Asymmetrically expressed axin required for anterior development in Tribolium

Canonical Wnt signaling has been implicated in an AP axis polarizing mechanism in most animals, despite limited evidence from arthropods. In the long-germ insect, Drosophila, Wnt signaling is not required for global AP patterning, but in short-germ insects including Tribolium castaneum, loss of Wnt signaling affects development of segments in the growth zone but not those defined in the blastoderm. To determine the effects of ectopic Wnt signaling, the expression and function of axin, which encodes a highly conserved negative regulator of the pathway, was analyzed. Tc-axin transcripts maternally localized to the anterior pole in freshly laid eggs. Expression spread toward the posterior pole during the early cleavage stages, becoming ubiquitous by the time the germ rudiment formed. Tc-axin RNAi produced progeny phenotypes that ranged from mildly affected embryos with cuticles displaying a graded loss of anterior structures, to defective embryos that condensed at the posterior pole in the absence of serosa. Altered expression domains of several blastodermal markers indicated anterior expansion of posterior fates. Analysis of other canonical Wnt pathway components and the expansion of Tc-caudal expression, a Wnt target, suggest that the effects of Tc-axin depletion are mediated through this pathway and that Wnt signaling must be inhibited for proper anterior development in Tribolium. These studies provide unique evidence that canonical Wnt signaling must be carefully regulated along the AP axis in an arthropod, and support an ancestral role for Wnt activity in defining AP polarity and patterning in metazoan development (Fu, 2012).

Double abdomen in a short-germ insect: Zygotic control of axis formation revealed in the beetle Tribolium castaneum
The distinction of anterior versus posterior is a crucial first step in animal embryogenesis. In Drosophila, this axis is established by morphogenetic gradients contributed by the mother that regulate zygotic target genes. This principle has been considered to hold true for insects in general. This study investigated symmetry breaking in the beetle Tribolium castaneum, which among insects represents the more ancestral short-germ embryogenesis. Maternal Tc-germ cell-less is required for anterior localization of maternal Tc-axin, which represses Wnt signaling and promotes expression of anterior zygotic genes. Both RNAi targeting Tc-germ cell-less or double RNAi knocking down the zygotic genes Tc-homeobrain and Tc-zen1 led to the formation of a second growth zone at the anterior, which resulted in double-abdomen phenotypes. Conversely, interfering with two posterior factors, Tc-caudal and Wnt, caused double-anterior phenotypes. These findings reveal that maternal and zygotic mechanisms, including Wnt signaling, are required for establishing embryo polarity and induce the segmentation clock in a short-germ insect (Ansari, 2018).

Protein interactions of Axin family members: Low-density lipoprotein receptor-related protein

To understand how the Wnt coreceptor LRP-5 is involved in transducing the canonical Wnt signals, Axin was identified as a protein that interacts with the intracellular domain of LRP-5. LRP-5, when expressed in fibroblast cells, shows no effect on the canonical Wnt signaling pathway by itself, but acts synergistically with Wnt. In contrast, LRP-5 mutants lacking the extracellular domain function as constitutively active forms that bind Axin and that induce LEF-1 activation by destabilizing Axin and stabilizing beta-catenin. Addition of Wnt causes the translocation of Axin to the membrane and enhances the interaction between Axin and LRP-5. In addition, the LRP-5 sequences involved in interactions with Axin are required for LEF-1 activation. Thus, it is concluded that the binding of Axin to LRP-5 is an important part of the Wnt signal transduction pathway (Mao, 2001).

Thus, expression of the cytoplasmic domain of LRP-5, with or without the transmembrane domain, acts as a constitutive activator of the Wnt pathway, activating LEF-1 transcription and stabilizing ß-catenin. This result suggests that the extracellular domain inhibits the function of the intracellular domain. Binding of a Wnt ligand presumably overcomes this inhibition, perhaps by inducing a conformational change in LRP-5 or through interactions with the Frizzled receptor. Intriguingly, a construct containing the intracellular domain and the transmembrane region (LRPC2) was significantly more active than one containing just the intracellular domain (LRPC3), indicating that recruitment of Axin to the membrane is important for the activation of the Wnt pathway by LRP-5. The finding that a LRPC3 variant containing a myristylation signal shows an activity similar to LRPC2 lends further supports to this idea. One of the roles that the translocation plays is Axin destabilization, which has previously been shown to be induced by Wnt, because expression of LRPC2, but not LRPC3, caused Axin degradation. Together with the results showing that Wnt stimulation causes Axin to be recruited to the membrane via LRP-5, it is proposed that Wnt induces Axin destabilization at least in part by stimulating the interaction of LRP-5 with Axin. Although the precise mechanism by which activated LRP-5 causes the destabilization of Axin is not clear, it is reasonable to conclude that Axin destabilization contributes to the stabilization of ß-catenin (Mao, 2001).

The degradation of Axin is not the only means of inhibiting its function. This is consistent with the results that a construct that does not degrade Axin (LRPC3) can still stimulate LEF-1 transcription. Thus, part of the mechanism by which the intracellular domain of LRP-5 inhibits the function of Axin may simply involve the binding of this region to Axin, thus preventing it from participating in the degradation of ß-catenin. The region necessary for binding Axin and for stimulating LEF-1 transcription was narrowed down to 40 C-terminal amino acids in LRP-5. This region contains three copies of a motif PPT/SP, which is conserved in human and mouse LRP-5 and LRP-6 and Drosophila Arrow. Although removal of the very C-terminal motif shows little effect, elimination of two of these motifs reduces both Axin binding and LEF-1 stimulation but still retains some activity. Truncation of an additional 10 amino acids, including the third motif, abolishes Axin binding and transcriptional activation. This repeated motif could function as a phosphorylation site for a serine/threonine kinase. In this light, it is interesting that the wild-type GSK3 strongly stimulates the binding of Axin to LRP-5, whereas a kinase-dead GSK3 does not. While the intracellular domain of LRP-5 cannot be phosphorylated by GSK3 when immunoprecipitated LRPC2 and GSK were tested in an in vitro kinase assay, GSK3 may stimulate an additional kinase. Alternatively, the phosphorylation of Axin by GSK3 might cause it to bind LRP-5 more effectively. For instance, the phosphorylation of Axin by GSK3 enhances Axin's ability to bind ß-catenin and to bind GSK3 itself. Given that GSK3 promotes ß-catenin degradation, while the LRP/Axin interaction has the opposite result, the stimulatory effect of GSK3 on the interaction between Axin and LRP-5 is somewhat surprising. However, such an effect may allow LRP-5 to interact only with the Axin molecules that are associated with GSK3. In this way, LRP-5 could specifically direct the degradation of only those (GSK3-associated) Axin molecules that would otherwise participate in the degradation of ß-catenin (Mao, 2001).

