Figure 6.
cGAS-associated PCK1 inhibits cGAS activation through competitive consumption of GTP, but not ATP. (A and B) Bacteria-purified cGAS proteins (2 mM) were incubated with HT-DNA (0.2 mg/ml), ATP (2 mM), and GTP (2 mM) in the presence or absence of 2 mM OAA and 2 mM bacteria-purified PCK1 proteins for the in vitro cGAMP production assay (n = 6 biological experiments) (A). After incubation, cGAS was heat inactivated, and products were subject to a cell-based cGAMP activity assay in BT-549 cells. Total cell lysates were harvested for immunoblotting analyses as indicated (B). (C and D) Bacteria-purified cGAS proteins (2 mM) were incubated with HT-DNA (0.2 mg/ml), ATP (2 mM), and different ATP/GTP titrations (2 mM, 4 mM, and 8 mM, respectively) in the presence or absence of 2 mM OAA and 2 mM bacteria-purified PCK1 proteins for the in vitro cGAMP production assay (C, left) (n = 6 biological experiments). The purity of bacteria-purified GST-PCK1 and His-PCK1 WT was identified by Coomassie blue staining (C, right). After incubation, cGAS was heat inactivated, and products were subject to a cell-based cGAMP activity assay in BT-549 cells. Total cell lysates were harvested for immunoblotting analyses as indicated (D). (E and F) BT-549 and MDA-MB-231 cells expressing PCK1 shRNA with reconstituted expression of Flag-rPCK1 WT or Flag-rPCK1 mutants treated with or without hypoxia in the presence or absence of ATP or GTP (0.1 or 0.5 mM) and digitonin (25 mg/ml) for 6 h were harvested for intracellular cGAMP measurement (E) (n = 6 biological experiments). Total cell lysates were harvested for immunoblotting analyses as indicated (F). (G) BT-549 cells expressing PCK1 shRNA with reconstituted expression of Flag-rPCK1 WT or Flag-rPCK1 mutants transfected with or without 2 μg/ml HT-DNA in the presence or absence of 0.5 mM ATP or GTP and digitonin (25 mg/ml) for 6 h were prepared for IF analyses as indicated (upper). Scale bar, 20 μm.The co-localization coefficients between the indicated proteins in the presence or absence of HT-DNA stimulation (lower). At least 10 cells from each independent experiment were analyzed and representative data are shown (n = 6 biological experiments). (H) BT-549 cells expressing PCK1 shRNA with reconstituted expression of Flag-rPCK1 WT or Flag-rPCK1 mutants transfected with or without 2 μg/ml HT-DNA in the presence or absence of 0.5 mM ATP or GTP and digitonin (25 mg/ml) for 6 h were harvested for immunoblotting analyses as indicated. (I) BT-549 cells expressing PCK1 shRNA with reconstituted expression of Flag-rPCK1 WT or Flag-rPCK1 mutants transfected with or without 2 μg/ml HT-DNA in the presence or absence of 0.5 mM ATP or GTP and digitonin (25 mg/ml) for 6 h were prepared for IF analyses as indicated (left). The co-localization coefficients between the indicated proteins in the presence or absence of hypoxia are shown (right). At least 10 cells from each independent experiment were analyzed, and the representative data are shown (n = 6 biological experiments). (J) BT-549 (left) and MDA-MB-231 (right) cells expressing PCK1 shRNA with reconstituted expression of Flag-rPCK1 WT or S151D mutant were transiently transfected with vectors expressing β-galactosidase and an IFN-β luciferase reporter. 24 h later, the aforementioned cells transfected with or without 2 μg/ml HT-DNA in the presence or absence of 0.5 mM ATP or GTP and digitonin (25 mg/ml) for 6 h were harvested for luciferase assay, and the relative IFN-β luciferase activity after normalization to β-galactosidase activity is shown (n = 4 biological experiments). (K) BT-549 cells expressing PCK1 shRNA with reconstituted expression of Flag-rPCK1 WT or S151D mutant transfected with or without 2 μg/ml HT-DNA in the presence or absence of 0.5 mM ATP or GTP and digitonin (25 mg/ml) for 12 h were harvested for qPCR analyses to measure CXCL10, IL6, IFNB1, and IFNA4 mRNA levels (n = 6 biological experiments). Experiments (B, D, F, and H) 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 (A, C, E, G, and I–K). Data are the mean ± SD; **P < 0.001 and ***P < 0.0001; NS, not significant. Source data are available for this figure: SourceData F6. Refer to the image caption for details.

