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FIGURE 1.
Primary structure of X. laevis NARF and association between NARF and NLK. A, Xenopus NARF (xNARF, accession number DQ011285) and its orthologue from human RING finger protein 138, isoform 1 (hRNF138, isoform 1, accession number NP 057355), mouse RNF138, isoform 1 (mRNF138, isoform 1, accession number NP 997506), and rat RIKEN cDNA (accession number XP 228926) are shown aligned. Identical residues are shaded and the RING finger motif is boxed. B, 293 cells were transfected with the indicated T7-NARF and FLAG-NLK expression plasmids. WT, wild-type NLK; ΔN, amino terminus-truncated NLK; ΔC, carboxyl terminus-truncated NLK. Immunoprecipitation (IP) was carried out with anti-FLAG antibody from whole cell lysates. Co-immunoprecipitation of T7-NARF with FLAG-NLK was visualized by Western blot (WB) analysis with anti-T7 antibody (top panel). Expression levels of each of the introduced recombinant proteins were apparently equivalent, as judged from Western blotting analysis using whole cell extracts with either anti-T7 antibody for T7-tagged NARF (second panel) or anti-FLAG antibody for FLAG-tagged NLKs (third panel). Immunoprecipitated FLAG-NLKs were also equivalent (bottom panel). C, cell lysates were prepared from CaCO-2 and SW480 cells and used for immunoprecipitation with anti-NLK rabbit polyclonal antibody. Rabbit normal IgG was used as control for immunoprecipitation. Co-immunoprecipitation of NARF was visualized by Western blotting with anti-NARF antibody.
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FIGURE 2.
NARF exhibits E3 ubiquitin-ligase activity in cooperation with the ubiquitin conjugating enzyme, E2-25K. A, E2 ubiquitin-conjugating enzyme E2-25K was identified as a NARF-associating protein by LC-MS/MS analysis. Interaction between NARF and E2-25K was confirmed in 293 cells transiently expressing FLAG-NARF and HA-E2-25K. Whole cell lysates were subjected to immunoprecipitation (IP) with anti-FLAG antibody, followed by Western blotting analysis (WB) with anti-HA antibody to detect HA-E2-25K co-immuno-precipitated with FLAG-NARF (top panel). B, in vitro ubiquitylation assay was performed with bacterially expressed GST-NARF wild-type (WT) or RING finger domain mutant (CA). GST-NARF was incubated with purified rabbit E1, bovine ubiquitin, and human E2-25K at 30 °C for 0, 30, 60, or 90 min. The reaction mixtures were resolved by SDS-PAGE, and Western blotting analysis with both anti-Ub antibody (left panel) and anti-GST antibody (right panel) detected the state of GST-NARF-WT poly-ubiquitylation. C, in vitro ubiquitylation assay of GST-NARF was performed with a panel of E2-conjugating enzymes including E2-25K, E2-14K, UbcH3, UbcH5a, UbcH5b, UbcH5c, UbcH6, UbcH7, and UbcH10. The reaction mixtures were incubated for 0 (–) or 120 min (+). Poly-ubiquitylated GST-NARF was detected by Western blotting analysis with anti-Ub antibody (left panel) and anti-GST antibody (right panel).
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FIGURE 3.
