Figure 2.

PRMT1 mediates arginine methylation of ASK1 in intact cells. (a) In vitro methylation of GST-ASK1(1–136) or its Arg-to-Lys mutants was examined in the presence of GST-PRMT1 and [³H]SAM. Reaction mixtures were subjected to SDS-PAGE, and ³H-labeled proteins were visualized by fluorography. The gel was also stained with Coomassie brilliant blue. (b) 293T cells were transfected for 48 h with expression vectors encoding ASK1–Myc, ASK1(R78K/R80K)–Myc, and Flag-PRMT1, as indicated. Cell lysates were then subjected to immunoprecipitation with anti-Myc antibody, and the resulting precipitates were examined by immunoblot with antibody to Arg⁷⁸-methylated ASK1 (α-Me-R78-ASK1). Cell lysates were also examined directly by immunoblot analysis with antibodies to Myc or to Flag. (c) 293T cells were transfected for 48 h with expression vectors encoding ASK1–Myc, Flag-PRMT1, and Flag-PRMT1(G80R), as indicated. The cells were then incubated with AdOx (10 μM) or dimethyl sulfoxide (DMSO, 0.1%) vehicle for 12 h, lysed, and subjected to immunoprecipitation with anti-Myc antibody. The resulting precipitates and cell lysates were subjected to immunoblot analysis with the indicated antibodies. (d and e) MCF7 cells were transfected for 48 h with control (GFP) or PRMT1 siRNAs (d) or were treated for 12 h with AdOx (10 μM) or DMSO (0.1%) (e). Cell lysates were then subjected to immunoprecipitation with antibody to Arg⁷⁸-methylated ASK1, and the resulting precipitates were subjected to immunoblot analysis with anti-ASK1 antibody. Cell lysates were also examined directly by immunoblot analysis with antibodies to ASK1 or to PRMT1. (f) HeLa cells were transfected for 48 h with an expression vector encoding ASK1–Myc or ASK1(R78K/R80K)–Myc without or with a vector for Flag-PRMT1. The cells were then fixed and immunostained with antibodies to Myc and to Arg⁷⁸-methylated ASK1. Nuclei were visualized by staining with 4′,6′-diamidino-2-phenylindole dihydrochloride (DAPI). Scale bar, 20 μm