Abstract
The E3 ubiquitin ligases RNF43 and ZNRF3 are key negative regulators of canonical WNT signaling, promoting turnover of the WNT receptors FRIZZLED and LRP5/6 at the plasma membrane. While their mechanism of action is well established, how RNF43/ZNRF3 themselves are regulated remains unclear. Here, we identify WNK kinases as novel upstream regulators of RNF43 through proximity labeling proteomics. Using gain- and loss-of-function approaches, we show that WNKs control RNF43 surface localization and thereby its ability to ubiquitinate and downregulate WNT receptors. Pharmacological inhibition of WNKs increases RNF43 membrane abundance and enhances WNT suppression - an effect abolished in RNF43/ZNRF3 double knockout cells and organoids. Mechanistically, WNK inhibition alters RNF43 trafficking and ubiquitination, revealing a role for WNKs in regulating its plasma membrane distribution. These findings define a new regulatory axis linking the pro-WNT activity of WNKs to RNF43/ZNRF3-mediated feedback inhibition. Targeting WNK now offers a novel therapeutic strategy to restore WNT pathway control in cancers with RSPO fusions or RNF43 mutations.