PRMT1 catalyzes FLT3-ITD protein methylation at R972/973. (A) Schematic model showing dimethylated (Di-Me) arginines identified following immunoprecipitation of endogenous FLT3 protein from MV4-11 cells. The precipitates were subjected to proteomic analysis. (B) FLT3 protein was immunoprecipitated from 293T cells ectopically expressing FLT3-ITD or FLT3-ITD methylation-deficient mutants (R655K, R845K, R972K, and R973K) and then analyzed for ADMA levels by western blotting. (C) Dot blot showing that the FLT3-R972/973 me2a antibody specifically binds asymmetric dimethylated R972/973 peptide. Amino acid sequence of peptides corresponding to the FLT3 966-980 region, in which R972 and R973 are unmodified, monomethylated, or dimethylated. Different amounts of peptides were spotted on polyvinylidene difluoride membranes and detected by control anti-FLT3 or anti-FLT3 asymmetric methylated Arg 972/973 (R972/973 me2a) antibodies. (D) 293T cells transfected with mock, WT FLT3-ITD, or FLT3-ITD methylation-deficient mutants (R655K, R845K, R972K and R973K) were analyzed by western blot using the FLT3 R972/973 me2a antibody. (E) In vitro methylation assay of GST-tagged FLT3 peptides (WT, R972K, R973K, and R972/973K) in the presence of PRMT1 protein and S-adenosyl-l-[methyl-3H] methionine (SAM). Peptide methylation was analyzed by western blotting using the methylation antibody. (F) 293T cells were transduced with lentiviral vector expressing FLT3-ITD and then transfected with mock or the indicated GFP-fused type I PRMTs. FLT3-ITD protein was immunoprecipitated from cells and analyzed for GFP by western blotting. MV4-11 cells transduced with ShCtrl, ShPRMT1 (G), or ShPRMT6 (H) were analyzed by western blotting for PRMT1 or PRMT6, FLT3 R972/973 me2a, total FLT3, and GAPDH. CT, C terminus; ECD, extracellular domain; JM juxtamembrane domain; KI, kinase insert; TK1, tyrosine kinase 1; TK2, tyrosine kinase 2; TM, transmembrane.