NRPB1 is a WNK-like pseudokinase that modulates the activity of the WNK4-SPAK/OSR1 pathway with implications in the physiology of the distal convoluted tubule

Abstract

The pseudokinase NRBP1 and the TSC22D protein family have been implicated in the regulation of cell proliferation and protein turnover. Preliminary observations from one of our laboratories (DA) revealed that, during osmotic stress, NRBP1 and TSC22D proteins interact with WNK1 in vitro. Mouse renal transcriptomic databases show that TSC22D1.1 and TSC22D2 expression is enriched in the distal convoluted tubule (DCT) of the nephron, where WNK kinases play an essential role in the regulation of Na+ reabsorption. WNK4 is the mayor catalytically active WNK in this nephron segment. It is known that WNK’s undergo liquid-liquid phase separation and form biomolecular condensates now called “WNK-bodies” that are observed in the DCT under certain conditions. In this work, our objective was to study the expression and subcellular localization of NRBP1 and TSC22D proteins in the kidney and to investigate their effect on the WNK4-SPAK pathway. We hypothesized that NRBP1 and TSC22D proteins may modulate the activity of the WNK-SPAK pathway. By immunofluorescent staining, we confirmed that TSC22D1.1 and TSC22D2 expression is enriched in the DCT. At baseline, they were localized to the apical membrane and, under conditions in which DCT WNK bodies are observed (mice in low K+ diet or mice with Familial Hyperkalemic Hypertension (FHHt)), they colocalized with WNKs and SPAK in the condensates. NRBP1 was also observed in condensates under these conditions. Increased TSC22D1.1 and TSC22D2 abundance was observed in the FHHt mouse model by immunoblot. In HEK293 transfected cells, the co-expression of NRBP1 and TSC22D1.1, TSC22D2, or TSC22D4 with WNK4 and SPAK increased pSPAK levels, as well as WNK4 T-loop phosphorylation. We then investigated the interactions between NRBP1, TSC22D2, and WNK4. The SPAK-WNK interaction is well characterized and involves RFXV motifs present in WNK kinases and a Conserved C-Terminal (CCT) domains present in SPAK. Long TSC22D proteins possess a conserved RFXV motif, NRBP1 possesses a conserved CCT domain, and WNK kinases possess two CCT domains in addition to the RFXV motifs. Thus, we tested the interaction of WNK4 with TSC22D2 and NRBP1 and observed that mutations on the CCT domain of WNK4 dramatically decreased WNK4’s activity and TSC22D2 binding. Interaction with NRBP1 was not affected. However, the overexpression of TSC22D2 and NRBP1 restored WNK4-CCT mutant activity. In conclusion, TSC22D1.1 and TSC22D2 proteins are enriched in the DCT and, under certain conditions that are known to activate the WNK pathway, colocalize with WNK4, SPAK, and NRPB1 in WNK bodies, demonstrating in vivo interaction of these proteins and suggesting a role in DCT physiology. Increased WNK activation is observed in the presence of NRBP1 and TSC22D1.1/TSC22D2. CONACyT, Mexico. IMIN, Mexico. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.