PRMT1-mediated R972/973 methylation regulates leukemia cell maintenance. Analysis of cell proliferation (A) and apoptosis (B) in MOCK-, FLT3-WT-, or FLT3-R972/973K-transduced BaF3 cells with FL stimulation. (C) Western blot analysis of FLT3 and indicated pFLT3 in BaF3-expressing indicated construct upon FL stimulation. (D) After incubation with a purified protein of GST-GRB2 SH2 domain, biotinylated peptides corresponding to FLT3 aa963-977 (non-modified) or containing pY969 or pY969-R972/973me2a were pulled down by streptavidin beads. SH2 domain/FLT3 peptide association, as revealed by immunoblotting with anti-GST antibodies (n = 3). Molecular mass markers (kD) are shown at left. (E) The binding pose of the dimethylated peptide from molecular dynamics simulation is shown on the SH2 domain protein surface. Peptide is shown in licorice mode (aa969-aa974). Hydrogen atoms are omitted for clarity. Protein surface is shown in white with residues within 5 Å of peptide shown in residue type color code: negative surface charge, red; positive surface charge, blue; nonpolar surface, cyan. (F) Western blotting of indicated total and phospho-proteins in FL-stimulated BaF3 cells expressing FLT3-WT, FLT3-Y969F, or FLT3-R972/973K. (G) Survival of NSG mice engrafted with indicated KOCL45 cells. (H) Western blotting of indicated total and phospho-proteins in SEM cells transduced with either shCtrl or shGBR10. Error bars represent standard error of the mean. *P < .05; **P < .01; ***P < .001; ****P < .0001.