Figure S1. JNK1/2-mediated PCK1 S151... | Rockefeller University Press

Figure S1.

$JNK1/2-mediated PCK1 S151 phosphorylation promotes its binding to cGAS upon hypoxic conditions. (A) BT-549 cells were treated with or without hypoxia for 2 h. An IP assay was performed using an anti-cGAS antibody, and immunoprecipitates of endogenous cGAS were separated using SDS-PAGE and stained with Coomassie brilliant blue. Selected peptide hits of proteins associated with cGAS identified through mass spectrometry are shown. (B) BT-549 and MDA-MB-231 cells treated with hypoxia for 2 h were harvested for IP and immunoblotting analyses as indicated. (C) BT-549 cells expressing Flag-PCK1 or Flag-PCK2 treated with or without hypoxia for 2 h were harvested for IP and immunoblotting analyses as indicated. (D) BT549 cells were treated with or without hypoxia for 2 h. Cytosolic and nuclear fractions were prepared for IP and immunoblotting analyses as indicated, and the relative PCK1 and cGAS protein level in different fractions was quantified by densitometric analysis of the blots. (E) Bacterially purified His-PCK1 incubated with bacterially purified GST or GST-JNK1 was prepared for the GST pull-down assay. Immunoblotting analyses were performed as indicated (upper), and Coomassie blue staining was performed to show the purity of proteins (lower). (F) Bacterially purified His-PCK1 proteins on Ni-NTA agarose beads were incubated with or without active GST-JNK1 in the presence of ATP for an in vitro kinase assay. Mass spectrometric analysis of a tryptic fragment at m/z 1002.98499 Da (+2.63 mmu/+2.62 ppm), which was matched with the +2 charged peptide 138-TMYVIPFSMGPLGSPLSK-155, suggested that S151 was phosphorylated. The XCorr score was 5.56. (G) Alignment of protein sequences spanning PCK1 S151 from different species. (H) BT-549 cells treated with or without hypoxia for 1 h for the IP assay using an anti-PCK1 antibody, and IP of endogenous PCK1 were separated using SDS-PAGE, stained with Coomassie brilliant blue, and subjected to LC-MS/MS analysis. Mass spectrometric analysis of a tryptic fragment at m/z 610.87012 Da (−4.69 mmu/−7.68 ppm), which was matched with the +5 charged peptide 130-FPGCMKGRTMYVIPFSMGPLGSPLSK-155, suggested that S151 was phosphorylated. The XCorr score was 3.2. (I) Bacterially purified His-PCK1 WT, S151A, or C288S on Ni-NTA agarose beads were incubated with or without purified active GST-JNK1 in the presence of ATP for an in vitro kinase assay. After washing His-PCK1 WT, S151A, or C288S-conjugated beads with PBS five times, the relative PCK1 activity was measured (upper), and Coomassie blue staining was performed to show the purity of bacterially purified proteins (lower). (J) The purity of bacterially purified proteins used in In vitro kinase assays was identified by Coomassie blue staining. (K) IHC analyses of human TNBC samples were performed with the indicated antibodies in the presence or absence of a blocking peptide for PCK1 S151. (L) BT-549 cells expressing Flag-PCK1 treated with or without hypoxia for 1 h were harvested for IP and immunoblotting analyses as indicated in the presence or absence of a blocking peptide for PCK1 S151. (M) BT-549 and MDA-MB-231 cells treated with hypoxia for 2 h were harvested for IP and immunoblotting analyses as indicated (upper), and the relative PCK1 protein level in different fractions was quantified by densitometric analysis of the blots (lower). (N) 4T-1 cells pretreated with or without SP600125 (20 μM) or for 30 min before treatment with or without hypoxia for the indicated time were harvested for immunoblotting analyses as indicated. (O) BT-549 and MDA-MB-231 cells expressing PCK1 shRNA with reconstituted expression of the indicated PCK1 proteins were harvested for immunoblotting analyses as indicated. (P) BT-549 cells expressing PCK1 shRNA with reconstituted expression of the indicated PCK1 proteins treated with or without hypoxia for 2 h were prepared for IF analyses as indicated (left). Scale bar, 20 μm. The intensity of green (PCK1 pS151) or red (PCK1) fluorescent signals along the oblique line were measured using Image J (right). At least n = 30 cells from the experiment were analyzed, and representative data are shown. (Q) Genomic DNA was extracted from two individual clones of parental BT-549 and MDA-MB-231 cells with knock-in expression of PCK1 S151A. PCR products amplified from the indicated DNA fragments were separated on an agarose gel. (R) Sequencing of parental BT-549 and MDA-MB-231 cells and the individual clones of parental cells with knock-in expression of PCK1 S151A. The red line indicates the sgRNA-targeting sequence. The black line indicates the protospacer adjacent motif (PAM). Blue arrows indicate mutated nucleotides. A mutated amino acid and its WT counterpart are indicated by the solid red box. Experiments (B–E and L–O) were repeated three times independently with similar results, and the representative data are shown. Data are representative of as mean ± SD. Statistical significance was determined by two-tailed Student’s t test (I and M); *P < 0.05 and ***P < 0.0001; NS, not significant. Source data are available for this figure: SourceData FS1. Refer to the image caption for details.$

