Fig. 2: Src-mediated tyrosine phosphorylation of lipin-1 is essential for PAP activity of lipin-1 and glycerolipid synthesis.

a Identification of phosphorylation sites on lipin-1. Flag-WT-lipin-1 or its tyrosine-to-phenylalanine (YF) mutants was co-expressed with or without Src in HEK293T cells. Cells were lysed and subjected to IP against Flag, followed by immunoblotting. b 3YF-lipin-1 mutant fails to be phosphorylated by Src in vitro. Bacterially expressed His-tagged WT-lipin-1 or its 3YF mutant was incubated with His-Src in a kinase assay buffer, followed by immunoblotting. c 3YF-lipin-1 mutant fails to be phosphorylated in EGF-stimulated in MDA-MB-231 cells. LPIN1-KO MDA-MB-231 cells stably expressing Flag-tagged WT-lipin-1 or 3YF-lipin-1 were maintained in a serum-free medium for 4 h, followed with or without EGF treatment. d Sequence alignment of the residues flanking Tyr795 across different species. Arrowhead points to the tyrosine residues corresponding to the Tyr795 residue in human lipin-1. e PAP activity in LPIN1-KO MDA-MB-231 cells reconstituted with WT-lipin-1 or 3YF-lipin-1. Cells were maintained in a serum-free medium for 4 h, and then treated with or without serum or EGF, followed by immunoprecipitation with anti-Flag. The enzymatic activities of immunoprecipitated Flag-lipin-1 were examined. f Knockdown of SRC does not affect oleic acid (OA)-induced lipin-1 translocation to the endoplasmic reticulum (ER). MDA-MB-231 cells expressing shRNA against SRC or Renilla as a control were maintained in complete medium containing 10% FBS and treated with or without OA for 2 h and homogenised and subjected to ultracentrifugation to collect microsome fractions, followed by immunoblotting. Calnexin, microsomal (Mic) marker. g Schematic diagram of phospholipids synthesised from glycerol-3-phosphate in mammalian cells. h DAG, TAG and phospholipid synthesis rates of LPIN1-KO MDA-MB-231 cells reconstituted with WT-lipin-1 or 3YF-lipin-1. Lipids from cells treated with ³H-labelled OA and EGF were extracted and resolved by thin-layer chromatography (TLC), followed by quantification with scintillation counting. DAG diglyceride, TAG triglyceride, PE phosphatidylethanolamine, PC phosphatidylcholine, PS phosphatidylserine. e, h were quantified in each independent experiment (n = 4). Data are mean ± s.e.m.; ordinary two-way ANOVA, followed by Tukey in e, or by Sidak in h; ***P < 0.001, N.S. not significant. Source data are provided as a Source Data file.