Axin: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

Gene name - Axin

Synonyms -

Cytological map position - 99D

Function - scaffolding protein

Keywords - segment polarity, wing, component of the ß-catenin destruction complex

Symbol - Axn

FlyBase ID: FBgn0026597

Genetic map position -

Classification - Axin family

Cellular location - cytoplasmic



NCBI links: Precomputed BLAST | Entrez Gene | UniGene
Recent literature
Anvarian, Z., Nojima, H., van Kappel, E.C., Madl, T., Spit, M., Viertler, M., Jordens, I., Low, T.Y., van Scherpenzeel, R.C., Kuper, I., Richter, K., Heck, A.J., Boelens, R., Vincent, J.P., Rüdiger, S.G. and Maurice, M.M. (2016). Axin cancer mutants form nanoaggregates to rewire the Wnt signaling network. Nat Struct Mol Biol [Epub ahead of print]. PubMed ID: 26974125
Summary:
Signaling cascades depend on scaffold proteins that regulate the assembly of multiprotein complexes. Missense mutations in scaffold proteins are frequent in human cancer, but their relevance and mode of action are poorly understood. This study shows that cancer point mutations in the scaffold protein Axin derail Wnt signaling and promote tumor growth in vivo through a gain-of-function mechanism. The effect is conserved for both the human and Drosophila proteins. Mutated Axin forms nonamyloid nanometer-scale aggregates decorated with disordered tentacles, which 'rewire' the Axin interactome. Importantly, the tumor-suppressor activity of both the human and Drosophila Axin cancer mutants is rescued by preventing aggregation of a single nonconserved segment. These findings establish a new paradigm for misregulation of signaling in cancer and show that targeting aggregation-prone stretches in mutated scaffolds holds attractive potential for cancer treatment.

Wang, Z., Tacchelly-Benites, O., Yang, E., Thorne, C. A., Nojima, H., Lee, E. and Ahmed, Y. (2016). Wnt/Wingless pathway activation is promoted by a critical threshold of Axin maintained by the tumor suppressor APC and the ADP-ribose polymerase Tankyrase. Genetics [Epub ahead of print]. PubMed ID: 26975665
Summary:
Wnt/β-catenin signal transduction directs metazoan development and is deregulated in numerous human congenital disorders and cancers. In the absence of Wnt stimulation, a multi-protein "destruction complex", assembled by the scaffold protein Axin, targets the key transcriptional activator β-catenin for proteolysis. Axin is maintained at very low levels that limit destruction complex activity, a property that is currently being exploited in the development of novel therapeutics for Wnt-driven cancers. This study used an in vivo approach in Drosophila to determine how tightly basal Axin levels must be controlled for Wnt/Wingless pathway activation, and how Axin stability is regulated. For nearly all Wingless-driven developmental processes, a three- to four-fold increase in Axin was found to be insufficient to inhibit signaling, setting a lower-limit for the threshold level of Axin in the majority of in vivo contexts. Further, both the tumor suppressor Adenomatous polyposis coli (APC) and the ADP-ribose polymerase Tankyrase (Tnks) were found to have evolutionarily conserved roles in maintaining basal Axin levels below this in vivo threshold, and separable domains were defined in Axin that are important for APC- or Tnks-dependent destabilization. Together, these findings reveal that both APC and Tnks maintain basal Axin levels below a critical in vivo threshold to promote robust pathway activation following Wnt stimulation.

