The tRNA methylase METTL1 is phosphorylated and inactivated by PKB and RSK in vitro and in cells

Abstract

A substrate for protein kinase B (PKB)alpha in HeLa cell extracts was identified as methyltransferase-like protein-1 (METTL1), the orthologue of trm8, which catalyses the 7-methylguanosine modification of tRNA in Saccharomyces cerevisiae. PKB and ribosomal S6 kinase (RSK) both phosphorylated METTL1 at Ser27 in vitro. Ser27 became phosphorylated when HEK293 cells were stimulated with insulin-like growth factor-1 (IGF-1) and this was prevented by inhibition of phosphatidyinositol 3-kinase. The IGF-1-induced Ser27 phosphorylation did not occur in 3-phosphoinositide-dependent protein kinase-1 (PDK1)-deficient embryonic stem cells, but occurred normally in PDK1[L155E] cells, indicating that the effect of IGF-1 is mediated by PKB. METTL1 also became phosphorylated at Ser27 in response to phorbol-12-myristate 13-acetate and this was prevented by PD 184352 or pharmacological inhibition of RSK. Phosphorylation of METTL1 by PKB or RSK inactivated METTL1 in vitro, as did mutation of Ser27 to Asp or Glu. Expression of METTL1[S27D] or METTL1[S27E] did not rescue the growth phenotype of yeast lacking trm8. In contrast, expression of METTL1 or METTL1[S27A] partially rescued growth. These results demonstrate that METTL1 is inactivated by PKB and RSK in cells, and the potential implications of this finding are discussed.

Figure 1

Detection of a 36 kDa substrate for PKBα in HeLa extracts. HeLa cell extracts were fractionated from 0–5% (w/v) PEG 6000, the 5% supernatant desalted and the material (950 mg protein) chromatographed on a 20 ml column of Source Q (see Materials and methods). Aliquots of each fraction were incubated for 4 min at 30°C as described previously (Knebel et al, 2001) at a further 10-fold dilution in the presence of 50 mM Tris–HCl pH 7.5, 1 mM EGTA, 0.1% (v/v) 2-mercaptoethanol, 10 mM MgCl₂ and 20 nM [γ-³²P]ATP (4 × 10⁶ cpm/pmol) in the presence (+) or absence (−) of PKBα or SGK1, each at 0.4 U/ml (Davies et al, 2000); 1 U is the amount that catalyses the phosphorylation of 1 nmol substrate peptide in 1 min. After SDS–PAGE and autoradiography, a 36 kDa protein eluting at about 0.2 M NaCl was detected that was phosphorylated by PKBα, but very poorly by SGK1. Autophosphorylation of PKB and SGK1 was negligible under the conditions used.

Figure 2

Identification of the residue in METTL1 phosphorylated by PKBα in vitro. (A) Following Mono S purification, METTL1 was phosphorylated with PKBα for 1 h as described in the legend to Figure 1, except that the concentration of [γ-³²P]ATP was increased to 0.1 mM and the specific radioactivity was 1000 cpm/pmol. After SDS–PAGE, the gel was stained with Sypro-orange, the phosphorylated band excised and digested with trypsin, and the peptides separated by reverse-phase hydrophobic interaction chromatography on a Vydac C₁₈ column equilibrated in 0.1% (v/v) trifluoroacetic acid. The column was developed with an acetonitrile gradient in 0.1% (v/v) trifluoroacetic acid (diagonal line) at a flow rate of 0.8 ml/min and fractions of 0.4 ml were collected. The two major peaks of ³²P radioactivity T1 and T2 were subjected to mass spectrometry (Supplementary Table 1). (B) Peptides T1 and T2 were subjected to Edman degradation to confirm their sequence and to solid-phase sequencing to identify the site(s) of phosphorylation (Stokoe et al, 1992). ³²P radioactivity released after each cycle of Edman degradation during solid-phase sequencing was analysed by Cerenkov counting.

Figure 3

The sequence of human METTL1 and the location of its functional domains. The phosphorylation site is at residue 27, a putative SAM-binding motif (GXGXG) between residues 58 and 65, and a motif similar to that found in an rRNA methylase (rRNA met) of C. trachomatis was found between residues 156 and 200 (Bahr et al, 1999).

