Extended Data Fig. 6: PHGDH interacts with S73 phosphorylated-c-Jun and represses c-Jun PARylation.
From: The alternative activity of nuclear PHGDH contributes to tumour growth under nutrient stress
In b-f, h, l and m, whole cellular extracts were subjected to immunoblotting analyses using the indicated antibodies. In g-k and n-p, data are presented as mean ± s.d. (n = 3 independent experiments), statistical analysis was performed using the two-tailed Student’s t-test. (a) SW1990 cells expressing Flag-PHGDH were cultured for 6 h in the glucose-free DMEM. Immunoprecipitation analysis of nuclear extracts was performed using the Flag antibody, and the extracts were analyzed by mass spectrometry. The results of a mass spectrometric analysis of a tryptic fragment at m/z 497.73694 (mass error, +6.08 ppm) matched those of the doubly charged peptide, suggesting that c-Jun Ser73 was phosphorylated. The Sequest score for the match was Xcorr =0.79. (b) SW1990 (upper panel) or LM3 cells (bottom panel) with depletion of c-Jun were reconstituted with expression of indicated Flag-tagged rc-Jun. (c) LM3 cells with depletion of c-Jun and reconstituted with indicated Flag-rc-Jun were cultured in the normal or glucose-free DMEM for 6 h. Whole cellular extracts were subjected to immunoprecipitation using the indicated antibodies. (d) The in vitro kinase assay was performed with incubation of indicated purified c-Jun with or without of immunoprecipitated JNK1; then c-Jun was collected and incubated with PARP1/DNA/NAD+ mixture to conduct in vitro PARylation assay (upper panel). The in vitro PARylation assay was first performed with incubation of indicated c-Jun with or without PARP1/DNA/NAD+ mixture and then c-Jun was collected, which was followed by in vitro kinase assay with immunoprecipitated JNK1 (bottom panel). (e) SW1990 cells with depletion of PHGDH and reconstituted with expression of indicated Flag-rPHGDH were cultured in the normal or glucose-free DMEM for 6 h. Whole cellular extracts were subjected to immunoprecipitation using the indicated antibodies. (f) SW1990 cells with depletion of PHGDH and reconstituted with expression of indicated Flag-rPHGDH were cultured in the normal or glucose-free DMEM for 12 h (upper panel). SW1990 cells expressing indicated Flag-rPHGDH were cultured in the glucose-free DMEM for 12 h, S73-phosphorylated c-Jun was depleted using the indicated antibodies (bottom panel). Chromatin extracts were collected. (g) SW1990 (left panel) or LM3 (right panel) cells transfected with or without c-Jun sgRNA were pretreated with or without NCT-503 (40 μM). Cells were cultured in the normal or glucose-free DMEM for 36 h. Cellular viability was examined by CCK-8 assay. (h) SW1990 (left panel) or LM3 (right panel) cells expressing indicated Flag-rPHGDH were transfected with or without c-Jun siRNA. Cells were cultured in the normal or glucose-free DMEM for 36 h. Cellular viability was examined by CCK-8 assay. (i) LM3 cells with depletion of c-Jun and reconstituted with indicated Flag-rc-Jun were pretreated with or without NCT-503 (40 μM). Cells were cultured in the normal or glucose-free DMEM for 36 h. Cellular viability was examined by CCK-8 assay. (j, k) SW1990 (j) or LM3 (k) cells with depletion of PHGDH and reconstituted with expression of indicated Flag-rPHGDH were pretreated with or without SP600125 (20 μM). Cells were cultured in the normal or glucose-free DMEM for 36 h. (l, m) SW1990 (l) or LM3 (m) cells pretreated with or without SP600125 (20 μM) were cultured in the normal or glucose-free DMEM for 12 h. (n) LM3 cells with depletion of c-Jun and reconstituted with expression of indicated Flag–rc-Jun were cultured in the normal or glucose-free DMEM for 36 h. Cellular viability was examined by CCK-8 assay. (o, p) SW1990 (o) or LM3 (p) cells with depletion of PHGDH were pretreated with or without PJ-34 (20 μM). Cells were cultured in the normal or glucose-free DMEM for 36 h. Cellular viability was examined by CCK-8 assay.
