Characterization of the kinase activity of a WNK4 protein complex
Characterization of the kinase activity of a WNK4 protein complex
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DOI：
10.1152/ajprenal.00358.2009
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发表时间：
2009-09-01
影响因子：
4.2
通讯作者：
Yu, Alan S. L.
中科院分区：
文献类型：
--
作者：
Ahlstrom, Robert;Yu, Alan S. L.
Ahlstrom R，Yu As.WNK4蛋白复合体的激酶活性表征。Am J Physiol Renal Physiol 297：F685-F692,2009。2009年7月8日首次出版；doi：10.1152/ajprenal.00358.2009。WNK4蛋白激酶突变导致假性低醛固酮增多症II型(PHAII)，这是一种遗传性疾病，其特征是肾脏的氯化钠和K+滞留导致高血压和高钾血症。与此一致的是，WNK4被认为可以调节几种肾小管转运蛋白，包括氯化钠共转运体NCC和K+通道ROMK，但其机制还不完全清楚，而且该激酶活性在其作用中的作用存在很大争议。为了检测WNK4的活性，我们现在已经成功地从HEK293细胞中表达并纯化了具有酶活性的全长WNK4蛋白。我们发现野生型全长WNK4在体外可磷酸化氧化应激反应蛋白1(OSR1)和Ste20/SPS1相关的富含脯氨酸/丙氨酸的蛋白激酶(SPAK)。将与PHAII相关的突变E559K、D561A和Q562E引入我们的蛋白质中，对这种磷酸化没有显著影响。我们得出结论，PHAII不太可能是由WNK4激酶活性异常引起的。我们还做了一个有趣的观察，WNK4激酶结构域的失活突变并不能完全消除OSR1/Spak的体外磷酸化。在此基础上，我们发现了一种新的40 kDa的蛋白激酶，它与WNK4的COOH末端部分特异结合，能够同时磷酸化WNK4和Spak/OSR1。我们认为这个40 kDa的激酶在WNK4信号转导通路中发挥作用，并可能介导WNK4的一些生理作用。
Ahlstrom R, Yu AS. Characterization of the kinase activity of a WNK4 protein complex. Am J Physiol Renal Physiol 297: F685-F692, 2009. First published July 8, 2009; doi:10.1152/ajprenal.00358.2009. Mutations in WNK4 protein kinase cause pseudohypoaldosteronism type II (PHAII), a genetic disorder that is characterized by renal NaCl and K+ retention leading to hypertension and hyperkalemia. Consistent with this, WNK4 is known to regulate several renal tubule transporters, including the NaCl cotransporter, NCC, and the K+ channel, ROMK, but the mechanisms are incompletely understood, and the role of the kinase activity in its actions is highly controversial. To assay WNK4 kinase activity, we have now succeeded in expressing and purifying full-length, enzymatically active WNK4 protein from HEK293 cells. We show that full-length wild-type WNK4 phosphorylates oxidative stress response kinase 1 (OSR1) and Ste20/SPS1-related proline/alanine-rich kinase (SPAK) in vitro. Introducing the PHAII-associated mutations, E559K, D561A, and Q562E, into our protein had no significant effect on this phosphorylation. We conclude that PHAII is unlikely to be caused by abnormal WNK4 kinase activity. We also made the intriguing observation that inactivating mutations of the WNK4 kinase domain did not completely abolish in vitro phosphorylation of OSR1/SPAK. Led by this, we identified a novel 40-kDa kinase that associates specifically with the COOH-terminal half of WNK4 and is able to phosphorylate both WNK4 and SPAK/OSR1. We suggest that this 40-kDa kinase functions in the WNK4 signal transduction pathway and may mediate some of the physiological actions attributed to WNK4.