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. 2018 Aug 21;293(39):15290–15303. doi: 10.1074/jbc.RA118.004512

Figure 3.

Figure 3.

CARM1 methylates PKM2 at Arg-445 and Arg-447 sites. A, PKM2 is methylated by CARM1. In vitro methylation assays were performed by incubating GST-tagged recombinant PRMTs (PRMT1, PRMT3, CARM1, Myc-PRMT5, and PRMT6) with purified His-tagged PKM2 proteins. A, B, and E, arrows indicate PKM2 methylation, and solid dots indicate PRMT automethylation. Membranes were stained with Ponceau S to ensure equal protein loading. B, recombinant human PKM1 (hPKM1) and PKM2 (hPKM2), mouse PKM2 (mPKM2), and tetramerization-deficient mutant mPKM2 (R399E) were subjected to in vitro methylation assays with CARM1. C, LC-MS/MS was performed to identify the arginine methylation sites of in vitro methylated His–mPKM2. Arginines 445 and 447 were found to be both mono- and dimethylated. D, PKM2 arginine methylation sites, Arg-445 and Arg-447, share sequence consensus with several known CARM1 substrates, including poly(A)-binding protein 1 (PABP1), histone H3R17, and cAMP-response element-binding protein-binding protein (CBP). E, in vitro methylation assays were performed using WT and R445K/R447K mutant PKM2, confirming that Arg-445 and Arg-447 are the major sites of methylation by CARM1.