Extended Data Fig. 4: AMPK phosphorylates PHGDH and promotes PHGDH-catalyzed malate oxidation.
From: The alternative activity of nuclear PHGDH contributes to tumour growth under nutrient stress
In b, c, d, f and h, immunoblotting analyses were performed using the indicated antibodies. In a, b, g, i-k, data are presented as mean ± s.d. (n = 3 independent experiments), statistical analysis was performed using the two-tailed Student’s t-test. (a, b) SW1990 cells with depletion of PHGDH and reconstituted with indicated Flag-tagged rPHGDH were cultured in the normal or glucose-free DMEM for 12 h. The enzymatic activity for 3-PG and 2-HG (a) or 3-PG, malate and 2-HG (b) oxidation of immunoprecipitated-rPHGDH proteins was determined. (c) SW1990 cells were cultured in the normal or glucose-free DMEM for 12 h. Ser55-phosphorylated (left panel) or Ser371-phosphorylated (right panel) PHGDH was depleted using the indicated antibodies. Whole cell lysates were collected. (d) SW1990 (left panel) or LM3 (right panel) cells were cultured in the normal or glucose-free DMEM for 12 h. Ser55-phosphorylated PHGDH was depleted using the indicated antibodies. Nuclear extracts were collected. (e) The cartoon showing the status of PHGDH nuclear accumulation and activity under glucose deficiency. When p38 and AMPK are both activated under glucose deficiency, PHGDH Ser371 and Ser55 can be independently phosphorylated at PHGDH, or concomitantly phosphorylated at PHGDH, among which PHGDH with both phosphorylation account for a larger part. PHGDH Ser55 phosphorylation by AMPK promotes the catalytic activity of PHGDH for malate oxidation. In this way, PHGDH with Ser371 and Ser55 phosphorylation can translocate into the nucleus and exhibits the enhanced activity for malate oxidation, which thereby represses nuclear NAD+. (f-i) In vitro kinase assays were performed by incubating bacterial purified recombinant His-WT PHGDH or His-PHGDH S55A (f and g) and His-WT PHGDH or PHGDH S371D (h and i) with or without purified AMPK complex. Immunoblotting analysis was performed (f and h). The enzymatic activity of pulldown PHGDH for malate (g and i, left panels) or 3-PG (g and i, right panels) oxidation was determined. (j, k) SW1990 cells expressing indicated Flag-rPHGDH was orderly supplemented with 0, 0.5 mM, 2.5 mM and 5 mM diethyl-malate. Cells were cultured for 12 h in the glucose-free DMEM. The relative level of malate (j), NAD+ (k, left panel) or NADH/ NAD+ (k, right panel) was measured.
