Fig. 4: ERK-mediated PFKFB3 phosphorylation at the O-GlcNAcylation site.
From: O-GlcNAcylation of PFKFB3 is required for tumor cell proliferation under hypoxia
a–c In vitro phosphorylation analyses were performed by mixing the purified ERK1 with the indicated purified GST-PFKFB3 proteins or mutant proteins in the presence of [γ-32P]ATP (a, b). d The indicated purified PFKFB3 WT or mutant proteins were analysed by enzymatic activity assay. e, f The indicated purified GST–PFKFB3 protein, or pre-incubated with purified OGT (f), was mixed with purified His–G3BP2 protein with or without purified ERK1. GST pulldown analyses were performed. g SW1990 cells with indicated shRNA expression were pretreated with or without PUGNAc (20 μM) for 24 h and cultured for 12 h under hypoxia or normoxia. In a–c, and e–g, immunoblotting analyses were performed using the indicated antibodies. In d, the values are presented as mean ± s.e.m. (n = 3 independent experiments).
