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.
MRC Protein Phosphorylation Unit, James Black Centre, College of Life Sciences, University of Dundee, Dundee DD1 5EH, UK. g.hoxhaj@dundee.ac.uk
Gerta
G
Hoxhaj
Ayaz
A
Najafov
Rachel
R
Toth
David G
DG
Campbell
Alan R
AR
Prescott
Carol
C
MacKintosh
D048948:14-3-3 Proteins / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism*; D003593:Cytoplasm / Q000187:drug effects / Q000378:metabolism; D055785:Gene Knockdown Techniques; D057809:HEK293 Cells; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000494:pharmacology*; D007425:Intracellular Membranes / Q000187:drug effects / Q000378:metabolism*; D007553:Isotope Labeling; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D010042:Ouabain / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects; D010768:Phosphoserine / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000378:metabolism; D021381:Protein Transport / Q000187:drug effects; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*; D044763:Ubiquitin-Conjugating Enzymes / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases; D054875:Ubiquitination / Q000187:drug effects
D048948:14-3-3 Proteins; D002352:Carrier Proteins; D036341:Intercellular Signaling Peptides and Proteins; D021122:Protein Subunits; D010768:Phosphoserine; D010042:Ouabain; C471607:UBE2N protein, human; D044763:Ubiquitin-Conjugating Enzymes; D044767:Ubiquitin-Protein Ligases; C544744:ZNRF1 protein, human; C580704:ZNRF2 protein, human; D000254:Sodium-Potassium-Exchanging ATPase