Figure 7.

Phosphorylation of GST-METTL1 inhibits its tRNA methylase activity. Assays were carried out in triplicate and error bars represent the standard error of the mean. (A) GST-METTL1 (3 μM) was phosphorylated in the standard assay buffer for the times indicated with 10 mM MgCl₂–0.1 mM [γ-³²P]ATP (1000 cpm/pmol) and 0.4 U/ml (about 0.01 μM) PKBα. The stoichiometry of phosphorylation was calculated from the ³²P radioactivity incorporated (determined after precipitation with trichloroacetic acid), the molecular mass of GST-METT1 and the amount of protein in the assay (estimated by the method of Bradford using BSA as a standard) after correction for the purity of GST-METTL1 determined by densitometric analysis of the Coomassie blue-stained gel. (B) GST-METTL1 was phosphorylated as in panel A, except that unlabelled Mg-ATP replaced Mg-[γ-³²P]ATP. At each time point, an aliquot was removed and METTL1 (80 nM) assayed for tRNA methylase activity as in Figure 6B. (C) GST-METTL1 was phosphorylated for 60 min as in panel B, in the presence (+) or absence (−) of PKB. Glutathione-Sepharose (5 μl) was added to each 0.05 ml reaction mix and left for 45 min at 4°C. After brief centrifugation, the supernatant was discarded and the pellet washed twice with 1 ml of 50 mM Tris–HCl, 0.1 mM EGTA, 0.03% (w/v) Brij 35, 0.1% (v/v) 2-mercaptoethanol pH 7.5 and 1 mM MnCl₂. The glutathione-Sepharose pellet was then incubated with 0.05 ml of the same buffer containing 20 mM glutathione to elute the GST-METTL1. After brief centrifugation, the supernatant was removed and incubated for 30 min at 30°C in the presence (+) or absence (−) of 50 U/ml PP1γ (where 1 U is the amount that catalyses the dephosphorylation of 1 nmol of phosphorylase a in 1 min). The PP1γ itself had been incubated previously for 10 min in the presence (+) or absence (−) of its inhibitor microcystin LR (MC-LR). Aliquots were then assayed for tRNA methylase activity (uppermost panel) or electrophoresed and immunoblotted with an antibody that recognises METTL1 phosphorylated at Ser27 (middle panel) and with an antibody that recognises all forms of METTL1 (lowest panel). (D) Purified GST-METTL1, GST-METTL1[S27A], GST-METTL1[S27D] and GST-METTL1[S27E], each at 80 nM, were assayed for 15 min as in Figure 6.