Figure 2. OGT activity is regulated by GSK3β.
(A) Top panels: Kinase assays for mouse brain extracts from WT-GSK3β or AS-GSK3β were visualized by silver staining (right panel) or by Western blot against thiophosphate ester antibody (left panel). Middle panels: Western blots of the same brain extracts probed with antibodies against GSK3β, OGT and PPP1R9B. Bottom panels: Kinase reactions of brain extracts were immunoprecipitated by antibodies for PPP1R9B or OGT, followed by Western blot against thiophosphate ester antibody. PPP1R9B and OGT were specifically phosphorylated by AS-GSK3β but not by WT GSK3β. (B) In vitro phosphorylation of OGT by GSK3β in the presence of radioactive ATP (left panel). Proteins used were detected by Western blotting probed with anti-OGT and anti-GSK3β antibodies (right panel). (C) Increased phosphorylation of OGT N-terminal peptide by GSK3β. Extracted ion chromatograms of the m/z of the phosphorylated version of the tryptic peptide spanning residues 2–17 of the protein OGT shows a significantly increased level upon GSK3β treatment (right panel). CID fragmentation spectrum determines the phosphorylation to be present on either serine 2 or serine 3 of the peptide (i.e. serine 3 or serine 4 of the OGT protein) (left panel). (D) OGT can be O-GlcNAc modified on serine 2 or serine 3 of the peptide. ETD Fragmentation spectrum of a singly GlcNAc-modified version of the tryptic peptide spanning residues 2–17 of the protein OGT shows a mixture spectrum, demonstrating modified versions where serine 2 and serine 3 of the peptide are both present. (E) O-GlcNAcylation in vitro assay. O-GlcNAc transferase activity was measured using affinity-purified OGT, in the presence or absence of GSK3β, and UDP-[3H]GlcNAc. OGT activity was enhanced by the presence of GSK3β (pOGT). S3 and S4 of OGT were mutated to either alanine (S3AS4A) or aspartate (S3DS4D) for the assay. CKII peptide and control peptide were used as substrates in the assay. 3HDPM: 3H Distintegrations per minute. Error bars indicate the mean ± standard error.