Ahlstrom Robert, Yu Alan S L
Department of Physiology and Biophysics, University of Southern California Keck School of Medicine, Los Angeles, California 90089, USA.
Am J Physiol Renal Physiol. 2009 Sep;297(3):F685-92. doi: 10.1152/ajprenal.00358.2009. Epub 2009 Jul 8.
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.
WNK4蛋白激酶的突变会导致II型假性醛固酮减少症（PHAII），这是一种遗传性疾病，其特征是肾脏对NaCl和K⁺的潴留，导致高血压和高钾血症。与此一致的是，已知WNK4可调节几种肾小管转运蛋白，包括NaCl共转运蛋白NCC和K⁺通道ROMK，但其机制尚未完全了解，并且激酶活性在其作用中的作用存在高度争议。为了检测WNK4激酶活性，我们现在已成功从HEK293细胞中表达并纯化了全长、具有酶活性的WNK4蛋白。我们表明，全长野生型WNK4在体外可磷酸化氧化应激反应激酶1（OSR1）和Ste20/SPS1相关的富含脯氨酸/丙氨酸的激酶（SPAK）。将与PHAII相关的突变E559K、D561A和Q562E引入我们的蛋白中，对这种磷酸化没有显著影响。我们得出结论，PHAII不太可能由WNK4激酶活性异常引起。我们还进行了有趣的观察，即WNK4激酶结构域的失活突变并未完全消除OSR1/SPAK在体外的磷酸化。由此，我们鉴定出一种新的40 kDa激酶，它与WNK4的COOH末端特异性结合，并且能够磷酸化WNK4和SPAK/OSR1。我们认为这种40 kDa激酶在WNK4信号转导途径中起作用，并且可能介导一些归因于WNK4的生理作用。
