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首页> 外文期刊>American Journal of Physiology >On the substrate recognition and negative regulation of SPAK, a kinase modulating Na +-K +-2Cl - cotransport activity
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On the substrate recognition and negative regulation of SPAK, a kinase modulating Na +-K +-2Cl - cotransport activity

机译：在SPAK的底物识别和负调节上，激酶调节Na + -K + -2Cl-共转运活性

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摘要

Threonines targeted by Ste20-related proline-alanine-rich kinase (SPAK) for phosphorylation have been identified in Na +-K +-2Cl - cotransporter type 1 (NKCC1), NKCC2, and Na +-Cl - cotransporter (NCC). However, what constitutes the substrate recognition of the kinase is still unknown. Using site-directed mutagenesis and functional measurement of NKCC1 activity in Xenopus laevis oocytes, we determined that SPAK recognizes two threonine residues separated by four amino acids. Addition or removal of a single residue abrogated SPAK activation of NKCC1. Although both threonines are followed by hydrophobic residues, in vivo experiments have determined that SPAK activation of the cotransporter only requires a hydrophobic residue after the first threonine. Interestingly, downstream of the second threonine residue, we have identified a conserved aspartic acid residue which is critical for NKCC1 function. Mouse SPAK activity requires phosphorylation of two specific residues by WNK [with no lysine (K)] kinases: a threonine (T243) in the catalytic domain and a serine (S383) in the regulatory domain. We found that mutating the threonine residue into a glutamic acid (T243E) combined with mutation of the serine into an aspartic acid (S383D) rendered SPAK constitutively active. Surprisingly, alanine substitution of S383 or mutation of residues surrounding this residue also resulted in a constitutively active kinase. Interestingly, deletion of amino acids 356-398 identified another serine residue in the catalytic domain (S321) as another putative target of WNK phosphorylation. We found that WNK4 is capable of stimulating the deletion mutant when S321 is present, but not when S321 is mutated into an alanine.
机译：已在Na + -K + -2Cl-共转运蛋白1型（NKCC1），NKCC2和Na + -Cl-共转运蛋白（NCC）中鉴定了被Ste20相关脯氨酸-丙氨酸丰富的激酶（SPAK）靶向的苏氨酸进行磷酸化。但是，还不清楚什么构成激酶的底物识别。使用定点诱变和功能测量非洲爪蟾卵母细胞中的NKCC1活性，我们确定SPAK识别由四个氨基酸分隔的两个苏氨酸残基。添加或去除单个残基消除了NKCC1的SPAK激活。尽管两个苏氨酸后面都带有疏水残基，但体内实验已确定共转运蛋白的SPAK活化仅在第一个苏氨酸之后仅需要一个疏水残基即可。有趣的是，在第二个苏氨酸残基的下游，我们确定了一个保守的天冬氨酸残基，它对NKCC1功能至关重要。小鼠SPAK活性需要WNK [无赖氨酸（K）]激酶将两个特定残基磷酸化：催化域中的苏氨酸（T243）和调节域中的丝氨酸（S383）。我们发现，将苏氨酸残基突变为谷氨酸（T243E）并与丝氨酸突变为天冬氨酸（S383D）相结合，使SPAK具有组成型活性。令人惊讶地，S383的丙氨酸取代或围绕该残基的残基的突变也产生了组成型活性激酶。有趣的是，氨基酸356-398的缺失将催化结构域（S321）中的另一个丝氨酸残基鉴定为WNK磷酸化的另一个推定靶标。我们发现，当存在S321时，WNK4能够刺激缺失突变体，但当S321突变为丙氨酸时，不能。

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