Nuclear localization and formation of beta-catenin-lymphoid enhancer factor 1 complexes are not sufficient for activation of gene expression

Abstract

In response to activation of the Wnt signaling pathway, beta-catenin accumulates in the nucleus, where it cooperates with LEF/TCF (for lymphoid enhancer factor and T-cell factor) transcription factors to activate gene expression. The mechanisms by which beta-catenin undergoes this shift in location and participates in activation of gene transcription are unknown. We demonstrate here that beta-catenin can be imported into the nucleus independently of LEF/TCF binding, and it may also be exported from nuclei. We have introduced a small deletion within beta-catenin (Delta19) that disrupts binding to LEF-1, E-cadherin, and APC but not axin. This Delta19 beta-catenin mutant localizes to the nucleus because it may not be efficiently sequestered in the cytoplasm. The nuclear localization of Delta19 definitively demonstrates that the mechanisms by which beta-catenin localizes in the nucleus are completely independent of LEF/TCF factors. beta-Catenin and LEF-1 complexes can activate reporter gene expression in a transformed T-lymphocyte cell line (Jurkat) but not in normal T lymphocytes, even though both factors are nuclear. Thus, localization of both factors to the nucleus is not sufficient for activation of gene expression. Excess beta-catenin can squelch reporter gene activation by LEF-1-beta-catenin complexes but not activation by the transcription factor VP16. Taken together, these data suggest that a third component is necessary for gene activation and that this third component may vary with cell type.

FIG. 1

Amino acid sequence and structure of the arm repeat array of wild-type β-catenin and deletion mutants. (A) Schematic of wild-type β-catenin, a 19-aa deletion mutant of β-catenin (Δ19); MT10 β-catenin, which contains only the first nine arm repeats and no flanking sequences; and ΔNΔ19, which is identical to Δ19 but contains a deletion of the first 148 aa. (B) Amino acid alignment of the arm repeats of β-catenin. According to the recently solved structure for β-catenin, each arm repeat consists of three alpha-helices, which are denoted by H1, H2, and H3 (28). The sequence deleted in the Δ19 β-catenin mutant is in boldface type and underlined.

FIG. 2

Deletion of 19 aa within the arm repeat array of β-catenin disrupts interaction with LEF-1, APC, and E-cadherin but not axin. (A) Wild-type (wt) β-catenin and a 19-aa deletion mutant of β-catenin (Δ19) were produced by a coupled transcription/translation system (Promega) in the presence of [³⁵S]methionine. These proteins were incubated with recombinant GST–LEF-1 or GST alone in a GST pull-down binding assay (see Materials and Methods). The first two lanes indicate the amount of β-catenin protein added (10% input). Glutathione beads are able to cosediment wild-type β-catenin with GST–LEF-1 but not GST alone. In contrast, Δ19 β-catenin is unable to bind to GST–LEF-1. (B) Whole-cell extracts were prepared from 293 cells that had been transiently transfected with the indicated expression plasmids (see Materials and Methods). Anti-Myc MAb was used to coimmunoprecipitate β-catenin-associated proteins. A Western blot of the immunoprecipitates was probed with LEF-1 antisera. LEF-1 can be detected in coimmunoprecipitates with wild-type (lane 1) but not Δ19 β-catenin (lane 2). Purified recombinant LEF-1 protein (5 μg) added to cell extract prior to immunoprecipitation (lanes 5 and 6) is able to interact only with wild-type β-catenin and not even weakly with Δ19 β-catenin. In lanes 3 and 4, a control eukaryotic expression plasmid was cotransfected with either LEF-1 (lane 3) or wild-type β-catenin (lane 4) to show that LEF-1 coimmunoprecipitates specifically by association with β-catenin and not nonspecifically with the anti-Myc MAb. The same Western blot was stripped and reprobed with anti-Myc MAb to determine that the protein levels of wild-type and Δ19 β-catenin were equivalent. (C) In vitro-translated wild-type or Δ19 β-catenin was incubated with two purified APC fragments (APC2 and APC3), an axin fragment (Axin), or no protein (Cnt). Anti-Glu-Glu antibody and protein G-Sepharose beads were added in a coimmunoprecipitation assay. Ten percent of the amount of β-catenin protein added is shown (Tln). APC2 and axin are coimmunoprecipitated with wild-type β-catenin but only axin is coimmunoprecipitated with Δ19 β-catenin. (D) In vitro-translated wild-type or Δ19 β-catenin and the C-terminal tail of E-cadherin were incubated with anti-Myc MAb and protein G-Sepharose beads in a coimmunoprecipitation assay. E-cadherin can be detected in coimmunoprecipitates with wild-type but not Δ19 β-catenin. The number of additional bands is due to partial translation products of β-catenin which are coimmunoprecipitated with anti-Myc MAb.

