Fig. 2: Arginine methylation at R236 elevates PHGDH catalytic activity by increasing substrate affinity. | Nature Communications

Fig. 2: Arginine methylation at R236 elevates PHGDH catalytic activity by increasing substrate affinity.

From: PHGDH arginine methylation by PRMT1 promotes serine synthesis and represents a therapeutic vulnerability in hepatocellular carcinoma

Fig. 2

a Endogenous PHGDH was immunoprecipitated in cells treated with AMI-1 or AdOx for 24 h. The mono-methylation (me1) level of PHGDH was determined by immunoblotting. PHGDH activity was measured and normalized to PHGDH protein. Data are presented as the mean ± SD (n  =  3 independent experiments). Statistical analysis was performed using the two-tailed Student’s t-test. b MS identification of R236 mono-methylation of FLAG-PHGDH immunopurified by FLAG beads. c Sequence conservation of PHGDH protein using the Consurf coloring scheme. Maroon and cyan indicate high and low conservation, respectively. d HEK293T cells expressing GFP-PHGDH WT, R236K or V83A were treated with AdOx for 24 h. The mono-methylation (me1) level of immunoprecipitated GFP-PHGDH was determined by immunoblotting. e FLAG-PHGDH WT, R236K or V83A was expressed in HEK293T cells. The mono-methylation of immunopurified FLAG-PHGDH was measured using a site-specific antibody against R236 mono-methylation (mePHGDH (R236me1)), which was pre-incubated with the R236 mono-methylated (R236me1) peptide or the unmodified peptide before use. f Endogenous PHGDH was immunoprecipitated in cells treated with AMI-1 or AdOx for 24 h. The R236 mono-methylation level of PHGDH was determined by immunoblotting using mePHGDH (R236me1) antibody. g HEK293T cells stably expressing FLAG-PHGDH WT, R236K or V83A were treated with AMI-1 or AdOx for 24 h. The R236 mono-methylation level of immunopurified FLAG-PHGDH was determined by immunoblotting using mePHGDH (R236me1) antibody. The activity of immunopurified FLAG-PHGDH was measured and normalized to FLAG-PHGDH protein. Data are presented as the mean ± SD (n  =  3 independent experiments). Statistical analysis was performed using the two-tailed Student’s t-test. h, i FLAG-PHGDH WT, R236K or V83A was expressed in HEK293T cells and immunopurified by FLAG beads. The Km value of FLAG-PHGDH for 3-PG (h) or NAD+ (i) was determined. Data are presented as the mean ± SD (n  =  3 independent experiments). Source data are provided as a Source Data file.

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