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. 2003 Dec;23(23):8462-70.
doi: 10.1128/MCB.23.23.8462-8470.2003.

Requirement for a nuclear function of beta-catenin in Wnt signaling

Feng Cong  1 , Liang Schweizer, Mario Chamorro, Harold Varmus
Affiliations
Free PMC article

Requirement for a nuclear function of beta-catenin in Wnt signaling

Feng Cong et al. Mol Cell Biol. 2003 Dec.
Free PMC article

Abstract

Wnt signaling stabilizes beta-catenin, which in turn influences the transcription of Wnt-responsive genes in conjunction with T-cell factor (TCF) transcription factors. At present, there are two models for the actions of beta-catenin. The conventional nuclear model suggests that beta-catenin acts in the nucleus to form a heterodimeric transcriptional factor complex with TCF, with TCF providing DNA-specific binding and the C and N termini of beta-catenin stimulating transcription. The alternative cytoplasmic model postulates that beta-catenin exports TCF from the nucleus to relieve its repressive activity or activates it in the cytoplasm. We have generated modified forms of beta-catenin and used RNA interference against endogenous beta-catenin to distinguish between these models in cultured mammalian and Drosophila cells. We show that the VP16 transcriptional activation domain can replace the C terminus of beta-catenin without loss of function and that the function of beta-catenin is compromised by fusion to a transcriptional repressor domain from histone deacetylase, favoring the direct effects of beta-catenin in the nucleus. Furthermore, membrane-tethered beta-catenin requires interaction with the adenomatous polyposis coli protein but not with TCF for its function, whereas untethered beta-catenin requires binding to TCF for its signaling activity. Importantly, by using RNA interference, we show that the signaling activity of membrane-tethered beta-catenin, but not free beta-catenin, requires the presence of endogenous beta-catenin, which is able to accumulate in the nucleus when stabilized by the binding of the beta-catenin degradation machinery to the membrane-tethered form. All of these data support a nuclear model for the normal function of beta-catenin.

