Figure 1.
JNK1/2-mediated PCK1 S151 phosphorylation promotes its binding to cGAS upon hypoxic conditions. (A) BT-549 and MDA-MB-231 cells treated with or without hypoxia for 2 h were harvested for IP and immunoblotting analyses as indicated. (B) BT-549 cells treated with or without hypoxia for 2 h were harvested for IP and immunoblotting analyses as indicated (upper). The relative cGAS protein levels in different groups were quantified by densitometric analysis of the blots (lower). (C) BT-549 cells pretreated with or without GSK2656157 (10 μM), SB203580 (10 μM), compound C (5 μM), or SP600125 (20 μM) for 30 min before treatment with or without hypoxia for 2 h were harvested for IP and immunoblotting analyses as indicated. (D) Parental and JNK1/2-KO BT-549 cells treated with or without hypoxia for 2 h were harvested for IP and immunoblotting analyses as indicated. (E) Bacterially purified His-PCK1 WT or S151A mutant was incubated with the bacterially purified GST-JNK1 (upper) or GST-JNK2 (lower) in the presence of [γ-32P]-ATP. Autoradiography and immunoblotting analyses were performed. (F) BT-549 cells pretreated with or without SP600125 (20 μM) for 30 min before treatment with or without hypoxia for the indicated time were harvested for immunoblotting analyses as indicated. (G) Bacterially purified His-PCK1 WT or S151A mutant on Ni-NTA agarose beads was incubated with or without active GST-JNK1 in the presence of ATP for an in vitro kinase assay. The His-tagged proteins were then incubated with or without CIP (10 U) for 30 min at 37°C; the proteins were collected for Ni-NTA pull-down, IP, and immunoblotting analyses as indicated. (H) BT-549 cells expressing PCK1 shRNA with reconstituted expression of Flag-rPCK1 WT, S151A, or S151D mutant treated with or without hypoxia for 2 h were harvested for IP and immunoblotting analyses as indicated. (I) BT-549 cells expressing PCK1 shRNA with reconstituted expression of Flag-rPCK1 WT and S151A mutant treated with or without hypoxia for 2 h were prepared for IF analyses as indicated (left). Scale bar, 20 μm. The intensity of green (cGAS) or red (PCK1) fluorescent signals along the oblique line was measured using Image J (right). At least n = 30 cells from the experiment were analyzed, and representative data are shown. (J) Parental BT-549 and MDA-MB-231 cells and the indicated clones with knock-in expression of PCK1 S151A mutants treated with or without hypoxia for 2 h were harvested for IP and immunoblotting analyses as indicated. Experiments (A–H and J) were repeated three times independently with similar results, and the representative data are shown. Statistical significance was determined by two-tailed Student’s t test (B), Data are the mean ± SD, *P < 0.05. CIP; calf intestinal alkaline phosphatase. Source data are available for this figure: SourceData F1. Refer to the image caption for details.

JNK1/2-mediated PCK1 S151 phosphorylation promotes its binding to cGAS upon hypoxic conditions. (A) BT-549 and MDA-MB-231 cells treated with or without hypoxia for 2 h were harvested for IP and immunoblotting analyses as indicated. (B) BT-549 cells treated with or without hypoxia for 2 h were harvested for IP and immunoblotting analyses as indicated (upper). The relative cGAS protein levels in different groups were quantified by densitometric analysis of the blots (lower). (C) BT-549 cells pretreated with or without GSK2656157 (10 μM), SB203580 (10 μM), compound C (5 μM), or SP600125 (20 μM) for 30 min before treatment with or without hypoxia for 2 h were harvested for IP and immunoblotting analyses as indicated. (D) Parental and JNK1/2-KO BT-549 cells treated with or without hypoxia for 2 h were harvested for IP and immunoblotting analyses as indicated. (E) Bacterially purified His-PCK1 WT or S151A mutant was incubated with the bacterially purified GST-JNK1 (upper) or GST-JNK2 (lower) in the presence of [γ-32P]-ATP. Autoradiography and immunoblotting analyses were performed. (F) BT-549 cells pretreated with or without SP600125 (20 μM) for 30 min before treatment with or without hypoxia for the indicated time were harvested for immunoblotting analyses as indicated. (G) Bacterially purified His-PCK1 WT or S151A mutant on Ni-NTA agarose beads was incubated with or without active GST-JNK1 in the presence of ATP for an in vitro kinase assay. The His-tagged proteins were then incubated with or without CIP (10 U) for 30 min at 37°C; the proteins were collected for Ni-NTA pull-down, IP, and immunoblotting analyses as indicated. (H) BT-549 cells expressing PCK1 shRNA with reconstituted expression of Flag-rPCK1 WT, S151A, or S151D mutant treated with or without hypoxia for 2 h were harvested for IP and immunoblotting analyses as indicated. (I) BT-549 cells expressing PCK1 shRNA with reconstituted expression of Flag-rPCK1 WT and S151A mutant treated with or without hypoxia for 2 h were prepared for IF analyses as indicated (left). Scale bar, 20 μm. The intensity of green (cGAS) or red (PCK1) fluorescent signals along the oblique line was measured using Image J (right). At least n = 30 cells from the experiment were analyzed, and representative data are shown. (J) Parental BT-549 and MDA-MB-231 cells and the indicated clones with knock-in expression of PCK1 S151A mutants treated with or without hypoxia for 2 h were harvested for IP and immunoblotting analyses as indicated. Experiments (A–H and J) were repeated three times independently with similar results, and the representative data are shown. Statistical significance was determined by two-tailed Student’s t test (B), Data are the mean ± SD, *P < 0.05. CIP; calf intestinal alkaline phosphatase. Source data are available for this figure: SourceData F1.

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