The Variable Domain of a Plant Calcium-dependent Protein Kinase (CDPK) Confers Subcellular Localization and Substrate Recognition for NADPH Oxidase
The Variable Domain of a Plant Calcium-dependent Protein Kinase (CDPK) Confers Subcellular Localization and Substrate Recognition for NADPH Oxidase
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DOI：
10.1074/jbc.m112.448910
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发表时间：
2013-05-17
影响因子：
4.8
通讯作者：
Yoshioka, Hirofumi
中科院分区：
文献类型：
--
作者：
Asai, Shuta;Ichikawa, Tatsushi;Yoshioka, Hirofumi
钙依赖性蛋白激酶（CDPKs）是植物和蜜蜂复合体中调节多种生物学过程的Ca 2+感受器。然而，CDPKs如何在体内区分特异性底物仍然是未知的。以前，我们发现马铃薯StCDPK 5主要定位于质膜，并通过N-末端区域的直接磷酸化激活质膜NADPH氧化酶（RBOH;呼吸爆发氧化酶同系物）StRBOHB。在这里，我们报告的贡献，StCDPK 5 N-末端可变（V）结构域激活StRBOHB在体内使用异源表达系统在本塞姆氏烟草。V结构域中的N-末端肉豆蔻酰化和棕榈酰化位点的突变消除了主要的质膜定位和StCDPK 5在体内激活StRBOHB的能力。番茄SlCDPK 2，它也包含肉豆蔻酰化和棕榈酰化位点在其N末端，磷酸化StRBOHB在体外，但不是在体内。通过交换StCDPK 5和SlCDPK 2之间每个结构域的区域来鉴定负责StRBOHB活化和磷酸化的功能结构域。用SlCDPK 2的V结构域取代StCDPK 5的V结构域废除了StRBOHB在体内的激活和磷酸化能力，并将嵌合CDPK重新定位到反式高尔基体网络，如对SlCDPK 2所观察到的。相反地，用StCDPK 5的V结构域取代的SlCDPK 2定位于质膜并激活StRBOHB。这些结果表明，V域赋予底物特异性在体内通过支配适当的亚细胞定位的CDPKs。
Calcium-dependent protein kinases (CDPKs) are Ca2+ sensors that regulate diverse biological processes in plants and api-complexans. However, how CDPKs discriminate specific substrates in vivo is still largely unknown. Previously, we found that a potato StCDPK5 is dominantly localized to the plasma membrane and activates the plasma membrane NADPH oxidase (RBOH; for respiratory burst oxidase homolog) StRBOHB by direct phosphorylation of the N-terminal region. Here, we report the contribution of the StCDPK5 N-terminal variable (V) domain to activation of StRBOHB in vivo using heterologous expression system in Nicotiana benthamiana. Mutations of N-terminal myristoylation and palmitoylation sites in the V domain eliminated the predominantly plasma membrane localization and the capacity of StCDPK5 to activate StRBOHB in vivo. A tomato SlCDPK2, which also contains myristoylation and palmitoylation sites in its N terminus, phosphorylated StRBOHB in vitro but not in vivo. Functional domains responsible for activation and phosphorylation of StRBOHB were identified by swapping regions for each domain between StCDPK5 and SlCDPK2. The substitution of the V domain of StCDPK5 with that of SlCDPK2 abolished the activation and phosphorylation abilities of StRBOHB in vivo and relocalized the chimeric CDPK to the trans-Golgi network, as observed for SlCDPK2. Conversely, SlCDPK2 substituted with the V domain of StCDPK5 localized to the plasma membrane and activated StRBOHB. These results suggest that the V domains confer substrate specificity in vivo by dictating proper subcellular localization of CDPKs.