Wnt-3a-induced binding of Axin to LRP-5 occurs within 4 min of the addition of ligand. This ligand-induced process occurs much sooner than the other processes that have thus far been reported. Wnt proteins induce the dephosphorylation of Axin and Dvl within 30 min, and the degradation of Axin starting at approximately 2 hr. Thus, the rapid stimulation of the interaction of LRP-5 and Axin by Wnt-3a suggests that the interaction between LRP-5 and Axin might be one of the first events in the Wnt canonical pathway (Mao, 2001).

In summary, a model is proposed describing the involvement of LRP proteins in the canonical Wnt signaling pathway. In this model, it is suggested that LRP, when activated by Wnt proteins, recruits Axin to the membranes. The translocation of Axin to the membrane prevents it from participating in the degradation of ß-catenin and plays an important role in the destabilization of Axin. LRP-6, a close homolog of LRP-5, may act in the same way as LRP-5, because a LRP-6 mutant that is equivalent to LRPC2 can also bind Axin and constitutively activates LEF-1. Thus, binding to Axin and activation of the canonical Wnt signaling pathway may occur with other LRP-5 homologs, including Arrow (Mao, 2001).

Current models of canonical Wnt signaling assume that a pathway is active if β-catenin becomes nuclearly localized and Wnt target genes are transcribed. In Xenopus, maternal LRP6 is essential in such a pathway, playing a pivotal role in causing expression of the organizer genes siamois and Xnr3, and in establishing the dorsal axis. Evidence iis provided that LRP6 acts by degrading axin protein during the early cleavage stage of development. In the full-grown oocyte, before maturation, axin levels are also regulated by Wnt11 and LRP6. In the oocyte, Wnt11 and/or LRP6 regulates axin to maintain β-catenin at a low level, while in the embryo, asymmetrical Wnt11/LRP6 signaling stabilizes β-catenin and enriches it on the dorsal side. This suggests that canonical Wnt signaling may not exist in simple off or on states, but may also include a third, steady-state, modality (Kofron, 2007)

Protein interactions of Axin family members: GSK3, beta-Catenin, APC, and Phosphatase 2A

The 'ß-catenin destruction complex' is central to canonical Wnt/ß-catenin signaling. The scaffolding protein Axin and the tumor suppressor adenomatous polyposis coli protein (APC) are critical components of this complex, required for rapid ß-catenin turnover. The crystal structure of a complex between ß-catenin and the ß-catenin-binding domain of Axin (Axin-CBD) was determined. The Axin-CBD forms a helix that occupies the groove formed by the third and fourth armadillo repeats of ß-catenin and thus precludes the simultaneous binding of other ß-catenin partners in this region. Biochemical studies demonstrate that, when ß-catenin is phosphorylated, the 20-amino acid repeat region of APC competes with Axin for binding to ß-catenin. It is proposed that a key function of APC in the ß-catenin destruction complex is to remove phosphorylated ß-catenin product from the Axin/GSK-3ß active site (Xing, 2003).

In the canonical Wnt/ß-catenin pathway, ß-catenin mediates the transmission of a Wnt signal into the nucleus and the subsequent activation of target genes. In the absence of a Wnt signal, a cytoplasmic protein complex containing glycogen synthase kinase-3ß (GSK-3ß), the adenomatous polyposis coli protein (APC), and the scaffolding protein Axin, among others, catalyzes the phosphorylation of ß-catenin. This complex has been termed the 'ß-catenin destruction complex' because phosphorylation of ß-catenin targets it for degradation by the proteasome. When the pathway is active, binding of Wnt to its receptors leads to the inactivation of the destruction complex and a consequent accumulation of ß-catenin. The ß-catenin translocates to the nucleus, where it binds to DNA-binding proteins of the Tcf/LEF family. Together they turn on the transcription of Wnt-responsive genes. Although ß-catenin levels may also be regulated by other Axin-independent pathways, phosphorylation of ß-catenin by the ß-catenin destruction complex is the central regulatory step of the canonical Wnt/ß-catenin signaling pathway (Xing, 2003 and references therein).

In the ß-catenin destruction complex, GSK-3ß phosphorylates the critical residues in the N terminus of ß-catenin, contingent upon priming phosphorylation by casein kinase I (CKI). By itself, GSK-3ß does not efficiently phosphorylate ß-catenin; thus Axin plays a critical role in bringing GSK-3ß, CKIalpha, and ß-catenin together to efficiently promote the phosphorylation reaction. The importance of Axin in ß-catenin destruction is underscored by the presence of mutations in the human AXIN1 gene in certain human cancers that are associated with increased ß-catenin levels (Xing, 2003 and references therein).

Another essential component of the destruction complex is the tumor suppressor APC. Mutations of APC cause the elevation of cytoplasmic ß-catenin levels and are found in ~85% of colon cancers. The function of APC in the ß-catenin destruction complex is connected with Axin, because the overexpression of Axin in APC-mutant cancer cells is sufficient to down-regulate ß-catenin levels in these cells. APC contains repetitive ß-catenin interaction motifs, including three 15-amino acid repeats (or possibly four) and seven 20-amino acid repeats. It has been shown that APC plays a role in the transportation of ß-catenin from the nucleus to the cytoplasm, where ß-catenin is phosphorylated and degraded. Although it has also been proposed that APC may attenuate ß-catenin levels by recruiting ß-catenin to the ß-catenin destruction complex, it remains unclear how APC plays an essential role in the ß-catenin destruction complex (Xing, 2003 and references therein).

In addition to Axin, APC, GSK-3ß, and CKI, many other proteins, such as protein phosphatase 2A (PP2A), have also been found to play a role in the ß-catenin destruction complex. A central question now is how these proteins interact to form a molecular machine that efficiently phosphorylates and degrades ß-catenin. Specifically, a catalytic machine must be efficient in both substrate recruitment and product release. How does the ß-catenin destruction complex keep ß-catenin in the complex long enough to be phosphorylated, yet release it quickly enough to maintain the efficiency of phosphorylation (Xing, 2003)?

The crystal structure was determined of a complex between the armadillo repeat region of ß-catenin and the ß-catenin-binding domain of Axin, thus revealing the structural basis of the ß-catenin/Axin interaction. This structure suggests that Axin and the 20-amino acid repeat region of APC compete for binding to ß-catenin when they are both involved in the ß-catenin destruction complex. Biochemical studies clearly show that these regions do compete for binding, but only when the 20-amino acid region is phosphorylated. Based on these data, it is suggested that APC is required for both the recruitment of ß-catenin and the removal of the phosphorylated ß-catenin from the Axin/GSK-3ß active site, which explains the critical role of APC in ß-catenin turnover (Xing, 2003).

Control of stability of beta-catenin (Drosophila homolog: Armadillo) is central in the Wnt signaling pathway. The protein Conductin is found to form a complex with both beta-catenin and the tumor suppressor gene product adenomatous polyposis coli (APC: see Drosophila APC). Conductin induces beta-catenin degradation, although conductin mutants that are deficient in complex formation stabilize beta-catenin. Fragments of APC that contain a conductin-binding domain also block beta-catenin degradation. Thus, conductin is a component of the multiprotein complex that directs beta-catenin to degradation and is located downstream of APC. In Xenopus embryos, conductin interfers with Wnt-induced axis formation (Behrens, 1998).

