Department of Biophysics, The University of Texas Southwestern Medical Center, Dallas, TX 75390.
Department of Biology, Chemistry, & Physical Sciences, APS/Illinois Institute of Technology, Argonne, IL 60439.
Mol Biol Cell. 2021 Aug 19;32(18):1614-1623. doi: 10.1091/mbc.E20-01-0089. Epub 2021 Mar 10.
With No Lysine (K) WNK kinases regulate electro-neutral cotransporters that are controlled by osmotic stress and chloride. We showed previously that autophosphorylation of WNK1 is inhibited by chloride, raising the possibility that WNKs are activated by osmotic stress. Here we demonstrate that unphosphorylated WNK isoforms 3 and 1 autophosphorylate in response to osmotic pressure in vitro, applied with the crowding agent polyethylene glycol (PEG)400 or osmolyte ethylene glycol (EG), and that this activation is opposed by chloride. Small angle x-ray scattering of WNK3 in the presence and absence of PEG400, static light scattering in EG, and crystallography of WNK1 were used to understand the mechanism. Osmosensing in WNK3 and WNK1 appears to occur through a conformational equilibrium between an inactive, unphosphorylated, chloride-binding dimer and an autophosphorylation-competent monomer. An improved structure of the inactive kinase domain of WNK1, and a comparison with the structure of a monophosphorylated form of WNK1, suggests that large cavities, greater hydration, and specific bound water may participate in the osmosensing mechanism. Our prior work showed that osmolytes have effects on the structure of phosphorylated WNK1, suggestive of multiple stages of osmotic regulation in WNKs.
无赖氨酸（K）WNK 激酶调节受渗透胁迫和氯离子控制的电中性协同转运蛋白。我们之前曾表明，氯离子抑制 WNK1 的自磷酸化，这表明 WNK 可能被渗透胁迫激活。在这里，我们证明未磷酸化的 WNK 同工型 3 和 1 在体外对渗透压有反应，渗透压是通过拥挤剂聚乙二醇（PEG）400 或渗透剂乙二醇（EG）施加的，氯离子对此有拮抗作用。使用小角 X 射线散射（PEG400 存在和不存在的情况下）、EG 中的静态光散射和 WNK1 的晶体学来了解机制。WNK3 和 WNK1 的渗透压感应似乎通过无活性、未磷酸化、结合氯离子的二聚体和具有自磷酸化能力的单体之间的构象平衡来发生。WNK1 无活性激酶结构域的改进结构，以及与 WNK1 的单磷酸化形式的结构比较，表明大空腔、更高的水合作用和特定的结合水可能参与渗透压感应机制。我们之前的工作表明，渗透剂对磷酸化 WNK1 的结构有影响，表明 WNK 中存在多个渗透压调节阶段。
