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Abstract: TH-PO514

Novel KLHL3-Binding Motif of WNK4 and Its Potential Role in Familiar Hyperkalemic Hypertension

Session Information

Category: Fluid and Electrolytes

  • 901 Fluid and Electrolytes: Basic


  • Wang, Lingyun, University of Alabama at Birmingham, Birmingham, Alabama, United States
  • Peng, Ji-Bin, University of Alabama at Birmingham, Birmingham, Alabama, United States

Gene mutations in with-no-lysine [K] kinase 4 (WNK4), and in kelch like 3 (KLHL3), have been found in patients with familiar hyperkalemic hypertension (FHHt, also known as pseudohypoaldosteronism type 2, PHAII). The level of WNK4 protein is controlled by KLHL3, which is the substrate adaptor in the cullin-RING ubiquitin E3 ligase complex for the degradation of WNK4. Mutations in either the acidic motif of WNK4 or the Kelch domain of KLHL3 impair their binding and raise the protein level of WNK4. The increased WNK4 protein abundance and total kinase activity stimulates the activity of sodium-chloride cotransporters (NCC) via SPAK/OSR1, and ultimately results in FHHt. Currently the acidic motif in WNK4 is recognized as the only binding motif for KLHL3. Our aim of this study is to determine whether additional KLHL3 binding site exists in WNK4, if so, whether it is relevant to FHHt.


WNK4 deletion constructs and KLHL3 FHHt mutants were made by PCR-based mutagenesis approaches. The constructs were expressed in Xenopus laevis oocytes. Western blotting and GST pull-down approaches were used to evaluate the interaction and degradation of WNK4 constructs.


In addition to the region containing acidic motif, the WNK4 C-terminal region (amino-acids 1046-1243) was capable of pulling down KLHL3, indicating that there is a new KLHL3-binding motif in this region. Using deletion constructs, the new binding motif was narrowed down to a 30 amino-acid stretch (amino-acids 1051-1080). WNK4 lacking either the acidic motif or the novel motif was rapidly degraded in the presence of KLHL3; however, WNK4 protein was stable when both motifs were deleted. Similar to the acidic motif, the new motif is also rich in Asp/Glu residues. Since the Kelch domain of KLHL3 has a positive surface electrostatic potential, similar to the acidic motif, the negatively charged residues in the new motif are likely important for the electrostatic interactions. Indeed, FHHt mutations in the Kelch domain of KLHL3 impaired the degradation of WNK4 lacking the acidic motif, and this effect was more pronounced for the FHHt mutations at the surface of the Kelch domain.


A new KLHL3-binding motif was identified in the C-terminal region of WNK4. The new motif is likely involved in the pathogenesis of FHHt.


  • NIDDK Support