Nuclear export of the APC tumour suppressor controls beta-catenin function in transcription - PubMed (original) (raw)

Nuclear export of the APC tumour suppressor controls beta-catenin function in transcription

Rina Rosin-Arbesfeld et al. EMBO J. 2003.

Abstract

The adenomatous polyposis coli (APC) protein is inactivated in most colorectal tumours. APC loss is an early event in tumorigenesis, and causes an increase of nuclear beta-catenin and its transcriptional activity. This is thought to be the driving force for tumour progression. APC shuttles in and out of the nucleus, but the functional significance of this has been controversial. Here, we show that APC truncations are nuclear in colorectal cancer cells and adenocarcinomas, and this correlates with loss of centrally located nuclear export signals. These signals confer efficient nuclear export as measured directly by fluorescence loss in photobleaching (FLIP), and they are critical for the function of APC in reducing the transcriptional activity of beta-catenin in complementation assays of APC mutant colorectal cancer cells. Importantly, targeting a functional APC construct to the nucleus causes a striking nuclear accumulation of beta-catenin without changing its transcriptional activity. Our evidence indicates that the rate of nuclear export of APC, rather than its nuclear import or steady-state levels, determines the transcriptional activity of beta-catenin.

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Figures

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Fig. 1. Nuclear export and import signals in APC. Top: structure of APC (drawn roughly to scale), with NESs and NLSs and binding motifs for β-catenin (15Rs, 20Rs) and for Axin indicated; ARD, armadillo repeat domain; MCR, mutation cluster region in which most type I truncations end; type II truncations end upstream of the first Axin-binding motif, but retain NES1506. Below: APC constructs used in this study; asterisks indicate alanine substitutions that inactivate the corresponding NESs in the mutants.

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Fig. 2. Subcellular distribution of APC in colorectal cancer cell lines. Confocal sections through colorectal cancer cells from lines with a wild-type (B and C) or truncated APC (A and D–F), stained with anti-M-APC and anti-β-catenin as indicated; staining against lamin was used to mark the nuclear envelopes (merges: green, APC; red, β-catenin or lamin). Arrowheads indicate nuclear APC or β-catenin. APC is nuclear in cells with APC type I truncations (D and E), but excluded from nuclei in cells with wild-type APC (B and C) or with APC type II truncations that retain NES1506 (F). Note that the short type I truncation of COLO320 cells is not recognized by anti-M-APC (see also Figure 4B).

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Fig. 3. Subcellular distribution of APC in colorectal carcinomas. Sections through colorectal carcinomas (D–F; two different tumours) and adjacent normal colonic mucosa (A–C), stained with anti-M-APC (red) and with hemalaun to mark the nuclei (blue); the image in (A) is taken at low magnification, to visualize the crypt axis. APC is cytoplasmic in normal cells above the crypt (A and C), but detectable in the crypt cell nuclei (B, arrowheads). In most carcinoma cells, APC is partly nuclear (D and E, arrowheads); high levels of granular cytoplasmic APC are seen in de-differentiated ‘mesenchymal’ cells at the invasive front (F, arrow).

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Fig. 4. Comparative TOPFLASH assays in colorectal cancer cell lines. (A) TCF-mediated transcription in colorectal cancer cell lines expressing wild-type or truncated APC, as indicated (codons of truncations are given above the bars); given are relative luciferase:Renilla values averaged from 3–6 independent experiments (error bars mark SDs; the Renilla values were obtained with pRL-CMV as an internal control, but essentially the same comparative values were obtained with pRL-TK and pRL-SV40). The TOPFLASH values are significantly lower in cell lines with activated β-catenin/wild-type APC or with APC type II truncations (retaining NES1506) compared with those with APC type I truncations (lacking NES1506). (B) Western blots of total cell extracts, probed with anti-M-APC (top) or with Ab-1 (bottom), to reveal full-length APC or APC truncations (arrowheads), and tubulin (arrows) as internal control. The short APC truncation in COLO320 cells is only detectable by Ab-1 (raised against an N-terminal peptide) but not by anti-M-APC (raised against the central third of APC). The levels of APC truncations in colorectal cancer cells are similar to one another, but appear to be generally higher than those of wild-type APC (though full-length APC is prone to degradation; see also Figure 5).

