Fig. 2: Y324 phosphorylation of PRMT5 suppresses its enzymatic activity. | Communications Biology

Fig. 2: Y324 phosphorylation of PRMT5 suppresses its enzymatic activity.

From: PRMT5 promotes DNA repair through methylation of 53BP1 and is regulated by Src-mediated phosphorylation

Fig. 2

a Analysis of AdoMet interaction with active site of PRMT5. AdoMet interacts with intact PRMT5 through five hydrogen bonds including Y324 (left, yellow cloud). Upon phosphorylation of the Y324 (pTYR324) site, two hydrogen bonds are lost (right, purple cloud). b In vitro PRMT5 methyltransferase assay. Immunoprecipitated myc-PRMT5 from HEK293T cells were incubated with MBP and3[H]-AdoMet. Methylated samples were analyzed by SDS-PAGE and autoradiography. Amounts of myc-PRMT5 and MBP were evaluated by western blotting and Coomassie Brilliant Blue (CBB) staining, respectively. c Interaction of PRMT5 with MEP50. U2OS cells were co-transfected with myc-PRMT5 (WT or Y324F) and vSrc plasmids. Co-immunoprecipitated endogenous MEP50 is shown. d, e Analysis of PRMT5 enzymatic kinetics. Enzyme activities of myc-PRMT5 proteins (WT, Y324F, Y324A, or GRAA (G367A-R367A, enzymatic dead-from)) were measured by in vitro methyltransferase assays with increasing concentrations of 3[H]-AdoMet. Amount of myc-PRMT5 protein was evaluated by Coomassie Blue staining. Radioactivity of the 3[H]-labeled MBP protein was analyzed by autoradiography (d) and then quantitated by Liquid scintillation counter (e). Error bars represent standard deviation (SD, n = 3). P-value of WT vs Y324F: 0.015, WT vs Y324A: 0.0013, WT vs GRAA: <0.001. f UV-crosslinking assay of PRMT5 with AdoMet. Recombinant GST-PRMT5 (WT or Y324F) was purified from E. coli and then cross-linked with 3 μCi 3[H]-AdoMet using UV crosslinker. 3[H]-DPM count represents the amount of 3[H]-AdoMet cross-linked with GST-PRMT5 (n = 3). g Suppression of PRMT5-targeting histone arginine methylation by Y324 phosphorylation. U2OS cells were transfected vSrc plasmid for 2 days and then nuclear histone fraction was extracted. Equal amount of histone proteins were analyzed by western blotting. The autoradiographs in (b, d), immunoblots in (b, c, g) and CBB in (b, d) are representative of three independent experiments with similar results, respectively. *P < 0.05 and **P < 0.01.

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