Department of Biomolecular Engineering, Graduate School of Engineering, Tohoku University, Aobayama 6-6-07, Sendai 980-8579, Japan.
Biochem J. 2009 Dec 10;424(3):439-48. doi: 10.1042/BJ20091221.
The Arabidopsis thaliana K+ channel KAT1 has been suggested to have a key role in mediating the aperture of stomata pores on the surface of plant leaves. Although the activity of KAT1 is thought to be regulated by phosphorylation, the endogenous pathway and the primary target site for this modification remained unknown. In the present study, we have demonstrated that the C-terminal region of KAT1 acts as a phosphorylation target for the Arabidopsis calcium-independent ABA (abscisic acid)-activated protein kinase SnRK2.6 (Snf1-related protein kinase 2.6). This was confirmed by LC-MS/MS (liquid chromatography tandem MS) analysis, which showed that Thr306 and Thr308 of KAT1 were modified by phosphorylation. The role of these specific residues was examined by single point mutations and measurement of KAT1 channel activities in Xenopus oocyte and yeast systems. Modification of Thr308 had minimal effect on KAT1 activity. On the other hand, modification of Thr306 reduced the K+ transport uptake activity of KAT1 in both systems, indicating that Thr306 is responsible for the functional regulation of KAT1. These results suggest that negative regulation of KAT1 activity, required for stomatal closure, probably occurs by phosphorylation of KAT1 Thr306 by the stress-activated endogenous SnRK2.6 protein kinase.
拟南芥 K+通道 KAT1 被认为在介导植物叶片表面气孔孔径的开启中起关键作用。尽管 KAT1 的活性被认为受到磷酸化调节，但内源性途径和这种修饰的主要靶位仍然未知。在本研究中，我们已经证明 KAT1 的 C 端区域是拟南芥钙非依赖性 ABA（脱落酸）激活蛋白激酶 SnRK2.6（Snf1 相关蛋白激酶 2.6）的磷酸化靶标。这通过 LC-MS/MS（液相色谱串联质谱）分析得到了证实，该分析表明 KAT1 的 Thr306 和 Thr308 被磷酸化修饰。通过单点突变和在非洲爪蟾卵母细胞和酵母系统中测量 KAT1 通道活性来检验这些特定残基的作用。 Thr308 修饰对 KAT1 活性的影响最小。另一方面，Thr306 的修饰降低了 KAT1 在这两个系统中的 K+转运摄取活性，表明 Thr306 负责 KAT1 的功能调节。这些结果表明，为了关闭气孔，可能通过应激激活的内源性 SnRK2.6 蛋白激酶对 KAT1 Thr306 的磷酸化来负调控 KAT1 活性。
