Abstract: FR-PO562
Heterotypic Site-Specific Interactions between WNK4, TSC22D2, and NRBP1 Promote Kinase Activity and Co-localization in WNK Bodies
Session Information
- Fluid, Electrolyte, and Acid-Base Disorders: Basic
October 25, 2024 | Location: Exhibit Hall, Convention Center
Abstract Time: 10:00 AM - 12:00 PM
Category: Fluid, Electrolytes, and Acid-Base Disorders
- 1101 Fluid, Electrolyte, and Acid-Base Disorders: Basic
Authors
- Magaña, German R., Universidad Nacional Autonoma de Mexico, Ciudad de Mexico, Ciudad de México, Mexico
- Carbajal-Contreras, Hector, Universidad Nacional Autonoma de Mexico, Ciudad de Mexico, Ciudad de México, Mexico
- Vázquez, Norma Hilda, Universidad Nacional Autonoma de Mexico, Ciudad de Mexico, Ciudad de México, Mexico
- Gamba, Gerardo, Universidad Nacional Autonoma de Mexico, Ciudad de Mexico, Ciudad de México, Mexico
- Castañeda-Bueno, Maria, Universidad Nacional Autonoma de Mexico, Ciudad de Mexico, Ciudad de México, Mexico
Group or Team Name
- Unidad de Fisiología Molecular.
Background
The WNK4 kinase regulates the activity of the thiazide-sensitive Na:Cl cotransporter, NCC, through the activation of the kinases SPAK/OSR1. Disregulation of this pathway is associated with altered sodium and potassium homeostasis. Recently, we found that WNK kinases associate in vivo with the pseudokinase NRBP1 and with proteins of the TSC22D family. These proteins co-localize with the kinase WNK4, KS-WNK1, and SPAK in biomolecular condensates called “WNK-bodies” that are observed in distal convoluted tubule (DCT) cells. The SPAK-WNK interaction is well characterized and involves RFXV motifs present in WNK kinases and a Conserved C-Terminal (CCT) domain present in SPAK. So how is WNK4 interacting with NRBP1 and TSC22Ds? Long TSC22D proteins have a conserved RFXV motif, NRBP1 has a conserved CCT domain, and WNK kinases have two CCT domains in addition to the RFXV motifs. In this work, our objective was to study the effect of these proteins in the WNK4-SPAK pathway and to investigate their interaction mechanisms.
Methods
HEK293 cells were transiently transfected with NRBP1, TSC22D1.1,TSC22D2, WNK4 and SPAK. Western blots of cell lysates and immunoprecipitated proteins and immunofluorescent stainings were performed.
Results
We observed that mutations in the CCT domain of WNK4 dramatically decreased WNK4’s activity, TSC22D2 binding, and colocalization of WNK4 with TSC22Ds in condensates. Interestingly, SPAK co-expression allowed for TSC22D2 recruitment into condensates formed with the WNK4-CCT mutant. The loss of both, the CCT domains and the RFXV motif in WNK4 produced an inactive WNK4 that was not activated by NRBP1/TSC22D2, and that was not able to co-localize in TSC22D condensates in the presence of SPAK.
Conclusion
In conclusion, our data show that TSC22D1.1, TSC22D2, and NRBP1 are novel components of WNK-bodies in vivo in the DCT. NRBP1 and long TSC22D isoforms positively modulate WNK activity. Heterotypic interactions established by the CCT domains and the RFXV motif of WNK kinases permit co-localization of WNKs with TSC22D proteins and NRBP1 in condensates and promote activation of the kinase. Redundancy in interactions between these proteins can preserve the activity of the pathway when one interacting motif or domain is affected.
Funding
- Government Support – Non-U.S.