Glycine N-methyltransferase (GNMT) is an abundant cytosolic enzyme that catalyses the methylation of glycine into sarcosine, coupled with conversion of the methyl donor, S-adenosylmethionine (AdoMet), into S-adenosylhomocysteine (AdoHcy). GNMT is believed to play a role in monitoring the AdoMet/AdoHcy ratio, and hence the cellular methylation capacity, but regulation of the enzyme itself is not well understood. In the present study, treatment of isolated rat hepatocytes with the protein phosphatase inhibitor okadaic acid, was found to induce an overphosphorylation of GNMT, as shown by proteomic analysis. The analysis comprised two-dimensional gel electrophoretic separation of 32P-labelled phosphoproteins and identification of individual protein spots by matrix-assisted laser-desorption ionization–time-of-flight mass spectrometry. The identity of GNMT was verified by N-terminal Edman sequencing of tryptic peptides. Chromatographic separation of proteolytic peptides and 32P-labelled amino acids suggested that GNMT was phosphorylated within a limited region, and only at serine residues. GNMT phosphorylation could be suppressed by naringin, an okadaic acid-antagonistic flavonoid. To assess the possible functional role of GNMT phosphorylation, the effect of okadaic acid on hepatocytic AdoMet and AdoHcy levels was examined, using HPLC separation for metabolite analysis. Surprisingly, okadaic acid was found to have no effect on the basal levels of AdoMet or AdoHcy. An accelerated AdoMet–AdoHcy flux, induced by the addition of methionine (1 mM), was likewise unaffected by okadaic acid. 5-Aminoimidazole-4-carboxamide riboside, an activator of the hepatocytic AMP-activated protein kinase, similarly induced GNMT phosphorylation without affecting AdoMet and AdoHcy levels. Activation of cAMP-dependent protein kinase by dibutyryl-cAMP, reported to cause GNMT phosphorylation under cell-free conditions, also had little effect on hepatocytic AdoMet and AdoHcy levels. Phosphorylation of GNMT would thus seem to play no role in regulation of the intracellular AdoMet/AdoHcy ratio, but could be involved in other GNMT functions, such as the binding of folates or aromatic hydrocarbons.
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July 2003
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Research Article|
July 15 2003
Okadaic acid-induced, naringin-sensitive phosphorylation of glycine N-methyltransferase in isolated rat hepatocytes
Michael T. N. MØLLER;
Michael T. N. MØLLER
1
∗Proteomics and Mammalian Cell Biology Section, Department of Cell Biology, Institute for Cancer Research, The Norwegian Radium Hospital, Montebello, N-0310 Oslo, Norway
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Hamid R. SAMARI;
Hamid R. SAMARI
1
∗Proteomics and Mammalian Cell Biology Section, Department of Cell Biology, Institute for Cancer Research, The Norwegian Radium Hospital, Montebello, N-0310 Oslo, Norway
2To whom correspondence should be addressed (e-mail per.seglen@labmed.uio.no).
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Monica FENGSRUD;
Monica FENGSRUD
∗Proteomics and Mammalian Cell Biology Section, Department of Cell Biology, Institute for Cancer Research, The Norwegian Radium Hospital, Montebello, N-0310 Oslo, Norway
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Per E. STRØMHAUG;
Per E. STRØMHAUG
∗Proteomics and Mammalian Cell Biology Section, Department of Cell Biology, Institute for Cancer Research, The Norwegian Radium Hospital, Montebello, N-0310 Oslo, Norway
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Anne C. ØSTVOLD;
Anne C. ØSTVOLD
†Neurochemical Laboratory, University of Oslo, N-0317 Oslo, Norway
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Per O. SEGLEN
Per O. SEGLEN
2
∗Proteomics and Mammalian Cell Biology Section, Department of Cell Biology, Institute for Cancer Research, The Norwegian Radium Hospital, Montebello, N-0310 Oslo, Norway
2To whom correspondence should be addressed (e-mail per.seglen@labmed.uio.no).
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Publisher: Portland Press Ltd
Received:
April 03 2003
Revision Received:
April 15 2003
Accepted:
April 16 2003
Accepted Manuscript online:
April 16 2003
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London ©2003
2003
Biochem J (2003) 373 (2): 505–513.
Article history
Received:
April 03 2003
Revision Received:
April 15 2003
Accepted:
April 16 2003
Accepted Manuscript online:
April 16 2003
Citation
Michael T. N. MØLLER, Hamid R. SAMARI, Monica FENGSRUD, Per E. STRØMHAUG, Anne C. ØSTVOLD, Per O. SEGLEN; Okadaic acid-induced, naringin-sensitive phosphorylation of glycine N-methyltransferase in isolated rat hepatocytes. Biochem J 15 July 2003; 373 (2): 505–513. doi: https://doi.org/10.1042/bj20030502
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