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Rethinking WNT signaling Nicholas S. Tolwinski and Eric Wieschaus Howard Hughes Medical Institute, Dept of Molecular Biology, Princeton University, Princeton, NJ 08544, USA
Recent research on the WNT signaling pathway warrants a reassessment of the basic mechanism that transmits signal from the membrane-bound receptor to the nucleus. This article incorporates these findings into a revised model for pathway activation. We propose that the control of Axin stability, rather than the control of ZW3 phosphorylation of the Armadillo protein, is the key step in signaling. Axin degradation is controlled by a stabilizing effect of ZW3-dependent phosphorylation, and a destabilizing effect of active Arrow. Removing Axin enables Armadillo to accumulate and re-localize to the nucleus. We argue that nuclear localization of Armadillo is required for transcriptional pathway activity. Finally, we speculate on the effects this revision will have on the major questions facing the WNT field of research.
were subsequently identified and shown to play a role in ARM degradation, leading to the theory that a large complex is necessary for ARM phosphorylation. Mutation of AXN or APC leads to high levels of ARM and activation of the WNT signaling pathway [4]. These studies did not address how the degradation pathway is regulated on binding of WNT to its cell-surface receptor. The key component involved in the degradation pathway is a cytoplasmic protein called Disheveled (DSH), which appears to deactivate the destruction complex on its activation by the receptor Frizzled (FZ) [1]. Two potentially significant findings are: (i) the role of DSH in ‘cycling’ AXN to the plasma membrane [5]; and (ii) DSH binds directly to the C-terminus of FZ [6]. However, how FZ activates DSH and how DSH, in turn, inactivates the destruction complex remain largely unanswered questions.
The wingless (WNT) signaling pathway has crucial roles in development and disease. Misregulation causes growth abnormalities in organisms as diverse as worms and humans and is responsible for tumorigenesis in adults [1,2]. Owing to the ubiquity and importance of this pathway, it has been the focus of much research over the past 20 years, and many of the genes and components are now understood in some detail. However, the manner in which these components are arranged in a pathway that connects activity at the cell surface with a transcriptional response in the nucleus remains controversial. In this article, we highlight some of the difficulties with the current textbook model and offer a new interpretation of the pathway mechanism. The initial characterization of the WNT pathway relied on classical genetics to identify the relevant gene products, and epistasis experiments to order them in a linear pathway. The observation that cells accumulate cytoplasmic b-catenin in response to WNT signaling suggested that the pathway is activated by controlling the levels of the Armadillo protein (ARM; known as b-catenin in vertebrates). In the absence of WNT signal, ARM levels are kept low through degradation. Following WNT ligand binding, this degradation is inhibited, which enables ARM to accumulate and activate a transcriptional response. This degradation depends on the phosphorylation of ARM by the Drosophila melanogaster kinase ZW3 [known as glycogen synthase kinase 3b (GSK3b) in mammals]. Phosphorylated ARM is recognized rapidly by the proteasome and destroyed. In zw3 mutants, ARM levels are high and uniformly distributed [3]. Other pathway components [i.e. Axin (AXN) and Adenomatous polyposis coli (APC)]
A central role for AXN phosphorylation and degradation How DSH deactivates the destruction complex in cells that receive WNT signal is unclear. The focus of the research field has been on the involvement of GSK3b kinase, understandably, because it is the component with an easily identifiable function: it phosphorylates ARM. However, we have found that, in certain genetic backgrounds, ZW3 can be eliminated without blocking the ability of the cell to respond to the WNT signal. Under these conditions, ARM accumulates to uniform levels in all cells, however, distinct ARM-dependent transcriptional responses are maintained in the subset of cells that receives the WNT signal [7]. The results clearly indicate the existence of a mechanism that is independent of ARM phosphorylation by ZW3. One possible component of this mechanism is AXN, which physically binds to ARM and has been thought to function as a scaffold, whose function is to bring together various components of the destruction complex [8,9]. This role is supported by in vitro biochemical data but it might reflect only one aspect of AXN’s function in WNT signaling. Various groups have demonstrated that the levels of AXN are downregulated in a WNT-dependent manner. Specifically, AXN is dephosphorylated after WNT stimulation, which leads to AXN destabilization over time [10 – 12]. Furthermore, unphosphorylated AXN has a lower affinity for ARM [10,11], whereas phosphorylated AXN binds to low-density lipoprotein receptor-related protein-5 (LRP-5) more effectively and is also degraded [13]. Drosophila embryos that uniformly transcribe AXN in all cells accumulate the protein at high levels only in cells that are not exposed to WNT [7]. Cells that receive WNT ligand have low levels of AXN. This decrease in AXN levels would prevent the formation of the destruction complex, leading
Corresponding author: Eric Wieschaus (
[email protected]).