JNK1/2-mediated PCK1 S151 phosphorylation promotes its binding to cGAS upon hypoxic conditions. (A) BT-549 cells were treated with or without hypoxia for 2 h. An IP assay was performed using an anti-cGAS antibody, and immunoprecipitates of endogenous cGAS were separated using SDS-PAGE and stained with Coomassie brilliant blue. Selected peptide hits of proteins associated with cGAS identified through mass spectrometry are shown. (B) BT-549 and MDA-MB-231 cells treated with hypoxia for 2 h were harvested for IP and immunoblotting analyses as indicated. (C) BT-549 cells expressing Flag-PCK1 or Flag-PCK2 treated with or without hypoxia for 2 h were harvested for IP and immunoblotting analyses as indicated. (D) BT549 cells were treated with or without hypoxia for 2 h. Cytosolic and nuclear fractions were prepared for IP and immunoblotting analyses as indicated, and the relative PCK1 and cGAS protein level in different fractions was quantified by densitometric analysis of the blots. (E) Bacterially purified His-PCK1 incubated with bacterially purified GST or GST-JNK1 was prepared for the GST pull-down assay. Immunoblotting analyses were performed as indicated (upper), and Coomassie blue staining was performed to show the purity of proteins (lower). (F) Bacterially purified His-PCK1 proteins on Ni-NTA agarose beads were incubated with or without active GST-JNK1 in the presence of ATP for an in vitro kinase assay. Mass spectrometric analysis of a tryptic fragment at m/z 1002.98499 Da (+2.63 mmu/+2.62 ppm), which was matched with the +2 charged peptide 138-TMYVIPFSMGPLGSPLSK-155, suggested that S151 was phosphorylated. The XCorr score was 5.56. (G) Alignment of protein sequences spanning PCK1 S151 from different species. (H) BT-549 cells treated with or without hypoxia for 1 h for the IP assay using an anti-PCK1 antibody, and IP of endogenous PCK1 were separated using SDS-PAGE, stained with Coomassie brilliant blue, and subjected to LC-MS/MS analysis. Mass spectrometric analysis of a tryptic fragment at m/z 610.87012 Da (−4.69 mmu/−7.68 ppm), which was matched with the +5 charged peptide 130-FPGCMKGRTMYVIPFSMGPLGSPLSK-155, suggested that S151 was phosphorylated. The XCorr score was 3.2. (I) Bacterially purified His-PCK1 WT, S151A, or C288S on Ni-NTA agarose beads were incubated with or without purified active GST-JNK1 in the presence of ATP for an in vitro kinase assay. After washing His-PCK1 WT, S151A, or C288S-conjugated beads with PBS five times, the relative PCK1 activity was measured (upper), and Coomassie blue staining was performed to show the purity of bacterially purified proteins (lower). (J) The purity of bacterially purified proteins used in In vitro kinase assays was identified by Coomassie blue staining. (K) IHC analyses of human TNBC samples were performed with the indicated antibodies in the presence or absence of a blocking peptide for PCK1 S151. (L) BT-549 cells expressing Flag-PCK1 treated with or without hypoxia for 1 h were harvested for IP and immunoblotting analyses as indicated in the presence or absence of a blocking peptide for PCK1 S151. (M) BT-549 and MDA-MB-231 cells treated with hypoxia for 2 h were harvested for IP and immunoblotting analyses as indicated (upper), and the relative PCK1 protein level in different fractions was quantified by densitometric analysis of the blots (lower). (N) 4T-1 cells pretreated with or without SP600125 (20 μM) or for 30 min before treatment with or without hypoxia for the indicated time were harvested for immunoblotting analyses as indicated. (O) BT-549 and MDA-MB-231 cells expressing PCK1 shRNA with reconstituted expression of the indicated PCK1 proteins were harvested for immunoblotting analyses as indicated. (P) BT-549 cells expressing PCK1 shRNA with reconstituted expression of the indicated PCK1 proteins treated with or without hypoxia for 2 h were prepared for IF analyses as indicated (left). Scale bar, 20 μm. The intensity of green (PCK1 pS151) or red (PCK1) fluorescent signals along the oblique line were measured using Image J (right). At least n = 30 cells from the experiment were analyzed, and representative data are shown. (Q) Genomic DNA was extracted from two individual clones of parental BT-549 and MDA-MB-231 cells with knock-in expression of PCK1 S151A. PCR products amplified from the indicated DNA fragments were separated on an agarose gel. (R) Sequencing of parental BT-549 and MDA-MB-231 cells and the individual clones of parental cells with knock-in expression of PCK1 S151A. The red line indicates the sgRNA-targeting sequence. The black line indicates the protospacer adjacent motif (PAM). Blue arrows indicate mutated nucleotides. A mutated amino acid and its WT counterpart are indicated by the solid red box. Experiments (B–E and L–O) were repeated three times independently with similar results, and the representative data are shown. Data are representative of as mean ± SD. Statistical significance was determined by two-tailed Student’s t test (I and M); *P < 0.05 and ***P < 0.0001; NS, not significant. Source data are available for this figure: SourceData FS1.