Fig. 4: Nuclear GCK-mediated TAZ phosphorylation recruits PIN1 and increases TAZ stability and transcriptional activity.

a The indicated cells were subjected to hypoxia for the indicated periods. WB analyses with the indicated antibodies were performed. b Parental MCF7 and BT549 cells and the indicated clones with knock-in expression of TAZ T346A mutants were stimulated with or without hypoxia for 8 h. WB analyses were performed with the indicated antibodies. c, d MCF7 and BT549 cells were transfected with the indicated plasmids (c). The indicated clones of these cells with knock-in expression of GCK S398D were constructed (d). These cells were treated with 100 μg/mL cycloheximide (CHX) prior to WB analysis. The quantification of TAZ protein levels relative to initial protein levels is shown. The data are the means ± SD, n = 3 independent experiments, by two-tailed Student’s t tests (c, d). e MCF7 cells with or without CK2α knockout were treated with or without hypoxia for 8 h. IP and WB analyses were performed with the indicated antibodies. f MCF7 cells transfected with the indicated plasmids were treated with or without hypoxia for 8 h before IP and WB analyses. g Purified GST-PIN1 WT or GST-PIN1 WW mutant was mixed with the indicated purified His-TAZ proteins in the presence or absence of purified His-GCK S398D and ATP. A GST pull-down assay was subsequently performed. h Cis–trans isomerization assays were carried out by mixing synthesized phosphorylated or nonphosphorylated oligopeptide of TAZ containing the T346P motif with purified WT GST-PIN1 or GST-PIN1 C113A mutant. Data represent the means ± SD of three independent experiments. i MCF7 and BT549 cells with or without PIN1 shRNA expression were treated with CHX (100 μg/mL) and harvested at the indicated times. WB analyses were performed with the indicated antibodies. The quantification of TAZ protein levels relative to initial protein levels is shown. The data are the means ± SD, n = 3 independent experiments, by two-tailed Student’s t tests. j MCF7 cells transfected with the indicated plasmids were treated with or without hypoxia for 8 h before WB analysis. k MCF7 cells transfected with the indicated plasmids were treated with 10 μM MG132 for 10 h before IP and WB analyses. l MCF7 cells transfected with the indicated plasmids were treated with or without hypoxia for 8 h before IP and WB analyses. m MCF7 cells were transfected with the indicated plasmids. IP and WB analyses were then performed. n MCF7 cells were transfected with the indicated plasmids. WB analyses were then performed. o Parental MCF7 and BT549 cells and the indicated clones with knock-in expression of TAZ T346A mutant stably expressing a luciferase reporter, which was driven by the CTGF promoter, were stimulated with or without hypoxia for 8 h or transfected with the indicated plasmids. A luciferase reporter assay was then performed. The data are the means ± SD, n = 3 independent experiments, by two-tailed Student’s t tests. 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.