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Fig. 5. Subcellular distribution of APC constructs in transfected SW480 cells. (A–D) Confocal sections through cells transfected with GFP-tagged APC constructs as indicated (green), stained with an antibody against β-catenin (red); one typical example of each construct is shown. HCala shows pronounced nuclear APC and β-catenin (arrowhead); the arrow indicates an APC microtubule tip cluster (seen only with full-length APC). Note the granular aggregates seen with NAPC (tagged with GFP, d; or with HA, not shown) that coincide with β-catenin staining. (E) Western blots of total cell extracts, probed with anti-M-APC and anti-tubulin, revealing relative expression levels of GFP-tagged APC constructs (arrowheads) and of endogenous APC (asterisks; in the case of full-length APC, a breakdown product is also observed); internal control, tubulin (arrow).

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Fig. 6. TOPFLASH assays in colorectal cancer cells transfected with APC constructs. SW480 or HCT116 cells were transfected with APC constructs as indicated, and relative luciferase:Renilla values were determined as in Figure 4. (A and C) GFP-tagged APC constructs, compared with a GFP control; relative expression levels of constructs in (A) are shown underneath (western blots probed with anti-GFP; this antibody is less sensitive than anti-M-APC, so does not permit detection of full-length APC under these conditions). (B) APC constructs with various tags as indicated, and a vector control (containing HA). Note that the GFP derivatives produce generally lower TOPFLASH values than the HA- and Flag-tagged constructs, so have to be compared with the GFP control, and with GFP-tagged APC (see also A).

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Fig. 7. Nuclear targeting of APC constructs causes nuclear accumulation of β-catenin. Confocal sections through HCT116 cells transfected with GFP-tagged APC constructs as indicated (green), stained with antibody against β-catenin (red); typical examples of each construct are shown (B and D show two fields each).

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Fig. 8. Nuclear export rates of GFP-tagged APC constructs measured by FLIP. Individual COS cells transfected with GFP-tagged HC constructs (as indicated) were selected, and their entire cytoplasm was subjected to repetitive photobleaching while their nuclear fluorescence was measured; graphs and images shown are of a representative cell from each group. (A, B, D and E) Confocal sections through transfected cells at various time points as indicated (pre, pre-bleaching). (C and F) Graphic representations of loss of nuclear fluorescence at each time point. Wild-type HC constructs (HC and NLS-HC) show significantly more loss of nuclear fluorescence than NES mutant constructs (HC-ala and NLS-HCala), demonstrating that their nuclear export activity depends on their NESs.

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References

    1. Behrens J., Jerchow,B.A., Wurtele,M., Grimm,J., Asbrand,C., Wirtz,R., Kuhl,M., Wedlich,D. and Birchmeier,W. (1998) Functional interaction of an axin homolog, conductin, with β-catenin, APC and GSK3β. Science, 280, 596–599. - PubMed
    1. Bienz M. (1999) APC: the plot thickens. Curr. Opin. Genet. Dev., 9, 595–603. - PubMed
    1. Bienz M. (2002) The subcellular destinations of APC proteins. Nat. Rev. Mol. Cell Biol., 3, 328–338. - PubMed
    1. Brabletz T., Jung,A., Dag,S., Hlubek,F. and Kirchner,T. (1999) β-Catenin regulates the expression of the matrix metalloproteinase-7 in human colorectal cancer. Am. J. Pathol., 155, 1033–1038. - PMC - PubMed
    1. Brabletz T., Jung,A., Reu,S., Porzner,M., Hlubek,F., Kunz-Schughart,L.A., Knuechel,R. and Kirchner,T. (2001) Variable β-catenin expression in colorectal cancers indicates tumor progression driven by the tumor environment. Proc. Natl Acad. Sci. USA, 98, 10356–10361. - PMC - PubMed

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