Identification of phosphorylation sites in glycine N-methyltransferase from rat liver.
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.
Luka
Zigmund
Z
Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA.
Ham
Amy-Joan L
AJ
Norris
Jeremy L
JL
Yeo
Eui-Ju
EJ
Yermalitsky
Valery
V
Glenn
Byron
B
Caprioli
Richard M
RM
Liebler
Daniel C
DC
Wagner
Conrad
C
D000595:Amino Acid Sequence; D000818:Animals; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D050938:Glycine N-Methyltransferase / Q000737:chemistry* / Q000378:metabolism; D022781:Hepatocytes / Q000378:metabolism; D008099:Liver / Q000166:cytology / Q000378:metabolism*; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010766:Phosphorylation; D020033:Protein Isoforms / Q000737:chemistry / Q000378:metabolism; D017433:Protein Structure, Secondary; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012694:Serine / Q000737:chemistry / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization
D020033:Protein Isoforms; D011994:Recombinant Proteins; D012694:Serine; D050938:Glycine N-Methyltransferase; D017868:Cyclic AMP-Dependent Protein Kinases