Axin antagonizes the developmental effects of Wnt in vertebrates. Axin simultaneously binds two components of the Wnt pathway: beta-catenin and its negative regulator, glycogen synthase kinase-3beta (see Drosophila Shaggy). In mammalian cells, Axin inhibits Wnt-1 stimulation of beta-catenin/lymphoid enhancer factor 1-dependent transcription. Axin also blocks beta-catenin-mediated transcription in colon cancer cells that have a mutation in the adenomatous polyposis coli gene. These findings suggest that Axin can block signaling stimulated by Wnt or by adenomatous polyposis coli mutations, by forming a complex with beta-catenin and glycogen synthase kinase-3beta (Sakanaka, 1998).

Axin is a negative regulator of Wnt signaling and dorsal axial development in vertebrates. It has been demonstrated that axin is associated with GSK3 in the Xenopus embryo and the GSK3-binding domain is located in a short region of axin. Binding of GSK3 correlates with the ability of axin to inhibit axial development and with the axis-inducing activity of its dominant-negative form (delta RGS). Wild-type axin, but not delta RGS, forms a complex with beta-catenin. Thus, axin may act as a docking station mediating negative regulation of beta-catenin by GSK3 during dorsoventral axis determination in vertebrate embryos (Itoh, 1998).

Using a yeast two-hybrid method, a novel protein has been identfied that interacts with glycogen synthase kinase 3beta (GSK-3beta) and has 44% amino acid identity with Axin, a negative regulator of the Wnt signaling pathway. This protein has been termed Axil, for Axin like. Like Axin, Axil ventralizes Xenopus embryos and inhibits Xwnt8-induced Xenopus axis duplication. Axil is phosphorylated by GSK-3beta. Axil binds not only to GSK-3beta but also to beta-catenin; the GSK-3beta-binding site of Axil is distinct from the beta-catenin-binding site. Axil enhances GSK-3beta-dependent phosphorylation of beta-catenin. These results indicate that Axil negatively regulates the Wnt signaling pathway by mediating the GSK-3beta-dependent phosphorylation of beta-catenin, thereby inhibiting axis formation (Yamamoto, 1998).

Axin antagonizes the developmental effects of Wnt in vertebrates. Axin simultaneously binds two components of the Wnt pathway: beta-catenin and its negative regulator, glycogen synthase kinase-3beta. In mammalian cells, Axin inhibits Wnt-1 stimulation of beta-catenin/lymphoid enhancer factor 1-dependent transcription. Axin also blocks beta-catenin-mediated transcription in colon cancer cells that have a mutation in the adenomatous polyposis coli gene. These findings suggest that Axin can block signaling stimulated by Wnt or by adenomatous polyposis coli mutations by forming a complex with beta-catenin and glycogen synthase kinase-3beta (Sakanaka, 1998).

Glycogen synthase kinase-3 (GSK-3) mediates epidermal growth factor, insulin and Wnt signals to various downstream events such as glycogen metabolism, gene expression, proliferation and differentiation. A GSK-3 beta-interacting protein has been isolated from a rat brain cDNA library using a yeast two-hybrid method. This protein consists of 832 amino acids and possesses Regulators of G protein Signaling (RGS) and Dishevelled (Dsh) homologous domains in its N- and C-terminal regions, respectively. The predicted amino acid sequence of this GSK-3beta-interacting protein shows 94% identity with mouse Axin, which recently has been identified as a negative regulator of the Wnt signaling pathway; therefore, this protein has been called rAxin (rat Axin). rAxin interacts directly with, and is phosphorylated by, GSK-3beta. rAxin also interacts directly with the armadillo repeats of beta-catenin. The binding site of rAxin for GSK-3beta is distinct from the beta-catenin-binding site; these three proteins formed a ternary complex. Furthermore, rAxin promotes GSK-3beta-dependent phosphorylation of beta-catenin. These results suggest that rAxin negatively regulates the Wnt signaling pathway by interacting with GSK-3beta and beta-catenin and mediating the signal from GSK-3beta to beta-catenin (Ikeda, 1998).

Inactivation of the adenomatous polyposis coli (APC) tumor suppressor protein is responsible for both inherited and sporadic forms of colon cancer. Growth control by APC may relate to its ability to downregulate beta-catenin post-translationally. In cancer, mutations in APC ablate APC's ability to regulate beta-catenin; mutations in beta-catenin prevent beta-catenin's downregulation by wild-type APC. Moreover, signaling by the protein product of the wnt-1 proto-oncogene upregulates beta-catenin and promotes tumorigenesis in mice. In a Xenopus developmental system, Wnt-1 signaling is inhibited by Axin, the product of the murine fused gene. This suggests a possible link between Axin, the Wnt-1 signaling components beta-catenin and glycogen synthase kinase 3 beta (GSK3 beta), and APC. Human Axin (hAxin) is shown to bind directly to beta-catenin, GSK3 beta, and APC in vitro; the endogenous proteins are found in a complex in cells. Binding sites for Axin map to a region of APC that is typically deleted due to cancer-associated mutations in the APC gene. Overexpression of hAxin strongly promotes the downregulation of wild-type beta-catenin in colon cancer cells, whereas mutant oncogenic beta-catenin is unaffected. The downregulation is increased by deletion of the APC-binding domain from Axin, suggesting that APC may function to derepress Axin activity. In addition, hAxin dramatically facilitates the phosphorylation of APC and beta-catenin by GSK3 beta in vitro. It is concluded that Axin acts as a scaffold upon which APC, beta-catenin and GSK3 beta assemble to coordinate the regulation of beta-catenin signaling (Hart, 1998).

Axin is a negative regulator of embryonic axis formation in vertebrates, which acts through a Wnt signal transduction pathway involving the serine/threonine kinase GSK-3 and beta-catenin. Axin has been shown to have distinct binding sites for GSK-3 and beta-catenin and to promote the phosphorylation of beta-catenin and its consequent degradation. This provides an explanation for the ability of Axin to inhibit signaling through beta-catenin. In addition, a more N-terminal region of Axin binds to adenomatous polyposis coli (APC), a tumor suppressor protein that also regulates levels of beta-catenin. These results report on a yeast two-hybrid screen for proteins that interact with the C-terminal third of Axin, a region in which no binding sites for other proteins have previously been identified. Axin can bind to the catalytic subunit of the serine/threonine protein phosphatase 2A (Drosophila homolog: Twins) through a domain between amino acids 632 and 836. This interaction has been confirmed by in vitro binding studies as well as by co-immunoprecipitation of epitope-tagged proteins expressed in cultured cells. These results suggest that protein phosphatase 2A might interact with the Axin.APC.GSK-3.beta-catenin complex, where PP2A could modulate the effect of GSK-3 on beta-catenin or other proteins in the complex. A region of Axin was identified that may allow it to form dimers or multimers. Through two-hybrid and co-immunoprecipitation studies, it has been demonstrated that the C-terminal 100 amino acids of Axin can bind to this same region (Hsu, 1999).

