WNK3-SPAK Interaction is Required for the Modulation of NCC and other Members of the SLC12 Family
WNK3-SPAK Interaction is Required for the Modulation of NCC and other Members of the SLC12 Family
复制标题
NCC 和 SLC12 家族其他成员的调节需要 WNK3-SPAK 相互作用
DOI：
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发表时间：
2012
影响因子：
--
通讯作者：
G. Gamba
中科院分区：
文献类型：
--
作者：
Diana Pacheco‐Alvarez;N. Vázquez;María Castañeda;Paola de;César Cortés;Erika Moreno;P. Meade;N. Bobadilla;G. Gamba
不含赖氨酸激酶3的丝氨酸/苏氨酸（WNK 3）调节电中性阳离子偶联氯离子协同转运蛋白（CCC）的活性，以促进Cl-内流并阻止Cl-外流，从而符合假定的“Cl-敏感激酶”的特征。Ste 20型激酶SPAK/OSR 1响应于细胞内氯离子浓度的降低而磷酸化，并调节NKCC 1的活性。一些研究表明，WNK在SPAK/OSR 1的上游发挥作用。本研究旨在分析WNK 3-SPAK相互作用在CCCs特别是NCC调控中的作用。在这项研究中，我们使用非洲爪蟾卵母细胞的功能性表达系统，以显示WNK 3中不同的SPAK结合位点（241，872，1336 RFxV）是激酶对CCCs产生作用所必需的。WNK 3-F1337 A不再激活NKCC 2，但对NCC、NKCC 1和KCC 4的作用得以保留。相反，WNK 3对这些协同转运蛋白的作用在WNK 3-F242 A中被阻止。F873的消除对WNK 3的作用没有影响。WNK 3促进NCC在苏氨酸58处的磷酸化，即使在NCC的独特SPAK结合位点不存在的情况下，但是这种作用在突变体WNK 3-F242 A中被消除。因此，我们的数据支持这样的假设，即WNK 3对NCC和其他CCC的作用需要SPAK激酶的相互作用和激活。该作用依赖于WNK 3中存在的SPAK的三个结合位点之一，但不依赖于CCC上的SPAK结合位点，这表明WNK 3能够结合SPAK和CCC以促进其磷酸化。
The serine/threonine with no lysine kinase 3 (WNK3) modulates the activity of the electroneutral cation-coupled chloride cotransporters (CCC) to promote Cl- influx and prevent Cl- efflux, thus fitting the profile for a putative “Cl--sensing kinase”. The Ste20-type kinases, SPAK/OSR1, become phosphorylated in response to reduction in intracellular chloride concentration and regulate the activity of NKCC1. Several studies have now shown that WNKs function upstream of SPAK/OSR1. This study was designed to analyze the role of WNK3-SPAK interaction in the regulation of CCCs with particular emphasis on NCC. In this study we used the functional expression system of Xenopus laevis oocytes to show that different SPAK binding sites in WNK3 (241, 872, 1336RFxV) are required for the kinase to have effects on CCCs. WNK3-F1337A no longer activated NKCC2, but the effects on NCC, NKCC1, and KCC4 were preserved. In contrast, the effects of WNK3 on these cotransporters were prevented in WNK3-F242A. The elimination of F873 had no consequence on WNK3 effects. WNK3 promoted NCC phosphorylation at threonine 58, even in the absence of the unique SPAK binding site of NCC, but this effect was abolished in the mutant WNK3-F242A. Thus, our data support the hypothesis that the effects of WNK3 upon NCC and other CCCs require the interaction and activation of the SPAK kinase. The effect is dependent on one of the three binding sites for SPAK that are present in WNK3, but not on the SPAK binding sites on the CCCs, which suggests that WNK3 is capable of binding both SPAK and CCCs to promote their phosphorylation.
鲨鱼直肠腺的 Na-K-Cl 共转运蛋白。
DOI：
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发表时间：
1992
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Lytle,C;Forbush3rd,B
通讯作者：
Forbush3rd,B
DOI：
10.1073/pnas.0508307102
发表时间：
2005-11-15
影响因子：
11.1
作者：
Kahle, KT;Rinehart, J;Lifton, RP
通讯作者：
Lifton, RP
影响因子：
5.5
作者：
Gagnon, KBE;England, R;Delpire, E
通讯作者：
Delpire, E