NARF ubiquitylates the transcription factors TCF/LEF. A, 35S-labeled recombinant TCF4 or LEF1 was prepared by an in vitro transcription/translation system, and used as a substrate for an in vitro ubiquitylation assay with GST-NARF wild-type (GST-NARF-WT) or RING finger domain mutant (GST-NARF-CA). Poly-ubiquitylated 35S-labeled TCF4 and LEF1 were detected by autoradiography (lanes 3 and 7). B, T7-TCF4 or T7-LEF1 were co-expressed with HA-ubiquitin (HA-Ub) and FLAG-NARF in 293 cells. T7-TCF4 or LEF1 were immunoprecipitated by anti-T7 antibody from whole cell lysates, and the poly-ubiquitylated states of each were detected by Western blot (WB) analysis with anti-HA antibody (top panels, lanes 3 and 7). Western blot analysis with anti-T7 antibody (middle panels) or anti-FLAG antibody (bottom panels) using whole cell lysates confirmed that there were equivalent levels of expressed recombinant proteins in each experiment. C, 293 cells were incubated with 10 μM of the proteasome inhibitor MG132 for 4 h, and whole cell lysates were analyzed by Western blot analysis with anti-TCF4 antibody (upper left panel) or anti-β-actin antibody (lower left panel). Endogenous TCF4 was immunoprecipitated (IP) with anti-TCF4 antibody, and analyzed with both anti-TCF4 antibody and anti-Ub antibody (two right panels).
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FIGURE 4.
NLK augments the ubiquitylation activity of NARF against TCF/LEF. A, T7-TCF4/LEF1, FLAG-NARF, HA-ubiquitin (Ub), and FLAG-NLK wild-type (WT) or kinase-negative mutant (KN) were co-expressed in 293 cells. T7-TCF4 or T7-LEF1 were immunoprecipitated (IP) by an anti-T7 antibody from whole cell lysates and poly-ubiquitylated T7-TCF4 or T7-LEF1 were detected by Western blotting analysis (WB) with anti-HA antibody (top panels). Western blotting analysis with anti-T7 antibody (middle panels) or anti-FLAG antibody (bottom panels) using whole cell lysates confirmed that there were equivalent levels of expressed recombinant proteins in each experiment. B, HA-NARF and T7-TCF4 or T7-LEF1 were co-expressed with FLAG-NLK wild type (WT) or kinase-negative mutant (KN) in 293 cells. T7-TCF4 or T7-LEF1 were immunoprecipitated with anti-T7 antibody from whole cell lysates, and the association of HA-NARF with T7-TCF4 or T7-LEF1 was detected by anti-HA antibody (top panels). Western blotting analysis with anti-FLAG antibody (second panels from top), anti-HA antibody (second panels from bottom), or anti-T7 antibody (bottom panels) using whole cell lysates confirmed that there were equivalent levels of expressed recombinant proteins in each experiment.
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FIGURE 5.
NLK and NARF coordinate the degradation of TCF/LEF. A, T7-TCF4 or T7-LEF1 was expressed with NARF and/or NLK in 293 cells. T7-TCF4 or T7-LEF1 were metabolically labeled with [35S]methionine and cysteine, and chased for the indicated time periods. Cells were lysed in RIPA buffer, and [35S]T7-TCF4 or [35S]T7-LEF1 were immunoprecipitated with anti-T7 antibody. [35S]T7-TCF4 or [35S]T7-LEF1 were detected by autoradiography (left panels) and quantified from the intensity of the visualized bands (right panels). The half-life of TCF4 was calculated from a linear plot of the rate of [35S]TCF4 decay in cells. B, the expression of endogenous NARF was suppressed by siRNA. 293 cells were transfected with siRNAs and treated with 10 μM MG132 or Me2SO for 4 h. Control siRNA is targeted for luciferase. Expression of endogenous TCF4 and NARF were examined by Western blotting (WB) analysis with anti-TCF4 antibody (top panel) and anti-NARF peptide antibody (middle panel), respectively. β-Actin was used as a loading control (bottom panel).
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FIGURE 6.