Croy, H. E., et al. (2016). The PARP enzyme Tankyrase antagonizes activity of the β-catenin destruction complex through ADP-ribosylation of Axin and APC2. J Biol Chem [Epub ahead of print]. PubMed ID: 27068743
Summary:
Most colon cancer cases are initiated by truncating mutations in APC. APC is a critical negative regulator of the Wnt signaling pathway that participates in a multi-protein destruction complex to target the key effector protein βcatenin for proteolysis. Poly ADP-ribose Polymerase (PARP) enzyme Tankyrase (TNKS) has been shown antagonizes destruction complex activity by promoting degradation of the scaffold protein Axin. A yeast two-hybrid (Y2H) screen uncovered TNKS as a putative binding partner of Drosophila APC2, suggesting that TNKS may play multiple roles in destruction complex regulation. TNKS was found to bind a C-terminal RPQPSG motif in Drosophila APC2, and that this motif is conserved in human APC2, but not human APC1. In addition, APC2 can recruit TNKS into the βcatenin destruction complex, placing the APC2/TNKS interaction at the correct intracellular location to regulate βcatenin proteolysis. TNKS directly PARylates both Drosophila Axin and APC2, but that PARylation does not globally regulate APC2 protein levels as it does for Axin. Moreover, TNKS inhibition in colon cancer cells decreases βcatenin signaling, which cannot be explained solely through Axin stabilization. Instead, these findings suggest that TNKS regulates destruction complex activity at the level of both Axin and APC2, providing further mechanistic insight into TNKS inhibition as a potential Wnt pathway cancer therapy.
Wang, Z., Tacchelly-Benites, O., Yang, E. and Ahmed, Y. (2016). Dual roles for membrane association of Drosophila Axin in Wnt signaling. PLoS Genet 12: e1006494. PubMed ID: 27959917
Summary:
Axin, a concentration-limiting scaffold protein, facilitates assembly of a "destruction complex" that prevents Wnt signaling in the unstimulated state and a plasma membrane-associated "signalosome" that activates signaling following Wnt stimulation. In the classical model, Axin is cytoplasmic under basal conditions, but relocates to the cell membrane after Wnt exposure. This study analyzed the subcellular distribution of endogenous Drosophila Axin in vivo and found that a pool of Axin localizes to cell membrane proximal puncta even in the absence of Wnt stimulation. Axin localization in these puncta is dependent on the destruction complex component Adenomatous polyposis coli (Apc). In the unstimulated state, the membrane association of Axin increases its Tankyrase-dependent ADP-ribosylation and consequent proteasomal degradation to control its basal levels. Furthermore, Wnt stimulation does not result in a bulk redistribution of Axin from cytoplasmic to membrane pools, but causes an initial increase of Axin in both of these pools, with concomitant changes in two post-translational modifications, followed by Axin proteolysis hours later. Finally, the ADP-ribosylated Axin that increases rapidly following Wnt stimulation is membrane associated. The study concludes that even in the unstimulated state, a pool of Axin forms membrane-proximal puncta that are dependent on Apc, and that membrane association regulates both Axin levels and Axin's role in the rapid activation of signaling that follows Wnt exposure.

Wang, Z., Tacchelly-Benites, O., Yang, E. and Ahmed, Y. (2016). Dual roles for membrane association of Drosophila Axin in Wnt signaling. PLoS Genet 12: e1006494. PubMed ID: 27959917
Summary:
Axin, a concentration-limiting scaffold protein, facilitates assembly of a "destruction complex" that prevents Wnt signaling in the unstimulated state and a plasma membrane-associated "signalosome" that activates signaling following Wnt stimulation. In the classical model, Axin is cytoplasmic under basal conditions, but relocates to the cell membrane after Wnt exposure. This study analyzed the subcellular distribution of endogenous Drosophila Axin in vivo and found that a pool of Axin localizes to cell membrane proximal puncta even in the absence of Wnt stimulation. Axin localization in these puncta is dependent on the destruction complex component Adenomatous polyposis coli (Apc). In the unstimulated state, the membrane association of Axin increases its Tankyrase-dependent ADP-ribosylation and consequent proteasomal degradation to control its basal levels. Furthermore, Wnt stimulation does not result in a bulk redistribution of Axin from cytoplasmic to membrane pools, but causes an initial increase of Axin in both of these pools, with concomitant changes in two post-translational modifications, followed by Axin proteolysis hours later. Finally, the ADP-ribosylated Axin that increases rapidly following Wnt stimulation is membrane associated. The study concludes that even in the unstimulated state, a pool of Axin forms membrane-proximal puncta that are dependent on Apc, and that membrane association regulates both Axin levels and Axin's role in the rapid activation of signaling that follows Wnt exposure.

