Chloride-Induced Monomer to Dimer Transition Controls WNK1 Scaffold Activity
- Fluid, Electrolyte, Acid-Base Disorders: Basic
November 03, 2023 | Location: Exhibit Hall, Pennsylvania 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
- Saha, Bidisha, University of California San Francisco, San Francisco, California, United States
- Takagi, Enzo, University of California San Francisco, San Francisco, California, United States
- Demko, John Eric, University of California San Francisco, San Francisco, California, United States
- Pearce, David, University of California San Francisco, San Francisco, California, United States
Although it is well established that WNK kinase activity is involved in controlling cation-chloride co-transporters, such as NCC and NKCC, recent findings demonstrate that WNK1 can also act as a scaffold, independent of its kinase function. It facilitates the interaction between mTORC2 and SGK1 and stimulates SGK1 phosphorylation thereby enhancing ENaC activity. Extracellular K+ stimulates this non-kinase scaffolding activity at the same time that it inhibits WNK kinase activity. Both of these effects are mediated by intracellular Cl-, which directly binds to WNKs. Previous studies have identified two conformational states of WNK kinases: a kinase inactive, unphosphorylated chloride-bound dimer and a kinase active monomer, which is not chloride-bound. The dimer conformation is maintained by salt bridges between the monomers, which dissociate in low chloride conditions. This study explores the conformational changes in WNK1 that contribute to its catalytic and non-catalytic functions.
Salt bridge mutations were generated using site-directed mutagenesis. WNK1 knockout (WNK1 KO) HEK293 cells were transfected with either wild-type (WT) WNK1 or the salt bridge mutants of WNK1. Subsequently, the cells were adapted to 1 mM [K+] and then subjected to an increase in [K+] to 5 mM. Following this, the cells were processed for co-immunoprecipitation (co-IP) and immunoblot analysis. Whole cell lysates were also assayed for OSR1/SPAK and SGK1 phosphorylation.
In cells transfected with wild-type (WT) WNK1, extracellular K+ stimulated mTORC2-dependent SGK1 phosphorylation, while inhibiting WNK kinase activity-dependent SPAK phosphorylation. In contrast, mutations disrupting the salt-bridge interface of WNK1 blocked the effect of K+ on mTORC2-dependent SGK1 phosphorylation but not SPAK phosphorylation, which became insensitive to changes in extracellular [K+]. Additionally, extracellular [K+] stimulated dimer formation in the WT WNK1, but it was significantly reduced or absent in mutant forms of WNK1.
Our data suggest that the Cl- induced shift of WNK1 from monomer to dimer conformations both inhibits WNK kinase activity and enhances scaffolding activity, providing a parsimonious mechanism for its opposite regulation of cation-chloride cotransporters and ENaC.
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