FIGURE 4.

SMYD2 methylates TRAF2 on Lys 115 and Lys 194. (A) SMYD2 methylated TRAF2. SMYD2 knockout 293T cells were transfected with TRAF2, either with or without SMYD2 and stimulated with TNF‐α, TRAF2 immunoprecipitate was subjected to western blot analysis with the methylation‐specific antibody. (B) In vitro methyltransferase assays. Recombinant TRAF2 protein was incubated with the concentration gradient of recombinant SMYD2 and S‐Adenosyl methionine (SAM) in a mixture of methylase buffer for 1 h at 30°C. After denaturing, samples were separated by SDS‐PAGE. (C) LLY‐507 reduced the methylation level of the substrate TRAF2. (D) The methylation degree induced by SMYD2 was elevated with increased substrate TRAF2 concentrations. After the methylation reaction using recombinant substrate TRAF2 and SMYD2, the concentration of AdoHcy generated by SAHN and SRHH was detected. (E) Identification of monomethylated lysine sites in TRAF2 at the K115 and K194 respectively by mass spectrometry. (F) TNF‐α stimulated the methylation of TRAF2 point mutants or two mutations at the K115 and K194. (G) Co‐immunoprecipitation (Co‐IP) assay of SMYD2 and the double sites mutated TRAF2 with indicated antibodies. (H) The dot‐blot assay showed that both 115K and 194K could be methylated by SMYD2 in vitro in concentration‐dependent manner