Impaired degradation of WNK1 and WNK4 kinases causes PHAII in mutant KLHL3 knock-in mice
Impaired degradation of WNK1 and WNK4 kinases causes PHAII in mutant KLHL3 knock-in mice
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DOI：
10.1093/hmg/ddu217
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发表时间：
2014-10-01
影响因子：
3.5
通讯作者：
Uchida, Shinichi
中科院分区：
文献类型：
--
作者：
Susa, Koichiro;Sohara, Eisei;Uchida, Shinichi
假性低醛固酮增多症（PHAII）是一种以盐敏感性高血压、高钾血症和代谢性酸中毒为特征的遗传性疾病，编码-no-赖氨酸激酶1 （WNK1）和WNK4激酶的基因是已知的致病基因。最近，KLHL3-Cullin3 E3连接酶的组分Kelch-like 3 （KLHL3）和Cullin3被新发现与PHAII有关。我们已经报道了WNK4是klhl3 - cullin3e3连接酶介导的泛素化的底物。然而，其他组也报道了WNK1和Isla CI共转运体（NCC）是klhl3 - cullin3e3连接酶的底物。因此，目前尚不清楚哪个分子是KLHL3的靶标。为了研究KLHL3突变引起PHAII的发病机制，我们构建并分析了KLHL3R528111+敲入小鼠。KLHL3（R52811/+）敲入小鼠表现为盐敏感性高血压、高钾血症和代谢性酸中毒。此外，KLHL3（R52811/+）小鼠肾脏中NCC的磷酸化水平升高，表明KLHL3R52811/敲入小鼠是一种理想的小鼠PHAII模型。有趣的是，在KLHL3（R52811/+）小鼠肾脏中，WNK1和WNK4的蛋白表达均显著增加，证实了这些WNK激酶的增加激活了KLHL3（R528111+）敲入小鼠的WNK- osr1 /SPAK-NCC磷酸化级联反应。为了研究突变体KLHL3 R528H是否可以与WNK激酶相互作用，我们使用荧光相关光谱法测量了tamra标记的WNK1和WNK4肽与全长KLHL3的结合，发现WNK1和WNK4都没有与突变体KLHL3 R528H结合。因此，我们发现WNK1和WNK4激酶的蛋白表达水平升高，由于KLHL3- cullin3介导的泛素化受损，KLHL3 R528H突变导致PHAII。
Pseudohypoaldosteronism type ll (PHAII) is a hereditary disease characterized by salt-sensitive hypertension, hyperkalemia and metabolic acidosis, and genes encoding with-no-lysine kinase 1 (WNK1) and WNK4 kinases are known to be responsible. Recently, Kelch-like 3 (KLHL3) and Cullin3, components of KLHL3-Cullin3 E3 ligase, were newly identified as responsible for PHAII. We have reported that WNK4 is the substrate of KLHL3-Cullin3 E3 ligase-mediated ubiquitination. However, WNK1 and Isla CI cotransporter (NCC) were also reported to be a substrate of KLHL3-Cullin3 E3 ligase by other groups. Therefore, it remains unclear which molecule is the target(s) of KLHL3. To investigate the pathogenesis of PHAII caused by KLHL3 mutation, we generated and analyzed KLHL3R528111+ knock-in mice. KLHL3(R52811/+) knock-in mice exhibited salt-sensitive hypertension, hyperkalemia and metabolic acidosis. Moreover, the phosphorylation of NCC was increased in the KLHL3(R52811/+) mouse kidney, indicating that the KLHL3R52811/ knock-in mouse is an ideal mouse model of PHAII. Interestingly, the protein expression of both WNK1 and WNK4 was significantly increased in the KLHL3(R52811/+) mouse kidney, confirming that increases in these WNK kinases activated the WNK-OSR1/SPAK-NCC phosphorylation cascade in KLHL3(R528111+) knock-in mice. To examine whether mutant KLHL3 R528H can interact with WNK kinases, we measured the binding of TAMRA-labeled WN K1 and WN K4 peptides to full-length KLHL3 using fluorescence correlation spectroscopy, and found that neither WNK1 nor WNK4 bound to mutant KLHL3 R528H. Thus, we found that increased protein expression levels of WNK1 and WNK4 kinases cause PHAII by KLHL3 R528H mutation due to impaired KLHL3-Cullin3-mediated ubiquitination.