Identification of features regulating OST1 kinase activity and OST1 function in guard cells
Identification of features regulating OST1 kinase activity and OST1 function in guard cells
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DOI：
10.1104/pp.106.079327
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发表时间：
2006-08-01
期刊：
影响因子：
7.4
通讯作者：
Thomine, Sebastien
中科院分区：
文献类型：
--
作者：
Belin, Christophe;de Franco, Pierre-Olivier;Thomine, Sebastien
植物激素脱落酸(ABA)介导植物的干旱反应，特别是触发气孔关闭。一些植物的Snf1-Related Kins2(SnRK2)蛋白参与了ABA信号通路。在拟南芥保卫细胞中，开放气孔1(OST1)/SRK2E/SnRK2-6是ABA信号转导的关键正调控因子。因此，更好地了解SnRK2蛋白激酶激活的机制是了解ABA信号转导的主要目标。在这里，我们报道了在大肠杆菌中产生的OST1的成功纯化：该蛋白是有活性的和自磷酸化的。利用质谱仪，我们在重组OST1中鉴定了5个自磷酸化的靶残基。序列分析表明，在OST1的催化结构域之后，有两个保守的盒：SnRK2特异性盒(谷氨酰胺-303到脯氨酸-318)和ABA特异性盒(亮氨酸-333到蛋氨酸-362)。定点突变和序列缺失表明，激活环和SnRK2特异盒中的丝氨酸(Ser)-175对重组OST1激酶的活性至关重要。在保卫细胞中靶向表达OST1激酶变异体发现了对OST1在ABA信号中的功能至关重要的其他特征，尽管对OST1激酶活性不是必需的：Ser-7、Ser-18、Ser-29和ABA特异性盒。Ser-7、Ser-18、Ser-29和Ser-43可能是调节磷酸化的靶标，ABA特异的box可能是保卫细胞中信号伙伴结合的靶标。
The phytohormone abscisic acid (ABA) mediates drought responses in plants and, in particular, triggers stomatal closure. Snf1-related kinase 2 (SnRK2) proteins from several plant species have been implicated in ABA-signaling pathways. In Arabidopsis ( Arabidopsis thaliana) guard cells, OPEN STOMATA 1 (OST1)/SRK2E/SnRK2-6 is a critical positive regulator of ABA signal transduction. A better understanding of the mechanisms responsible for SnRK2 protein kinase activation is thus a major goal toward understanding ABA signal transduction. Here, we report successful purification of OST1 produced in Escherichia coli: The protein is active and autophosphorylates. Using mass spectrometry, we identified five target residues of autophosphorylation in recombinant OST1. Sequence analysis delineates two conserved boxes located in the carboxy-terminal moiety of OST1 after the catalytic domain: the SnRK2-specific box (glutamine-303 to proline-318) and the ABA-specific box (leucine-333 to methionine-362). Site-directed mutagenesis and serial deletions reveal that serine (Ser)-175 in the activation loop and the SnRK2-specific box are critical for the activity of recombinant OST1 kinase. Targeted expression of variants of OST1 kinase in guard cells uncovered additional features that are critical for OST1 function in ABA signaling, although not required for OST1 kinase activity: Ser-7, Ser-18, and Ser-29 and the ABA-specific box. Ser-7, Ser-18, Ser-29, and Ser-43 represent putative targets for regulatory phosphorylation and the ABA-specific box may be a target for the binding of signaling partners in guard cells.