Fig. 6: DGCR8 is phosphorylated by ATM at S677 upon radiation.
a MYC-GFP-, DGCR8-, and Δ692-DGCR8-overexpressing LM2 cells with or without ATM inhibitor (ATMi) Ku55933 pretreatment (10 μM, 1 h) were treated with IR (8 Gy) and cultured for 30 min, followed by pulldown with anti-MYC beads and immunoblotting with antibodies against p-S/TQ and MYC. LE long exposure, SE short exposure. b LM2 and LM2-R cells were treated with IR (8 Gy) and cultured for 30 min, followed by immunoprecipitation with a DGCR8-specific antibody and immunoblotting with antibodies against p-S/TQ and DGCR8. c Consensus ATM phosphorylation site on human DGCR8 (S677) and alignment with the conserved site on Dgcr8 from other species. d Annotated tandem mass spectrometry (MS/MS) spectrum of the peptide encompassing phosphorylated serine 677 (QLASphosQKILQLLHPHVK) of DGCR8. MYC-DGCR8-overexpressing LM2 cells with or without IR (8 Gy followed by 1-h incubation) were lysed, denatured, and subjected to immunoprecipitation with anti-MYC beads, followed by MS analysis. PH indicates phosphorylation. NH3, H2O, and ++ indicate ammonia, water, and double charges, respectively. The graph shows the mass-to-charge ratios (m/z) of the doubly charged precursor peptide ions. The x and y axes represent m/z and relative ion intensity, respectively. e The extracted ion chromatograms for S677phos (peptide QLAS(phos)QKILQLLHPHVK) in non-irradiated and irradiated LM2 cells based on high-performance liquid chromatography (HPLC)-MS/MS analysis. The x and y axes represent the retention time of HPLC/MS analysis and the MS intensity, respectively. The area under the curve is used to indicate the relative abundance of S677phos with or without IR treatment. f HEK293T cells were transfected with DGCR8 (full-length or Δ692), S677A, or S677D mutant, treated with IR (8 Gy), and cultured for 30 min, followed by immunoprecipitation with anti-MYC beads and immunoblotting with antibodies against p-S/TQ and MYC. g In vitro kinase assay. Purified WT DGCR8 and Δ692-DGCR8 or their S677A mutants were incubated with purified wild-type ATM or the kinase-dead mutant in kinase buffer. After the reaction, proteins were resolved by SDS-PAGE and subjected to immunoblotting with the indicated antibodies. Purified MBP-p53 was used as a positive control for ATM kinase activity. h, i HEK293T (h) and LM2 (i) cells were transfected with MYC-tagged WT DGCR8 or the S677A mutant, treated with IR (8 Gy), and cultured for 30 min, followed by immunoprecipitation with anti-MYC beads and immunoblotting with antibodies against p-DGCR8 (S677), DGCR8, and MYC.
