Figure 6.

OGT Y976-phosphorylation is important for the association of representative phosphotyrosine-binding proteins.A, PKM2 K433 is required for OGT binding. (left) PKM2-associated proteins immunoprecipitated from A549 cells were analyzed by WB with indicated antibodies. (right) Relative protein levels of IP. pY976 and OGT was normalized to OGT and/or Flag (PKM2). Quantification shows mean ± SD (n = 3) with significance determined by two-way ANOVA, ∗∗p < 0.01, ∗∗∗p < 0.001, ns, nonsignificant. B, mutation at Y976 disrupts OGT interaction with selected phosphotyrosine-binding proteins. A549 cells transfected with Flag-tagged OGT^WT or OGT^Y976F were incubated with or without EGF (100 ng/ml) for 1 h. (top) The whole lysates and OGT-associated proteins from IP were analyzed by WB. (bottom) Relative protein levels of IP. Indicated proteins were normalized to Flag-OGT. Quantification shows mean ± SD (n = 3) with significance determined by two-way ANOVA, ∗∗∗p < 0.001, ns, nonsignificant. C, mutation at OGT Y976 impairs O-GlcNAcylation on selected phosphotyrosine-binding proteins. (top) A549 cells treated and transfected as in (B) were subjected to Click-iT O-GlcNAc Enzymatic Labeling System. O-GlcNAcylated proteins were biotinylated, precipitated, and analyzed by WB. (bottom) Relative protein levels of elution. O-GlcNAcylation level for each protein (elution) was normalized to its total protein (input). Quantification shows mean ± SD (n ≥ 3) with significance determined by two-way ANOVA, ∗∗∗p < 0.001, ns, nonsignificant. EGF, epidermal growth factor; EGFR, IP, immunoprecipitation; WB, western blotting; OGT, O-GlcNAc transferase.