The stabilization of beta-catenin is a key regulatory step during cell fate changes and transformations to tumor cells. Several interacting proteins, including Axin, APC, and the protein kinase GSK-3beta are implicated in regulating beta-catenin phosphorylation and its subsequent degradation. Wnt signaling stabilizes beta-catenin, but it has not been clear whether and how Wnt signaling regulates the beta-catenin complex. Axin has been shown to be dephosphorylated in response to Wnt signaling. The dephosphorylated Axin binds beta-catenin less efficiently than the phosphorylated form. Thus, Wnt signaling lowers Axin's affinity for beta-catenin, thereby disengaging beta-catenin from the degradation machinery (Willert, 1999a).

A model is presented for the role of Axin in Wnt signal transduction. In an unstimulated cell, GSK-3beta is active and phosphorylates Axin, which in turn, recruits beta-catenin into the Axin/GSK-3beta complex. By virtue of its proximity to GSK-3beta, beta-catenin is then phosphorylated. Phosphorylated beta-catenin is then targeted for degradation. Upon transduction of the Wnt signal through the Frizzled (Fz) receptors to Dishevelled, GSK-3beta kinase activity is inhibited so that PP2A dephosphorylates Axin. Unphosphorylated Axin, in turn, no longer recruits beta-catenin to the complex. Failure of beta-catenin to associate with the Axin/GSK-3beta complex prevents beta-catenin's phosphorylation by GSK-3beta so that beta-catenin can accumulate to high levels in the cytoplasm and nucleus and activate transcription in concert with the Tcf/Lef-1 family of transcription factors. GSK-3beta also phosphorylates APC, and in turn APC may facilitate beta-catenin recruitment into the complex; however, this event has not been shown to be regulated by Wnt signaling (Willert, 1999a).

An implication of these results is that the primary target for GSK-3beta phosphorylation is Axin. Previous models have argued that GSK-3beta directly phosphorylates beta-catenin and thereby targets it for degradation. However, GSK-3beta does not bind directly to beta-catenin, and efficient in vitro phosphorylation of beta-catenin by GSK-3beta requires the presence of Axin, which binds both proteins. In contrast, efficient Axin phosphorylation by GSK-3beta does not require additional proteins. Thus, in a Wnt-stimulated cell, beta-catenin fails to be phosphorylated by GSK-3beta because it is not recruited into the Axin/GSK-3beta complex. It should also be noted that phosphorylation of APC by GSK-3beta increases beta-catenin binding to APC; however, in contrast to Axin, phosphorylation of APC has not been shown to be regulated by Wnt signaling (Willert, 1999a and references).

Glycogen synthase kinase 3 (GSK-3) is a constitutively active kinase that negatively regulates its substrates, one of which is beta-catenin, a downstream effector of the Wnt signaling pathway that is required for dorsal-ventral axis specification in the Xenopus embryo. GSK-3 activity is regulated through the opposing activities of multiple proteins. Axin, GSK-3, and beta-catenin form a complex that promotes the GSK-3-mediated phosphorylation and subsequent degradation of beta-catenin. Adenomatous polyposis coli (APC) joins the complex and downregulates beta-catenin in mammalian cells, but its role in Xenopus is less clear. In contrast, GSK-3 binding protein (GBP or FRAT1 ), which is required for axis formation in Xenopus, binds and inhibits GSK-3. It has been shown that GBP inhibits GSK-3, in part, by preventing Axin from binding GSK-3. Similarly, a dominant-negative GSK-3 mu- tant, which causes the same effects as GBP, keeps endogenous GSK-3 from binding to Axin. GBP also functions by preventing the GSK-3-mediated phosphorylation of a protein substrate without eliminating GSK-3's catalytic activity. The previously demonstrated axis-inducing property of overexpressed APC is attributable to its ability to stabilize cytoplasmic beta-catenin levels, demonstrating that APC is impinging upon the canonical Wnt pathway in this model system. These results contribute to a growing understanding of how GSK-3 regulation in the early embryo leads to regional differences in beta-catenin levels and establishment of the dorsal axis (Farr, 2000).

The coordinated regulation of GSK-3 activity by both positive and negative regulators is critical for a wide range of downstream effects, from insulin regulation to developmental processes. The experiments presented here advance the understanding of GSK-3 regulation and suggest the following model for Xgsk-3 regulation in the fertilized Xenopus egg. On the ventral side, beta-catenin levels are kept low by Xgsk-3-dependent phosphorylation in a complex including Axin and APC. Phosphorylation of Axin and APC by Xgsk-3 may be required to assemble this complex. On the dorsal side, GBP inhibits phosphorylation of beta-catenin either by displacing Xgsk-3 from the Axin/APC/Xgsk-3 complex or by prebinding GSK-3 and preventing its association with the Axin-APC complex. GBP binding to Xgsk-3 may block access of protein substrates to the active site. GBP might also prevent Xgsk-3 from phosphorylating Axin and APC, and thereby further inhibit complex formation. The dominant-negative Xgsk-3 acts in a manner analogous to GBP since it either displaces wild-type Xgsk-3 from the complex, or binds endogenous Xgsk-3 and prevents its binding to Axin, thus, inhibiting beta-catenin degradation. The complete chain of molecular events linking fertilization and the dorsal enrichment of beta-catenin has yet to be determined, but this study brings closer an understanding of the interactions of key players. It will ultimately be very interesting to elucidate the relative dorso-ventral abundance of each of these proteins in the Xenopus oocyte and fertilized embryo. However, it may not be just the relative abundance of each of these players that specifies the endogenous axis. Alternatively, posttranslational modifications and/or the presence of yet unidentified factors could affect dorso-ventral differences in known players. GBP is a candidate for the dorsal determinant, and the reagents to examine the possible dorsal localization or dorsal modification of GBP are being developed. Alternatively, GBP may be ubiquitous, but be activated or recruited to function by another factor that becomes dorsally localized in response to sperm entry. The determinant might, for example, bind to the Axin/APC/Xgsk-3 complex and alter its conformation in such a way as to allow GBP to bind Xgsk-3 and remove it from the complex. In this regard, the observation that Dishevelled interacts with GBP and Axin and is dorsally enriched in the early Xenopus embryo is very suggestive of such a mechanism. Alternatively, GBP might function together with Dishevelled to prevent the initial formation of complexes between Axin and GSK-3 in the dorsal cortical cytoplasm, rather than functioning to disrupt complexes once they have formed (Farr, 2000).

Regulation of beta-catenin degradation by intracellular components of the wnt pathway was reconstituted in cytoplasmic extracts of Xenopus eggs and embryos. The ubiquitin-dependent beta-catenin degradation in extracts displays a biochemical requirement for axin, GSK3, and APC. Axin dramatically accelerates while dishevelled inhibits beta-catenin turnover. Through another domain, dishevelled recruits GBP/Frat1 to the APC-axin-GSK3 complex. These results confirm and extend models in which inhibition of GSK3 has two synergistic effects: (1) reduction of APC phosphorylation and loss of affinity for beta-catenin and (2) reduction of beta-catenin phosphorylation and consequent loss of its affinity for the SCF ubiquitin ligase complex. Dishevelled thus stabilizes beta-catenin, which can dissociate from the APC/axin complex and participate in transcriptional activation (Salic, 2000).

