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

Article14 April 2005free access The tRNA methylase METTL1 is phosphorylated and inactivated by PKB and RSK in vitro and in cells Robert A Cartlidge Robert A Cartlidge MRC Protein Phosphorylation Unit, School of Life Sciences, MSI/WTB complex, University of Dundee, Dundee, UK Search for more papers by this author Axel Knebel Axel Knebel MRC Protein Phosphorylation Unit, School of Life Sciences, MSI/WTB complex, University of Dundee, Dundee, UK Search for more papers by this author Mark Peggie Mark Peggie Division of Signal Transduction Therapy, School of Life Sciences, MSI/WTB complex, University of Dundee, Dundee, UK Search for more papers by this author Andrei Alexandrov Andrei Alexandrov Department of Biochemistry and Biophysics, University of Rochester School of Medicine, Rochester, NY, USA Search for more papers by this author Eric M Phizicky Eric M Phizicky Department of Biochemistry and Biophysics, University of Rochester School of Medicine, Rochester, NY, USA Search for more papers by this author Philip Cohen Corresponding Author Philip Cohen MRC Protein Phosphorylation Unit, School of Life Sciences, MSI/WTB complex, University of Dundee, Dundee, UK Division of Signal Transduction Therapy, School of Life Sciences, MSI/WTB complex, University of Dundee, Dundee, UK Search for more papers by this author Robert A Cartlidge Robert A Cartlidge MRC Protein Phosphorylation Unit, School of Life Sciences, MSI/WTB complex, University of Dundee, Dundee, UK Search for more papers by this author Axel Knebel Axel Knebel MRC Protein Phosphorylation Unit, School of Life Sciences, MSI/WTB complex, University of Dundee, Dundee, UK Search for more papers by this author Mark Peggie Mark Peggie Division of Signal Transduction Therapy, School of Life Sciences, MSI/WTB complex, University of Dundee, Dundee, UK Search for more papers by this author Andrei Alexandrov Andrei Alexandrov Department of Biochemistry and Biophysics, University of Rochester School of Medicine, Rochester, NY, USA Search for more papers by this author Eric M Phizicky Eric M Phizicky Department of Biochemistry and Biophysics, University of Rochester School of Medicine, Rochester, NY, USA Search for more papers by this author Philip Cohen Corresponding Author Philip Cohen MRC Protein Phosphorylation Unit, School of Life Sciences, MSI/WTB complex, University of Dundee, Dundee, UK Division of Signal Transduction Therapy, School of Life Sciences, MSI/WTB complex, University of Dundee, Dundee, UK Search for more papers by this author Author Information Robert A Cartlidge1, Axel Knebel1, Mark Peggie2, Andrei Alexandrov3, Eric M Phizicky3 and Philip Cohen 1,2 1MRC Protein Phosphorylation Unit, School of Life Sciences, MSI/WTB complex, University of Dundee, Dundee, UK 2Division of Signal Transduction Therapy, School of Life Sciences, MSI/WTB complex, University of Dundee, Dundee, UK 3Department of Biochemistry and Biophysics, University of Rochester School of Medicine, Rochester, NY, USA *Corresponding author. MRC Protein Phosphorylation Unit, School of Life Sciences, MSI/WTB complex, University of Dundee, Dow Street, Dundee DD1 5EH, UK. Tel.: +44 1382 344238; Fax: 44 1382 223778; E-mail: [email protected] The EMBO Journal (2005)24:1696-1705https://doi.org/10.1038/sj.emboj.7600648 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info A substrate for protein kinase B (PKB)α 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. Introduction Protein kinase B (PKB, also called Akt) plays a central role in mediating the intracellular actions of signals that trigger the activation of class 1 phosphatidyinositol (PtdIns) 3-kinases and elevate the intracellular level of PtdIns (3,4,5) trisphosphate. The PtdIns (3,4,5) trisphosphate generated in response to such signals binds to the pleckstrin homology (PH) domains of PKB and its activator 3-phosphoinositide-dependent protein kinase-1 (PDK1), colocalising them at the plasma membrane and allowing PDK1 to activate PKB. PKB then performs multiple functions by phosphorylating many intracellular proteins. In this way, it mediates many of the metabolic actions of insulin, as well as regulating the growth, proliferation and survival of cells, including tumour cells (reviewed by Mora et al, 2004). For these reasons, the identification of its substrates is of great importance for our understanding of both diabetes and cancer. The discovery that PKB phosphorylates serine and threonine residues that lie in Arg-Xaa-Arg-Xaa-Xaa-Ser/Thr- motifs (where Xaa is any amino acid) (Alessi et al, 1996) has facilitated the identification of potential substrates for PKB. However, such motifs can only be phosphorylated in vivo if they lie on accessible regions of proteins, while other residues in the vicinity of the phosphorylation site may be negative or positive specificity determinants. Moreover, other members of the AGC subfamily of