title: Significance of Ser-188 in human mitochondrial NAD kinase as determined by phosphomimetic and phosphoresistant amino-acid substitutions.
abstract: 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.
authors.affiliation: Laboratory of Basic and Applied Molecular Biotechnology, Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Uji, Kyoto, Japan.
authors.forename: Yutaka
authors.initials: Y
authors.lastname: Kawabata
authors.affiliation: Faculty of Science and Engineering, Department of Life Science, Setsunan University, Neyagawa, Osaka, Japan.
authors.forename: Kousaku
authors.initials: K
authors.lastname: Murata
authors.affiliation: Laboratory of Basic and Applied Molecular Biotechnology, Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Uji, Kyoto, Japan. Electronic address: kawais@kais.kyoto-u.ac.jp.
authors.forename: Shigeyuki
authors.initials: S
authors.lastname: Kawai
mesh_terms: D019943:Amino Acid Substitution; D001665:Binding Sites; D002384:Catalysis; D004789:Enzyme Activation; D006801:Humans; D024101:Mitochondrial Proteins / Q000737:chemistry* / Q000235:genetics; D009249:NADP / Q000737:chemistry* / Q000235:genetics; D010766:Phosphorylation; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000737:chemistry* / Q000235:genetics; D011485:Protein Binding; D012694:Serine / Q000737:chemistry*; D013329:Structure-Activity Relationship
chemical_list: D024101:Mitochondrial Proteins; D012694:Serine; D009249:NADP; D017853:Phosphotransferases (Alcohol Group Acceptor); C021844:NAD kinase
keywords: Mitochondria; NAD kinase; Phosphomimetic mutation; Phosphoproteomics; Phosphorylation