Schaefer, K. N., Bonello, T. T., Zhang, S., Williams, C. E., Roberts, D. M., McKay, D. J. and Peifer, M. (2018). Supramolecular assembly of the beta-catenin destruction complex and the effect of Wnt signaling on its localization, molecular size, and activity in vivo. PLoS Genet 14(4): e1007339. PubMed ID: 29641560
Summary:
Wnt signaling provides a paradigm for cell-cell signals that regulate embryonic development and stem cell homeostasis. The tumor suppressors Axin form the core of the multiprotein destruction complex, which targets the Wnt-effector beta-catenin for phosphorylation, ubiquitination and destruction. This study manipulated Axin and APC2 levels. Endogenous Axin and APC2 proteins and their antagonist Dishevelled accumulate at roughly similar levels, suggesting competition for binding may be critical. In the absence of Wnt signals, Axin and APC2 co-assemble into large cytoplasmic complexes containing tens to hundreds of Axin proteins. Wnt signals trigger recruitment of these to the membrane, while cytoplasmic Axin levels increase, suggesting altered assembly/disassembly. Glycogen synthase kinase3 regulates destruction complex recruitment to the membrane and release of Armadillo/beta-catenin from the destruction complex. Manipulating Axin or APC2 levels had no effect on destruction complex activity when Wnt signals were absent, but, surprisingly, had opposite effects on the destruction complex when Wnt signals were present. Elevating Axin made the complex more resistant to inactivation, while elevating APC2 levels enhanced inactivation. These data suggest both absolute levels and the ratio of these two core components affect destruction complex function, supporting models in which competition among Axin partners determines destruction complex activity.
BIOLOGICAL OVERVIEW

The vertebrate Axin protein, the product of the mouse fused gene, binds to beta-catenin to inhibit Wnt signaling. A homolog of vertebrate Axin, Drosophila Axin (here termed Axin in conformity to FlyBase usage) has been identified. Axin knockout produces phenotypes that are similar to overexpression of the Drosophila Wnt gene wingless (wg). Overexpression of Axin produces phenotypes similar to loss of wg. Axin overexpression can modify phenotypes elicited by wg and another Drosophila Wnt gene, Wnt oncogene analog 2. Using immunoprecipitation of endogenous Axin embryonic protein, it has been shown that Axin interacts with Armadillo and Zeste-white 3. The loss-of-function and overexpression phenotypes show that Axin, like its mammalian counterpart, acts as a negative regulator of wg/Wnt signaling (Willert, 1999b). Together, Axin, APC and Shaggy/Zeste-white 3 form a protein complex termed the "ß-catenin destruction complex."

Two approaches have been used to clone Axin. Hamada (1999) performed a yeast two-hybrid screen of a Drosophila embryo cDNA library using the Armadillo repeat domain of Armadillo as target and identified Axin as an Arm-interacting protein. Willert (1999b) identified Axn by searching the EST database with the protein sequence of mouse Axin. An EST with significant homology to the DIX (a domain similar between Axin and Dishevelled) domain of Axin was identified and used to isolate the full-length clone from an embryonic Drosophila cDNA library (Willert, 1999b).

Loss-of-function mutant phenotypes can be mimicked by the injection of double stranded RNA. This method, referred to as RNA interference (RNAi), was initially successfully used in C. elegans and more recently in Drosophila. RNAi was used to disrupt the function of Axn during patterning of the embryonic cuticle, a process that requires Wg signaling. The wild-type embryo secretes a cuticle consisting of a repeated pattern of denticle belts with intervening naked regions. Loss of wg leads to a cuticle covered with denticles -- one lacking any naked areas. Overexpression of wg in the embryo leads to loss of all denticle structures, i.e. a naked cuticle. Disruption of AXN expression by RNAi leads to a similar naked cuticle, suggesting that Axn functions to down-regulate Wg signaling. Virtually all injected embryos had extra naked cuticle, ranging from partially to nearly completely naked. Control injection of either sense or antisense single stranded AXN mRNA does not cause any phenotypic changes. As expected, wg RNAi produces a partial wg-like cuticle. Thus, mimicking loss of Axn function by RNAi leads to phenotypes similar to overexpressing wg, consistent with the model that Axn is a negative regulator of Wg signaling (Willert, 1999b).

