Chloride Sensing by WNK Kinases

描述（由申请人提供）：氯是离子转运蛋白的已知调节剂，但尚未在哺乳动物中鉴定出控制转运的氯传感器。我们发现WNK [不含赖氨酸（K）]激酶WNK 1，已知是盐敏感的，直接受氯离子调节，并在结构研究中发现了氯离子结合位点。在这里，我们将确定是否一个或所有WNK（亚型1，2，3和4）是由氯化物调节，以及在什么浓度范围内，使用测定，热稳定性测量和等温研磨量热法。我们将通过与现有晶体的氯化物结合和活性WNK 1的结构比较，确定氯化物如何影响WNK 1的激酶结构域的结构。WNK受磷酸化和自身抑制调节。将通过试验和晶体学评估氯化物抑制与这些额外机制的关系。将设计和评价影响氯离子结合的突变体。将在细胞中测试基于WNK的氯离子敏感机制。

DESCRIPTION (provided by applicant): Chloride is a known regulator of ion transporters, yet chloride sensors that control transport have not been identified in mammals. We discovered that the WNK [with no lysine (K)] kinase, WNK1, known to be salt sensitive, is directly regulated by chloride, and found the chloride binding site in structural studies. Here we will determine whether one or all WNKs (isoforms 1, 2, 3, and 4) are regulated by chloride, and over what concentration ranges, using assays, thermal stability measurements and isothermal tritration calorimetry. We will determine how chloride affects the structure of the kinase domain of WNK1 through structural comparisons of chloride-bound and active WNK1, with existing crystals. WNKs are regulated by phosphorylation and autoinhibition. How the chloride inhibition relates to these additional mechanisms will be assessed by assays and crystallography. Mutants affecting chloride binding will be designed and evaluated. The WNK-based chloride sensing mechanism will be tested in cells.