protein kinases, such as SGK (Kobayashi and Cohen, 1999), RSK and S6K isoforms (Alessi et al, 1996), also phosphorylate the same consensus motif as PKB. Indeed, some substrates phosphorylated by PKB, such as glycogen synthase kinase 3 (GSK3), are also phosphorylated by RSK in vivo (Shaw and Cohen, 1999). We have developed a method for finding new physiological substrates of protein kinases, termed KESTREL (Knebel et al, 2001). This method is especially useful for identifying proteins that are phosphorylated by one particular protein kinase, but not by closely related protein kinases with similar specificity requirements. For example, we have exploited KESTREL to identify elongation factor 2-kinase as a protein that is phosphorylated at Ser359 and inactivated by p38δ MAP kinase, but not by the closely related p38α, p38β and p38γ (Knebel et al, 2001). More recently, we used the same method to identify filamin gamma as a physiological substrate for PKBα and not SGK1 (Murray et al, 2004b) and NDRG isoforms as physiological substrates for SGK1 and not PKBα (Murray et al, 2004a). In this paper, we have detected a further protein that is phosphorylated much more efficiently by PKBα than SGK1 and identify it as methyltransferase-like protein-1 (METTL1) (Bahr et al, 1999), an enzyme that catalyses the 7-methylguanosine modification at position 46 of tRNAPhe in vitro (Alexandrov et al, 2002). We have also found that phosphorylation inactivates METTL1. To our knowledge, the present study is the first to suggest that any tRNA modification is regulated by insulin and growth factors. Results PKB phosphorylates a 36 kDa protein in HeLa cell extracts Desalted HeLa cell extract was fractionated from 0–5% polyethylene glycol 6000 and the 5% supernatant chromatographed on Source Q. Aliquots of each fraction collected were phosphorylated for 4 min with MgATP in the absence or presence of PKBα or SGK1. A protein of apparent molecular mass 36 kDa was detected, eluting at about 0.2 M NaCl, that was phosphorylated by PKBα, but very poorly by SGK1 (Figure 1). This protein was purified further by successive chromatographies on HiTrap-heparin-Sepharose and Mono S. At this stage, the 32P radioactivity comigrated with the major protein-staining band in the preparation, which was excised from the gel and identified by tryptic mass fingerprinting (see Supplementary Table 1) as methyltransferase-like protein-1 (METTL1), the catalytic subunit of an enzyme that catalyses the m7G46 modification of tRNAPhe (Alexandrov et al, 2002). To investigate whether the PKBα substrate and METTL1 were the same protein, the purified material was maximally phosphorylated (Figure 2), digested with trypsin and chromatographed to reveal two phosphopeptides T1 and T2 (Figure 2A). Peptide T2 was examined by mass spectrometry, which revealed it to be a mixture of two peptides comprising residues 25–35 and 23–35 of METTL1, each containing one phosphate group, the latter arising from incomplete tryptic digestion of the Arg–Ala bond between residues 24 and 25. A similar analysis of T1 showed that it also comprised residues 25–35 of METTL1 plus one phosphate, but in which the methionine residue of the peptide had become oxidised. Edman and solid-phase sequencing of T1 and T2 confirmed the identity of each peptide and established that there was a single site of phosphorylation at Ser27 (Figure 2B). This experiment also showed that the 36 kDa substrate for PKBα was indeed METTL1. The identification of Ser27 as the site of phosphorylation was anticipated, as it is the only residue in METTL1 that lies in the canonical consensus sequence for phosphorylation by PKB, Arg-Xaa-Arg-Xaa-Xaa-Ser/Thr- (where Xaa is any amino acid). The amino-acid sequence and domain structure of METTL1 are shown in Figure 3. 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. Download figure Download PowerPoint 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. Download figure Download PowerPoint 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). Download figure Download PowerPoint Phosphorylation of METTL1 in cells To examine whether METTL1 became phosphorylated in cells, we generated antibodies capable of immunoprecipitating the human protein and a phospho-specific antibody that recognised METTL1 only when phosphorylated at Ser27 (see Supplementary data). To investigate whether METTL1 was a physiological substrate for PKBα, we stimulated human embryonic kidney (HEK) 293 cells with insulin-like growth factor-1 (IGF-1), a potent activator of this protein kinase, which at 20 ng/ml does not activate extracellular signal-regulated protein kinases 1 and 2 (ERK1/ERK2) of the classical MAP kinase cascade in these cells. METTL1 was not phosphorylated significantly in unstimulated cells, but became phosphorylated at Ser27 after stimulation by IGF-1. Phosphorylation was maximal after about 15 min, a time at which the activation of PKB was also maximal (data not shown). The phosphorylation of METTL1, like the phosphorylation of PKB itself, was prevented by the PtdIns 3-kinase inhibitor wortmannin (Figure 4A) but not by PD 184352, a specific inhibitor of MKK1, the activator of ERK1/ERK2 (Seebolt-Leopold et al, 1999; Davies et al, 2000) (Figure 4C), consistent with METTL1 being phosphorylated by PKB in cells. PD 184352 did not alter the IGF-1-induced phosphorylation of PKB at Thr308 significantly when normalised to the total level of expression of PKB in either this (Figure 4A) or several other similar experiments (data not shown). 