The CUL3-KLHL3 E3 ligase complex mutated in Gordon's hypertension syndrome interacts with and ubiquitylates WNK isoforms: disease-causing mutations in KLHL3 and WNK4 disrupt interaction.
The CUL3-KLHL3 E3 ligase complex mutated in Gordon's hypertension syndrome interacts with and ubiquitylates WNK isoforms: disease-causing mutations in KLHL3 and WNK4 disrupt interaction.
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DOI：
10.1042/bj20121903
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发表时间：
2013-04-01
期刊：
影响因子：
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通讯作者：
Kurz T
中科院分区：
文献类型：
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作者：
Ohta A;Schumacher FR;Mehellou Y;Johnson C;Knebel A;Macartney TJ;Wood NT;Alessi DR;Kurz T
WNK（不含赖氨酸激酶）-SPAK（SPS 1相关脯氨酸/富含丙氨酸的激酶）/OSR 1（氧化应激反应激酶1）信号通路通过调节离子共转运蛋白的活性在控制哺乳动物血压方面发挥着重要作用。肾脏。最近的研究已经确定戈登高血压综合征患者具有CUL 3（Cullin-3）或BTB蛋白KLHL 3（Kelch样3）的突变。CUL 3与BTB蛋白组装形成Cullin-RING E3泛素连接酶复合物。为了探索CUL 3-KLHL 3复合物如何运作，我们免疫沉淀KLHL 3，发现它与WNK亚型和CUL 3强烈相关，但与途径的其他组分[SPAK/OSR 1或NCC（Na+/Cl−共转运蛋白）/NKCC 1（Na+/K+/2Cl−共转运蛋白1）]无关。引人注目的是，分析的15种主要KLHL 3疾病突变中有13种抑制了与WNK 1或CUL 3的结合。重组野生型CUL 3-KLHL 3 E3连接酶复合物，但不是致病CUL 3-KLHL 3 [R528 H]突变体复合物，在体外泛素化WNK 1。此外，siRNA（小干扰RNA）介导的CUL 3敲低增加HeLa细胞中WNK 1蛋白水平和激酶活性。我们将WNK 1中的KLHL 3相互作用位点定位到非催化区（残基479-667）。有趣的是，WNK 4中的等同区域包含戈登综合征患者中突变的残基。令人惊讶的是，我们发现戈登氏病引起的WNK 4 [E562 K]和WNK 4 [Q565 E]突变，以及WNK 1 [479-667]片段中的等同突变，消除了与KLHL 3相互作用的能力。这些结果表明，CUL 3-KLHL 3 E3连接酶复合物通过其与WNK同种型相互作用并泛素化WNK同种型的能力来调节血压。本研究的结果还强调，导致戈登综合征的WNK 4中的错义突变强烈抑制了与KLHL 3的相互作用。这可以通过增加WNK 4的表达来升高血压，从而通过促进NCC/NKCC 2离子共转运蛋白的激活来刺激肾脏中的不适当的盐潴留。本研究揭示了破坏E3连接酶与关键细胞底物（如WNK亚型）相互作用和泛素化的能力的突变如何引发慢性疾病，如高血压。
The WNK (with no lysine kinase)–SPAK (SPS1-related proline/alanine-rich kinase)/OSR1 (oxidative stress-responsive kinase 1) signalling pathway plays an important role in controlling mammalian blood pressure by modulating the activity of ion co-transporters in the kidney. Recent studies have identified Gordon's hypertension syndrome patients with mutations in either CUL3 (Cullin-3) or the BTB protein KLHL3 (Kelch-like 3). CUL3 assembles with BTB proteins to form Cullin–RING E3 ubiquitin ligase complexes. To explore how a CUL3–KLHL3 complex might operate, we immunoprecipitated KLHL3 and found that it associated strongly with WNK isoforms and CUL3, but not with other components of the pathway [SPAK/OSR1 or NCC (Na+/Cl− co-transporter)/NKCC1 (Na+/K+/2Cl− co-transporter 1)]. Strikingly, 13 out of the 15 dominant KLHL3 disease mutations analysed inhibited binding to WNK1 or CUL3. The recombinant wild-type CUL3–KLHL3 E3 ligase complex, but not a disease-causing CUL3–KLHL3[R528H] mutant complex, ubiquitylated WNK1 in vitro. Moreover, siRNA (small interfering RNA)-mediated knockdown of CUL3 increased WNK1 protein levels and kinase activity in HeLa cells. We mapped the KLHL3 interaction site in WNK1 to a non-catalytic region (residues 479–667). Interestingly, the equivalent region in WNK4 encompasses residues that are mutated in Gordon's syndrome patients. Strikingly, we found that the Gordon's disease-causing WNK4[E562K] and WNK4[Q565E] mutations, as well as the equivalent mutation in the WNK1[479–667] fragment, abolished the ability to interact with KLHL3. These results suggest that the CUL3–KLHL3 E3 ligase complex regulates blood pressure via its ability to interact with and ubiquitylate WNK isoforms. The findings of the present study also emphasize that the missense mutations in WNK4 that cause Gordon's syndrome strongly inhibit interaction with KLHL3. This could elevate blood pressure by increasing the expression of WNK4 thereby stimulating inappropriate salt retention in the kidney by promoting activation of the NCC/NKCC2 ion co-transporters. The present study reveals how mutations that disrupt the ability of an E3 ligase to interact with and ubiquitylate a critical cellular substrate such as WNK isoforms can trigger a chronic disease such as hypertension.