Fig. 1: GCK S398 phosphorylation by CK2 induces nuclear translocation of GCK.

a MCF7 and BT549 cells were stimulated with or without hypoxia for 8 h. Immunofluorescence analyses were performed with an anti-GCK antibody. DAPI (blue) was used to stain the nuclei. b MCF7 (luminal A) and BT549 (basal) cells were treated with or without hypoxia for 8 h. Whole-cell lysates (WCLs) were collected and cytosolic (Cyto) and nuclear (Nuc) fractions were prepared. WB, western blot. c MCF7 cells were pretreated with or without the indicated inhibitors for 30 min and then treated with or without hypoxia and the indicated inhibitors for 8 h. Cytoplasm and nucleus were prepared. Whole-cell lysates were collected. d MCF7 cells with or without CK2α knockout were subjected to hypoxia for 8 h. Cytoplasm and nucleus were prepared. WCLs were collected. e MCF7 cells with or without CK2α knockout were subjected to hypoxia for 8 h. Immunoprecipitation (IP) with an anti-GCK antibody was performed. f A GST pulldown assay was performed by mixing purified His-CK2α with purified GST or GST-GCK. Immunoblotting analyses were performed as indicated. g An in vitro kinase assay was performed by incubating GST-CK2α WT or GST-CK2α K68M with His-GCK WT or His-GCK S398A in the presence of active ERK2 and ATP-γ-S. The samples were then alkylated with p-nitrophenyl mesylate (PNBM). h MCF7 cells expressing Flag-GCK WT or Flag-GCK S398A were treated with or without hypoxia for 8 h. IP analyses were performed with an anti-Flag antibody. i MCF7 cells pretreated with or without TBB (10 µM) for 30 min were subjected to hypoxia or/and TBB for the indicated times. Total cell lysates were prepared. j MCF7 and BT549 cells transfected with the indicated plasmids were treated with or without hypoxia for 8 h. Cytoplasm and nucleus were prepared. WCL was collected. k MCF7 and BT549 cells transfected with the indicated plasmids were stimulated with or without hypoxia for 8 h. Immunofluorescence analyses were performed with an anti-Flag antibody. DAPI (blue) was used to stain the nuclei. All immunoblotting and immunofluorescence experiments were performed in at least three independent biological replicates, with consistent results observed across repetitions. Representative data are shown. Source data are provided as a Source Data file.