Phosphorylation-dependent binding of 14-3-3 to ZNRF2 in response to agonists. ZNRF2-GFP (A) and endogenous ZNRF2 (B), isolated from HEK293 cells that had been exposed to various stimuli and inhibitor combinations, were tested for binding to 14-3-3s in a far-western overlay and for co-immunoprecipitation with endogenous 14-3-3s. ZNRF2 expression and phosphorylation (pSer19, pSer82 and pSer145) was analyzed by western blotting of cell lysates (30 µg), as was the phosphorylation status of AMPK, PKB, ERK1/2, ACC and VASP. SS, serum-starved conditions. (C) ZNRF2-GFP, transiently expressed in HEK293 cells grown in the presence of 10% FBS, was immunoprecipitated and dephosphorylated with lambda-phosphatase (with or without 50 mM EDTA to inhibit dephosphorylation). Binding of 14-3-3 to ZNRF2 was analyzed by 14-3-3 far-western overlay. (D) ZNRF2-GFP isolated from transfected HEK293 cells stimulated with IGF1 in the presence or absence of the PI3K inhibitors (PI-103, Wortmannin, LY294002), PKB inhibitor (AKTi1/2), mTORC1 (rapamycin), p90RSK inhibitor (BI-D1870), PKC inhibitor (Gö6983), an ERK1/2 pathway inhibitor (PD184352) and an mTOR kinase inhibitor (Ku-0063794) was tested for binding to 14-3-3 in a far-western assay, and for co-immunoprecipitation with endogenous 14-3-3s (K19 antibody). Western blotting was performed as in B. (E) GFP-transfected cells were stimulated with PMA in the presence or absence of the PKC inhibitors (Gö6983 and Gö6976), p90RSK inhibitor (BI-D1870), ERK1/2 pathway inhibitors (PD184352 and U0126) and p38 MAPK inhibitors (SB203580 and Birb0796).
Effect of mutations in ZNRF2 on its binding to 14-3-3. (A) Wild-type ZNRF2-GFP or single/double serine to alanine mutants were isolated from transfected HEK293 cells that had been stimulated with IGF1, PMA or forskolin as indicated. The ZNRF2-GFP proteins were tested for their phosphorylation and 14-3-3 binding, as for Fig. 1. (B) Similar to A, but including mutated GST-ZNRF2 phosphorylated with PKBα, SGK1, p90RSK, PKA, PKCα and PKCζ, as indicated. WT, wild type.
N-myristoylation of ZNRF2 and its effect on phosphorylation, activity, subcellular localization and protein-protein interactions. (A) Alignment of the N-terminus of ZNRF2 homologues showing the myristoylation consensus motif. (B) Detection of myristic acid in ZNRF2 by mass spectrometric analysis (Orbitrap). MS data from a tryptic digest of ZNRF2-GFP isolated from HEK293 cells was searched using Mascot allowing for the myristoyl N-terminal glycine modification, and the scores for the peptide shown, found in two samples, were 43 and 66 with the significance cutoff (P<0.05) being 19. The difference between the expected mass of unmodified GAKQSGPAAANGR (1183.6058 Da) and observed mass (m/z = 697.9107:2+ = 1393.8068 Da) is 210.2010 Da, which corresponds to the mass of the myristoyl modification (210.1984 Da). The y-ions generated by MS2 fragmentation indicate the sequence from the C-terminal end of the peptide, whereas the b-ions are consistent with an N-myristoyl group. (C) Subcellular localization of ZNRF2 in HeLa and HEK293 cells transfected with GFP-tagged wild-type or Gly2Ala-ZNRF2 (G2A). Cells were stained with DAPI (to label nuclei). At least five sets of cells were analyzed and representative images are shown. Scale bar: 10 µm. (D) HEK293-Flp-In-Trex cells stably expressing GFP-tagged wild-type or Gly2Ala-ZNRF2 were fractionated as described in the Materials and Methods, and cell lysates were subjected to western blotting with the indicated antibodies.
Reversible membrane-to-cytosol translocation of ZNRF2. (A) HEK293 cells exposed to various stimuli and inhibitor combinations were fractionated into cytosol (CE) and membrane (ME) extracts by ultracentrifugation and the extracts 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 various 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 used for live cell imaging. Cells were treated with 1 µM GDC-0941 (C) or 10 µM MK-2206 for 60 min (D). Images were taken before and after the treatments. Scale bars: 10 µm.
