Luka Zigmund, Ham Amy-Joan L, Norris Jeremy L, Yeo Eui-Ju, Yermalitsky Valery, Glenn Byron, Caprioli Richard M, Liebler Daniel C, Wagner Conrad
Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA.
Protein Sci. 2006 Apr;15(4):785-94. doi: 10.1110/ps.051906706. Epub 2006 Mar 7.
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.
先前的研究表明，大鼠甘氨酸N-甲基转移酶（GNMT）在体内会发生磷酸化，并且在体外可在丝氨酸残基上被磷酸化，酶活性显著增加，但未鉴定出磷酸化位点。在这项工作中，报告了大鼠GNMT特异性磷酸化位点的鉴定。分析了三种不同的大鼠GNMT制剂：（1）通过标准蛋白质纯化方法从肝脏中纯化得到；（2）通过免疫沉淀从分离的肝细胞和肝脏组织中制备得到；（3）在大肠杆菌中表达的重组蛋白。我们使用电喷雾质谱法测量了蛋白质异构体的分子量，并对胰蛋白酶和糜蛋白酶消化产生的肽段进行了液相色谱-串联质谱法（LC-MS/MS）分析。我们还进行了磷酸氨基酸的化学分析和蛋白质测序。在所有样品中，均发现了磷酸化的丝氨酸残基71、182和241。在从肝脏组织和肝细胞制备的GNMT中，发现另外一个S9残基被磷酸化。在肝细胞和重组GNMT中检测到S139。丝氨酸9在体外也被鉴定为环磷酸腺苷（cAMP）依赖性蛋白激酶的作用靶点。这些磷酸化残基在GNMT三级结构中的位置表明它们可能对酶的构象和活性产生影响。