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. Download figure Download PowerPoint PKBα and SGK1 are not the only protein kinases that phosphorylate Arg-Xaa-Arg-Xaa-Xaa-Ser- motifs. Other members of the AGC subfamily of protein kinases, such as isoforms of p90 ribosomal protein S6 kinase (RSK) and p70 ribosomal S6 kinase (S6K), also phosphorylate this motif preferentially (Alessi et al, 1996). Indeed, RSK2 can phosphorylate METTL1 at Ser27 in vitro (Figure 4B). To investigate whether RSK phosphorylated METTL1 in cells, we exposed 293 cells to the tumour-promoting phorbol ester phorbol-12-myristate 13-acetate (PMA), which does not activate PKB in these cells but is a potent activator of the classical MAP kinase cascade, and hence the activation of RSK (Figures 4A, C and D). METTL1 became maximally phosphorylated at Ser27 30 min after stimulation with PMA, a time at which the activation of the classical MAP kinase cascade was also maximal, as judged by the phosphorylation of ERK1 and ERK2. The PMA-induced phosphorylation of both ERK1/ERK2 and METTL1 were prevented by PD 184352 (Figure 4C), but not by wortmannin (Figure 4A), consistent with phosphorylation of METTL1 being catalysed by one or more RSK isoforms. The activation of S6K isoforms requires the protein kinase mTOR (mammalian target of rapamycin), which is potently and specifically inhibited by rapamycin. The activation of mTOR itself requires phosphorylation of the TSC2 component of the tubersclerosis complex, which can be catalysed by either PKB or RSK (Roux et al, 2004). We found that rapamycin prevented the phosphorylation/activation of S6K1 by either IGF-1 or PMA, as expected, but had no significant effect on the phosphorylation of METTL1 or the activation of PKB induced by either agonist in this (Figure 4D) and several other experiments (data not shown). This excluded the involvement of S6K isoforms in the phosphorylation of METTL1 under these conditions. Further evidence that the IGF-1-induced phosphorylation of METTL1 at Ser27 is catalysed by PKB To obtain further evidence that the IGF-1-mediated phosphorylation of Ser27 is mediated by PKB, and not by another protein kinase that lies 'downstream' of PtdIns 3-kinase, we made use of embryonic stem (ES) cells that do not express PDK1 (Williams et al, 2000) or that express the PDK1[L155E] mutant instead of the wild-type enzyme (Collins et al, 2003). This mutation disrupts a hydrophobic pocket in PDK1 required for its interaction with, and activation of, every known PDK1 substrate except PKB (Mora et al, 2004). Thus the PDK1[L155E] mutant can activate PKB normally, but cannot activate substrates such as S6K, SGK and atypical PKCs (Collins et al, 2003). IGF-1 does not activate PKB or induce the phosphorylation of METTL1 at Ser27 in ES cells that do not express PDK1, but activates PKB and induces METTL1 Ser27 phosphorylation similarly in ES cells expressing wild-type PDK1 or PDK1[L155E] (Figure 5). These results indicate the IGF-1-mediated phosphorylation of METTL1 is indeed catalysed by PKB. 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. Download figure Download PowerPoint Phosphorylation of Ser27 inactivates METTL1 To examine the effect of METTL1 phosphorylation on its activity, we expressed and purified human METTL1 as a GST fusion protein in 293 cells (see Materials and methods). This preparation was active as a tRNA methylase and activity was not altered by cleavage of the GST tag with PreScission protease (Figure 6A). For this reason, GST-METTL1 was used in subsequent experiments. As a control, GST was expressed and purified, and this preparation was devoid of tRNA methylase activity (Figure 6A). At a concentration of 80 nM GST-METTL1, tRNA methylation was approximately linear for 15 min (Figure 6B), and so the effect of phosphorylation on activity was studied under these conditions. Figure 6.Human METTL1 is a tRNAPhe 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-tRNAPhe 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. Download figure Download PowerPoint The phosphorylation of METTL1 at Ser27 was found to inactivate its tRNA methylase activity, inhibition correlating well with the extent of phosphorylation (Figure 7A and B). The inactive phosphorylated enzyme could be reactivated by dephosphorylation in vitro catalysed by protein phosphatase 1 (PP1) and reactivation was prevented by microcystin LR, a specific inhibitor of PP1 (Figure 7C). 