SPAK Isoforms and OSR1 Regulate Sodium-Chloride Co-transporters in a Nephron-specific Manner
SPAK Isoforms and OSR1 Regulate Sodium-Chloride Co-transporters in a Nephron-specific Manner
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DOI：
10.1074/jbc.m112.402800
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发表时间：
2012-11-02
影响因子：
4.8
通讯作者：
Welling, Paul A.
中科院分区：
文献类型：
--
作者：
Grimm, P. Richard;Taneja, Tarvinder K.;Welling, Paul A.
STE20/SPS-1相关的富含脯氨酸的丙氨酸蛋白激酶(SPAK)和氧化应激相关的蛋白激酶(OSR1)在体外激活钾依赖的氯化钠共转运蛋白NKCC2和硫氮化物敏感的氯化钠共转运蛋白NCC，两者都与激酶调节分子Cab39/MO25α共同定位于粗大升支(TAL)和远端曲管(DCT)的顶膜。然而，在小鼠中，Spak的遗传消融导致NCC功能的选择性丧失，而NKCC2则变得过度磷酸化。在这里，我们探索野生型和Spak缺失小鼠的潜在机制。与DCT不同，OSR1保留在KO小鼠的TAL顶膜上，在那里它伴随着活性的、磷酸化的AMP激活的激酶的增加。我们发现，在体外适度抑制辅转运蛋白活性的另一种SPAK亚型(可能是SPAK2亚型)在髓质中比皮质中更丰富。因此，在SPAK基因敲除中，NKCC2的磷酸化增强可能是通过去除抑制性SPAK2、OSR1的持续活性和激活其他激酶来解释的。相比之下，OSR1/SPAK/M025α信号装置在DCT中被破坏。OSR1在顶膜上基本上不活跃，从M025α和NCC移位，并重新分布到细胞质内含有WNK1的致密点状结构中。与这些变化平行的是NCC磷酸化的减少和DCT1所独有的远端曲小管质量的减少。由于OSR1依赖于DCT中的Spak，因此无法激活NCC。因此，Spak(-/-)小鼠对饮食盐限制高度敏感，表现出长期的负钠平衡和低血压。
STE20/SPS-1-related proline-alanine-rich protein kinase (SPAK) and oxidative stress-related kinase (OSR1) activate the potassium-dependent sodium-chloride co-transporter, NKCC2, and thiazide-sensitive sodium-chloride cotransporter, NCC, in vitro, and both co-localize with a kinase regulatory molecule, Cab39/MO25 alpha, at the apical membrane of the thick ascending limb (TAL) and distal convoluted tubule (DCT). Yet genetic ablation of SPAK in mice causes a selective loss of NCC function, whereas NKCC2 becomes hyperphosphorylated. Here, we explore the underlying mechanisms in wild-type and SPAK-null mice. Unlike in the DCT, OSR1 remains at the TAL apical membrane of KO mice where it is accompanied by an increase in the active, phosphorylated form of AMP-activated kinase. We found an alterative SPAK isoform (putative SPAK2 form), which modestly inhibits cotransporter activity in vitro, is more abundant in the medulla than the cortex. Thus, enhanced NKCC2 phosphorylation in the SPAK knock-out may be explained by removal of inhibitory SPAK2, sustained activity of OSR1, and activation of other kinases. By contrast, the OSR1/SPAK/M025 alpha signaling apparatus is disrupted in the DCT. OSR1 becomes largely inactive and displaced from M025 alpha and NCC at the apical membrane, and redistributes to dense punctate structures, containing WNK1, within the cytoplasm. These changes are paralleled by a decrease in NCC phosphorylation and a decrease in the mass of the distal convoluted tubule, exclusive to DCT1. As a result of the dependent nature of OSR1 on SPAK in the DCT, NCC is unable to be activated. Consequently, SPAK(-/-) mice are highly sensitive to dietary salt restriction, displaying prolonged negative sodium balance and hypotension.