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

MRC Protein Phosphorylation Unit, School of Life Sciences, MSI/WTB complex, University of Dundee, Dundee, UK.
The EMBO Journal (Impact Factor: 10.43). 06/2005; 24(9):1696-705. DOI: 10.1038/sj.emboj.7600648
Source: PubMed


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.

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    • "Bacterial genes have also been experimentally identified and named trmB (classical name, yggH) from Escherichia coli (8), Bacillus subtilus (9) and Aquifex aeolicus (10). There is a clear structural difference between eukaryotic and bacterial tRNA (m7G46) methyltransferases: the eukaryotic enzyme is a heterodimer (7,11,12), while the bacterial enzyme is a monomer (8) or homodimer (9). Recently, crystal structures of the eukaryotic (13) and bacterial (9) enzymes have been reported. "
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    ABSTRACT: N(7)-methylguanine at position 46 (m(7)G46) in tRNA is produced by tRNA (m(7)G46) methyltransferase (TrmB). To clarify the role of this modification, we made a trmB gene disruptant (DeltatrmB) of Thermus thermophilus, an extreme thermophilic eubacterium. The absence of TrmB activity in cell extract from the DeltatrmB strain and the lack of the m(7)G46 modification in tRNA(Phe) were confirmed by enzyme assay, nucleoside analysis and RNA sequencing. When the DeltatrmB strain was cultured at high temperatures, several modified nucleotides in tRNA were hypo-modified in addition to the lack of the m(7)G46 modification. Assays with tRNA modification enzymes revealed hypo-modifications of Gm18 and m(1)G37, suggesting that the m(7)G46 positively affects their formations. Although the lack of the m(7)G46 modification and the hypo-modifications do not affect the Phe charging activity of tRNA(Phe), they cause a decrease in melting temperature of class I tRNA and degradation of tRNA(Phe) and tRNA(Ile). (35)S-Met incorporation into proteins revealed that protein synthesis in DeltatrmB cells is depressed above 70 degrees C. At 80 degrees C, the DeltatrmB strain exhibits a severe growth defect. Thus, the m(7)G46 modification is required for cell viability at high temperatures via a tRNA modification network, in which the m(7)G46 modification supports introduction of other modifications.
    Full-text · Article · Nov 2009 · Nucleic Acids Research
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    • "Homodimerization has been proposed to be an initial step toward hetero-oligomerization, but in the case of tRNA m7G methylation, the hypothetical bacterial dimer is totally unrelated to the Trm8-Trm82 heterodimer, which raises interesting questions as to the reason for evolving a multisubunit structure in eukaryotes. METTL1, the human homolog of Trm8, was shown to be phosphorylated on Ser27 (corresponding to Ser59 in Trm8) by the PKB and RSK kinases, and results in inactivation of the enzyme (Cartlidge et al., 2005). Ser59 was also shown to be phosphorylated in yeast (Li et al., 2007). "
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    ABSTRACT: Loss of N7-methylguanosine (m7G) modification is involved in the recently discovered rapid tRNA degradation pathway. In yeast, this modification is catalyzed by the heterodimeric complex composed of a catalytic subunit Trm8 and a noncatalytic subunit Trm82. We have solved the crystal structure of Trm8 alone and in complex with Trm82. Trm8 undergoes subtle conformational changes upon Trm82 binding which explains the requirement of Trm82 for activity. Cocrystallization with the S-adenosyl-methionine methyl donor defines the putative catalytic site and a guanine binding pocket. Small-angle X-ray scattering in solution of the Trm8-Trm82 heterodimer in complex with tRNA(Phe) has enabled us to propose a low-resolution structure of the ternary complex which defines the tRNA binding mode of Trm8-Trm82 and the structural elements contributing to specificity.
    Full-text · Article · Feb 2008 · Structure
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    • "Moreover, recently, we have reported that a gene involved in m 7 G modification of tRNA is required for infection by the phytopathogenic fungus Colletotrichum lagenarium [19]. Although eukaryote tRNA (m 7 G46) methyltransferases contain two protein subunits (Trm8 and Trm82 in yeast [15]; METTL1 and WDR4 in human [15] [20]), the enzymes from eubacteria are composed of only TrmB protein [16] [17] [21]. "
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    ABSTRACT: Yeast tRNA (m(7)G46) methyltransferase contains two protein subunits (Trm8 and Trm82). To address the RNA recognition mechanism of the Trm8-Trm82 complex, we investigated methyl acceptance activities of eight truncated yeast tRNA(Phe) transcripts. Both the D-stem and T-stem structures were required for efficient methyl-transfer. To clarify the role of the D-stem structure, we tested four mutant transcripts, in which tertiary base pairs were disrupted. The tertiary base pairs were important but not essential for the methyl-transfer to yeast tRNA(Phe) transcript, suggesting that these base pairs support the induced fit of the G46 base into the catalytic pocket.
    Full-text · Article · May 2007 · FEBS Letters
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