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Published online before print March 7, 2006, 10.1110/ps.051906706
Protein Science (2006), 15:785-794. Published by Cold Spring Harbor Laboratory Press. Copyright © 2006 The Protein Society
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Identification of phosphorylation sites in glycine N-methyltransferase from rat liver

Zigmund Luka1, Amy-Joan L. Ham1,2, Jeremy L. Norris1,2, Eui-Ju Yeo1,3, Valery Yermalitsky1, Byron Glenn1, Richard M. Caprioli1,2, Daniel C. Liebler1,2 and Conrad Wagner1

1 Department of Biochemistry
2 Mass Spectrometry Research Center, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA

(RECEIVED October 11, 2005; FINAL REVISION January 11, 2006; ACCEPTED January 13, 2006)

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


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