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. Download figure Download PowerPoint The METTL1[S27A] mutant, which had similar activity to the wild-type enzyme, was not phosphorylated at all by PKBα or RSK2 (data not shown), confirming that Ser27 was the only site of phosphorylation. Conversely, the mutation of Ser27 to Asp or Glu to mimic the effect of phosphorylation greatly decreased activity (Figure 7D). Expression of METTL1 in the presence of WDR4 complements a yeast trm8 growth phenotype in vivo Saccharomyces cerevisiae express a homologue of METTL1 termed tRNA modifier 8 (trm8) complexed to another protein trm82, which is essential for the stability and function of trm8 (Alexandrov et al, 2005). Human METTL1 can replace trm8 in catalysing the m7G46 modification in yeast and this requires WDR4, the human homologue of trm82 (Alexandrov et al, 2002). We recently showed that yeast trm8 and trm82 mutants have a temperature-sensitive growth defect in minimal media containing glycerol, and that complementation of this phenotype was correlated with m7G methyltransferase activity (Alexandrov et al, 2005). As we also showed that expression of METTL1 and WDR4 in yeast lacking Trm8 and Trm82 yielded extracts with m7G methyltransferase activity (Alexandrov et al, 2002), we speculated that we might be able to demonstrate complementation of yeast mutants with METTL1/WDR4. As shown in Figure 8A, expression of METTL1/WDR4 under PGAL control complements the temperature-sensitive growth defect of a yeast strain lacking trm8 and containing an additional deletion in trm4 to enhance the phenotype (A Alexandrov and EM Phizicky, unpublished work). In row g of Figure 8A, expression of yeast Trm8p efficiently complements the growth defect at both 33°C (panel II) and 37°C (panel III) in media containing galactose, in which Trm8p is expressed, but not in media containing dextrose (panel V), in which Trm8p is not expressed. In row a, coexpression of wild-type METTL1 and WDR4 also complemented the growth defect at 33 and 37°C, albeit about 10-fold less effectively than Trm8p (row a, panels II and III). 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 (PGAL 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. Download figure Download PowerPoint Expression of METTL1[S27A], but not METTL1[S27D] or METTL1[S27E], complements a yeast trm8-Δ mutant in vivo To begin to address the possibility that phosphorylation of METTL1 at Ser27 inhibits its function in vivo, we mutated this site to Ala, Asp or Glu, and examined complementation of yeast trm8-Δ mutants after expression in yeast. In Figure 8A (row b), expression of METTL1[S27A] complemented the growth phenotype (panel II), similarly to wild-type METTL1 protein (row a). In contrast, the S27D and S27E mutants completely failed to complement (rows c and d). Similar expression of METTL1 variants was confirmed by immunoblot analysis (Figure 8B) demonstrating that expression of each METTL1 variant was similar (lanes a–d). Lack of effect of METTL1 phosphorylation on its interaction with WDR4 We cloned the human homologue of Trm82 (Alexandrov et al, 2002), termed WD-repeat protein 4 (WDR4) (Michaud et al, 2000), and investigated whether it could interact with METTL1 in cells. These experiments demonstrated that the two proteins do indeed form a complex and that interaction is not disrupted by the IGF-1-stimulated phosphorylation of METTL1 at Ser27 or by the mutation of Ser27 to Asp or Glu (see Supplementary data and Supplementary Figure 2). The PKBα-catalysed phosphorylation of the METTL1–WDR4 complex inhibited the tRNA methylase activity similarly to METTL1. PKBα did not phosphorylate the WDR4 component, consistent with its lack of an Arg-Xaa-Arg-Xaa-Xaa-Ser/Thr- motif (data not shown). Ser27 of METTL1 lies within a canonical mode I consensus sequence for 14-3-3 binding (Rittinger et al, 1999). However, we have been unable to detect significant binding of 14-3-3 to Ser27-phosphorylated METTL1 in vitro, or in cell extracts (R Cartlidge and C MacKintosh, unpublished results). This is consistent with the inability of IGF-1 to trigger the nuclear exit of METTL1. METTL1 and WDR4 were nuclear in control or IGF-1-stimulated cells (see Supplementary Figure 3). Discussion We have identified METTL1 as a protein that is phosphorylated rapidly and stoichiometrically at Ser27 by PKB (Figure 7A), and established that METTL1 becomes phosphorylated at Ser27 in cells in response to agonists that activate PtdIns 3-kinase or the classical MAP kinase cascade. The finding that IGF-1-induced Ser27 phosphorylation is prevented by the PI 3-kinase inhibitors wortmannin (Figure 4A) and LY 294002 (data not shown), but not by PD 184352 or rapamycin, and that it occurs in cells expressing the PDK1[L155E] mutant, but not in PDK1-deficient mouse ES cells (Figure 5), demons