FIG 8.

Two serine residues, S448/S451, are critical for O-GlcNAcylation of NRF1. (A) Detection of O-GlcNAcylation of NRF1 protein. Nuclear extracts of 293T cells expressing NRF1-3×FLAG were prepared and then pulled down with an anti-FLAG antibody. Immunoprecipitated samples were subjected to immunoblot analysis with antibodies against O-GlcNAc peptides and the FLAG tag. (B) Motifs in NRF1, which mediate interaction with Fbw7 (left) and β-TrCP (right). Serine residues that were replaced with alanine in subsequent experiments are underlined. The numbers beneath the underlines denote positions of the serine residues in the amino acid sequence of the NRF1 protein. (C) Summary of serine-to-alanine substitutions in the two motifs of NRF1. (D and E) Nuclear accumulation of NRF1 and its mutant molecules in response to enhanced cellular O-GlcNAcylation induced by high-glucose condition (D) or PugNAc treatment (E). 293T cells expressing wild-type or mutant NRF1-3×FLAG (WT, A-SS, S-AA, and A-AA) were cultured in low- or high-glucose culture medium (D) or with or without 100 μM PugNAc (E) for 24 h. Nuclear extracts were prepared and subjected to immunoblot analysis with antibodies against the FLAG tag and lamin B. (F) Effects of OGA expression on the protein levels of NRF1 and its mutant molecules. 293T cells expressing wild-type or mutant NRF1-3×FLAG were transfected with an empty or a Myc-OGA expression vector. At 24 h after transfection, the cells were cultured in high-glucose medium for another 24 h and harvested. Nuclear extracts were prepared and subjected to immunoblot analysis with antibodies against the FLAG tag, Myc tag, and lamin B. (G) Identification of critical serine residues for O-GlcNAcylation of the NRF1 protein. Nuclear extracts of 293T cells expressing the WT or S-AA mutant of NRF1-3×FLAG were prepared and pulled down with an anti-FLAG antibody. Immunoprecipitated samples were subjected to immunoblot analysis with antibodies against O-GlcNAc peptides and the FLAG tag. (H) Effects of PugNAc on protein interactions with NRF1. 293T cells that were stably transfected with an empty vector or an expression vector of the WT or S-AA mutant of NRF1-3×FLAG were pretreated with 100 μM PugNAc or left untreated for 24 h before nuclear extracts were prepared for immunoprecipitation with an anti-FLAG antibody. Immunoprecipitated proteins were subjected to immunoblot analysis with antibodies against β-TrCP, OGT, HCF-1, and the FLAG tag. (I) Effects of PugNAc on ubiquitination of NRF1. 293F cells that were stably transfected with an empty vector or an expression vector of NRF1-3×FLAG were pretreated with or without 100 μM PugNAc for 24 h and incubated with 10 μM MG132 for another 4 h before whole-cell extracts were prepared for immunoprecipitation with an anti-FLAG antibody. Immunoprecipitated proteins were subjected to immunoblot analysis with antibodies against NRF1 and the FLAG tag.