cGAS-associated PCK1 inhibits cGAS activation through competitive consumption of GTP, but not ATP. (A and B) Bacteria-purified cGAS proteins (2 mM) were incubated with HT-DNA (0.2 mg/ml), ATP (2 mM), and GTP (2 mM) in the presence or absence of 2 mM OAA and 2 mM bacteria-purified PCK1 proteins for the in vitro cGAMP production assay (n = 6 biological experiments) (A). After incubation, cGAS was heat inactivated, and products were subject to a cell-based cGAMP activity assay in BT-549 cells. Total cell lysates were harvested for immunoblotting analyses as indicated (B). (C and D) Bacteria-purified cGAS proteins (2 mM) were incubated with HT-DNA (0.2 mg/ml), ATP (2 mM), and different ATP/GTP titrations (2 mM, 4 mM, and 8 mM, respectively) in the presence or absence of 2 mM OAA and 2 mM bacteria-purified PCK1 proteins for the in vitro cGAMP production assay (C, left) (n = 6 biological experiments). The purity of bacteria-purified GST-PCK1 and His-PCK1 WT was identified by Coomassie blue staining (C, right). After incubation, cGAS was heat inactivated, and products were subject to a cell-based cGAMP activity assay in BT-549 cells. Total cell lysates were harvested for immunoblotting analyses as indicated (D). (E and F) BT-549 and MDA-MB-231 cells expressing PCK1 shRNA with reconstituted expression of Flag-rPCK1 WT or Flag-rPCK1 mutants treated with or without hypoxia in the presence or absence of ATP or GTP (0.1 or 0.5 mM) and digitonin (25 mg/ml) for 6 h were harvested for intracellular cGAMP measurement (E) (n = 6 biological experiments). Total cell lysates were harvested for immunoblotting analyses as indicated (F). (G) BT-549 cells expressing PCK1 shRNA with reconstituted expression of Flag-rPCK1 WT or Flag-rPCK1 mutants transfected with or without 2 μg/ml HT-DNA in the presence or absence of 0.5 mM ATP or GTP and digitonin (25 mg/ml) for 6 h were prepared for IF analyses as indicated (upper). Scale bar, 20 μm.The co-localization coefficients between the indicated proteins in the presence or absence of HT-DNA stimulation (lower). At least 10 cells from each independent experiment were analyzed and representative data are shown (n = 6 biological experiments). (H) BT-549 cells expressing PCK1 shRNA with reconstituted expression of Flag-rPCK1 WT or Flag-rPCK1 mutants transfected with or without 2 μg/ml HT-DNA in the presence or absence of 0.5 mM ATP or GTP and digitonin (25 mg/ml) for 6 h were harvested for immunoblotting analyses as indicated. (I) BT-549 cells expressing PCK1 shRNA with reconstituted expression of Flag-rPCK1 WT or Flag-rPCK1 mutants transfected with or without 2 μg/ml HT-DNA in the presence or absence of 0.5 mM ATP or GTP and digitonin (25 mg/ml) for 6 h were prepared for IF analyses as indicated (left). The co-localization coefficients between the indicated proteins in the presence or absence of hypoxia are shown (right). At least 10 cells from each independent experiment were analyzed, and the representative data are shown (n = 6 biological experiments). (J) BT-549 (left) and MDA-MB-231 (right) cells expressing PCK1 shRNA with reconstituted expression of Flag-rPCK1 WT or S151D mutant were transiently transfected with vectors expressing β-galactosidase and an IFN-β luciferase reporter. 24 h later, the aforementioned cells transfected with or without 2 μg/ml HT-DNA in the presence or absence of 0.5 mM ATP or GTP and digitonin (25 mg/ml) for 6 h were harvested for luciferase assay, and the relative IFN-β luciferase activity after normalization to β-galactosidase activity is shown (n = 4 biological experiments). (K) BT-549 cells expressing PCK1 shRNA with reconstituted expression of Flag-rPCK1 WT or S151D mutant transfected with or without 2 μg/ml HT-DNA in the presence or absence of 0.5 mM ATP or GTP and digitonin (25 mg/ml) for 12 h were harvested for qPCR analyses to measure CXCL10, IL6, IFNB1, and IFNA4 mRNA levels (n = 6 biological experiments). Experiments (B, D, F, and H) 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 (A, C, E, G, and I–K). Data are the mean ± SD; **P < 0.001 and ***P < 0.0001; NS, not significant. Source data are available for this figure: SourceData F6.

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