A P-element insertion near the beginning of the Axn gene has been shown to disrupt expression of the gene to produce a loss-of-function allele of Axn. Embryos lacking zygotic Axn are still wild type and only upon removal of the maternally contributed Axn gene product is a naked cuticle revealed. Thus, the RNAi experiments successfully disrupt the maternally contributed Axn gene product and produce a phenotype identical to that of a loss-of-function mutation in the gene (Willert, 1999b).

To address further whether Axn regulates Wg signaling, the UAS-Gal4 system was used to overexpress Axn in various tissues. The Daughterless (Da)-Gal4 driver expresses early during embryogenesis; when combined with UAS-wg, it produces a completely naked cuticle. Overexpression of Axn using the Da-Gal4 driver produces a loss of wg-like phenotype, a cuticle covered with denticles, consistent with overexpression of Axn blocking Wg signaling in the embryonic epidermis. To extend this study Axn was misexpressed in the wing using the 69B-Gal4 driver. wg is expressed in a narrow stripe along the presumptive wing margin where it is required for proneural achaete-scute complex gene expression and for the formation of margin bristles. Loss of Wg signaling along the wing margin, as in the case of dsh loss-of-function clones, leads to loss of these margin bristles and notches along the wing. Overexpression of Axn in the wing also produces this wing notching effect, a result consistent with Axn interfering with the Wg signaling pathway at the wing margin (Willert, 1999b).

Deletion of the RGS domain of mouse Axin produces a dominant negative Axin protein, as assayed by its ability to induce a secondary axis when overexpressed in Xenopus embryos (Itoh, 1998 and Zeng, 1997). An analogous mutation was constructed in Axn that deletes the entire RGS domain (DaxinDeltaRGS; deletion of amino acids 50 to 172). Surprisingly, when overexpressed, this Axn mutant produces the same phenotypes in the embryo and in the wing as does wild-type Axn overexpression. Thus, the RGS domain is dispensable in overexpression assays for Axn. Thus, the RGS domain, which interacts with the APC protein (Behrens, 1998 and Hamada, 1999), is dispensable for overexpressed Axin’s ability to block Wnt signaling. The discrepancy between the Xenopus and fly data with respect to the RGS deleted Axin may be explained in a number of ways. In analogy to APC, Axin overexpression in various organisms may produce distinct phenotypes. APC overexpression in mouse cell culture is known to down regulate Wnt signaling, while overexpression of APC in Xenopus embryos leads to axis duplication, a hallmark phenotype of genes that activate Wnt signaling. As another exception to the model of APC being a negative factor, the C. elegans APC homolog Apr-1 has been shown to positively regulate Wnt signaling. The reasons for the inconsistent phenotypes for APC loss- and gain-of-function are not yet understood (Willert, 1999b and references).

In support of the findings presented by Willert (1999b), Hart (1998) has shown that overexpression of human Axin lacking the APC binding domain (i.e. the RGS domain) promotes Axin’s ability to downregulate beta-catenin levels. Although both mutant forms of mouse Axin (Zeng, 1997) and Axn primarily delete the RGS domain, there are subtle differences in the deletions: DaxinDeltaRGS deletes the entire RGS domain (amino acids 50-172 in Axn corresponding to amino acids 89-216 in mouse Axin) while mouse AxinDeltaRGS deletes part of the RGS domain and some N-terminally flanking sequence (amino acids 124-227 in Axin corresponding to amino acids 87-183 in Axn). In sum, it remains to be established whether the interaction between Axin and APC is critical for Axin to inhibit Wnt signaling. A genetic analysis of the Axn gene and identification of mutations that disrupt this interaction will likely provide insights into this question (Willert, 1999b).