The central mechanistic features of beta-catenin phosphorylation and ubiquitination have been well described in several systems by genetic approaches and by overexpression in cell lines and embryos. These studies have identified dsh, axin, GSK3, APC, and GBP/FRAT1 as important regulators of beta-catenin levels. However, there is disagreement as to whether APC acts in a positive or negative fashion; whether GBP plays an important role, or whether it just buffers GSK3; how the components interact; what the precise function of axin and APC is in regulating beta-catenin phosphorylation, and finally, the role of dsh in this process. To address these issues, cytoplasmic extracts from Xenopus eggs were used to reconstitute the regulated degradation of beta-catenin. By measuring beta-catenin stability directly rather than steady-state levels of the protein, the complicating effects of transcription and translation present in other systems were avoided. By adding known amounts of purified proteins to extracts and measuring degradation kinetics, a quantitative analysis of wnt signaling can be performed. This system also allows an examination of mutant proteins that might not show phenotypes if expressed in vivo, due to rapid turnover. In extracts, beta-catenin was degraded with a half-life of 1 hr, dropping to less than 15 min in the presence of added axin, which suggestes that axin is rate limiting for degradation. Addition of dsh, a positive regulator epistatically upstream of GSK3, stabilizes beta-catenin, suggesting that the wnt pathway from dsh to beta-catenin had been recapitulated in extracts (Salic, 2000).

This in vitro system allowed for a test of the biochemical requirement for several different components of the wnt pathway, ruling out the necessary involvement of any machinery upstream of protein translation. beta-catenin degradation depends on axin, GSK3, and APC; extracts depleted of any of these proteins cannot degrade beta-catenin. Of the four defined domains in axin, only the C-terminal DIX domain is dispensable for activity. The DIX domain of axin binds a related domain in dsh, and this interaction is required for dsh function; hence, binding of axin and dsh through the DIX domains confers signal-dependent regulation of beta-catenin. Another domain of dsh required for activity is PDZ, which binds GBP. Dsh and GBP synergize to inhibit beta-catenin degradation in extracts and beta-catenin phosphorylation in vitro, which are both axin-dependent processes. The inhibitory effect of GBP and dsh on beta-catenin degradation in extracts is reversed by high concentrations of GSK3 due to titration of dsh/GBP on axin. These observations further support a role for dsh, as an adaptor protein recruiting GBP to the axin/GSK3/APC/beta-catenin complex and inhibiting locally the enzymatic activity of GSK3. These recent results suggest that the axin-GSK3 interaction is highly dynamic and that increasing the local GBP concentration at the level of the complex efficiently promotes the dissociation of GSK3 from its site on axin (Salic, 2000).

The simplest model would have beta-catenin and GSK3 binding to their sites on axin and GBP binding to its site on dsh. When dsh and axin bind via their DIX domains, GSK3 would be inhibited. How does APC fit into this model? In addition to the mechanistic confusions about the role of APC, there are suggestions that APC can be an inhibitor of beta-catenin degradation in some circumstances or an activator in others. Specifically in Xenopus, injection of APC can cause secondary axis formation, the opposite of the ventralizing effects of axin injections. In an in vitro system, there is an absolute requirement for APC in beta-catenin degradation, consistent with genetic experiments in Drosophila and experiments in cultured cells. The confusion is likely to be simply due to the tendency of overexpressed APC to act as a dominant inhibitor. In Xenopus extracts, overexpressed APC is a potent inhibitor of beta-catenin degradation, although these experiments yield a mixture of truncated and full-length APC proteins. The role of APC in beta-catenin degradation is clear from the in vitro experiments. An interaction of APC with the RGS domain of axin is essential for beta-catenin degradation, demonstrating that endogenous APC is in fact a negative regulator of beta-catenin levels in Xenopus. Although beta-catenin binds to axin beads in a purified system, the two proteins show little interaction in the nanomolar range. Extracts contain an activity that promotes the binding of beta-catenin to axin at low concentrations. The activity is inhibited by the RGS domain of axin, suggesting that it is either APC or APC complexed to other proteins. Also, axinDeltaRGS (which cannot bind APC) does not bind beta-catenin in extracts. With purified components in vitro, APC accelerates the binding of beta-catenin to axin, recapitulating the stimulating effect of extracts on binding between the two proteins. Taken together, these results identify APC as the activity stimulating the axin-beta-catenin interaction in extracts (Salic, 2000).

beta-catenin interacts with numerous cellular proteins, some not directly involved in wnt signaling. The requirement for an APC-like molecule makes sense if one realizes that not only the stability of beta-catenin is important but also its availability for other interactions, both transcriptional and cytoskeletal. Aside from participating in beta-catenin degradation, APC also prevents the interaction of beta-catenin with Tcf3, thus inhibiting the transcriptional activation function of beta-catenin. Additionally, APC maintains a pool of beta-catenin in a state that can either lead to its degradation or release in response to a wnt signal. The phosphorylation state of APC is maintained by rapid futile cycles of opposing phosphorylation and dephosphorylation reactions. Changes in the phosphorylation of APC by GSK3 acts as a switch to trigger either beta-catenin degradation or its discharge in the cytoplasm. Although it has been suggested that phosphorylation of axin modulates binding to beta-catenin, no evidence could be found for such a regulation in extracts because (1) APC is the principal mediator of the axin-beta-catenin interaction and (2) using purified axin and beta-catenin, an increase in the affinity of beta-catenin for axin due to phosphorylation of the latter by GSK3 could not be detected. These data suggests that the APC/axin/GSK3/beta-catenin complex exists in two states. In one state, beta-catenin is bound tightly to axin via APC; beta-catenin and APC are both phosphorylated by GSK3. In this situation, phosphorylated beta-catenin can only be removed through SCF-dependent degradation while phosphorylated APC continues to bind beta-catenin molecules avidly. Upon binding of active dsh to axin, GBP interacts with GSK3, removing it from axin competitively. This inhibits phosphorylation on both beta-catenin and APC. The former stabilizes beta-catenin to ubiquitination, and the latter releases intact beta-catenin so that it can interact with other partners. The beta-catenin-binding site on axin is also important because its deletion impairs the function of axin. Although this scenario is written as a set of irreversible steps, it is likely that all binding events are dynamic. The fact that dominant-negative GSK3 blocks degradation of beta-catenin in extracts suggests that the interaction between endogenous axin and GSK3 must be dynamic. The dissociation rate for GSK3 bound to axin must be fast enough to allow its displacement by the dominant-negative mutant on a time scale of perhaps less than 1 hr. The same must be true for the axin-APC interaction, since the RGS domain of axin (which interacts with APC) blocks beta-catenin degradation (Salic, 2000).

