modulating its targeting to distinct subcellular locations and protein complexes.; Further investigation of the kinetic mechanisms of O-GlcNAcylation of OIP106 revealed it to be a high affinity substrate for OGT (K_m = 3.35 μM), and its glycosylation was dependant upon its interaction with TPRs 2–6 of OGT. The isolated TPR domain of OGT competitively inhibited the glycosylation of OIP106, providing kinetic evidence for the TPR domain's role as a protein substrate docking site.