Amnekar Ramchandra V, Dite Toby, Lis Pawel, Bell Sebastian, Brown Fiona, Johnson Clare, Wilkinson Stuart, Raggett Samantha, Dorward Mark, Wightman Mel, Macartney Thomas, Soares Renata F, Lamoliatte Frederic, Alessi Dario R
MRC Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, UK.
Sci Adv. 2025 Jul 18;11(29):eadv4636. doi: 10.1126/sciadv.adv4636. Epub 2025 Jul 16.
WNK family kinases are regulated by osmotic stress and control ion homeostasis by activating SPAK and OXSR1 kinases. Using a proximity labeling approach, we found that osmotic stress promotes the association of WNK1 with the NRBP1 pseudokinase and TSC22D2/4 adaptor proteins, results that are confirmed by immunoprecipitation, mass spectrometry, and immunoblotting studies. NRBP1 pseudokinase is closely related to WNK isoforms and contains a RΦ-motif-binding conserved C-terminal (CCT) domain, like the CCT domains in WNKs, SPAK, and OXSR1. Knockdown or knockout of NRBP1 markedly inhibited basal as well as sorbitol-induced activation of WNK1 and downstream components. We demonstrate that recombinant NRBP1 can directly induce the activation of WNK4 in vitro. AlphaFold-3 modeling predicts that WNK1, SPAK, NRBP1, and TSC22D4 form a complex, in which two TSC22D4 RΦ-motifs interact with the CCTL1 domain of WNK1 and the CCT domain of NRBP1. Our data indicate that NRBP1 and likely its close homolog NRBP2 function as an upstream activator of the WNK pathway.
WNK家族激酶受渗透压应激调节，并通过激活SPAK和OXSR1激酶来控制离子稳态。使用邻近标记方法，我们发现渗透压应激促进WNK1与NRBP1假激酶和TSC22D2/4衔接蛋白的结合，免疫沉淀、质谱和免疫印迹研究证实了这一结果。NRBP1假激酶与WNK亚型密切相关，并且像WNK、SPAK和OXSR1中的CCT结构域一样，含有一个RΦ基序结合保守C末端（CCT）结构域。敲低或敲除NRBP1显著抑制WNK1及下游组分的基础激活以及山梨醇诱导的激活。我们证明重组NRBP1在体外可直接诱导WNK4的激活。AlphaFold-3建模预测WNK1、SPAK、NRBP1和TSC22D4形成一个复合物，其中两个TSC22D4 RΦ基序与WNK1的CCTL1结构域和NRBP1的CCT结构域相互作用。我们的数据表明NRBP1及其可能的紧密同源物NRBP2作为WNK途径的上游激活剂发挥作用。
