title: NDPK2 as a signal transducer in the phytochrome-mediated light signaling.
abstract: Nucleoside-diphosphate kinase (NDPK) 2 in Arabidopsis has been identified as a phytochrome-interacting protein by using the C-terminal domain of phytochrome A (PhyA) as the bait in yeast two-hybrid screening. The far-red light-absorbing form of phytochrome (Pfr) A stimulates NDPK2 gamma-phosphate exchange activity in vitro. To better understand the multiple functions of NDPK and its role in phytochrome-mediated signaling, we characterized the interaction between phytochrome and NDPK2. Domain studies revealed that PER-ARNT-SIM domain A in the C-terminal domain of phytochrome is the binding site for NDPK2. Additionally, phytochrome recognizes both the NDPK2 C-terminal fragment and the NDPK2 hexameric structure to fulfill its binding. To illustrate the mechanism of how the Pfr form of phytochrome stimulates NDPK2, His-197-surrounding residue mutants were made and tested. Results suggested that the H-bonding with His-197 inside the nucleotide-binding pocket is critical for NDPK2 functioning. The pH dependence profiles of NDPK2 indicated that mutants with different activities from the wild type have different pK(a) values of His-197 and that NDPK2 hyperactive mutants possess lower pK(a) values. Because a lower pK(a) value of His-197 accelerates NDPK2 autophosphorylation and the phospho-transfer between the phosphorylated NDPK2 and its kinase substrate, we concluded that the Pfr form of phytochrome stimulates NDPK2 by lowering the pK(a) value of His-197.
authors.affiliation: Department of Chemistry, University of Nebraska, Lincoln, NE 68588-0304, USA.
authors.forename: Yu
authors.initials: Y
authors.lastname: Shen
authors.forename: Jeong-Il
authors.initials: JI
authors.lastname: Kim
authors.forename: Pill-Soon
authors.initials: PS
authors.lastname: Song
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D017360:Arabidopsis / Q000201:enzymology / Q000378:metabolism* / Q000528:radiation effects*; D001665:Binding Sites; D019281:Dimerization; D004795:Enzyme Stability; D006153:Guanosine Diphosphate / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008027:Light; D009154:Mutation / Q000235:genetics; D009701:Nucleoside-Diphosphate Kinase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010710:Phosphates / Q000378:metabolism; D010766:Phosphorylation; D010834:Phytochrome / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D015398:Signal Transduction / Q000528:radiation effects*
chemical_list: D010710:Phosphates; D010834:Phytochrome; D006153:Guanosine Diphosphate; D000255:Adenosine Triphosphate; C121988:nucleoside diphosphate kinase 2; D009701:Nucleoside-Diphosphate Kinase