Alternatively spliced PY cassette exons in WNK1 enhance sensitivity to aldosterone via the E3 ubiquitin ligase Nedd4‐2

Abstract

Aldosterone stimulates NaCl reabsorption in the distal nephron in part by increasing thiazide‐sensitive cotransporter (NCC) phosphorylation. Through undefined upstream mechanisms, WNK1 kinase participates in this process by activating SPAK and OSR1, which phosphorylate NCC directly. In the kidney, WNK1 is expressed as kinase active (L‐WNK1) and defective (KS‐WNK1) isoforms; exons 11 and 12 are differentially spliced into both gene products, but the role of these events in WNK1 function is unclear. Here, we explore how exons 11 and 12 link WNK1 to aldosterone action. Both exons harbor PY motifs ([L/P]PXY); these conserved sequences are absent from the rest of WNK1 and are binding sites for Nedd4‐2, an E3 ligase which is inhibited by the aldo‐induced kinase SGK1. In mammalian cells, Nedd 4‐2 associated with WNK1, accelerating its degradation by the ubiquitin proteasome pathway in a PY motif dependent manner. In mpkCCD cells, 100nM aldosterone increased L‐WNK and KS‐WNK1 protein abundance within 2h, and both isoforms formed an endogenous complex with Nedd 4‐2 and SGK1. Antisera specific to exon 12 confirmed the enrichment of Nedd4‐2 sensitive WNK1 isoforms in the distal nephron. These results suggest that exons 11 and 12 function as modular “PY cassettes” that alter WNK1 abundance in response to aldosterone via Nedd4‐2, revealing a mechanism that directly connects aldosterone signaling to NCC phosphorylation.