NARF negatively regulates the Wnt signaling pathway. A, 293 cells were transiently transfected with the expression vectors for Wnt-1, a luciferase reporter linked to the Wnt-responsive TCF-binding sites (3x(TCF)/Luc) or the mutated sites (3x(mTCF)/Luc), and either NARF wild-type (NARF-WT) or RING finger domain mutant (NARF-CA). Cells were harvested after 24 h post-transfection and assayed for luciferase activity. The transfection efficiency was normalized with the activity of co-transfected Renilla luciferase vector controlled by the EF-1α promoter. Values are expressed as the –fold increase in luciferase activity relative to the level of activity with reporter plasmid alone. B, synthetic mRNAs encoding β-catenin (100 pg) or Siamois (0.5 pg) were microinjected with NARF-WT mRNA (500 and 700 pg) or RING finger domain mutant mRNA (500 and 750 pg) into the ventral equatorial region of the 4-cell stage Xenopus embryos to induce secondary axis formation as indicated. The representative embryos are shown (left panels). The ectopic axis formations were counted at the tadpole stage and expressed as the ratio of axis-formed embryos to total examined numbers (n = 30, axis duplication %, right panel). C, RT-PCR analysis in animal caps. Synthetic mRNA encoding β-catenin (100 pg) was microinjected with NARF-WT mRNA (50 and 300 pg) or RING finger domain mutant mRNA (50 and 300 pg) into the animal pole of two blastomeres at the 2-cell stage Xenopus embryos. Animal cap explants were removed at the blastula stage. Total RNAs were prepared and analyzed by RT-PCR for the expression of Wnt target genes, Xnr3 and Siamois. Histone was used as a loading control. Emb indicates whole embryo control with (+RT) or without (–RT) at the RT step.
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FIGURE 7.
Knockdown of the endogenous NARF enhances Wnt-3a-dependent DKK-1 and Axin2 gene expression. A, HeLaS3, transiently transfected with control (siControl) or specific human NARF siRNA (siNARF), were treated with Wnt-3A conditioned medium for the indicated periods, and total cell lysates were subjected to SDS-PAGE and Western blotting (WB) analysis with anti-NARF antibody (upper panel). The loading control was carried out with an anti-β-actin antibody (lower panel). B and C, the effect of siNARF on Wnt-3A-induced DKK-1 and Axin2 mRNA expression. siRNA-treated HeLaS3 cells were incubated with Wnt-3A CM for the indicated time periods. Semiquantitative multiplex RT-PCR was performed for 30 cycle amplifications with DKK-1 primers or 35 cycle amplifications with Axin2 primers, and Quantum RNA 18 S internal standards II. Representative SYBR Green I-stained PCR products are shown (left panel). DKK-1 and Axin2 expression were normalized to expression of the 18 S ribosomal RNA and presented as the ratio of fluorescence intensity of the DKK-1 or Axin2 versus 18 S bands (right panel). Data are shown as the mean ± S.D. of the three separate experiments.
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FIGURE 8.
A proposal model for the roles of NARF in the suppression of the Wnt-β-catenin signaling pathways.
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Supplemental Figure 1 Synthetic mRNAs encoding ~-catenin (100 pg) and NLK (500,
1000 pg) were microinjected with control or NARF morpholino-antisense oligonucleotide
(Mo, 40ng) into the ventral equatorial region offour-cell-stageXenopus embryos to induce
secondary axis formation as indicated. The ectopic axis formations were counted at the
tadpole stage and expressed as the ratio of axis-formed embryos to total examined numbers
(n=30, Axis duplication%).
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Supplemental Figure 2. NARF Mo specifically inhibits NARF protein synthesis.
Flag-tagged wild-type NARF (NARF-WT-Flag) mRNA (1000 pg) or Flag-tagged NARF bearing silent
mutations (NARF-mut-Flag) mRNA (1000 pg) were microinjected with Flag-tagged globin mRNA (250
pg) and either control Mo (20 ng) or NARF Mo (20 ng) into the animal pole of two blastomeres at the
two-cell stage Xenopus embryos. Animal cap explants were removed at blastula stage. Western blotting
analysis (WB) with anti-Flag antibody using whole celllysates confirmed that NARF Mo specifically
inhibited the translation ofNARF-WT, but not ofNARF-mut or globin. The Mo sequences were as
follows: NARF Mo, 5'-AACATGACATCGCTTCAGCCATAAG-3'; control Mo, 5'CCTCTTACCTCAGTTACAATTTATA-
3' (Gene Tools, LLC).
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