Glover Mark, Zuber Annie Mercier, O'Shaughnessy Kevin M
Clinical Pharmacology Unit, Department of Medicine, University of Cambridge, Cambridge, United Kingdom.
J Am Soc Nephrol. 2009 Jun;20(6):1314-22. doi: 10.1681/ASN.2008050542. Epub 2009 May 21.
Mutations in the WNK kinases WNK1 and WNK4 cause a rare familial form of hypertension (Gordon syndrome) by increasing expression of the thiazide-sensitive co-transporter NCCT in the kidney. Regulation of NCCT expression involves a scaffold of proteins composed of several kinases, including the third member of the WNK kinase family, WNK3. This protein, expressed in several tissues including kidney and brain, displays splice variation around exons 18 and 22. We expressed these proteins in Xenopus oocytes and found that the renal isoform of WNK3 increased but the brain isoform decreased NCCT expression and activity. Introduction of a kinase-inactivating mutation into renal WNK3 reversed its action on NCCT, and the same mutation in the brain isoforms led to loss of function. We also studied the effect of phosphorylation of a key NCCT threonine (T58) on the effects of WNK3/4 coexpression; NCCT mutants with a T58A or T58D substitution had the same surface expression as T58 but had significantly altered transporter activity; however, both isoforms of WNK3 as well as WNK4 still modulated expression of these NCCT mutants. Finally, experiments using kinase-dead STE20/SPS1-related proline/alanine-rich kinase (SPAK), a putative downstream target for WNKs, revealed that brain WNK3 acts in tandem with SPAK, whereas renal WNK3 seems to upregulate NCCT through a SPAK-independent pathway. Taken together, these results suggest that the C-terminal motifs contributed by exons 18 and 22 play an important role in the actions of WNK3 isoforms on NCCT.
WNK激酶WNK1和WNK4的突变通过增加肾脏中噻嗪类敏感共转运蛋白NCCT的表达，导致一种罕见的家族性高血压（戈登综合征）。NCCT表达的调节涉及由几种激酶组成的蛋白质支架，包括WNK激酶家族的第三个成员WNK3。这种蛋白质在包括肾脏和大脑在内的多种组织中表达，在第18和22外显子周围存在剪接变异。我们在非洲爪蟾卵母细胞中表达了这些蛋白质，发现WNK3的肾脏异构体增加了NCCT的表达和活性，而大脑异构体则降低了其表达和活性。将激酶失活突变引入肾脏WNK3可逆转其对NCCT的作用，大脑异构体中的相同突变导致功能丧失。我们还研究了关键的NCCT苏氨酸（T58）磷酸化对WNK3/4共表达效应的影响；T58A或T58D替代的NCCT突变体与T58具有相同的表面表达，但转运体活性显著改变；然而，WNK3的两种异构体以及WNK4仍然调节这些NCCT突变体的表达。最后，使用激酶失活的STE20/SPS1相关脯氨酸/丙氨酸丰富激酶（SPAK，WNK的一个假定下游靶点）进行的实验表明，大脑WNK3与SPAK协同作用，而肾脏WNK3似乎通过一条不依赖SPAK的途径上调NCCT。综上所述，这些结果表明，第18和22外显子贡献的C末端基序在WNK3异构体对NCCT的作用中起重要作用。