FIG. 3

Δ19 β-catenin localizes constitutively to the nucleus. Plasmids encoding Myc epitope-tagged wild-type β-catenin (A), MT10 (C), and Δ19 (D) were transiently transfected into Cos-1 cells. Subcellular localization was determined 48 h later by immunofluorescence with anti-Myc MAb to detect transfected β-catenin expression plasmids or anti-β-catenin MAb to detect endogenous β-catenin. In some cases, FITC antimouse or Texas red antimouse secondary antibody was used. (A to C) Wild-type β-catenin, endogenous β-catenin, and MT10 localize to various sites in the cytoplasm and the plasma membrane. Background levels are higher for MT10, since immunofluorescence of MT10 was weak and exposure times were longer than for the other β-catenin proteins. (D) In contrast to wild-type and MT10 β-catenin, Δ19 β-catenin localizes predominantly to the nucleus. DAPI staining of DNA indicates the locations of nuclei.

FIG. 4

β-Catenin does not interact with importin α2 or importin β. A GST pull-down experiment with ³⁵S-labeled, in vitro-translated wild-type β-catenin, importin α2, and recombinant GST-importin β was performed. An N-terminal deletion mutant of importin α2 that removes the importin β interaction domain (ΔN importin α2) was included as a negative control for nonspecific binding. The first three lanes indicate the amount of importin α2 and β-catenin proteins added (10% input). While full-length (FL) importin α2 readily interacts with GST-importin β, β-catenin does not, either in the presence or absence of importin α2.

FIG. 5

Localization of β-catenin in normal human peripheral blood lymphocytes and Jurkat T lymphocytes. (A and B) PBMCs were transfected with the indicated expression constructs. Four hours after transfection, cell preparations were treated with PMA and ionomycin or treated with ethanol as a mock control (untreated) for 3 h. (C) Jurkat cells were transiently transfected with the indicated β-catenin constructs, and subcellular localization was determined 24 h later. Transiently expressed β-catenin proteins were detected with anti-Myc MAb and FITC-antimouse secondary antibody. DAPI staining of DNA indicates the locations of nuclei. (A) In untreated cells, both the wild type and MT10 are cytoplasmic while Δ19 is nuclear. (B) When cells are treated with PMA and ionomycin, both wild-type β-catenin and MT10 localize to the nucleus. (C) Wild-type, MT10, and Δ19 β-catenin all localize to the nucleus in Jurkat cells.

FIG. 6

Wild-type and Δ19 β-catenins are exported from the nucleus in normal human peripheral blood lymphocytes. PBMCs were transfected with the indicated expression constructs. (A) Six hours prior to harvest, cell preparations were treated with PMA and ionomycin (Control). (B) Three hours prior to harvest, cell preparations were treated with actinomycin D and cycloheximide. Transiently expressed β-catenin was detected with anti-Myc MAb and FITC–anti-mouse secondary antibody. DAPI staining of DNA indicates the locations of nuclei. In cells treated with PMA and ionomycin, both wild-type and Δ19 β-catenins are localized to the nucleus. Subsequent treatment with actinomycin D and cycloheximide inhibits nuclear import and protein synthesis. With these treatments, wild-type and Δ19 β-catenins are exported to the cytoplasm.