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to a stabilization of ARM, and would enable it to enter the nucleus. These effects of WNT signaling on AXN are supported by a recent biochemical study characterizing the kinetics of WNT pathway activation [14]. Lee and colleagues found that the intracellular levels of AXN are ,1000-times lower than those of other destruction complex components, consistent with the possibility that AXN might be the limiting component in the pathway. Consequently, degradation of AXN gives a much more robust and rapid increase in b-catenin transcriptional activity than lowering the levels of other components. The authors propose a novel, stabilizing role for ZW3 phosphorylation of AXN, contrary to the previously characterized destabilizing effect of ZW3 phosphorylation on ARM [15]. In their model for WNT signaling, receptor activation causes DSH to prevent ZW3 from phosphorylating AXN, leading to AXN degradation [14]. Because embryos that lack ZW3 accumulate stripes of AXN protein in response to WNT signaling, some additional mechanism must contribute to AXN stability in vivo. One possibility is the single-pass transmembrane protein Arrow (ARR; known as LRP-5 or -6 in mammals). ARR was identified as a co-receptor for WNT and is required for pathway activation [16– 18]. Biochemical analyses showed that activated ARR can recruit AXN to the membrane and promote its degradation [13]. Using a GFP-labeled AXN in living embryos, Cliffe et al. have confirmed that WNT signaling causes a relocalization of AXN from the cytoplasm to the plasma membrane and that this relocalization depends on DSH [5]. Fusion of FZ and ARR is sufficient to activate the pathway in the absence of WNT but DSH is required for this activation [7]. In addition, FZ binds DSH through its C-terminus [6]. How DSH affects ARR-mediated degradation of AXN in the absence of ZW3 is unknown, although the protein might (a)
simply be necessary to maintain the physical association of the receptor– AXN complex. These observations can be integrated into a revised model of WNT signaling where AXN functions as the key element in transmitting the WNT signal. After WNT binds to the FZ receptor, DSH is recruited to the plasma membrane where it transports AXN to ARR. This then brings the entire destruction complex to the membrane where DSH inhibits ZW3 phosphorylation of AXN, and might also promote the degradation of AXN directly, through ubiquitin-mediated proteolysis [14] (Figure 1). Because AXN is degraded in a WNT-dependent manner even when ZW3 is absent [7], ARR must act as an activativator of degradation when AXN is brought to the membrane [13]. As the levels of AXN (and therefore the degradation complex) decline, ARM accumulates. Because AXN also functions as a cytoplasmic anchor for ARM [19], when the anchor is absent ARM is free to enter the nucleus where it partners with T-cell factor (TCF) to activate the transcription of WNT targets. A caveat for the anchoring activity of AXN is that it is present at such low levels. Our results suggest that AXN is necessary for the anchoring function but it might not be the anchor itself. One possible candidate is APC, which is much more abundant than AXN and mimics all of the AXN phenotypes [14,20,21]. Although overall levels of ARM are significantly higher than those of AXN, it is also possible that only a fraction of ARM protein is signaling competent, and it is this fraction that is anchored by AXN. In wild-type epidermal cells, ARM levels are kept low by the degradation machinery, a necessity given that ARM mRNA is abundant and efficiently translated [22]. Thus, the association of AXN with any single ARM molecule must be transient and its anchoring activity can not be due to static complex formation. Anchoring would therefore depend on the constant flow of newly synthesized ARM (b)
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Figure 1. The two-state model of WNT signaling. (a) In the absence of the WNT ligand, the cytoplasmic destruction-complex – composed of Axin (AXN), Adenomatous polyposis coli (APC) and glycogen synthase kinase (GSK3b), which is the invertebrate ZW3 – causes the degradation of Armadillo (ARM), maintains low ARM levels and keeps ARM out of the nucleus. (b) After WNT binds to the Frizzled (FZ) receptor, the destruction complex moves to the membrane where an FZ-activated Disheveled (DSH) prevents GSK3b from phosphorylating AXN. This in turn leads to the degradation of AXN protein, dissociating the destruction complex, and enables ARM to accumulate and translocate to the nucleus. www.sciencedirect.com
