Identification of phosphorylation sites in glycine N‐methyltransferase from rat liver

Abstract

Previous studies have shown that rat glycine N-methyltransferase (GNMT) is phosphorylated in vivo, and could be phosphorylated in vitro on serine residues with a significant increase of enzyme activity, but no phosphorylation sites were identified. In this work the identification of the specific phosphorylation sites of rat GNMT is reported. Three different preparations of rat GNMT were analyzed: (1) purified from liver by standard methods of protein purification, (2) prepared from isolated hepatocytes and from liver tissue by immunoprecipitation, and (3) recombinant protein expressed in Escherichia coli. We measured the molecular weights of protein isoforms using electrospray mass spectrometry and used liquid chromatography-tandem mass spectrometry (LC-MS/MS) of peptides resulting from tryptic and chymotryptic digests. We also performed chemical analysis of phosphoamino acids and protein sequencing. In all samples, the phosphorylated serine residues 71, 182, and 241 were found. In GNMT prepared from liver tissue and hepatocytes an S9 additional residue was found to be phosphorylated. In hepatocytes and in recombinant GNMT S139 was detected. Serine 9 was also identified as a target for cAMP-dependent protein kinase in vitro. The positions of these phosphorylated residues in the tertiary structure of GNMT indicate their possible effect on enzyme conformation and activity.Keywords: glycine N-methyltransferase; rat; phosphorylation Glycine N-methyltransferase is a mammalian enzyme, which catalyzes the transfer of a methyl group from Sadenosylmethionine (SAM) to glycine producing S-adenosylhomocysteine (SAH) and N-methylglycine (sarcosine) (Heady and Kerr 1973;Takusagawa et al. 1999). The biological role of this enzyme was proposed as regulating the ratio of SAM/SAH, which has been considered to be an index of the methylation capacity of the cell (Farrar and Clarke 2002). GNMT is also a major liver folate-binding protein (Cook and Wagner 1984), which binds 5-methyltetrahydrofolate pentaglutamate (5-CH 3 -H 4 Pte-Glu 5 ) in vivo and in vitro, and acts as a potent inhibitor of GNMT activity. This serves to link the role of GNMT in the de novo synthesis of methyl groups to the synthesis of preformed methyl groups in the form of methionine (Cook and Wagner 1984;Wagner et al. 1985;Balaghi et al. 1993). It has been pointed out that GNMT is a key enzyme in the degradative conversion of methionine to pyruvate, and as such, can be considered to be involved in gluconeogenesis (Yeo and Wagner 1994;Rowling and Schalinske 2003). The activity of GNMT has been shown to be increased during starvation and diabetes. The activity of cAMP-dependent protein kinase is increased under these conditions (Gorin and Rosenblum 1974;Karasik et al. 1990). Article published online ahead of print. Article and publication date are at

Acetylation of N-terminal valine of glycine N-methyltransferase affects enzyme inhibition by folate

“…Purity of the final protein samples was at least 98% as determined by scanning of the gel after SDS electrophoresis and Coomassie staining. As reported previously, the N-terminal residue in liver GNMT was acetylated valine but in recombinant protein the N-terminal valine was not acetylated [23,25].…”

“…In an earlier study we showed that both purified native liver and recombinant GNMT proteins contain small amounts of phosphorylated serine residues [25]. Only a single species with a mass corresponding to the N-terminal acetylated form was found by LC-MS/MS analysis of the native liver enzyme.…”

“…In the recombinant protein the N-terminal residue is valine as in native liver enzyme but it is not acetylated [25].…”

Glycine N-Methyltransferase and Regulation of S-Adenosylmethionine Levels

“…The initial methionine residue is removed, and GNMT proteins from liver or expressed in E. coli have an N-terminal valine. The N-terminal valines in rat and human GNMTs are acetylated, but when recombinant rat or human GNMT is expressed in E. coli, they are not acetylated (16,17). The only known posttranslational modification of liver GNMT is serine phosphorylation.…”

“…The only known posttranslational modification of liver GNMT is serine phosphorylation. Serines 9, 71, 139, 182, and 241 may be partially phosphorylated in rat liver and recombinant GNMTs, but in the purified rat liver enzyme, the amount of phosphorylation is very low (17). Ser 9 of rat GNMT can be phosphorylated in vitro by the glucagon-activated cAMP-dependent protein kinase (18).…”