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A role of NDPK2 as a signal transducer in the phytochrome -mediated light signaling: NDPK:phytochrome and NDPK:small G -protein interactions
Nucleoside diphosphate kinase 2 (NDPK2) in Arabidopsis has been identified as a phytochrome interacting protein by using the C-terminal domain of phytochrome A as the bait in yeast two-hybrid screening. The Pfr form of phytochrome A stimulates NDPK2 γ-phosphate exchange activity in vitro. To better understand the multiple functions of NDPK and its role in the signal transduction pathway mediated by phytochrome, the interaction between phytochrome and NDPK2 was characterized. Domain studies revealed that the phytochrome C-terminus alone is sufficient to bind and stimulate NDPK2. Furthermore, PAS domain A was determined as the NDPK2 binding site. Additionally, phytochrome recognizes both NDPK2 C-terminal fragment and NDPK2 hexameric structure to fulfill its binding with NDPK2. ^ To illustrate the mechanism of how the Pfr form of phytochrome stimulates NDPK2, His197-surrounding residue mutants were made using site-directed mutagenesis. The results of the γ-phosphate exchange activity studies suggested that the H-bonding with His197 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 p Ka values of His197. The results also indicated that higher activities of NDPK2 mutants correspond to lower pKa values of the active histidine. Since a lower pKa value of His197 accelerates both phosphorylation and phospho-transferring process of NDPK2, the Pfr form of phytochrome stimulates NDPK2 activity by modulating the H-bonding of His197 and lowering its pKa value. ^ In addition, autophosphorylation of NDPK2 was investigated. His197 was determined as the only residue responsible for NDPK2 autophosphorylation. A direct interaction between NDPK2 and small G-proteins in plants was also demonstrated, including Pra2 and Pra3. NDPK2 served as a GTPase activating protein (GAP) to both Pra2 and Pra3 by stimulating their GTPase activities. These results established the relationship between phytochrome-mediated signaling and G-protein-mediated signaling via NDPK2. ^
Shen, Yu, "A role of NDPK2 as a signal transducer in the phytochrome -mediated light signaling: NDPK:phytochrome and NDPK:small G -protein interactions" (2004). ETD collection for University of Nebraska - Lincoln. AAI3147155.