Specific Armadillo Repeat Sequences Facilitate β-Catenin Nuclear Transport in Live Cells via Direct Binding to Nucleoporins Nup62, Nup153, and RanBP2/Nup358

Abstract

Background:The nuclear localization of ␤-catenin is directly linked to its cancer causing activity. Results: Armadillo repeats (10 -12) mediate nuclear transport of ␤-catenin through direct interaction with specific nuclear pore complex proteins. Conclusion: ␤-Catenin can function like a nuclear transport receptor in its ability to translocate independently through the nuclear pore complex. Significance: ␤-Catenin may transport specific binding partners between the nucleus and cytoplasm in response to Wnt signaling.␤-Catenin transduces the Wnt signal from the membrane to nucleus, and certain gene mutations trigger its nuclear accumulation leading to cell transformation and cancer. ␤-Catenin shuttles between the nucleus and cytoplasm independent of classical Ran/transport receptor pathways, and this movement was previously hypothesized to involve the central Armadillo (Arm) domain. Fluorescence recovery after photobleaching (FRAP) assays were used to delineate functional transport regions of the Arm domain in living cells. The strongest nuclear import/export activity was mapped to Arm repeats R10 -12 using both in vivo FRAP and in vitro export assays. By comparison, Arm repeats R3-8 of ␤-catenin were highly active for nuclear import but displayed a comparatively weak export activity. We show for the first time using purified components that specific Arm sequences of ␤-catenin interact directly in vitro with the FG repeats of the nuclear pore complex (NPC) components Nup62, Nup98, and Nup153, indicating an independent ability of ␤-catenin to traverse the NPC. Moreover, a proteomics screen identified RanBP2/Nup358 as a binding partner of Arm R10 -12, and ␤-catenin was confirmed to interact with endogenous and ectopic forms of Nup358. We further demonstrate that knock-down of endogenous Nup358 and Nup62 impeded the rate of nuclear import/export of ␤-catenin to a greater extent than that of importin-␤. The Arm R10 -12 sequence facilitated transport even when ␤-catenin was bound to the Arm-binding partner LEF-1, and its activity was stimulated by phosphorylation at Tyr-654. These findings provide functional evidence that the Arm domain contributes to regulated ␤-catenin transport through direct interaction with the NPC.The canonical Wnt signaling pathway regulates normal tissue development and maintenance through its effector molecule, ␤-catenin. In the absence of Wnt signaling ␤-catenin accumulates at adherens junctions. ␤-Catenin levels are regulated through degradation by a destruction complex, comprising of adenomatous polyposis coli (APC), 4 axin, casein kinase I, and glycogen synthase kinase-3␤, which phosphorylates and subsequently ubiquitinates ␤-catenin at the N terminus, leading to its degradation by the proteosome (1, 2). On receiving Wnt signals or through misregulation of the degradation pathway, ␤-catenin is stabilized and accumulates in the nucleus where it binds to lymphoid enhancer factor-1 (LEF/ TCF) transcription factors and activates transcription of tumor promoting genes involved in cell migration...

Regulation of the phosphorylation and nuclear import and export of β-catenin by APC and its cancer-related truncated form

“…Although APC (and axin) was proposed to mediate the nuclear export of b-catenin (Bienz, 2002), there is strong evidence that bcatenin can freely diffuse into and out of the nucleus (Fagotto et al, 1998;Kose et al, 1997;Wiechens and Fagotto, 2001;Henderson and Fagotto, 2002;Sharma et al, 2012), and that APC and other binding partners of b-catenin all negatively affect bcatenin translocation (Krieghoff et al, 2006). Our observation of very fast transport kinetics further corroborates the notion of selective diffusion, and the analysis of transport in APC-depleted cells confirms that APC is not required for translocation.…”

“…5). The import and export kinetics in parental SW480 cells were roughly similar to those measured in HEK293 and NIH 3T3 cells (Krieghoff et al, 2006;Sharma et al, 2012). However, the use of the spinning-disk confocal microscope allowed us to obtain information about the initial phase of recovery, which had not been studied so far.…”

“…However, the nuclear localization of truncated APC was later contested (Henderson and Fagotto, 2002), and it might be an artifact caused by unspecific antibody staining (Brocardo et al, 2005). An alternative mechanism was proposed in which b-catenin freely shuttles through the nuclear pore (Fagotto et al, 1998;Koike et al, 2004;Sharma et al, 2012). Kinetic analysis of transport shows that the overexpression of APC or other binding partners, such as axin, actually decreases the nuclear import of b-catenin (Krieghoff et al, 2006), supporting the hypothesis that these proteins influence the distribution of b-catenin through its sequestration in particular compartments (Krieghoff et al, 2006;Roberts et al, 2011).…”

Wnt/beta-Catenin Signaling and Small Molecule Inhibitors

“…c-Met, the tyrosine kinase receptor of HGF has been linked to -catenin signaling [379,380] and it has been shown that HGF/c-Met can activate -catenin signaling independent from Wnt signaling [381] at the site of E-cadherin containing junctions. Binding of HGF to c-Met triggers an autophosphorylation at Tyr1234 and Tyr1235, which in turn mediates a tyrosine kinasemediated Tyr654 and Tyr670 phosphorylation of -catenin [382] inducing the dissociation of -catenin from E-cadherin [352,383]. Similarly, Tyr142 and Tyr654 phosphorylation by the FLT3/ITD kinase (Fms-like Tyrosine Kinase-3) leads to a dissociation ofcatenin from its complex with E-cadherin [344,384].…”

Bioinformatics Analysis of the Ribosomal Proteins,RPL27, RPL37a and RPL41: 3-D Protein Modeling and Protein-protein Interaction Prediction

“…A recent study also indicates ARM domains 10-12 of the C-terminal tail of β-catenin to be active in the bidirectional transport activity when bound to specific nuclear pore complex (NPC) components [54]. Therefore, we predict that RPL37a might play another role in inhibiting migration of β-catenin within the cell.…”