FIGURE 3.

Methylation of mammalian eEF2. A, human and rabbit eEF2 is trimethylated on Lys-525. Shown is MS/MS fragmentation of a trypsin-generated human (HEK293) (Hs, top) and rabbit (Oc, bottom) eEF2-derived peptide, supporting Lys-525 as the target of methylation. B, eEF2 is trimethylated on Lys-525 in the rat. Shown is MS/MS fragmentation of AspN-generated trimethylated (top) and unmethylated (bottom) eEF2-derived peptides from the rat brain. C, eEF2 methylation status in a panel of rat tissues. Chromatograms were gated for different methylation states of AspN-generated Lys-525-containing peptides (as in B) from partially purified eEF2 from rat brain, kidney, and spleen. Relative intensities of signals for the different methylation states are indicated in percentages. A.u., arbitrary units. D, eEF2 from a hypomethylated human embryonic kidney cell line is efficiently methylated by both FAM86A and Yjr129c. A protein extract from HEK293 cells that had either been left untreated (HEK293) or treated with 20 μm AdOx to induce hypomethylation (HEK293(AdOx)) was incubated with recombinant FAM86A or Yjr129c in the presence of [³H]AdoMet. The MTase reactions were separated by SDS-PAGE and subjected to fluorography (top) and Ponceau S staining (bottom). E, Lys-525 in human eEF2 is subject to FAM86A- and Yjr129c-mediated trimethylation in vitro. eEF2 was partially purified from extracts from untreated and AdOx-treated cells and then subjected to LC-MS/MS analysis. When indicated, eEF2 had been incubated with recombinant Yjr129c or FAM86A in the presence of AdoMet. Chromatograms were gated for different methylation states of trypsin-generated Lys-525-containing eEF2 peptides (as shown in A). Arrows, peptides of interest; *, unrelated peptides. Relative intensities of signals for the different methylation states are indicated in percentages for the hypomethylated cell line.