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. 2018 Jun 19;32(12):6760–6770. doi: 10.1096/fj.201800244RR

Figure 3.

Figure 3

PRMT5-induced DUSP14 methylation is required for its phosphatase activity. A) Arginine 17, 38, and 45 residues of DUSP14 were methylated in vivo. Flag-tagged DUSP14 wild-type or mutant (3R→K) was transfected into HEK293T cells. Flag-tagged DUSP14 was immunoprecipitated with anti-Flag antibody and then immunoblotted with anti–methyl-arginine antibody. Methylated DUSP14 proteins were fractionated on 9% SDS-PAGE. B) Multiple ladder bands detected in A were posttranslationally modified DUSP14 proteins. Multiple ladder bands of Flag-tagged DUSP14, but not DUSP14 mutant (3R→K), proteins were detected by immunoprecipitation and then immunoblotting, both using anti-Flag antibody. C) Methylation of DUSP14 proteins was enhanced by PRMT5 overexpression. Myc-PRMT5 plus either Flag-DUSP14 wild-type or mutant (3R→K) were cotransfected into HEK293T cells. Flag-tagged DUSP14 was immunoprecipitated with anti-Flag antibody and then immunoblotted with anti–methyl-arginine, anti-PRMT5, or anti-Flag antibody. Methylated DUSP14 proteins were fractionated on 6% SDS-PAGE; the <50 kDa methyl-arginine bands were run off the gel. D) The high-MW ladder bands were due to DUSP14 ubiquitination. Myc-PRMT5 plus either Flag-DUSP14 wild-type or mutant (K103R) were cotransfected into HEK293T cells. Flag-tagged DUSP14 proteins were immunoprecipitated with anti-Flag antibody and then immunoblotted with anti–methyl-arginine, anti-PRMT5, or anti-Flag antibody. Methylated DUSP14 proteins were fractionated on 6% SDS-PAGE; the <50 kDa methyl-arginine bands were run off the gel. E) DUSP14 was methylated by PRMT5 in vitro. Purified Flag-tagged DUSP14 wild-type, Flag-tagged DUSP14 mutant (3R→K), and Myc-tagged PRMT5 proteins were used for in vitro methylation assay. PRMT5-methylated DUSP14 wild-type, but not DUSP14 mutant (3R→K), proteins were detected by immunoblotting using anti–methyl-arginine antibody. F) DUSP14 methylation was essential for its phosphatase activity. Flag-DUSP14 wild-type, mutant C111S, or 3R→K was transfected into HEK293T cells. DUSP14 proteins were immunoprecipitated with anti-Flag antibody, and DUSP14 phosphatase activity was determined by in vitro phosphatase assay. Arrowhead indicates the DUSP14 protein (upper band of the doublet). Data shown are representative of 3 independent experiments.