The above data clearly demonstrate that Axn loss-of-function or overexpression produces phenotypes similar to wg overexpression or loss-of-function, respectively. However, it fails to demonstrate that Axn can modify the effects of Wnt signaling. To examine whether ectopic Axn expression can interfere with the effects of Wnt signaling, wg was misexpressed in the eye and DWnt-2 was misexpressed in the ovary. When overexpressed in the eye, wg produces a glassy eye that is greatly reduced in size compared to a wild-type eye. Misexpression of Axn in the eye produces a weakly rough eye. When Axn and wg are both overexpressed, the morphology of the eye is intermediate to that of a wild-type eye and that of a wg eye, indicating that Axn can interfere with the Wg signal (Willert, 1999b).

In order to address whether Axin inhibits only Wg, or whether it can function as a more general inhibitor of Wnt signals, an examination was carried out to see whether Axn could interfere with a different Wnt signal. Four Wnt genes have been identified in Drosophila (see The World Wide Web Wnt Window ), but mutations have been described only for two of them: wg and DWnt-2. Loss of DWnt-2 produces a muscle migration defect in the male gonads, resulting in male sterility, and a lack of the characteristic pigment cells that migrate over the male testis. Ovaries are normally not surrounded by pigment cells, but misexpression of DWnt-2 in females can induce ectopic male-specific pigment cells. This dominant phenotype of DWnt-2 misexpression was used to address whether Daxin can block ectopic pigment cell formation. Overexpression of Axn strongly reduces the frequency of DWnt-2-mediated pigment cell formation in ovaries, demonstrating that Axn can block the DWnt-2 signal in addition to the Wg signal. Overexpression of a dominant-negative Pangolin/dTCF lacking the amino-terminal Arm binding domain (DN-dTCF) also blocks the ectopic DWnt-2 induced pigment cells in the ovary (Willert, 1999b).

The Adenomatous polyposis coli tumour suppressor is essential for Axin complex assembly and function and opposes Axin's interaction with Dishevelled

Most cases of colorectal cancer are linked to mutational inactivation of the Adenomatous polyposis coli (APC) tumour suppressor. APC downregulates Wnt signalling by enabling Axin to promote the degradation of the Wnt signalling effector β-catenin (Armadillo in flies). This depends on Axin's DIX domain whose polymerization allows it to form dynamic protein assemblies ('degradasomes'). Axin is inactivated upon Wnt signalling, by heteropolymerization with the DIX domain of Dishevelled, which recruits it into membrane-associated 'signalosomes'. How APC promotes Axin's function is unclear, especially as it has been reported that APC's function can be bypassed by overexpression of Axin. Examining apc null mutant Drosophila tissues, it was discovered that APC is required for Axin degradasome assembly, itself essential for Armadillo downregulation. Degradasome assembly is also attenuated in APC mutant cancer cells. Notably, Axin becomes prone to Dishevelled-dependent plasma membrane recruitment in the absence of APC, indicating a crucial role of APC in opposing the interaction of Axin with Dishevelled. Indeed, co-expression experiments reveal that APC displaces Dishevelled from Axin assemblies, promoting degradasome over signalosome formation in the absence of Wnts. APC thus empowers Axin to function in two ways-by enabling its DIX-dependent self-assembly, and by opposing its DIX-dependent copolymerization with Dishevelled and consequent inactivation (Mendoza-Topaz, 2011).

Drosophila apc null mutants wer used for stringent in vivo function tests to show that APC is indispensable in Drosophila tissues for Axin's activity in assembling functional degradasomes that destabilize Armadillo. The evidence suggests that the same is also true for APC mutant colorectal cancer cells in which Axin–GFP, if expressed at low levels, shows a marked tendency to fail in assembling functional degradasomes. This provides a new insight into how APC promotes the destabilization of Armadillo/β-catenin—namely by enabling Axin to assemble degradasomes. The failure of this assembly step explains why Axin fails to destabilize Armadillo/β-catenin, given that this function of Axin crucially depends on its DAX-dependent polymerization. It is emphasized that previous studies have shown that APC loss-of-function can be bypassed by Axin overexpression, which indicated a non-obligatory role of APC in the destabilization of β-catenin/Armadillo. However, these studies underestimated APC's essential role in this process since Axin was assayed under conditions of residual APC function, and possibly also since high Axin overexpression levels were used for complementation (Mendoza-Topaz, 2011).