Effect of Ser19Ala mutation on the reversible membrane-to-cytosol translocation of ZNRF2. (A) As in 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 stimulation. Scale bar: 10 µm. (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) extracts by ultracentrifugation, and the extracts were analyzed by western blotting with the indicated antibodies.
Interactions of ZNRF1 and ZNRF2 with Na+/K+ATPase subunit α1, Ubc13 and 14-3-3. (A) HEK293 cells were transiently transfected with either N- or C-terminally tagged ZNRF1 or ZNRF2. Proteins were immunoprecipitated from 50 mg of lysates, resolved on a 4-12% gradient NuPAGE gel and stained with colloidal Coomassie Brilliant Blue. The excised gel pieces were digested with trypsin and analyzed by mass spectrometry. Identified proteins are shown on the right-hand side of the gel. (B) Extracts of cells stably expressing GFP or C-terminally GFP-tagged ZNRF1 or ZNRF2 were subjected to immunoprecipitation and precipitates were analyzed by western blotting with the indicated antibodies. (C) Full-length ZNRF2 or ZNRF2 deletion fragments (the amino acid numbers of the fragment are indicated) with a C-terminal GFP tag were tested for the binding to endogenous Na+/K+ATPase α1, Ubc13 and 14-3-3s. (D) As in C, except that ZNRF1 plasmids were used. (E) Deletion fragments of Na+/K+ATPase subunit α with a C-terminal FLAG tag were tested for the binding to ZNRF1-GFP or ZNRF2-GFP. (F) Extracts of HEK293 cells were analyzed by size exclusion chromatography on a HiLoad 26/60 Superdex 200 column in buffer containing 0.2 M NaCl, and every third fraction was denatured and analyzed by western blotting with the indicated antibodies. The elution positions of Dextran blue (2000 kDa, in the void volume of this column), thyroglobulin (670 kDa), bovine gamma-globulin (158 kDa) and chicken ovalbumin (44 kDa) are shown.
ZNRF1 can ubiquitylate Na+/K+ATPase α1 in vitro. (A) In vitro ubiquitylation reactions of ZNRF2 and ZNRF1 with (WT) ubiquitin, K48R ubiquitin or K63R ubiquitin were analyzed by western blotting (left) or Coomassie Blue staining (CBB, right). (B) The indicated proteins were immunoprecipitated from HEK293 cells and used for in vitro ubiquitylation reactions. (C) In vitro ubiquitylation reaction of FLAG-Na+/K+ATPase α1 purified from HEK293-Flp-In-TRex cells using GST-ZNRF1, GST-ZNRF2 or GST-ZNRF2-C199A. (D) As in C, except that the ZNRF2-GFP or GFP control was purified from HEK293-Flp-In-TRex cells treated with DMSO or PI-103 (1 µM, 30 min). UbcH5a was used as the E2. Reactions were analyzed by western blotting using anti-ubiquitin antibody (E) As in D, except Ubc13-Uev1a was used as the E2. The arrows indicate ubiquitylated FLAG-Na+/K+ATPase α1. Reactions were analyzed as in D. (F) The indicated fragments of Na+/K+ATPase α1 were purified either from HEK293 cells or E. coli and used for in vitro ubiquitylation reactions using either UbcH5a or Ubc13-Uev1a as the E2 and GFP-ZNRF2 as the E3. Reactions were analyzed by western blotting using K48- or K63-specific polyubiquitin chain antibodies.
ZNRF2 knockdown rescues ouabain-induced decrease of total and plasma membrane levels of Na+/K+ATPase α1. (A) HeLa cells were transfected with control (scrambled) or ZNRF2 siRNA and 56 h post-transfection, cells were treated with 50 nM ouabain for 16 h. Na+/K+ATPase α1 levels and ZNRF2 knockdown levels were analyzed by western blotting of cell lysates. GAPDH was used as an internal control. (B) As in A, except membrane surface proteins were biotinylated, purified using streptavidin-agarose and analyzed by western blotting for Na+/K+ATPase α1 levels. Total lysate fractions were analyzed for ZNRF2 and ZNRF1 levels as in A. (C) Signals from three experiments similar to B, with ouabain concentrations from 50 to 70 nM for 12 to 15 h, were quantified using the Image J program. **P<0.005 (as shown by a Student's t-test). (D) Working model for the regulation of ZNRF2 and its potential role in the regulation of Na+/K+ATPase α1.