Autophosphorylation of Arabidopsis nucleoside diphosphate kinase 2 occurs only on its active histidine residue.
Arabidopsis nucleoside diphosphate kinase 2 (NDPK2) is a component in the phytochrome-mediated light signaling. In the present study, its autophosphorylation was investigated. Acid-stable and alkali-stable phosphorylated residues were analyzed under two different conditions. Results revealed that NDPK2 is phosphorylated only on its active histidine residue His197 and the presence of serine/threonine phosphorylation is an experimental artifact due to the harsh condition applied in the treatment of the phosphorylated protein sample. To resolve the controversy of whether serine/threonine phosphorylation of NDPK occurs as has been suggested by other NDPK studies, NDPK2 putative phosphorylation site mutants were generated and examined. No serine/threonine phosphorylation was identified in NDPK2 or implicated in its enzymatic activity. Further studies indicated that the low enzymatic activity and autophosphorylation level of NDPK2 mutant S199A are shown to be due to a damaged H-bonding with the active histidine residue His197 in the nucleotide-binding pocket. In addition, NDPK2 Kpn loop mutant T182A was found to possess an extremely low enzymatic activity and almost no autophosphorylation, suggesting the importance of the oligomeric states of NDPK2 in NDPK2 functioning.
Shen
Yu
Y
Department of Chemistry, University of Nebraska-Lincoln, Lincoln, Nebraska 68588-0304, USA.
Kim
Jeong-Il
JI
Song
Pill-Soon
PS
D017360:Arabidopsis / Q000201:enzymology* / Q000378:metabolism / Q000528:radiation effects; D004789:Enzyme Activation; D006639:Histidine / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008027:Light; D009154:Mutation; D009701:Nucleoside-Diphosphate Kinase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010710:Phosphates / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation; D010834:Phytochrome / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D012694:Serine / Q000737:chemistry; D015398:Signal Transduction / Q000528:radiation effects; D013912:Threonine / Q000737:chemistry
D010710:Phosphates; D010834:Phytochrome; D013912:Threonine; D012694:Serine; D006639:Histidine; C121988:nucleoside diphosphate kinase 2; D009701:Nucleoside-Diphosphate Kinase