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through the degradation complex. We suspect that for any low-abundance anchor to function effectively, it must be coupled to protein degradation. Does ARM function in the nucleus? For AXN to function as an anchor, we must assume that the nuclear localization of ARM is essential for pathway activation. Consistent with this view, the final step in WNT signaling has long been thought to be the formation of a transactivating complex, in which the transcription factor TCF provides the specific DNA-binding activity and ARM provides the transcriptional activation. TCF is found predominantly in the nucleus but ARM is not: it is only after WNT activation that ARM is detectable in the nucleus. Given that ARM appears to cross the nuclear envelope freely [23], its localization must be controlled by a balance between its various nuclear and cytoplasmic binding partners. In addition to its interaction with TCF, ARM might also play a role in chromatin remodeling because physical interactions with a growing number of chromatin-associated factors have been demonstrated [24]. Furthermore, the recent identification of pygopus, legless and chibby, as part of the ARM-dependent transcription machinery, appear to confirm a definite nuclear role for ARM [25– 29]. In this model, the effects of ARM depend on its entry into the nucleus, which, along with stability, is the most obvious feature of WNT activation. The relevance of the nuclear localization of ARM has recently been challenged, based primarily on the observation that membrane-tethered ARM can activate expression of WNT transcriptional targets without entering the nucleus. One interpretation of this finding is that the effects of ARM on transcription might be indirect; for example, ARM might function in the cytoplasm by modifying other transcription factors or by facilitating their movement into, or out of, the nucleus [30]. An alternative interpretation of the same results is that the high-level expression of a membrane-tethered form titrates out the negative regulators of the pathway. An obvious candidate would be a cytoplasmic anchor (e.g. AXN) – given that expression of high levels of the membrane-tethered form enables endogenous ARM to enter the nucleus [19]. Other negative regulators are possible targets of membrane-tethered ARM, for example, TCF/Pangolin (PAN) [30]. Distinguishing between these interpretations has been difficult because mutations that remove all endogenous ARM activity are invariably lethal to the cell, as a result of the role of ARM in cell adhesion. Two strategies that might circumvent this technical problem could involve (i) obtaining either point-mutation alleles that affect WNT signaling but not adhesion or (ii) supplying low levels of membrane-tethered ARM in otherwise null backgrounds [31]. Using these strategies, preliminary results in our laboratory indicate that the membrane-tethered forms alone can not signal in the absence of endogenous free ARM, suggesting that ARM must be untethered to enter the nucleus and function [32]. Although overexpression studies are, by their nature, uninformative about where in the cell ARM functions, identifying the target of membrane-tethered ARM might www.sciencedirect.com
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still shed some light into the physical interactions that regulate the WNT pathway. Future directions Although the WNT pathway has been a subject of intense study, since the discovery of WNT1 as the causal oncogene in mammary tumors [33], some vital questions remain unanswered. For example, what role does b-catenin play in the nucleus? Various functions have been proposed, including transactivation and chromatin remodeling [24]. The transcription factor most closely associated with WNT signaling is TCF, which is known to function in activation [34] and repression [35] of transcription. However, the contrasting results obtained with a dominant-negative form of TCF [34], and the recently published TCF maternal and zygotic null phenotype [36], point to an interesting feature of the pathway. WNT transcription targets are activated to some extent in the absence of TCF but not in the presence of the dominant-negative TCF, which can not bind ARM. One possibility is that ARM binding to TCF removes TCF repression and activates transcription [35]. Different WNT targets might be differentially sensitive to the two phenomena. Taking this view, genetic removal of TCF would eliminate TCF repression but not allow for the transactivation normally produced by the TCF – ARM complex on certain targets. The dominant-negative TCF construct would continue to bind WNT targets such that these targets would not only fail to be activated but remain in a repressed state. This would not only suggest that transcriptional activation can be rather complicated, but also that careful analysis might reveal the intricate combinatorial codes that WNT uses to pattern Drosophila segments, and many other developmental processes. Another important question concerns the fact that many components of the WNT signaling pathway play significant roles in other cell structures and signaling pathways. ARM is a major component of adherens junctions, GSK3b functions in both the hedgehog and insulin signaling pathway, the Frizzled receptors maintain planar cell polarity