Why does Axin fail to assemble functional degradasomes in the absence of APC? This is believed to be true due to a combination of two different effects of APC loss on Axin. First, Axin is destabilized in the absence of APC, so its levels may fall below the minimal cellular concentration required for DAX-dependent polymerization: note that the DIX domain auto-affinity is in the micromolar range, and so the DAX-dependent polymerization may not occur spontaneously at low cellular Axin concentrations, but might require a co-factor capable of clustering Axin, increasing its local concentration and nucleating polymerization. APC is a candidate for such a co-factor, given its relatively high cellular abundance and affinity to Axin which allow it to associate efficiently with Axin at physiological concentrations. APC might cluster Axin directly, by binding simultaneously to multiple Axin molecules through its multiple Axin-binding sites, or indirectly through additional factors (such as CtBP, itself capable of clustering APC. Notably, Axin would become independent of this co-factor if overexpressed at high enough levels, as this would allow it to overcome its low auto-affinity and to polymerize spontaneously (Mendoza-Topaz, 2011).

Second, the absence of APC (or of binding to APC) renders Axin prone to Dsh-dependent relocation from the cytoplasm to the PM, into signalosome-like particles. Since the recruitment of Axin into signalosomes normally blocks its function in promoting the phosphorylation and destabilization of β-catenin/Armadillo, its relocation to the PM might also explain its inactivity in the absence of APC. The evidence indicates that APC shields Axin from interaction with Dsh in the absence of Wnt signalling, to ensure Axin's function in the cytoplasmic degradasomes. This shielding function of APC may be particularly important in cells experiencing non-canonical Wnt signalling (promoting PM-association of Dishevelled), such as in third-larval instar wing discs.The observations in Drosophila tissues suggested that APC may compete with Dishevelled for association with Axin, which is strongly supported by evidence from co-expression experiments in mammalian cells (carried out in the absence of Wnt stimulation): these indicate that Axin cannot interact simultaneously with APC and Dvl2, and that APC is capable of displacing Dvl2 from Axin protein assemblies. The notion of a competition between APC and Dishevelled for their association with Axin is consistent with previous evidence from epistasis experiments in Drosophila embryos, which indicated that APC acts at the same level as Dishevelled rather than below it (Mendoza-Topaz, 2011).

Evidently, the function of APC that shields Axin from its interaction with Dishevelled is somehow antagonized by Wnt stimulation, which enables Dishevelled to bind to Axin and recruit it to the PM into signalosomes. Indeed, Wnt signalling may overcome the competition between APC and Dishevelled for their binding to Axin, allowing simultaneous interaction of all three proteins. Consistent with this, Axin–GFP appears to co-localize with E-APC in the Wg signalling zones of Drosophila embryos within the PM-associated signalosomes that are likely to also contain Dsh (although this has not been confirmed directly owing to the lack of a suitable Dsh antibody), suggesting that Wg signalling allows all three proteins to coincide in signalosomes (Mendoza-Topaz, 2011).

It is noted that a previous study in Drosophila uncovered a positive role of APC in antagonizing Axin (rather than promoting its function), thereby stimulating signalling through Armadillo. The key evidence supporting this rather unexpected conclusion was that the levels of Axin were upregulated in apc null mutant wing disc clones, as shown by immunofluorescence. This contrasts with the current result from apc mutant embryos, which shows much reduced levels of Axin–GFP, as judged by Western blotting. Although this quantitative biochemical approach is difficult to apply to apc mutant wing disc clones (owing to insufficient apc mutant material), the dramatic PM relocation of Axin–GFP observed in these clones may have been misled by the high levels of apical Axin, and mistaken for a general upregulation rather than simply a relocation (as has been shown for Axin–GFP). The current evidence reaffirms the negative role of APC in Wg/Armadillo signalling, demonstrating an essential function of APC in keeping Axin in the cytoplasm, where it enables it to assemble functional degradasomes (Mendoza-Topaz, 2011).

