Calcineurin dephosphorylates Kelch-like 3, reversing phosphorylation by angiotensin II and regulating renal electrolyte handling
Calcineurin dephosphorylates Kelch-like 3, reversing phosphorylation by angiotensin II and regulating renal electrolyte handling
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DOI：
10.1073/pnas.1817281116
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发表时间：
2019-02-19
影响因子：
11.1
通讯作者：
Shibata, Shigeru
中科院分区：
文献类型：
--
作者：
Ishizawa, Kenichi;Wang, Qin;Shibata, Shigeru
钙调神经磷酸酶是一种钙/钙调素调节的磷酸酶，在T细胞受体参与T细胞活化过程中起重要作用。钙调神经磷酸酶抑制剂(CNI)被广泛用作免疫抑制药，其常见的不良反应是高血压和高钾血症。虽然先前的研究表明肾脏远端曲管(DCT)的Na-Cl协同转运体(NCC)激活与这种毒性有关，但这种作用的分子机制尚不清楚。CNI的肾脏效应模拟高血压和高钾血症，这些高血压和高钾血症是由非赖氨酸(WNK)激酶和Kelch-like 3(KLHL3)-CUL3泛素连接酶复合体的胚系突变引起的。WNK4是NCC的激活剂，通过与KLHL3结合、WNK4的S泛素化和蛋白酶体降解而被降解。这种结合是通过蛋白激酶C使丝氨酸433位的KLHL3(KLHL3(S433-P))磷酸化而阻止的，导致WNK4水平增加和NCC活性增加。到目前为止，介导KLHL3(S433-P)去磷酸化的机制尚不清楚。我们现在证明在DCT中表达的钙调神经磷酸酶是一种有效的KLHL3(S433-P)磷酸酶。在哺乳动物细胞中，钙调神经磷酸酶激活剂离子载体离子霉素可降低KLHL3(S433-P)水平，这种作用可被钙调神经磷酸酶抑制剂他克莫司和siRNA介导的钙调神经磷酸酶的下调所逆转。在体内，他克莫司增加KLHL3(S433-P)的水平，导致WNK4、磷酸化的SPAK和NCC的水平增加。此外，他克莫司减弱了KLHL3介导的WNK4泛素化和降解，而S433A替换的KLHL3没有这种作用。此外，细胞外K+增加可诱导依赖钙调神经磷酸酶的KLHL3去磷酸化(S433-P)。这些发现表明KLHL3(S433-P)是一种钙调神经磷酸酶底物，在他克莫司诱导的病理过程中KLHL3的磷酸化增加。
Calcineurin is a calcium/calmodulin-regulated phosphatase known for its role in activation of T cells following engagement of the T cell receptor. Calcineurin inhibitors (CNIs) are widely used as immuno-suppressive agents; common adverse effects of CNIs are hypertension and hyperkalemia. While previous studies have implicated activation of the Na-Cl cotransporter (NCC) in the renal distal convoluted tubule (DCT) in this toxicity, the molecular mechanism of this effect is unknown. The renal effects of CNIs mimic the hypertension and hyperkalemia that result from germ-line mutations in with-no-lysine (WNK) kinases and the Kelch-like 3 (KLHL3)-CUL3 ubiquitin ligase complex. WNK4 is an activator of NCC and is degraded by binding to KLHL3 followed by WNK4's ubiquitylation and proteasomal degradation. This binding is prevented by phosphorylation of KLHL3 at serine 433 (KLHL3(S433-P)) via protein kinase C, resulting in increased WNK4 levels and increased NCC activity. Mechanisms mediating KLHL3(S433-P) dephosphorylation have heretofore been unknown. We now demonstrate that calcineurin expressed in DCT is a potent KLHL3(S433-P) phosphatase. In mammalian cells, the calcium ionophore ionomycin, a calcineurin activator, reduces KLHL3(S433-P) levels, and this effect is reversed by the calcineurin inhibitor tacrolimus and by siRNA-mediated knock-down of calcineurin. In vivo, tacrolimus increases levels of KLHL3(S433-P), resulting in increased levels of WNK4, phosphorylated SPAK, and NCC. Moreover, tacrolimus attenuates KLHL3-mediated WNK4 ubiquitylation and degradation, while this effect is absent in KLHL3 with S433A substitution. Additionally, increased extracellular K+ induced calcineurin-dependent dephosphorylation of KLHL3(S433-P). These findings demonstrate that KLHL3(S433-P) is a calcineurin substrate and implicate increased KLHL3 phosphorylation in tacrolimus-induced pathologies.