The Wnt–β-catenin pathway is pivotal for numerous important cellular events during embryonic development, tissue homeostasis, and tumorigenesis. The key component of this pathway, β-catenin, exerts its signaling activity only in the nucleus. Therefore, nuclear import/export of β-catenin represents a crucial step in regulating signaling competent β-catenin levels and serves as an attractive target for anti-cancer therapies, but the underlying molecular mechanisms are poorly defined.
In this study, we have identified 14-3-3 proteins as novel Cby-binding partners. We demonstrated that 14-3-3 proteins specifically bind to serine 20 within the 14-3-3–binding consensus motif of Cby. Their interaction entirely depends upon phosphorylation of this critical serine residue by Akt kinase. Furthermore, we found that association with 14-3-3 triggers cytoplasmic relocation of Cby protein. Intriguingly, Cby and 14-3-3 form a tripartite complex with β-catenin and act cooperatively to sequester β-catenin into the cytoplasmic compartment. In support of our model, Cby collaborates with nuclear Akt to repress β-catenin–dependent transcriptional activation. Our results, therefore, suggest that inhibition of β-catenin signaling by Cby involves at least two distinct molecular mechanisms (); i.e., competing with Tcf/Lef transcription factors for binding to β-catenin in the nucleus (Takemaru et al., 2003
) and facilitating nuclear export of β-catenin via interaction with 14-3-3 upon its phosphorylation by Akt. Consistent with this, Cby mutants defective in 14-3-3 binding (S18A/S20A, 20A, and Δ1-22) unexpectedly exhibit significantly reduced ability to repress TopFlash activation by β-catenin () even though they accumulate in the nucleus (). Hence, the nuclear export and repressor activities of Cby may equally contribute to the rapid down-regulation of nuclear β-catenin activity. However, it is also plausible that one mechanism predominates over the other, depending on the cellular context.
Figure 9. Dual mechanism model for inhibition of β-catenin signaling activity by Cby. In the nucleus, Cby binds to β-catenin and competes with Tcf/Lef transcription factors, leading to repression of target gene expression. In addition, phosphorylation (more ...)
14-3-3ζ has been found to associate with β-catenin (Tian et al., 2004
). Later, it was found that Akt phosphorylates β-catenin at serine 552, which appears to enhance its interaction with 14-3-3ζ (Fang et al., 2007
). In both cases, ectopic expression of 14-3-3ζ resulted in a moderate activation (two- to fourfold) of β-catenin–dependent transcription in TopFlash assays. We found that 14-3-3ζ enhances, whereas 14-3-3η and ε isoforms repress, β-catenin activation of the TopFlash reporter (). One possible explanation for this observation is that 14-3-3 overexpression exerts complex biological effects, which makes our interpretation of the TopFlash results difficult. In fact, 14-3-3 proteins have been shown to interact with a plethora of target proteins ranging from transcription factors to various signaling molecules (Dougherty and Morrison, 2004
; Pozuelo Rubio et al., 2004
). However, it is interesting to note that, consistent with our results (), ectopic expression of 14-3-3ζ was found to cause the cytoplasmic enrichment of β-catenin (Tian et al., 2004
), potentially by interacting with endogenous Cby. Nonetheless, our findings indicate that the Cby–14-3-3 interaction significantly contributes to their stable complex formation with β-catenin (). More importantly, 14-3-3 itself is not sufficient to sequester β-catenin into the cytoplasm, as 14-3-3 binding–defective CbyS20A retains β-catenin in the nucleus even in the presence of excess 14-3-3 (). Therefore, we speculate that phosphorylation of β-catenin and Cby by Akt provokes 14-3-3 binding to form a stable ternary complex followed by β-catenin nuclear export and termination of its signaling (). Similarly, it has been shown that Akt phosphorylates FoxO Forkhead transcription factors, and this promotes their interaction with 14-3-3, leading to their nuclear exit and cytoplasmic retention (Brunet et al., 1999
; Van Der Heide et al., 2004
). It is also noteworthy that, in Caenorhabditis elegans
, 14-3-3/PAR-5 binds to and mediates nuclear export of TCF/POP-1 (Lo et al., 2004
The key negative regulators of the Wnt–β-catenin pathway, APC and Axin, have been shown to affect the nuclear cytoplasmic shuttling of β-catenin (Henderson, 2000
; Rosin-Arbesfeld et al., 2000
; Cong and Varmus, 2004
; Wiechens et al., 2004
). Both proteins carry functional NLS and NES sequences and export β-catenin from the nucleus to the cytoplasm in a CRM-1–dependent manner, most likely for degradation. The Cby–14-3-3 pathway represents yet another mechanism that controls β-catenin subcellular distribution. In addition to two putative NLS sequences (Takemaru et al., 2003
), Cby harbors a potential NES, and its localization is sensitive to leptomycin B (unpublished data), which is indicative of a CRM-1–dependent process. At present, the relationship between APC–, Axin–, and Cby–14-3-3–dependent β-catenin nuclear export pathways is unclear. However, Cby does not form a complex with APC or Axin (unpublished data), which implies that the Cby–14-3-3 route operates independently of that of APC and Axin.
Akt is a major effector of the phosphatidylinositol-3-kinase signaling, which is activated by a diverse array of extracellular stimuli, and is known to promote cell growth, survival, and tumor formation (Hennessy et al., 2005
). Several lines of evidence indicate that Akt plays a positive role in Wnt–β-catenin signaling, most likely through phosphorylation and inactivation of GSK3, although its precise mechanism of action remains largely debatable (Brazil et al., 2002
). Here, we provide evidence that nuclear-targeted Akt inhibits, whereas membrane-tethered Akt stimulates, β-catenin–mediated transcriptional activation (). It is possible that membrane/cytoplasmic activated Akt favorably phosphorylates and thus inactivates GSK3, whereas nuclear Akt phosphorylates β-catenin and Cby, which in turn facilitates 14-3-3 binding and subsequent nuclear exclusion of the ternary complex. The opposing roles of Akt in the Wnt–β-catenin pathway may be temporally and spatially controlled in an in vivo scenario. Once activated at the plasma membrane in response to diverse stimuli, Akt translocates into the nucleus, where it phosphorylates and modulates the activity of nuclear factors including Forkhead transcription factors and p300/CBP coactivators (Andjelkovic et al., 1997
; Brunet et al., 1999
; Borgatti et al., 2003
; Huang and Chen, 2005
). However, little is known about the physiological functions of nuclear Akt. Our results reveal that the subcellular compartmentalization of Akt differentially influences β-catenin signaling. This is reminiscent of GSK3, as it both positively and negatively affects Wnt–β-catenin signaling depending on its intracellular location (Zeng et al., 2005
). It is also conceivable that phosphorylation of Cby at serine 20 is catalyzed by additional kinases because 14-3-3–binding motifs have been shown to be substrates for various protein kinases (Dougherty and Morrison, 2004
; Aitken, 2006
). Clearly, further work will be required to define the cross-talk between the Wnt–β-catenin and Akt signaling pathways.
In conclusion, our study reveals a new molecular mechanism for the regulation of β-catenin subcellular distribution by the combinatorial action of Cby and 14-3-3 proteins. As deregulation of 14-3-3 has been implicated in the development of several types of human cancer (Hermeking, 2003
; Wilker and Yaffe, 2004
), it would be of great interest to determine if β-catenin signaling is aberrantly activated in these tumor cells.