The stability of beta-catenin is subject to a system of weak protein interactions and posttranslational modifications. The ability to dissect this pathway in vitro without altering appreciably its kinetics and without simplifying essential steps should aid an understanding of these weak interactions and their physiological consequences. Several remaining questions should be clarified by studies in partially purified systems and ultimately by reconstitution from purified components. These include the mechanism of signal transmission from frizzled to dsh and the mechanism of dsh activation. In particular, does the wnt signal regulate the GBP-dsh or the dsh-axin interaction? It will also be of interest to know whether APC suffices by itself to mediate beta-catenin degradation or whether associated proteins are also required. Finally, it will be of interest to know whether signaling pathways cross-regulate the wnt pathway and how that might work. In vitro reconstitution should be useful in deciphering the molecular events involved in transducing the wnt signal (Salic, 2000).

Axin and the adenomatous polyposis coli (APC) tumor suppressor protein are components of the Wnt/Wingless growth factor signaling pathway. In the absence of Wnt signal, Axin and APC regulate cytoplasmic levels of the proto-oncogene beta-catenin through the formation of a large complex containing these three proteins, glycogen synthase kinase 3beta (GSK3beta) and several other proteins. Both Axin and APC are known to be critical for beta-catenin regulation, and human cancers result from truncations in APC that eliminate the Axin-binding site. A protease-resistant domain of Axin that contains the APC-binding site is a member of the regulators of G-protein signaling (RGS) superfamily. The crystal structures of this domain alone, and also in complex with an Axin-binding sequence from APC, reveal that the Axin-APC interaction occurs at a conserved groove on a face of the protein that is distinct from the G-protein interface of classical RGS proteins. The RGS-SAMP3 structure also provides insights into the conservation of Axin-binding repeats in diverse species. Previous work has identified sequences from Drosophila APC (dAPC) and APC2 (eAPC) that have homology to the SAMP repeats of vertebrate APC. However, the distant homology between these sequences and those of vertebrate SAMP repeats have made it difficult to identify these sequences unambiguously as Axin-binding sites in the absence of functional studies. The conservation of those residues that have been observed to make contacts in the RGS-SAMP3 complex supports the theory that three such sequences from dAPC and one from eAPC are Axin-binding sites. Thus the molecular interactions observed in the Axin-APC complex provide a rationale for the evolutionary conservation seen in both proteins (Spink, 2000).

Zebrafish embryos homozygous for the masterblind (mbl) mutation exhibit a striking phenotype in which the eyes and telencephalon are reduced or absent and diencephalic fates expand to the front of the brain. mbl minus embryos carry an amino-acid change at a conserved site in the Wnt pathway scaffolding protein, Axin1. The amino-acid substitution present in the mbl allele abolishes the binding of Axin to Gsk3 and affects Tcf-dependent transcription. Therefore, Gsk3 activity may be decreased in mbl minus embryos and in support of this possibility, overexpression of either wild-type Axin1 or Gsk3ß can restore eye and telencephalic fates to mbl minus embryos. These data reveal a crucial role for Axin1-dependent inhibition of the Wnt pathway in the early regional subdivision of the anterior neural plate into telencephalic, diencephalic, and eye-forming territories (Heisenberg, 2001).

This study reveals a novel role for Axin1 in the regional subdivision of prospective forebrain territories. It is proposed that Wnt signaling must be suppressed to allow the development of telencephalic and optic fates and that if this fails to occur, prospective forebrain cells adopt a more caudal, diencephalic identity. It is intriguing that headless/tcf3 mutant zebrafish embryos, in which the canonical Wnt pathway is also likely to be overactivated, show expansion of midbrain (or midbrain/hindbrain boundary) fates at the expense of forebrain fates. Similarly, graded overexpression of Wnt8 can lead to expansion of midbrain-specific gene expression into prospective forebrain territories. These observations raise the possibility that thresholds of Wnt activity may specify different posterior to anterior fates within the neural plate in a manner analogous to that proposed for graded Bmp activity in the allocation of fates along the dorsoventral axis of the neural plate (Heisenberg, 2001).

Axin, APC, and the kinase GSK3ß are part of a destruction complex that regulates the stability of the Wnt pathway effector ß-catenin. In C. elegans, several Wnt-controlled developmental processes have been described, but an Axin ortholog has not been found in the genome sequence and SGG-1/GSK3ß, and the APC-related protein APR-1 have been shown to act in a positive, rather than negative fashion in Wnt signaling. EGL-20/Wnt-dependent expression of the homeobox gene mab-5 in the Q neuroblast lineage requires BAR-1/ß-catenin and POP-1/Tcf. How BAR-1 is regulated by the EGL-20 pathway has been investigated. First, a negative regulator of the EGL-20 pathway, pry-1, has been characterized. pry-1 encodes an RGS and DIX domain-containing protein that is distantly related to Axin/Conductin. These results demonstrate that despite its sequence divergence, PRY-1 is a functional Axin homolog. PRY-1 interacts with BAR-1, SGG-1, and APR-1 and overexpression of PRY-1 inhibits mab-5 expression. Furthermore, pry-1 rescues the zebrafish axin1 mutation masterblind, showing that PRY-1 can functionally interact with vertebrate destruction complex components. Finally, SGG-1, in addition to its positive regulatory role in early embryonic Wnt signaling, may function as a negative regulator of the EGL-20 pathway. It is concluded that a highly divergent destruction complex consisting of PRY-1, SGG-1, and APR-1 regulates BAR-1/ß-catenin signaling in C. elegans (Korswagen, 2002).

The Wnt pathway controls numerous developmental processes via the ß-catenin-TCF/LEF transcription complex. Deregulation of the pathway results in the aberrant accumulation of ß-catenin in the nucleus, often leading to cancer. Normally, cytoplasmic ß-catenin associates with APC and axin and is continuously phosphorylated by GSK-3ß, marking it for proteasomal degradation. Wnt signaling is considered to prevent GSK-3ß from phosphorylating ß-catenin, thus causing its stabilization. However, the Wnt mechanism of action has not been resolved. The regulation of ß-catenin phosphorylation and degradation by the Wnt pathway has been studied. Using mass spectrometry and phosphopeptide-specific antibodies, it has been shown that a complex of axin and casein kinase I (CKI) induces ß-catenin phosphorylation at a single site: serine 45 (S45). Immunopurified axin and recombinant CKI phosphorylate ß-catenin in vitro at S45; CKI inhibition suppresses this phosphorylation in vivo. CKI phosphorylation creates a priming site for GSK-3ß and is both necessary and sufficient to initiate the ß-catenin phosphorylation-degradation cascade. Wnt3A signaling and Dvl overexpression suppress S45 phosphorylation, thereby precluding the initiation of the cascade. Thus, a single, CKI-dependent phosphorylation event serves as a molecular switch for the Wnt pathway (Amit, 2002).

