Kawabata Yutaka, Murata Kousaku, Kawai Shigeyuki
Laboratory of Basic and Applied Molecular Biotechnology, Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Uji, Kyoto, Japan.
Faculty of Science and Engineering, Department of Life Science, Setsunan University, Neyagawa, Osaka, Japan.
Biochem Biophys Res Commun. 2015 Dec 25;468(4):691-5. doi: 10.1016/j.bbrc.2015.11.017. Epub 2015 Nov 12.
Human mitochondrial NAD kinase is a crucial enzyme responsible for the synthesis of mitochondrial NADP(+). Despite its significance, little is known about the regulation of this enzyme in the mitochondria. Several putative and known phosphorylation sites within the protein have been found using phosphoproteomics, and here, we examined the effect of phosphomimetic mutations at six of these sites. The enzymatic activity was downregulated by a substitution of an Asp residue at Ser-289 and Ser-376, but not a substitution of Ala, suggesting that the phosphorylation of these residues downregulates the enzyme. Moreover, the activity was completely inhibited by substituting Ser-188 with an Asp, Glu, or in particular Ala, which highlights two possibilities: first, that Ser-188 is critical for catalytic activity, and second, that phosphorylation of Ser-188 inhibits the activity. Ser-188, Ser-289, and Ser-376 were found to be highly conserved in the primary structures of mitochondrial NAD kinase homologs in higher animals. Moreover, Ser-188 has been frequently detected in human and mouse phosphorylation site studies, whereas Ser-289 and Ser-376 have not. Taken together, this indicates that Ser-188 (and perhaps the other residues) is an important phosphorylation site that can downregulate the NAD kinase activity of this critical enzyme.
人类线粒体NAD激酶是一种负责合成线粒体NADP(+)的关键酶。尽管其具有重要意义，但关于该酶在线粒体内的调控机制却知之甚少。利用磷酸化蛋白质组学已在该蛋白内发现了几个假定的和已知的磷酸化位点，在此，我们研究了其中六个位点的模拟磷酸化突变的影响。将Ser-289和Ser-376处的丝氨酸残基替换为天冬氨酸会下调酶活性，但替换为丙氨酸则不会，这表明这些残基的磷酸化会下调该酶的活性。此外，将Ser-188替换为天冬氨酸、谷氨酸，尤其是丙氨酸会完全抑制酶活性，这凸显了两种可能性：其一，Ser-188对催化活性至关重要；其二，Ser-188的磷酸化会抑制酶活性。已发现Ser-188、Ser-289和Ser-376在高等动物线粒体NAD激酶同源物的一级结构中高度保守。此外，在人和小鼠的磷酸化位点研究中经常检测到Ser-188，而Ser-289和Ser-376则未被检测到。综上所述，这表明Ser-188（或许还有其他残基）是一个重要的磷酸化位点，可下调这种关键酶的NAD激酶活性。
