Fig. 3: c-SRC phosphorylates ACSS2 at Tyr530 and Tyr562.

a, b U87 cells were transfected with ACSS2 and c-SRC as indicated. After 24 h of transfection, cells were lysed and subjected to anti-Flag M2 beads for reciprocal immunoprecipitation (IP) assays, followed by Western blot to analyze the interaction between ACSS2 and c-SRC. c Endogenous IP between ACSS2 and c-SRC was performed in U87 cells, with ACSS2 knockdown U87 cells serving as a control. d GST, GST-ACSS2 and His-c-SRC were expressed and purified from BL21 E.coli competent cells. GST-pulldown assay of GST-ACSS2 and His-c-SRC was performed with GST as control. e U87 cells were transfected with HA-ACSS2 and Flag-c-SRC, followed by immunoprecipitation with anti-HA beads. The tyrosine phosphorylation of ACSS2 was detected with or without (w/wo) calf-intestinal alkaline phosphatase (CIAP) treatment. f Different forms of HA-c-SRC (WT, CA, and KD) were individually co-transfected with Flag-ACSS2 into U87 cells. The tyrosine phosphorylation levels of ACSS2 were determined after immunoprecipitation with anti-Flag M2 beads. WT: wild-type; CA: constitutive activated (Y529F); KD: kinase-dead (K297R) g, h Representative mass spectra of ACSS2 peptides, showing phosphorylation residues at Tyr530 (g) and Tyr562 (h). i Flag-ACSS2-WT or Flag-ACSS2-DMut (double mutation, Y530F + Y562F) were co-transfected with HA-c-SRC in U87 cells. Phosphorylation levels of Tyr530, Tyr562, and total tyrosine (p-Tyr) of ACSS2 were detected with corresponding antibodies after immunoprecipitation with anti-Flag M2 beads. j U87 cells transfected with c-SRC-WT and c-SRC-KD were analyzed for the phosphorylation levels of ACSS2-Tyr530 and ACSS2-Tyr562 with specific antibodies. The samples derive from the same experiment but different gels for p-Tyr530, p-Tyr562, ACSS2, and Flag. KD: kinase dead. k GST-ACSS2 and His-c-SRC were purified from BL21 E.coli competent cells. In vitro kinase assay was performed to determine ACSS2 phosphorylation by c-SRC. The samples derive from the same experiment but different gels for p-Tyr530, p-Tyr562, and p-Tyr. l Phosphorylation levels of ACSS2-Tyr530, ACSS2-Tyr562, and c-SRC in U87 cells were analyzed after treatment with c-SRC inhibitor Saracatinib (10 μM) for 24 h. The samples derive from the same experiment but different gels for p-Tyr530, p-Tyr562, ACSS2, and p-c-SRC. m c-SRC is further knocked down in ACSS2 knockdown U87 cells, followed by phosphorylation analysis of p-Tyr530 and p-Tyr562 on ACSS2. The samples derive from the same experiment but different gels for p-Tyr530, p-Tyr562, ACSS2, and c-SRC. n, o The alignments of the residues (Tyr530 and Tyr562 of ACSS2 in homo sapiens) across different species were analyzed by Snapgene software (6.0.2). Experiments in this figure were performed three times, except those in (a, b, e, g, h, and i). Most of these experiments were also repeated in 293 T cells (Supplementary Fig. 3).