Shen Yu, Kim Jeong-Il, Song Pill-Soon
Department of Chemistry, University of Nebraska-Lincoln, Lincoln, Nebraska 68588-0304, USA.
Biochemistry. 2006 Feb 14;45(6):1946-9. doi: 10.1021/bi051868a.
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.
拟南芥核苷二磷酸激酶2（NDPK2）是光敏色素介导的光信号传导中的一个组成部分。在本研究中，对其自身磷酸化进行了研究。在两种不同条件下分析了酸稳定和碱稳定的磷酸化残基。结果表明，NDPK2仅在其活性组氨酸残基His197上发生磷酸化，丝氨酸/苏氨酸磷酸化的存在是由于在处理磷酸化蛋白样品时采用的苛刻条件导致的实验假象。为了解决其他NDPK研究中所提出的NDPK是否发生丝氨酸/苏氨酸磷酸化的争议，构建并检测了NDPK2推定磷酸化位点突变体。在NDPK2中未鉴定出丝氨酸/苏氨酸磷酸化，也未涉及其酶活性。进一步的研究表明，NDPK2突变体S199A的低酶活性和自身磷酸化水平是由于核苷酸结合口袋中与活性组氨酸残基His197的氢键受损所致。此外，发现NDPK2 Kpn环突变体T182A具有极低的酶活性且几乎没有自身磷酸化，这表明NDPK2的寡聚状态在NDPK2功能中的重要性。
