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Abstract: TH-PO318

PKA Regulates the WNK-SPAK-NCC/NKCC2 Signaling Pathway Through Phosphorylation of WNK4 and I-1

Session Information

Category: Fluid‚ Electrolyte‚ and Acid-Base Disorders

  • 1001 Fluid‚ Electrolyte‚ and Acid-Base Disorders: Basic

Authors

  • Carbajal-Contreras, Hector, Universidad Nacional Autonoma de Mexico, Ciudad de Mexico, Ciudad de México, Mexico
  • Murillo-de-Ozores, Adrian R., Universidad Nacional Autonoma de Mexico, Ciudad de Mexico, Ciudad de México, Mexico
  • Magaña, German R., Universidad Nacional Autonoma de Mexico, Ciudad de Mexico, Ciudad de México, Mexico
  • Bourqui, Laurent, Universitat Zurich, Zurich, ZH, Switzerland
  • Vázquez, Norma Hilda, Universidad Nacional Autonoma de Mexico, Ciudad de Mexico, Ciudad de México, Mexico
  • Loffing, Johannes, Universitat Zurich, Zurich, ZH, Switzerland
  • 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

We have previously shown that vasopressin signaling in the kidney increases the phosphorylation of the Na+-Cl- cotransporter (NCC) and the Na+-K+-Cl- cotransporter 2 (NKCC2) through an increase in the phosphorylation of WNK4’s RRxS motifs. Previous work has also highlighted the importance of Inhibitor-1 (I-1) and protein phosphatase 1 (PP1) in the regulation of these transporters’ phosphorylation in response to cAMP. Since WNK4, SPAK and NCC/NKCC2 have all been linked to PP1-mediated dephosphorylation, we sought to dissect the individual importance of each component in this proposed pathway.

Methods

We transfected HEK293 cells with NKCC2, NCC, PP1, I-1, SPAK, WT WNK4 or a WNK4 mutant which lacks a bona fide PP1-binding site (WNK4 PP1b) in distinct combinations. Cells were stimulated with 30 nM forskolin for 30 minutes. Protein extracts were subjected to immunoblot to assess the abundance of these proteins and their phosphorylated forms. Immunoprecipitation was carried out in order to assess pNCC and pNKCC2.

Results

We found an increase in pSPAK with forskolin exclusively in the presence of WNK4. While the presence of I-1 by itself is not sufficient to upregulate pSPAK in cells lacking WNK4, it increases the dynamic range of SPAK phosphorylation in the presence of WT WNK4. WNK4 PP1b is highly active at baseline and can be further phosphorylated and activated with forskolin, but is not phosphorylated to a greater degree in the presence of I-1. NKCC2’s phosphorylation is decreased with the cotransfection of PP1 and can be regulated by forskolin in the presence of I-1, independently of WNK-SPAK upregulation. The addition of WNK4 and I-1 to NKCC2-expressing cells further increased the dynamic range of pNKCC2 regulation by forskolin. pNCC was not increased by forskolin with I-1 alone, but was upregulated in the presence of WNK4.

Conclusion

WNK4 is a node in cAMP signaling where kinase and phosphatase signaling converge in the modulation of its phosphorylation-dephosphorylation balance. NKCC2 and NCC show distinct regulatory patterns by PP1, which hints towards specific regulatory mechanisms for each transporter. These findings add to the understanding of the transduction of cAMP signaling in the kidney in response to hormones such as ADH, PTH and epinephrine.