Figure 4

Effects of IGF1 and PMA on the phosphorylation of endogenous METTL1 in HEK293 cells. (A) Cells were serum starved for 8 h and then incubated for 20 min without (−) or with (+) 100 nM wortmannin before stimulation for a further 15 min with 20 ng/ml IGF-1 or for 30 min with 400 ng/ml PMA. METTL1 was immunoprecipitated from 1.5 mg cell lysate protein, denatured in SDS, subjected to SDS–PAGE and, after transfer to Immobilon-P membranes, immunoblotted with the antibody that recognises METTL1 phosphorylated at Ser27 and with the antibody that recognises phosphorylated and unphosphorylated METTL1. A further 20 μg of lysate protein was subjected to SDS–PAGE and immunoblotted with an antibody that recognises PKB phosphorylated at Thr308, an antibody that recognises phosphorylated and unphosphorylated PKBα equally well, an antibody that recognises ERK1 and ERK2 phosphorylated at the Thr-Glu-Tyr motif (pTEpY) and an antibody that recognises all forms of the ERK1 and ERK2 proteins. (B) Bacterially expressed human GST-METTL1 was left unphosphorylated (no kinase, NK) or maximally phosphorylated with PKBα or RSK2 and 100 ng aliquots spotted onto a nitrocellulose membrane and immunoblotted with the antibody that recognises METTL1 phosphorylated at Ser27 and with the antibody that recognises unphosphorylated and phosphorylated METTL1 equally well. (C) Same as panel A, except that after serum starvation for 8 h, the cells were incubated for 1 h in the absence or presence of 2 μM PD 184352 (instead of wortmannin) prior to stimulation with IGF-1 or PMA. (D) Same as panel A, except that following serum starvation the cells were incubated for 1 h in the absence or presence of 100 nM rapamycin (rather than wortmannin) prior to stimulation with IGF-1 or PMA. In addition, 20 μg cell lysate was immunoblotted with an antibody that recognises S6K1 phosphorylated at Thr389 and with an antibody that recognises phosphorylated and unphosphorylated S6K1 equally well.

Figure 5

METTL1 is phosphorylated in mouse ES cells expressing wild-type PDK1 or PDK1[L155E], but not in PDK1 null cells. Mouse ES cells expressing PDK1[+/+], PDK1[L155E/L155E] or PDK1[−/−] were serum starved for 4 h, incubated for 20 min without (−) or with (+) 100 nM wortmannin, and then stimulated for a further 15 min with 20 ng/ml IGF-1. METTL1 was immunoprecipitated from 2.5 mg cell lysate protein using the antibody raised against mouse METTL1 residues 258–268, denatured in SDS, subjected to SDS–PAGE and, after transfer to Immobilon-P membranes, immunoblotted as in panel A.

Figure 6

Human METTL1 is a tRNA^Phe methylase. Assays were carried out in triplicate and error bars represent the standard error of the mean. Purified GST, GST-METTL1 and METTL1 (each at 80 nM) were incubated for 15 min with [α-³²P]GTP-labelled pre-tRNA^Phe in the presence of 1 mM SAM. The reactions were terminated by phenol extraction of the tRNA and after complete digestion with P1 nuclease, the m⁷G₄₆ modification was detected after separation from unmodified guanosine by thin-layer chromatography (TLC), followed by phosphorimager analysis and quantification of the % conversion to m⁷G₄₆ (see Materials and methods). (B) Same as panel A, except that only GST-METTL1 was used and the assays were performed for the times indicated.

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.

Figure 8

Expression of METTL1[S27A], but not S27D or S27E, complements a yeast trm8-Δ mutant. (A) Complementation of yeast trm8 mutants by METTL1 and Ser27 variants. A trm8-Δ yeast strain AA0636 (Alexandrov et al, 2005), containing a trm4 mutation to enhance its phenotype (see Materials and methods), was transformed with plasmids expressing galactose-inducible (P_GAL controlled) WDR4, METTL1, METTL1 variants, TRM8 or vector controls as indicated (Alexandrov et al, 2002), and tested for growth by serial 10-fold dilution of cultures (starting from 4 × 10³ cells), followed by incubation at the indicated temperatures on synthetic media containing galactose or glucose, as described in Materials and methods. (B) Analysis of expression of METTL1 and Ser27 variants. Strains described in panel A were grown as described, harvested after 5 h induction in media containing galactose, and crude lysates analysed by immunoblotting using the antibody recognising both phosphorylated and unphosphorylated forms of METTL1. Lanes a–d and f contain the METTL1 variants indicated.