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Figures

FIG. 1.
FIG. 1.
Functional substitution of the C terminus of β-catenin by the VP16 transcriptional activation domain. (A) Schematic representation of β-catenin constructs. β-Catenin is composed of the N-terminal domain (□), 12 armadillo repeats (▨), and the C-terminal domain (░⃞). Amino acid residues 675 to 781 were deleted to form β-catenin ΔC, and residues 422 to 490 of VP16 were added to the amino terminus of β-catenin ΔC to form VP16-β-catenin ΔC. All β-catenin derivatives were tagged with HA epitopes at their C termini. (B) Activation of TOP-FLASH by β-catenin (β), β-catenin ΔC (βΔC), and VP16-β-catenin ΔC (VP16-βΔC). Human 293 cells were transfected with indicated plasmids, together with TOP-FLASH and a CMV-Renilla luciferase reporter, and the luciferase activities were determined as described in Materials and Methods. Expression of β-catenin constructs was monitored by immunoblotting with anti-HA antibodies (below the chart). (C) Induction of the DKK1 gene by β-catenin proteins. 293 cells were transfected with indicated plasmids. The abundance of DKK1 mRNA was determined by reverse transcription and quantitative real-time PCR. The results were normalized by the level of GAPDH. vec, Vector.
FIG. 2.
FIG. 2.
Loss of signaling activity by fusing the transcriptional repression domain from an HDAC to β-catenin. (A) Schematic representation of HDAC4-β-catenin chimeras. The catalytic domain (amino acids 663 to 1083) of HDAC4 was fused to the N terminus of β-catenin (HDAC4-β). Two residues (His-802 and His-803) essential for the catalytic activity of HDAC4 were mutated in HDAC4mut-β. All β-catenin derivatives are tagged with HA epitopes at their C termini. (B) Activation of TOP-FLASH by β-catenin and HDAC4-β-catenin chimeras. 293 cells were transfected with TOP-FLASH together with indicated expression constructs. β-Catenin expression was determined by immunoblotting with anti-HA antibodies (below the chart). vec, Vector.
FIG. 3.
FIG. 3.
Differential requirements of TCF and APC binding for activities of untethered and membrane-tethered β-catenins. (A) Stabilization of endogenous β-catenin by membrane-tethered β-catenin. HA-tagged connexin-β-catenin ΔC (CnxβΔC) was expressed in COS cells. Cells were incubated with rabbit polyclonal anti-HA antibody and mouse monoclonal anti-β-catenin antibody. The epitope recognized by anti-β-catenin antibody was missing in CnxβΔC. Cells were next stained with FITC-conjugated, anti-rabbit immunoglobulin and Texas red-conjugated, anti-mouse immunoglobulin antibodies. Note that β-catenin is localized on the plasma membrane in cells without CnxβΔC (the left side of pictures), whereas β-catenin accumulated in the nucleus in cells with CnxβΔC (the right side of pictures). (B) Schematic representation of β-catenin mutants with defective TCF or APC binding sites. In βm1, Lys 435, crucial for TCF binding, was changed to Ala. In βm2, W383 and R386, crucial for APC binding, were both changed to Ala. Wild-type β-catenin, βm1, and βm2 were also fused to the C terminus of the transmembrane domain of connexin to form Cnxβ, Cnxβm1, and Cnxβm2, respectively. All β-catenin constructs were fused with HA epitopes at their C termini. (C) Differential requirements of TCF and APC binding for untethered and membrane-tethered β-catenin. 293 cells were transfected with TOP-FLASH, and the indicated expression constructs and luciferase activities were assayed. The expression of β-catenin mutants was followed by immunoblotting with anti-HA antibodies (below the chart). vec, Vector.
FIG. 4.
FIG. 4.
Requirement of endogenous β-catenin for the stimulatory effect of membrane-tethered β-catenin/plakoglobin on luciferase reporters. (A) Effect of β-catenin-specific siRNA on the expression of endogenous β-catenin. 293 cells or NIH 3T3 cells were treated with control (c) and human or mouse-specific β-catenin (hβ or mβ) siRNA, and the expression of endogenous β-catenin was determined by immunoblotting with anti-β-catenin antibodies. Equal loading was confirmed by Western blotting with anti-α-tubulin antibodies. (B) Requirement of endogenous β-catenin for the signaling activity of membrane-tethered plakoglobin (CnxPkg) in 293 cells. 293 cells were transfected with control (▪) or human β-catenin-specific (░⃞) siRNA, together with TOP-FLASH and the indicated expression constructs, and the luciferase activities were determined 48 h after transfection. (C) Requirement of endogenous β-catenin for the signaling activity of membrane-tethered β-catenin in NIH 3T3 cells. To achieve high sensitivity in NIH 3T3 cells, β-catenin was cotransfected with a mouse LEF-1 expression plasmid and a LEF-1 luciferase reporter that contains multiple LEF-1 binding sites. NIH 3T3 cells were transfected with control (▪) or mouse β-catenin-specific (░⃞) siRNA, together with the indicated expression constructs, and assayed for luciferase activity. Amino acid residues 675 to 781 of β-catenin were deleted from Cnxβ to form CnxβΔC. (D) Requirement of endogenous armadillo for the signaling activity of membrane-tethered β-catenin in Drosophila S2 cells. S2 cells were grown in six-well plates and treated with control (▪) or armadillo-specific (░⃞) dsRNA at a concentration of 15 μg/well. Cells were then transfected with LEF-luciferase and LEF-1 expression constructs with the indicated plasmids that encode various forms of plakoglobin and β-catenin. Amino acid residues 677 to 781 and 583 to 781 of β-catenin were removed from Cnxβ to form CnxβΔC1 and CnxβΔC2. (E) Requirement of endogenous β-catenin for membrane-tethered plakoglobin-induced DKK1 expression. 293 cells were transfected with control siRNA (▪) or human β-catenin-specific siRNA (░⃞) with the indicated expression plasmids. The expression of DKK1 gene was determined by quantitative real-time PCR after reverse transcription. vec, Vector.
FIG. 5.
FIG. 5.
Signaling activities of β-catenin C-terminal truncation mutants. (A) Residual signaling activity of β-catenin C-teminal truncation mutants. Human β-catenin C-terminal truncation mutants were generated. The approximate truncation sites in arm043 and armXM19 mutant alleles from Drosophila are labeled (upper panel). In βΔC1, the C terminus of β-catenin (amino acids 677 to 781) was removed. In βΔC2, the C terminus and the last two armdillo repeats (amino acid 583 to 781) were removed. β-Catenin mutants were also fused to the C terminus of the transmembrane domain of connexin. All β-catenin derivatives in Drosophila expression constructs were fused with GFP at their C termini. S2 cells were treated with control (▪) or armadillo-specific (░⃞) dsRNA and transfected with the indicated plasmids as in Fig. 4D. (B) The N terminus of β-catenin contains transcriptional activation activity. β-Catenin-LEF1ΔN chimeric proteins were generated (upper panel). A LEF-1 N-terminal truncation mutant (lacking amino acids 1 to 57) was fused at its N terminus with either the N terminus (amino acids 1 to 218) or C terminus (amino acids 659 to 781) of β-catenin. LEF1-β-catenin chimeras were fused with an HA epitope at the C termini. The effects of LEF1-β-catenin chimera on TOP-FLASH were tested in 293 cells; expression of these chimeras was examined by Western blotting with anti-HA antibodies (below the chart). vec, Vector.
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