On membranes, ZNRF1 and ZNRF2 interact with the Na+/K+ATPase 1 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. regions of the Na+/K+ATPase 1 subunit. Ouabain, a Na+/K+ATPase inhibitor and restorative cardiac glycoside, decreases ZNRF1 protein levels, whereas knockdown of ZNRF2 inhibits the ouabain-induced decrease of cell surface and total Na+/K+ATPase 1 levels. Therefore, ZNRF1 and ZNRF2 are fresh players in rules of the ubiquitous Na+/K+ATPase that is tuned to changing demands in many physiological contexts. ?=? 697.9107:2+ ?=? 1393.8068?Da) is 210.2010?Da, which corresponds to the mass of the myristoyl changes (210.1984?Da). The y-ions generated by MS2 fragmentation indicate the sequence from your C-terminal end of the peptide, whereas the b-ions are consistent with L-Valine an N-myristoyl group. (C) Subcellular localization of ZNRF2 in HeLa and HEK293 cells transfected with L-Valine GFP-tagged wild-type or Gly2Ala-ZNRF2 (G2A). Cells were stained with DAPI (to label nuclei). At least five units of cells were analyzed and representative images are demonstrated. Scale pub: 10?m. (D) HEK293-Flp-In-Trex cells stably expressing GFP-tagged wild-type or Gly2Ala-ZNRF2 were fractionated as explained in the Materials and Methods, and cell lysates were subjected to western blotting with the indicated antibodies. The proportion of ZNRF2-GFP in the membrane fraction improved when cells were serum deprived; or when cells cultivated in serum were treated with PI-103, G?6983 and H-89 before activation with IGF1, PMA and forskolin, respectively (Fig.?4A). Conversely, the amount of ZNRF2-GFP in the membrane fractions decreased when cells were stimulated with IGF1, PMA, forskolin and serum (Fig.?4A). Consistent with these fractionation data, live cell imaging showed that ZNRF2-GFP relocated from intracellular membranes to the cytosol upon IGF1 activation (Fig.?5A), and returned to the membranes when cells were treated with PI3K inhibitors GDC-0941 (Fig.?4C) and PI-103 (supplementary material Fig. S4B), and PKB inhibitors MK-2206 (Fig.?4D) and AKTi-1/2 (supplementary material Fig. S4B), which decreased ZNRF2 phosphorylation at Ser19 and Ser145 (Fig.?4B). These membranal constructions associated with dephosphorylated ZNRF2 co-localized having a Golgi tracker stain (Fig.?4C,D). Similarly, the association of ZNRF2 with membranes was lost when cells were stimulated with PMA or forskolin, and regained when cells were given the kinase inhibitors G?6983 and H-89, respectively (Fig.?4A). Open in a separate windowpane Fig. 4. Reversible membrane-to-cytosol translocation of L-Valine ZNRF2. (A) HEK293 cells exposed to numerous stimuli and inhibitor mixtures were fractionated into cytosol (CE) and membrane (ME) components by ultracentrifugation and the components were analyzed by western blotting with the indicated antibodies. Ubiquitylation activity of endogenous ZNRF2 from cytosol and membrane fractions was analyzed in vitro using Ubc13-Uev1a as E2. (B) HEK293-Flp-In-TRex cells stably expressing ZNRF2-GFP were exposed to numerous PI3K and PKB inhibitors and phosphorylation ZNRF2 phosphorylation was analyzed by western blotting. (C,D) HEK293-Flp-In-TRex cells stably expressing ZNRF2-GFP were utilized for live cell imaging. Cells were treated with 1?M GDC-0941 (C) or 10?M MK-2206 for 60?min (D). Images were taken before and after the treatments. Scale bars: 10 m. Open in a separate windowpane Fig. 5. Effect of Ser19Ala mutation within the reversible membrane-to-cytosol translocation of ZNRF2. (A) As with Fig.?4C, except that before imaging the cells were serum starved for 12?h and then stimulated with IGF1. Images were taken before and after the activation. Scale pub: 10?m. (B) HEK293-Flp-In-TRex cells stably expressing wild-type or the indicated mutants of ZNRF2-GFP were fractionated into cytosol (CE) and membrane (ME) components by ultracentrifugation, and the components L-Valine were analyzed by western blotting with the indicated antibodies. Interestingly, in cells growing under 10% FBS conditions, ZNRF2-Ser19Ala (and to a lesser degree ZNRF2-Ser82Ala and ZNRF2-Ser145Ala) showed higher membrane localization than the wild-type control (Fig.?5B; supplementary material Fig. S4E). Strikingly, ZNRF2-Ser19Ala stayed on membranes even when cells were stimulated with IGF1 (Fig.?5A). Collectively these findings display that as well as advertising binding to 14-3-3, the phosphorylation of Ser19 counteracts the N-myristoyl-mediated focusing on of ZNRF2 to membranes. Much like ZNRF2, ZNRF1-GFP was in both membrane and soluble fractions of transfected cell lysates, while GFP-ZNRF1 lacking the N-myristoyl group was entirely soluble (supplementary material Fig. S3F). However, in contrast to ZNRF2, we observed no reversible membrane-to-cytosol shuttling upon IGF1 activation of either ZNRF1-GFP (Fig.?5A) or endogenous ZNRF1 (supplementary material Fig. S3C). ZNRF1-GFP showed a more membranal localization in cells growing under 10% FBS conditions, compared to ZNRF2, whose localization was more diffuse (supplementary SEMA3F material Fig. S4E). ZNRF1 and ZNRF2 are co-purified with Ubc13 and Na+/K+ATPase Towards understanding the cellular functions of ZNRF2 and ZNRF1, we aimed to identify their interacting proteins. N-myristoylated ZNRF2-GFP and ZNRF1-GFP, non-myristoylated GFP-ZNRF2 and GFP-ZNRF1, and GFP only, were isolated from lysates of transiently-transfected HEK293 cells using GFP-Trap beads. After SDS-PAGE, strong bands in the molecular weights expected for the GFP-tagged proteins and.