and DSH, among others, is involved in the non-canonical WNT signaling pathways [37]. How can pathway specificity be maintained if the components have various functions? The current view is that pathways avoid cross talk by assembling into multi-component complexes. Lee et al. proposed that by maintaining low levels of a key regulator (AXN), other pathways using the more abundant WNT components would not be affected by WNT-signaling levels [14]. This model should be readily testable because AXN overexpression should affect the non-WNT dependent functions of DSH, APC and GSK3b. Furthermore, a degradation-resistant-AXN allele could function as a potent tumor suppressor. Complex formation offers a satisfying answer as to how signaling molecules maintain their specificity in a crowded cellular environment. However, the logical question to ask is: once the complexes form, how do they break apart? To function in signaling, these complexes must be dynamic. The work of Lee and colleagues suggests one possible explanation [14]: to dissociate a complex, it might be more effective to quickly remove the component that first assembles it. Because AXN is present at low concentrations
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and is eliminated rapidly after pathway activation, it seems likely that the relatively stable destruction complex dissociates in this manner and ARM is released. An intriguing piece of evidence for this model is that one of the main transcriptional targets of WNT is Axin 2 [38,39], indicating that negative-feedback regulation functions through an increase in Axin 2 levels. Finally, although we have presented a case for how we believe this pathway operates, there is no reason to assume that only one method of signal activation exists. With the ever-growing list of pathway components with various functions, cross talk possibilities, feedback regulation and so on [40] (for more examples, see http:// www.stanford.edu/~rnusse/wntwindow.html), the WNT pathway must obviously be far more complex than we currently understand. In this article, we have outlined what we believe to be the modifications necessary to bring the textbook pathway up to date but, despite the possible cross-interactions and myriad components, the pathway remains linear. This might appear surprising considering the number of complicating factors that have been discovered by those working in the WNT signaling field. The question then becomes: does this linearity of pathways reflect a limitation in our thinking or does signaling occur through linear pathways in real cells? Conclusion We propose the following pathway for WNT activation. Extracellular WNT binds to FZ and this activated receptor– ligand complex recruits DSH and ARR. DSH and ARR localize AXN along with the other destructioncomplex components to the membrane. At this point, DSH prevents the phosphorylation of AXN and ARR promotes the degradation of AXN, which rapidly disappears. The loss of AXN prevents the assembly of the destruction complex, enabling ARM levels to increase and also relieving the cytoplasmic anchoring that prevents b-catenin nuclear localization. Therefore, b-catenin accumulates and translocates to the nucleus where it activates transcription. Because of the important role played by WNT in development and disease, these new insights might be significant for the development of therapeutics. It is important to note that a drug that blocks AXN degradation might be more specific and effective at blocking WNT signaling than drugs that affect GSK3b, which plays a role in various multifunctional pathways. Acknowledgements We are indebted to E. Lee for sharing his manuscript with us ahead of publication. We thank J. Goodliffe, A. Nouri and L. Manji for critical reading of the manuscript. This work was supported by the Howard Hughes Medical Institute and the National Institutes of Health grant P01CA41086 to E. Wieschaus. N. Tolwinski is supported by a New Jersey Commission of Cancer Research predoctoral fellowship.
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Articles of interest in Trends and Current Opinion journals In search of antisense Giovanni Lavorgna, Dvir Dahary, Ben Lehner, Rotem Sorek, Christopher M. Sanderson and Giorgio Casari Trends in Biochemical Sciences 29, 88–94 Tuberous sclerosis complex: from Drosophila to human disease Duojia Pan, Jixin Dong, Yong Zhang and Xinsheng Gao Trends in Cell Biology 14, 78–85 Mobile elements and mammalian genome evolution Prescott L. Deininger, John V. Moran, Mark A. Batzer and Haig H. Kazazian Jr Current Opinion in Genetics and Development 13, 651–658 Proteomic approaches in cancer risk and response assessment Emanuel F. Petricoin and Lance A. Liotta Trends in Molecular Medicine 10, 5–64 Mycobacterium tuberculosis: a model system for structural genomics Clare V. Smith and James C. Sacchettini Current Opinion in Structural Biology 13, 658–664 www.sciencedirect.com
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