Abstract: TH-PO347
WNK1 Is a Chloride-Sensing Scaffold That Potently Regulates mTORC2 Activity and ENaC
Session Information
- Fluid, Electrolyte, and Acid-Base Disorders: Basic
November 03, 2022 | Location: Exhibit Hall, Orange County Convention Center‚ West Building
Abstract Time: 10:00 AM - 12:00 PM
Category: Fluid‚ Electrolyte‚ and Acid-Base Disorders
- 1001 Fluid‚ Electrolyte‚ and Acid-Base Disorders: Basic
Authors
- Saha, Bidisha, University of California San Francisco, San Francisco, California, United States
- Leite-Dellova, Deise C A, University of California San Francisco, San Francisco, California, United States
- Takagi, Enzo, University of California San Francisco, San Francisco, California, United States
- Demko, John E., University of California San Francisco, San Francisco, California, United States
- Shabbir, Waheed, University of California San Francisco, San Francisco, California, United States
- Pearce, David, University of California San Francisco, San Francisco, California, United States
Background
mTORC2 is a multiprotein signaling complex that regulates a variety of cellular processes induced by hormones and growth factors. Recent evidence suggests that mTORC2 is also involved in cell autonomous action, as it responds to local extracellular K+ concentration to rapidly stimulate SGK1 phosphorylation and downstream target, ENaC, an epithelial sodium channel, in renal epithelial cells. Here we show that WNK1 has a pivotal role in mediating cell autonomous responses to local K+ concentration that activates mTORC2.
Methods
We generated WNK1 knockout mpkCCD cells and WNK1 and SIN1 (a component of mTORC2 complex) double knockout HEK293 cells using CRISPR/Cas9 system. mpkCCD cells were grown on Transwell filters and incubated in media containing different [K+], with or without WNK kinase inhibitor. Amiloride-sensitive current was measured before the cells were processed for immunoblot analysis. For intracellular Cl- measurement, HEK293 cells were transfected with mCl-YFP plasmid and fluorescence signals were measured and analyzed. For modulation of intracellular [Cl-], cells were incubated in media containing different [Cl-] and ionophores. Cells were then processed for immunoblot and Co-IP analysis.
Results
Our results showed that the effect of extracellular K+ on SGK1 phosphorylation and ENaC activity is dependent on elevated intracellular [Cl-] and on presence of chloride-binding protein kinase WNK1, but not on its kinase activity. Intracellular Cl- stimulates the scaffolding activity of WNK1, which physically interacts with both SGK1 and mTORC2 and enhances SGK1 recruitment to mTORC2. This results in a selective increase in SGK1 phosphorylation, and activation of ENaC which drives K+ secretion.
Conclusion
These findings establish a novel mode of chloride-regulated WNK1 action that regulates mTORC2 activity selectively towards SGK1 phosphorylation and ENaC activation, independently of its kinase activity and reveals a dual role of WNK1’s Cl--sensing property on epithelial ion transport regulation: one, low [Cl-]-dependent activation of NCC through catalytic mechanism and the second, high [Cl-]-dependent activation of ENaC through non-catalytic scaffolding mechanism; the coupling of both pathways is significantly implicated in DCT2, controlling K+ secretion.
Funding
- NIDDK Support