APC-mediated downregulation of beta-catenin activity involves nuclear sequestration and nuclear export

Abstract

Mutational inactivation of adenomatous polyposis coli (APC) initiates most colon carcinomas. APC functions include targeting cytoplasmic beta-catenin, a Wnt pathway mediator, for proteolysis. Although APC shuttles between cytoplasm and nucleus, the role of nuclear APC protein, particularly with respect to nuclear beta-catenin levels and activity, remains unclear. Here, we demonstrate that APC lacking functional nuclear localization signals (NLSs) or nuclear export signals (NESs) does not effectively downregulate nuclear beta-catenin levels; neither does wild-type APC when nuclear export is blocked. While APC bearing mutated NLSs could not downregulate beta-catenin-mediated transcriptional activation, APC lacking NESs remained active. Consistent with the hypothesis that nuclear APC lacking NESs can inhibit beta-catenin function by sequestration, we show that endogenous APC and beta-catenin proteins interact within the nucleus. These data demonstrate that nuclear APC binding to beta-catenin, and then inducing its nuclear export, plays a critical role in the control of nuclear beta-catenin levels and activity.

Fig. 1. (A) Schematic representation of full-length APC. Protein interaction domains are indicated, with axin binding sites represented by ovals. Oligo, oligomerization; MT, microtubule. (B) APC protein localization is dependent on functional NESs, and nuclear β-catenin protein levels are not downregulated by APC protein with mutant NES or NLS. SW480 cells were transfected with expression constructs for various Flag-APC proteins. At 24 h post-transfection, cells were prepared for immunofluorescence microscopy using antibodies against the Flag epitope and β-catenin. Flag-APC protein was visualized with FITC (green), β-catenin with Texas-Red (red) and nuclei with 4′-6-diamidine-2-phenylindole (DAPI) counterstain (blue). Whereas APC protein expression correlated with a decrease in β-catenin staining (see arrowheads), the expression of APC protein lacking functional NESs or NLSs resulted in a predominantly nuclear localization of β-catenin. (C) APC and β-catenin protein localization is sensitive to LMB treatment. SW480 cells were transfected and scored as described in Figure 1B, with the addition of LMB 16 h post-transfection. LMB shifted the staining patterns of APC and APC(mNES1,2)PKI proteins from a cytoplasmic to a more nuclear distribution, with affiliated nuclear localization of β-catenin. Scale bar, 10 µm.

Fig. 2. APC protein with mutant NESs, but not with mutant NLSs, is able to downregulate β-catenin/LEF-1 activity. Expression of either APC or APC(mNES1,2) in SW480 cells caused a ∼4-fold reduction in endogenous β-catenin/LEF-1 activity. APC(mNLS1,2) did not have a significant effect. Differences between APC(mNLS1,2) and either APC or APC(mNES1,2) were significant, p <0.001.

Fig. 3. APC and β-catenin interact in the nucleus. (A) Antibodies against APC, β-catenin or a non-specific isotype control (IgG1) were used to immunoprecipitate proteins from the nuclear lysate of HCT116 cells. Proteins were separated by SDS–PAGE and analyzed for the presence of APC and β-catenin by immunoblotting. (B) The relative purity of the nuclear and cytoplasmic lysates was confirmed by sequentially probing for the nuclear (N) marker lamin, the membrane marker adaptin and the cytoplasmic (C) marker tubulin. The immunoblot probed for all three proteins is shown.

Fig. 4. Model of the APC shuttling effect on nuclear β-catenin. (A) Nuclear β-catenin, in conjunction with T cell-factor/lymphoid-enhancer-factor (TCF/LEF-1), activates the transcription of genes such as cyclin D1 and c-myc. (B) Nuclear APC protein interacts with nuclear β-catenin, displacing it from the LEF-1 transcription complex. Nuclear β-catenin exports to the cytoplasm where it is degraded by the proteasomal machinery.