A Novel Ste20-related Proline/Alanine-rich Kinase (SPAK)-independent Pathway Involving Calcium-binding Protein 39 (Cab39) and Serine Threonine Kinase with No Lysine Member 4 (WNK4) in the Activation of Na-K-Cl Cotransporters
A Novel Ste20-related Proline/Alanine-rich Kinase (SPAK)-independent Pathway Involving Calcium-binding Protein 39 (Cab39) and Serine Threonine Kinase with No Lysine Member 4 (WNK4) in the Activation of Na-K-Cl Cotransporters
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DOI：
10.1074/jbc.m113.540518
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发表时间：
2014-06-20
影响因子：
4.8
通讯作者：
Delpire, Eric
中科院分区：
文献类型：
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作者：
Ponce-Coria, Jose;Markadieu, Nicolas;Delpire, Eric
依赖钠离子的氯离子共转运体(NKCC1、NKCC2和NCC)被磷酸化激活，在多种生理反应中发挥关键作用，包括肾盐平衡、听力、上皮液分泌和容量调节。丝氨酸苏氨酸激酶WNK4(No K=赖氨酸成员4)和Ste20激酶家族的成员，即Spak和OSR1(Ste20相关的丙氨酸/丙氨酸激酶，氧化应激响应激酶)控制着磷酸化。根据目前的理解，WNK4磷酸化Spak/OSR1中的关键残基，导致激酶激活，使Spak/OSR1与NKCC1、NKCC2和NCC结合并磷酸化。最近，钙结合蛋白39(CAB39)被认为是Spak/OSR1活性的结合伙伴和增强子，促进了激酶的自活化和辅转运体的磷酸化。在本研究中，我们提供的证据表明，Cab39与WNK4和Spak/OSR1有不同的相互作用，将经典的两个激酶级联转换为信号激酶转导机制。我们发现WNK4与Cab39联合激活NKCC1的方式不依赖于Spak/OSR1。我们发现WNK4具有一个与Spak/OSR1 C末端CCT/PF2结构域非常相似的结构域，该结构域是Ste20激酶与Na+驱动的氯共转运蛋白之间物理相互作用所必需的。模拟、酵母双杂交和功能数据表明，位于WNK4催化结构域下游的这个类似PF2的结构域促进了该激酶与NKCC1之间的直接相互作用。我们的结论是，除了SPAK和OSR1外，WNK4还能够将自己锚定在NKCC1的N-末端结构域上，并促进共转运蛋白的激活。
Na+-dependent chloride cotransporters (NKCC1, NKCC2, and NCC) are activated by phosphorylation to play critical roles in diverse physiological responses, including renal salt balance, hearing, epithelial fluid secretion, and volume regulation. Serine threonine kinase WNK4 (With No K = lysine member 4) and members of the Ste20 kinase family, namely SPAK and OSR1 (Ste20-related proline/alanine-rich kinase, Oxidative stress-responsive kinase) govern phosphorylation. According to present understanding, WNK4 phosphorylates key residues within SPAK/OSR1 leading to kinase activation, allowing SPAK/OSR1 to bind to and phosphorylate NKCC1, NKCC2, and NCC. Recently, the calcium-binding protein 39 (Cab39) has emerged as a binding partner and enhancer of SPAK/OSR1 activity, facilitating kinase autoactivation and promoting phosphorylation of the cotransporters. In the present study, we provide evidence showing that Cab39 differentially interacts with WNK4 and SPAK/OSR1 to switch the classic two kinase cascade into a signal kinase transduction mechanism. We found that WNK4 in association with Cab39 activates NKCC1 in a SPAK/OSR1-independent manner. We discovered that WNK4 possesses a domain that bears close resemblance to the SPAK/OSR1 C-terminal CCT/PF2 domain, which is required for physical interaction between the Ste20 kinases and the Na+-driven chloride cotransporters. Modeling, yeast two-hybrid, and functional data reveal that this PF2-like domain located downstream of the catalytic domain in WNK4 promotes the direct interaction between the kinase and NKCC1. We conclude that in addition to SPAK and OSR1, WNK4 is able to anchor itself to the N-terminal domain of NKCC1 and to promote cotransporter activation.