Wnt and Dickkopf (Dkk) regulate the stabilization of beta-catenin antagonistically in the Wnt signaling pathway; however, the molecular mechanism is not clear. In this study, Wnt3a was foud to act in parallel to induce the caveolin-dependent internalization of low-density-lipoprotein receptor-related protein 6 (LRP6), as well as the phosphorylation of LRP6 and the recruitment of Axin to LRP6 on the cell surface membrane. The phosphorylation and internalization of LRP6 occurred independently of one another, and both were necessary for the accumulation of beta-catenin. In contrast, Dkk1, which inhibits Wnt3a-dependent stabilization of beta-catenin, induced the internalization of LRP6 with clathrin. Knockdown of clathrin suppressed the Dkk1-dependent inhibition of the Wnt3a response. Furthermore, Dkk1 reduced the distribution of LRP6 in the lipid raft fraction where caveolin is associated. These results indicate that Wnt3a and Dkk1 shunt LRP6 to distinct internalization pathways in order to activate and inhibit the beta-catenin signaling, respectively (Yamamoto, 2008).

The role of microtubule actin cross-linking factor 1 (MACF1) in the Wnt signaling pathway

MACF1 (microtubule actin cross-linking factor 1) is a multidomain protein that can associate with microfilaments and microtubules. MACF1 was highly expressed in neuronal tissues and the foregut of embryonic day 8.5 (E8.5) embryos and the head fold and primitive streak of E7.5 embryos. MACF1-/- mice die at the gastrulation stage and displaye developmental retardation at E7.5 with defects in the formation of the primitive streak, node, and mesoderm. This phenotype was similar to Wnt-3-/- and LRP5/6 double-knockout embryos. In the absence of Wnt, MACF1 associated with a complex that contains Axin, ß-catenin, GSK3ß and APC. Upon Wnt stimulation, MACF1 appears to be involved in the translocation and subsequent binding of the Axin complex to LRP6 at the cell membrane. Reduction of MACF1 with small interfering RNA decreased the amount of ß-catenin in the nucleus, and led to an inhibition of Wnt-induced TCF/ß-catenin-dependent transcriptional activation. Similar results were obtained with a dominant-negative MACF1 construct that contained the Axin-binding region. Reduction of MACF1 in Wnt-1-expressing P19 cells resulted in decreased T (Brachyury) gene expression, a DNA-binding transcription factor that is a direct target of Wnt/ß-catenin signaling and required for mesoderm formation. These results suggest a new role of MACF1 in the Wnt signaling pathway (Chen, 2006).

The roles of APC and Axin derived from experimental and theoretical analysis of the Wnt pathway

Wnt signaling plays an important role in both oncogenesis and development. Activation of the Wnt pathway results in stabilization of the transcriptional coactivator ß-catenin. Recent studies have demonstrated that axin, which coordinates ß-catenin degradation, is itself degraded. Although the key molecules required for transducing a Wnt signal have been identified, a quantitative understanding of this pathway has been lacking. This study developed a mathematical model for the canonical Wnt pathway that describes the interactions among the core components: Wnt, Frizzled, Dishevelled, GSK3ß, APC, axin, ß-catenin, and TCF. Using a system of differential equations, the model incorporates the kinetics of protein-protein interactions, protein synthesis/degradation, and phosphorylation/dephosphorylation. Initially a reference state of kinetic, thermodynamic, and flux data was defined from experiments using Xenopus extracts. Predictions based on the analysis of the reference state were used iteratively to develop a more refined model from which the effects of prolonged and transient Wnt stimulation were analyzed on ß-catenin and axin turnover. Several unusual features of the Wnt pathway were predicted, some of which were tested experimentally. An insight from this model, which was confirmed experimentally, is that the two scaffold proteins axin and APC promote the formation of degradation complexes in very different ways. This study could also explain the importance of axin degradation in amplifying and sharpening the Wnt signal, and it was shown that the dependence of axin degradation on APC is an essential part of an unappreciated regulatory loop that prevents the accumulation of ß-catenin at decreased APC concentrations. By applying control analysis to the mathematical model, this study demonstrates the modular design, sensitivity, and robustness of the Wnt pathway and derives an explicit expression for tumor suppression and oncogenicity (Lee, 2003).

Theory and quantitation are mutually dependent activities. It would seem unlikely that one would go to the trouble to measure detailed kinetic quantities without a specific model to test, and it is equally unlikely that realistic models can be constructed without the constraints of quantitative experimental data. The intent in trying to reproduce a substantial part of the Wnt pathway in Xenopus egg extracts was to acquire the kind of detailed kinetic data required to build a realistic model. There are several unusual advantages to the extract system that contributed to this effort. The Xenopus egg extract is essentially neat cytoplasm; it reproduces the in vivo rate of β-catenin degradation and responds to known regulators as expected from in vivo experiments. Kinetic experiments with high time resolution are possible in this system, since a well-stirred extract is presumably synchronous in ways in which collections of cells may not be. In extracts it is possible to precisely set the level of components by depletion or addition. The direct output of the canonical Wnt pathway is an easily measured cytoplasmic event, the degradation of β-catenin. Thus, in this unusual system it is possible to acquire quantitative information about signaling pathways, not achievable in vivo. At the same time, these extracts have limitations. The receptor events were not considered, and it is likely that reactions at the plasma membrane contribute to dynamic features. Also, the analysis is incomplete, since there are other components of Wnt signaling, such as casein kinase Iδ, casein kinase Iɛ, and PAR1, as well as cross-talk from other pathways, that influence the behavior of the system. The multiple phosphorylation steps were oversimplified. A simple interconversion of the phosphorylated and unphosphorylated complex of axin, APC, and GSK3β was assumed, whereas in reality multiple phosphorylation states exist within the complex; the states may be random or sequential. The information needed to provide a much more specific model of phosphorylation interconversions is not available at this time, although the model could easily be extended. Finally, there is the question of what Wnt process are being studied. Events in the cytoplasm of unfertilized eggs are being examined. Though endowed with all of the core components of the Wnt pathway, the egg is, as far as is known, transcriptionally silent and not involved in Wnt signaling, though this system is active very soon in embryogenesis. Thus, there is no biological in vivo behavior with which to compare the in vitro behavior. Nevertheless, the basic core circuitry is intact and is presumably prepared for the early Wnt events in the embryo. All the properties of the egg extract system are very similar to that circuitry in vertebrate somatic cells (Lee, 2003).

To build a mathematical description of the Wnt signaling system, Lee (2003) started with the basic circuitry discerned from previous studies in Xenopus embryos and mammalian cells, whose similarity to the in vitro system they had already confirmed. A system of differential equations was derived that described the time-dependent variations of the system variables, i.e., the concentrations of the pathway components and their complexes. Parameters of the model are binding constants of proteins, rate constants of phosphorylations and dephosphorylations, rate constants of protein degradation, and rates of protein synthesis. Model reduction was achieved by considering conservation relations and by applying rapid equilibrium approximations for selected binding processes. Despite these simplifications, the model consists of a nonlinear system of differential equations whose solution requires the use of computers. Not all of these parameters were accessible to measurement. To circumvent this problem, not only kinetic parameters characterizing individual steps were used as primary inputs, but quantities that are more easily accessible from experiment, such as the overall flux of β-catenin degradation. This allowed rate constants to be derived as well as protein concentrations in a reference state, where there was no Wnt signal. This state serves as a starting point for predicting the system behavior during Wnt signaling as well as after experimental perturbations (Lee, 2003).

