In this study, we identify RanBP3 as a novel inhibitor of Wnt signaling that acts on β-catenin directly by enhancing nuclear export of its active form. We show that RanBP3 binds directly to β-catenin and that the interaction is increased in the presence of RanGTP. Expression of RanBP3 represses Wnt signaling both in vitro and in X. laevis embryonic development. Inhibition of RanBP3 by RNAi causes overactivation of Wnt signaling in tissue culture cells and in D. melanogaster embryos. In addition, expression of RanBP3 in human cells specifically reduces active β-catenin levels in the nucleus and relocates ΔGSK3–β-catenin from the nucleus to the cytoplasm, independently of CRM1.
RanBP3 was originally identified as a nuclear protein that contains FG repeats and a RanGTP-binding domain (Mueller et al., 1998
). RanBP3 can directly bind the nuclear export receptor CRM1, stimulating the formation of nuclear export complexes and increasing the export rate of certain CRM1 substrates (Englmeier et al., 2001
; Lindsay et al., 2001
). One mechanism by which RanBP3 could influence β-catenin activity would therefore be increased nuclear export via the CRM1 pathway. Although the nuclear export mechanisms of β-catenin are not fully understood, two pathways have been proposed (Henderson and Fagotto, 2002
). In the first, β-catenin exits the nucleus independently of nuclear export receptors by interacting directly with proteins of the nuclear pore complex (Eleftheriou et al., 2001
; Wiechens and Fagotto, 2001
). In the second pathway, β-catenin exits the nucleus via the CRM1 pathway, but because β-catenin does not contain NESs of its own, it uses binding to APC to exit the nucleus. The APC tumor suppressor does contain functional NESs and has been shown to be exported by CRM1 (Henderson, 2000
; Neufeld et al., 2000
; Rosin-Arbesfeld et al., 2000
). Therefore, RanBP3 could inhibit β-catenin by stimulating its export via APC and CRM1. However, four lines of evidence argue against this. First, in a CRM1 export complex, RanBP3 would bind to the complex via CRM1. Instead, we find that RanBP3 interacts directly with β-catenin. Second, β-catenin activity is RanBP3 sensitive in the colon carcinoma cell line COLO320 (Quinn et al., 1979
) that expresses a short type I APC truncation lacking all β-catenin interaction sites (Rosin-Arbesfeld et al., 2003
). We cannot formally exclude the possibility that the neuronal APC-like protein APC2 (van Es et al., 1999
), which is expressed in certain colon carcinoma cell lines, compensates for the loss of APC. However, in luciferase reporter assays, CRM1 overexpression does not reverse stimulation of β-catenin activity caused by depletion of RanBP3. Finally, RanBP3-mediated relocalization of active β-catenin is insensitive to LMB, a potent CRM1 inhibitor (Wolff et al., 1997
). Therefore, we conclude that the mechanism by which RanBP3 inhibits β-catenin is independent of CRM1 and APC.
It was recently suggested that nuclear β-catenin signaling is performed mainly by β-catenin dephosphorylated at serine 37 and threonine 41, which are main target sites of GSK3β (Staal et al., 2002
; van Noort et al., 2002
). Depletion of RanBP3 by RNAi specifically increases the amount of dephosphorylated β-catenin in nuclear fractions, whereas RanBP3 overexpression has the opposite effect. No concomitant increase, but rather a small decrease, of cytoplasmic endogenous active β-catenin was observed by overexpression of RanBP3. We attribute this to cytoplasmic phosphorylation and subsequent degradation of wt β-catenin.
Endogenous active β-catenin was visualized in situ, using the anti–active β-catenin antibody recognizing dephosphorylated β-catenin. This was only possible in SW480 colon carcinoma cells that contain a high level of active β-catenin because of severely defective APC function (Korinek et al., 1997
). RanBP3 overexpression reduced active β-catenin levels in the nucleus but had no effect on total β-catenin. This suggests that only a small proportion of total β-catenin is active in SW480 cells and confirms the specificity of RanBP3 for active β-catenin. Apparently, absence of proper β-catenin phosphorylation and degradation is not sufficient for β-catenin to be in an active, dephosphorylated state. Also, we infer that the modulation by RanBP3 of β-catenin activity as measured in our luciferase reporter assays acts on a small dephosphorylated pool, explaining why RanBP3 modulates wt and ΔGSK3–β-catenin to a similar extent ( and ).
To determine whether RanBP3 enhances β-catenin NH2
-terminal phosphorylation or nuclear export, we have visualized both nuclear and cytoplasmic distribution of active β-catenin. For this, we used a fluorescently tagged β-cateninΔGSK3
that is resistant to NH2
-terminal phosphorylation and degradation. As shown in , RanBP3 causes a clear and significant shift of β-cateninΔGSK3
from the nucleus to the cytoplasm. We therefore conclude that RanBP3 directly enhances nuclear export of active β-catenin. How does RanBP3 perform this task? Recent studies have indicated that the interactions of nuclear factors with chromatin or with each other are dynamic (Dundr et al., 2002
; Phair et al., 2004
). This suggests that RanBP3 does not need to actively remove β-catenin from the TCF/LEF–chromatin complexes. We therefore favor the possibility that association with RanBP3 prevents association of active β-catenin with chromatin and keeps it in a more soluble state. In itself, this would be sufficient to allow CRM1-independent nuclear exit. We do not know whether RanBP3 accompanies β-catenin to the cytoplasm and acts as a true nuclear export factor. The stimulatory effect of RanGTP on the β-catenin–RanBP3 interaction and the consistently weaker inhibitory effects on β-catenin of a RanBP3 mutant unable to bind RanGTP would argue in favor of this possibility. Hydrolysis of RanGTP in the cytoplasm would increase the efficiency of release of β-catenin for subsequent interactions with the cytoplasmic interacting proteins, such as E-cadherin or the APC–Axin–GSK3β complex.
We studied the effect of RanBP3 in X. laevis and D. melanogaster embryogenesis. Overexpression of the X. laevis homologue of RanBP3 during early embryogenesis inhibits β-catenin–dependent dorsoventral axis formation. RNAi of the D. melanogaster homologue of RanBP3 causes naked cuticle phenotypes and a broader Engrailed expression domain because of overactivation of the Wnt signaling pathway. Therefore, the results obtained in these two model organisms support the results obtained in cultured human cell lines and indicate that the inhibitory function of RanBP3 is highly conserved in metazoan evolution.
Wnt signaling plays an important role in tumor initiation and progression in a variety of human solid tumors, including colon carcinomas, hepatocellular carcinomas, and melanomas (Bienz and Clevers, 2000
; Polakis, 2000
). As a negative modulator of Wnt signaling, RanBP3 is a novel candidate tumor suppressor protein. Interestingly, the RanBP3 gene is located in 19p13.3, a region that is commonly deleted in several types of cancer and in which multiple tumor suppressor genes are likely to be present (Lee et al., 1998
; Oesterreich et al., 2001
; Tucci et al., 2001
; Yanaihara et al., 2003
; Kato et al., 2004
; Miyai et al., 2004
; Yang et al., 2004
). Further work is required to determine whether the loss of the RanBP3 gene contributes to these or other types of cancer.
In conclusion, we have identified an unexpected role for RanBP3 as a novel inhibitor of Wnt signaling that enhances nuclear export of active β-catenin. This function is separate from its role in CRM1-mediated nuclear export. The structural similarities between CRM1 and β-catenin suggest that RanBP3 may be a more general cofactor for nuclear export of ARM repeat proteins.