FIG. 7

A direct interaction between β-catenin and LEF-1 is necessary but not sufficient to activate gene expression. Wild-type, Δ19, or MT10 β-catenin expression plasmids were cotransfected with the Gal4 DNA binding domain (DBD) fused to LEF-1 aa 1 to 256 (G4LEF) and a reporter plasmid containing five tandemly repeated Gal4 UAS directing luciferase reporter gene (G4 UAS/Luc) expression in Cos-1 and Jurkat cells and PBMCs. A summary of the β-catenin localization in each of these cell lines is shown in the upper right corner of each panel. (A) Five micrograms of β-catenin, 5 μg of G4LEF expression plasmids, and 1 μg of reporter plasmid were transfected into Cos-1 cells. (B) Seven hundred fifty nanograms of β-catenin and 50 ng of G4LEF expression plasmids or 750 ng of G4VP16 expression plasmid and 1 μg of reporter plasmid were transfected into Jurkat cells. In the absence of β-catenin, the range of luciferase activity varied from 574 to 825. (C) The indicated expression plasmids (10 μg) were cotransfected with 10 μg of reporter plasmid into PBMCs. P + I, treatment of cell preparations with PMA plus ionomycin for 3 h prior to harvest of cells; ∗, use of the Top TK reporter plasmid in place of the G4 UAS/Luc reporter plasmid. A plasmid (0.5 μg) containing the lacZ gene under the control of the CMV promoter was cotransfected as an internal control for transfection efficiency. G4LEF (aa 80 to 256), which does not bind to β-catenin, was used as a negative control (A and B). An empty eukaryotic expression plasmid was used to equalize the amount of DNA used in each transfection. Cells were harvested at 15 to 24 h posttransfection, and cell lysates were analyzed for luciferase and β-galactosidase activity. Wild-type β-catenin and LEF-1 transactivated the reporter gene construct in Cos-1 and Jurkat cells, while neither Δ19 nor MT10 cooperated with LEF-1 to activate gene expression. In addition, the level of transcription activation of β-catenin–LEF-1 complexes in Jurkat cells was higher than that observed with G4VP16 (750 ng). β-Catenin and G4LEF-1 or full-length LEF-1 (FL LEF-1) did not transactivate reporter gene expression in PBMCs, although G4VP16 (10 μg) was able to transactivate the reporter gene in PBMCs. Error bars were calculated by using the average of duplicate points (A and C) or triplicate points (B). Values shown are from one of three replicate experiments.

FIG. 8

Excess β-catenin squelches transactivation of β-catenin and LEF-1 but not transactivation by VP16. (A) Wild-type β-catenin expression plasmid (750 ng) was cotransfected with 50 ng of G4LEF (aa 1 to 256), 1 μg of G4 UAS/Luc reporter plasmid, and the following plasmids used as competitors: 4- and 20-fold (3 and 15 μg, respectively) excess amounts of Δ19 β-catenin, control plasmid (empty eukaryotic expression plasmid), and ΔNΔ19 β-catenin (see Fig. 1) and a 60-fold (3 μg) excess amount of ΔNLEF (hLEF-1 aa 67 to 399). Excess amounts of Δ19 and ΔNΔ19 β-catenin, but not control plasmid or ΔNLEF, were able to squelch wild-type β-catenin–LEF-1 reporter gene activation. (B) G4VP16 (750 ng) was cotransfected with 1 μg of G4 UAS/Luc reporter plasmid and the following plasmids used as competitors: 4- and 20-fold (3 and 15 μg, respectively) excess amounts of control plasmid or a wild-type β-catenin construct. Wild-type β-catenin does not squelch G4VP16 transactivation. A plasmid (0.5 μg) containing the lacZ gene under the control of the CMV promoter was cotransfected as an internal control for transfection efficiency. Error bars were calculated by using the average of duplicate points. Values shown are from one of three replicate experiments. Fold activation was calculated by comparing levels of luciferase activity to the G4 UAS/Luc reporter alone.