Figure 2: ROK and MP regulate the methyltransferase activity of PRMT5 through phosphorylation/dephosphorylation at Thr80. | Scientific Reports

Figure 2: ROK and MP regulate the methyltransferase activity of PRMT5 through phosphorylation/dephosphorylation at Thr80.

From: Myosin phosphatase and RhoA-activated kinase modulate arginine methylation by the regulation of protein arginine methyltransferase 5 in hepatocellular carcinoma cells

Figure 2

(A) Autoradiograms of PRMT5 phosphorylated in the absence or in the presence of 0.1 μg/ml protein kinase A (PKA, left panel), 0.1 μg/ml protein kinase C (PKC, middle panel) or 0.4 U/ml Rho-associated kinase (ROK, right panel) with 32P-ATP. (B) Western blot analysis of ROK-phosphorylated PRMT5 using antibody specific for phospho-Thr. After stripping the membrane anti-PRMT5 antibody was applied to detect PRMT5 as an input control. (C) Ion trap collision-induced dissociation (CID) spectra of PRMT5 phosphopeptides. CID of m/z: 656.338 (3+) identified as SDLLLSGRDWNpTLIVGK representing [69–85] of the wild type protein. Thr80 was identified as the modification site (see fragment ion y11 (phosphorylated)). Peptide fragments are labeled according to the nomenclature by Biemann56. (D) Effect of ROK inhibitor (10 μM H1152) on the phosphorylation level of PRMT5 during in vitro ROK assay. Control samples were prepared in the absence of ROK, positive control samples were prepared in the presence of ROK without ROK inhibitor. Relative phosphorylation level of Thr80 was judged by Western blot using anti- pPRMT5T80 antibody and blots for PRMT5 served as loading control. (E) Effect of 25 nM FT-MYPT1 and 5 nM rPP1cδ or their combination on the phosphorylation level of PRMT5 at Thr8080 as judged by Western blot. Data were compared to ROK-phosphorylated PRMT5. (F,G) Amount of MEP50 bound to FT-PRMT5 during ROK-phosphorylation (F) and dephosphorylation by MP (G) compared to unphosphorylated control samples. MEP50 was detected by anti-MEP50 antibody during Western blot and relative amount was normalized to the level of PRMT5. (H,I) In vitro arginine methyltransferase assay of unphosphorylated and ROK-phosphorylated PRMT5 measured by the symmetric dimethylation level of histone H2A Arg3 (H2AR3me2s, F) or histone H4 Arg3 (H4R3me2s, G) in the presence of 25 nM FT-MYPT1, 5 nM rPP1cδ or their combinations. Gels have been processed under the same experimental conditions. Values represents mean ± SEM; **p < 0.01, ***p < 0.001, ****p < 0.0001, #p < 0.05, one-way ANOVA followed by Tukey’s multiple comparison test, n = 3.

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