The basic model reproduced quantitatively the behavior of the reference state, including perturbations of this state achieved by varying the concentration of axin, GSK3β, and TCF. It also reproduced extensions of this to the signaling state. A wide variety of different sets of experimental data could be simulated by the same model, employing the same sets of kinetic parameters. This process was approached iteratively. For example, the early model did not include nonaxin-dependent degradation of β-catenin, but inclusion of this process improved the fit to the experimental data. More significantly, addition of this process had interesting biological implications, which are discussed (Lee, 2003).

In many ways, one of the most peculiar findings was the very low concentration of axin in the Xenopus extracts. Axin levels in other organisms may similarly be very low: Drosophila axin can be detected by Western blotting only following its immunoprecipitation. Although theoretical and experimental studies have shown that axin is inhibitory at high concentrations, both indicate that axin is not present at the optimal concentration for the highest rate of β-catenin degradation. Therefore, axin levels are not set for optimality of β-catenin degradation, but are presumably optimized for some other purpose. Theoretically, axin levels must be held below the very sharp threshold of Dsh inhibition. Experimentally, these thresholds, which blunt Wnt signaling, are observed but are not as sharp as expected, and this may indicate some other compensatory effects. These thresholds would limit axin concentration to well below 1 nM if activated Dsh were constrained to concentrations of below 1 μM. Under these circumstances, it can be expected that axin would never be found at concentrations approaching those of other Wnt pathway components (50-100 nM) (Lee, 2003).

The low concentration of axin relative to other components (such as GSK3β, Dsh, and APC) has another design feature potentially very general and important for the modularity of metazoan signaling pathways. Axin is a critical node point for controlling β-catenin levels, but it also interacts with components shared with several other important pathways. The interaction of these components with axin fluctuates due to Wnt signals (reflecting changes in binding as well as changes in axin levels), yet because the concentration of axin is so low, there will be no appreciable change in the overall levels of GSK3β, Dsh, or APC (all these components important in other pathways would otherwise be driven to fluctuate). The very low axin concentration thus isolates the Wnt pathway from perturbing other systems, a simple mechanism to achieve modularity. Other scaffold proteins may serve similar functions in other pathways. These insights follow from a very simple measurement of axin concentration and suggest the utility of measuring the levels of signaling pathway components in different cell types and circumstances. Since quantitative and kinetic features may be important in defining modules, it suggests that qualitative circuit diagrams of signal transduction may overlook very important design features. Modularity within the Wnt pathway can be defined by an extension of a summation theorem which argues that the steady state of an entire pathway would have control coefficients that added to zero. When the Wnt pathway is broken down to several subpathways, it is found that within these subpathways the control coefficients would sum to zero at steady state. While some of this subdivision is obvious (i.e., the kinase phosphatase module involving the phosporylation of APC and axin complexed to GSK3β), in other cases, such as the β-catenin module, it is much less obvious. Here the reactions include the phosphorylation of β-catenin in the APC/axin/GSK3β complex, the release and degradation of β-catenin, and the synthesis and nonaxin-dependent degradation of β-catenin. Balanced perturbation of these subpathways as a whole will not affect the overall flux of β-catenin degradation. It is not clear whether this concept of modularity might be extended usefully in two other directions: modularity in systems not at steady state, i.e., with transients, and estimates of linkage between pathways by some definition of nonzero summations expressing the degree of independence or modularity (Lee, 2003).

This paper marks one of the first extensions of metabolic control theory to signal transduction. Metabolism and signal transduction seem very different, the former involving the transfer of mass and the latter the transfer of information. In addition, metabolic pathways generally involve dedicated components and the specificity of interaction of substrates and enzymes is very high. Signaling pathways share many components; interactions are often weak. Metabolism, which has had a long history of quantitative study, was a natural field for the development of control theory, and this theory has been successful in converting the specific information about the behavior of enzymes in a pathway to the overall behavior of metabolic circuits. Control coefficients are useful measures of the impact of one process or quantity on another. In its application to metabolism, it allowed erroneous concepts, such as the notion of a rate-determining step, to be disposed. In signal transduction, control coefficients might play a similar role. Here they can be used to indicate quantitively the effects of a particular reaction on some other property, such as flux through the pathway or concentration of another component. By this definition, certain rate constants, such as the phosphorylation and dephosphorylation of APC and axin, have a major influence on the levels of β-catenin, while others, such as the degradation rate of phosphorylated β-catenin, have little effect. The sign and magnitude of these control coefficients give some indication what gene products could be oncogenes or tumor suppressors. By this criterion APC, GSK3β, and axin are potent tumor suppressors, whereas β-catenin is an oncogene. Dsh would be expected to exert only moderate oncogenic effects. Clearly the effects of certain gene products are dependent on context, including their rate of synthesis and steady-state concentration. As understanding of pathways improves, the effect of mutation or pharmacologic inhibition could be estimated quantitatively using control coefficients. The differences between cell types and organisms could be exploited to better predict mutagenic and pharmacologic impact on signal propagation (Lee, 2003).

Despite considerable progress in identifying components of the Wnt pathway, many important mechanistic details are still lacking. This analysis has shown that Dsh seems to act to prevent the phosphorylation of the axin/APC complex, not the phosphorylation of β-catenin. Dsh (complexed to the GSK-3 binding protein FRAT1 or GBP, which has no Drosophila homolog) does not seem to be a general GSK3β inhibitor, like Li+, but rather is focused on the two scaffolding proteins. This was apparent from the biphasic nature of both the theoretical and experimental curves, which suggests that Dsh inhibits the rephosphorylation of axin/APC, but still allows many cycles of β-catenin phosphorylation, ubiquitination, and degradation. This mechanism was further proven by a timing-of-addition experiment. It needs to be further confirmed and extended by looking specifically at individual phosphorylation sites on all the components of the complex. Another insight into the mechanisms of complex formation and control of β-catenin degradation concerns the inhibition of β-catenin degradation at concentrations of axin approaching those of other components. This suggests that axin binds APC, GSK3β, and β-catenin in random order. The axin concentration is limited by other factors; owing to the low concentration of axin, random binding is not likely ever to be a problem. The situation for APC seems very different. The concentration of APC is comparable to that of the other components. Overexpression studies show no inhibitory effects. These theoretical and experimental observations suggest that APC as a scaffold must be very different from axin as a scaffold. Most likely, APC binds components in an ordered manner (Lee, 2003).

Protein interactions of Axin family members: Dishevelled proteins

Axin Evolutionary homologs part 2/2 |

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

Home page: The Interactive Fly © 1995, 1996 Thomas B. Brody, Ph.D.

The Interactive Fly resides on the
Society for Developmental Biology's Web server.