W342F Mutation in CCaMK Enhances Its Affinity to Calmodulin But Compromises Its Role in Supporting Root Nodule Symbiosis in Medicago truncatula.
W342F Mutation in CCaMK Enhances Its Affinity to Calmodulin But Compromises Its Role in Supporting Root Nodule Symbiosis in Medicago truncatula.
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DOI：
10.3389/fpls.2017.01921
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发表时间：
2017
影响因子：
5.6
通讯作者：
Poovaiah BW
中科院分区：
文献类型：
--
作者：
Jauregui E;Du L;Gleason C;Poovaiah BW
钙/钙调素依赖性蛋白激酶（CCaMK）受游离Ca ~（2+）和负载Ca ~（2+）的钙调素调节。这种双重结合被认为参与其调节和相关的生理功能，尽管缺乏直接的实验证据。在这里，我们的文件中的钙调素结合结构域的CCaMK的定点突变改变其结合能力的钙调素，提供了一个有效的方法来研究钙调素如何调节CCaMK的激酶活性和调节根瘤菌共生在蒺藜苜蓿。我们观察到，在位置342的色氨酸突变为苯丙氨酸（W342 F）显着增加的突变体的钙调素结合能力。突变体CCaMK在缺乏钙和钙调素的情况下进行自磷酸化和催化底物磷酸化。当突变体W342 F在ccamk-1根中表达时，转基因根表现出改变的生长表型。这些结果表明，改变钙调素结合结构域的CCaMK可以产生一个组成型激活的激酶，在CCaMK的生理功能的负面作用。
The calcium/calmodulin-dependent protein kinase (CCaMK) is regulated by free Ca2+ and Ca2+-loaded calmodulin. This dual binding is believed to be involved in its regulation and associated physiological functions, although direct experimental evidence for this is lacking. Here we document that site-directed mutations in the calmodulin-binding domain of CCaMK alters its binding capacity to calmodulin, providing an effective approach to study how calmodulin regulates CCaMK in terms of kinase activity and regulation of rhizobial symbiosis in Medicago truncatula. We observed that mutating the tryptophan at position 342 to phenylalanine (W342F) markedly increased the calmodulin-binding capability of the mutant. The mutant CCaMK underwent autophosphorylation and catalyzed substrate phosphorylation in the absence of calcium and calmodulin. When the mutant W342F was expressed in ccamk-1 roots, the transgenic roots exhibited an altered nodulation phenotype. These results indicate that altering the calmodulin-binding domain of CCaMK could generate a constitutively activated kinase with a negative role in the physiological function of CCaMK.
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