ZNRF2 is released from membranes by growth factors and, together with ZNRF1, regulates the Na⁺/K⁺ATPase

Here, we describe a phosphorylation-based reverse myristoyl switch for mammalian ZNRF2, and show that this E3 ubiquitin ligase and its sister protein ZNRF1 regulate the Na⁺/K⁺ pump (Na⁺/K⁺ATPase). N-myristoylation localizes ZNRF1 and ZNRF2 to intracellular membranes and enhances their activity. However, when ZNRF2 is phosphorylated in response to agonists including insulin and growth factors, it binds to 14-3-3 and is released into the cytosol. On membranes, ZNRF1 and ZNRF2 interact with the Na⁺/K⁺ATPase α1 subunit via their UBZ domains, while their RING domains interact with E2 proteins, predominantly Ubc13 that, together with Uev1a, mediates formation of Lys63-ubiquitin linkages. ZNRF1 and ZNRF2 can ubiquitylate the cytoplasmic loop encompassing the nucleotide-binding and phosphorylation regions of the Na⁺/K⁺ATPase α1 subunit. Ouabain, a Na⁺/K⁺ATPase inhibitor and therapeutic cardiac glycoside, decreases ZNRF1 protein levels, whereas knockdown of ZNRF2 inhibits the ouabain-induced decrease of cell surface and total Na⁺/K⁺ATPase α1 levels. Thus, ZNRF1 and ZNRF2 are new players in regulation of the ubiquitous Na⁺/K⁺ATPase that is tuned to changing demands in many physiological contexts.