The main corollary of these results from Drosophila tissues is that APC promotes the DAX-dependent homopolymerization of Axin (required for degradasome assembly), and that it antagonizes the heteropolymerization between Axin and Dishevelled (mediated by DIX–DAX interaction). The latter is further supported by the evidence from co-expression experiments in mammalian cells that APC displaces Dvl2 from Axin puncta. This creates a mechanistic conundrum: APC binds to the N-terminal RGS domain of Axin, but appears to control the DAX-dependent interactions at its C-terminus. Although it is conceivable that APC achieves this at 'long range', given its unusually large size it is more likely that APC relies on additional factors, perhaps even on enzymes, to promote Axin's self-assembly at the expense of its heteropolymerization with Dishevelled. Future work will be required to determine the precise molecular mechanism by which APC enables Axin to assemble functional degradasomes and opposes its recruitment by Dishevelled, and how this is overcome during Wnt signalling (Mendoza-Topaz, 2011).


GENE STRUCTURE

Sequence analysis has identified two Axn species, termed A1 and A2. These forms of Axn are revealed by amino acid differences between two independent Axin isolates: Willert (1999b) and Hamada (1999). Form A2 (Willert, 1999b) includes the six amino acids SRSGSS while Form A1 of Hamada, 1999, does not.

cDNA clone length - 4669 bp


PROTEIN STRUCTURE

Amino Acids - 739

Structural Domains

The Axin cDNA contains two alternative polyadenylation sites and an open reading frame encoding a 739 amino acid protein (form A1). Several clones (3 out of 5) contain an 18 basepair (bp) insert (form A2). This insert in Drosophila is in a similar, albeit not identical, region where in mouse, Axin contains a 108 bp insert to give rise to two different forms (form 1 and 2). The 18 nucleotides in the fly gene code for six amino acids, four of which are serine residues, directly amino-terminal to the DIX domain. No difference in the activities of the two isoforms of either mouse Axin or Drosophila Axn have been identified. The recently published sequence of Drosophila Axin (Hamada, 1999) is identical to form A2, but lacks two serine residues at position 644 and 645. The differences in sequences, including forms A1 and A2, may reflect naturally occurring polymorphisms in the Axn gene. Overall, the identity between mouse and Axn is 21% , although several domains are more highly conserved. The Axn protein is more distantly related (17%) to Conductin (Behrens, 1998), suggesting that it is the vertebrate Axin ortholog. Like the vertebrate Axin proteins, Axn contains an RGS domain near the amino terminus. These domains are found in a family of proteins that regulate G-proteins. Although no such G-protein signaling function has been attributed to the mouse Axin RGS domain (Mao, 1998), the RGS has been shown to be required for the interaction with the APC protein (Behren, 1998). Deletion of this domain in mouse Axin produces a dominant negative Axin protein, as assayed by axis duplication activity in Xenopus embryos (Itoh, 1998; Zeng, 1997). In contrast, overexpression of wild-type mouse Axin inhibits axis duplication, suggesting that interaction with APC is crucial for Axin’s ability to block the Wnt signaling pathway. The region between the RGS and DIX domains, where both GSK-3beta and beta-catenin binding sites are located in Axin and Conductin, contains stretches of lower similarity. Despite this low similarity, Axn does in fact interact with both Arm and Zw3. Hamada (1999) mapped the Arm binding domain in Axn to amino acids 459-538 by precipitating in vitro translated Arm with GST-Axn fusion proteins, a region which contains significant homology with Axin and Conductin. The Axn protein contains four potential nuclear localization signals, but the mouse Axin protein, that also contains a nuclear localization signal, has been localized predominantly to the cytoplasm (Behrens, 1998 and Fagotto, 1999) (Willert, 1999b).


Axin: Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

date revised: 6 October 99

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