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Molecular and Cellular Biology, October 1999, p. 7147-7157, Vol. 19, No. 10
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Regulation of Glycogen Synthase Kinase 3beta and Downstream Wnt Signaling by Axin

Chester M. Hedgepeth,1 Matthew A. Deardorff,1 Kathleen Rankin,2,3 and Peter S. Klein1,2,3,*

Cell and Molecular Biology Graduate Group,1 Department of Medicine,2 and Howard Hughes Medical Institute,3 University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104-6148

Received 24 February 1999/Returned for modification 4 April 1999/Accepted 22 June 1999


    ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Axin is a recently identified protein encoded by the fused locus in mice that is required for normal vertebrate axis formation. We have defined a 25-amino-acid sequence in axin that comprises the glycogen synthase kinase 3beta (GSK-3beta ) interaction domain (GID). In contrast to full-length axin, which has been shown to antagonize Wnt signaling, the GID inhibits GSK-3beta in vivo and activates Wnt signaling. Similarly, mutants of axin lacking key regulatory domains such as the RGS domain, which is required for interaction with the adenomatous polyposis coli protein, bind and inhibit GSK-3beta in vivo, suggesting that these domains are critical for proper regulation of GSK-3beta activity. We have identified a novel self-interaction domain in axin and have shown that formation of an axin regulatory complex in vivo is critical for axis formation and GSK-3beta activity. Based on these data, we propose that the axin complex may directly regulate GSK-3beta enzymatic activity in vivo. These observations also demonstrate that alternative inhibitors of GSK-3beta can mimic the effect of lithium in developing Xenopus embryos.


    INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Glycogen synthase kinase 3beta (GSK-3beta ) (zeste white-3/shaggy in Drosophila) was first identified as an inhibitor of glycogen synthase (39, 48) and subsequently identified as a negative regulator of Wnt signaling (2, 32). GSK-3beta also plays an essential role in protozoans such as Dictyostelum discoideum and Saccharomyces cerevisiae, where it is required for sporulation (12, 34). In metazoans, Wnt signaling causes inhibition of GSK-3beta (3), which in turn leads to stabilization of cytoplasmic beta -catenin (armadillo in Drosophila) and activation of Wnt target genes (2, 32). Epistasis experiments in Drosophila have suggested that zeste white-3/GSK-3beta functions downstream of disheveled and upstream of armadillo/beta -catenin, but the molecules that directly regulate GSK-3beta in this pathway have not been defined.

Recent data from several labs (1, 11, 16, 20, 21, 43) have shown the interaction of vertebrate GSK-3beta with axin, the product of the fused locus in mice (51). Mice homozygous for certain axin/fused alleles die at embryonic days 8 to 10 with ectopic dorsal axes and other developmental abnormalities (7, 23). In addition, analysis in Xenopus embryos, using mouse axin, showed that axin can function as a negative regulator of the Wnt pathway, since overexpression blocks endogenous dorsal development as well as dorsalization by ectopic Wnt expression. Based on these observations, axin was proposed to be an inhibitor of dorsal axis formation (51).

Molecular cloning of axin revealed that the gene encodes a protein with an amino-terminal domain similar to RGS proteins, which regulate heterotrimeric G-protein function, although it has not yet been reported that axin can regulate G-protein function. Also, axin contains at its C terminus a domain with similarity to disheveled (DIX). We have recently identified a Xenopus homologue of axin that is 69% identical to mammalian axin and also binds to GSK-3beta . Unlike mouse axin, Xenopus axin (Xaxin) shows remarkably high expression in the anterior midbrain during early development of the central nervous system in addition to a lower level of ubiquitous expression (16).

Ventral expression of a dominant inhibitory mouse axin (Delta RGS) in Xenopus causes dorsalization and axis duplication (51). However, a Delta RGS mutant of human axin does not behave as a dominant negative in SW480 cells but rather appears to facilitate the turnover of beta -catenin (11). The mechanism by which the Delta RGS mutant exerts its dominant negative effects in Xenopus has not been studied. However, it has recently been reported that the tumor suppressor APC (adenomatous polyposis coli protein) is able to bind to the RGS domain of axin (1, 11, 25), suggesting that the binding of APC to this region may be important for normal axis formation.

Recent data from several laboratories have demonstrated that axin is part of a multimeric complex containing GSK-3beta , beta -catenin, and APC (11, 20, 21, 43), which act together to regulate beta -catenin stability. Recent work indicates that axin also interacts with protein phosphatase 2A and with axin itself (19), although the functional significance of this self-interaction remains to be elucidated. Axin binds to GSK-3beta strongly in vitro, in COS cells (20), and in Xenopus embryos (reference 21 and this work). This binding facilitates the phosphorylation of beta -catenin by GSK-3beta in vitro (20). Furthermore, overexpression of full-length axin in SW480 cells increases beta -catenin turnover and blocks downstream TCF/LEF-1-mediated transcriptional activity (11, 43). The GSK-3beta and beta -catenin binding sites lie close together in axin, suggesting that axin acts as a scaffold bringing enzyme and substrate into close proximity (20). However, binding of GSK-3beta to axin has not been shown to modulate the enzymatic activity of GSK-3beta .

In addition to axin, another GSK-3beta binding protein (GBP) has recently been identified in Xenopus (49). In addition to binding GSK-3beta , GBP inhibits GSK-3beta activity in vivo. Furthermore, expression of GBP in ventral blastomeres of Xenopus embryos potently induces ectopic dorsal axes, and antisense depletion studies show that GBP is required for dorsal axis formation. The mechanism by which GBP regulates GSK-3beta activity has not yet been elucidated.

Axin appears to act as a Wnt antagonist by binding both GSK-3beta and beta -catenin and facilitating the phosphorylation of beta -catenin by GSK-3beta . Here, we have investigated whether axin or axin mutants directly regulate GSK-3beta activity in Xenopus. Using in vitro and in vivo assays for GSK-3beta activity and Wnt signaling, we have narrowed the GSK-3beta interaction domain (GID) to 25 residues and have shown that this short sequence is a potent in vivo inhibitor of GSK-3beta activity. Similarly, a mutant of axin lacking the RGS domain binds and inhibits GSK-3beta , providing an explanation for the dominant inhibitory activity of the Delta RGS mutant in embryos. In addition, we identify a novel axin self-interaction domain (AID) and provide evidence that axin-axin interactions, as well as the RGS domain, are necessary to maintain GSK-3beta activity and to antagonize Wnt signaling in vivo. These observations also show that alternative inhibitors of GSK-3beta mimic the effects of lithium on embryonic development, providing strong additional support that GSK-3beta is the target of lithium action in this setting.


    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Materials. Recombinant GSK-3beta was purchased from New England Biolabs. beta -Catenin plasmid and antibody were provided by Barry Gumbiner (Memorial Sloan Kettering). Phosphospecific PHF antibodies were provided by Peter Davies (Albert Einstein School of Medicine) (9), and T14/46 antibodies were provided by Virginia M. Y. Lee (University of Pennsylvania). Xenopus GSK-3beta plasmids were provided by David Kimelman (University of Washington). [gamma -32P]ATP was from Amersham. Western analysis was performed by using enhanced chemiluminescence (Amersham). DNA sequencing was performed by the Center for Research on Reproduction and Women's Health at the University of Pennsylvania.

DNA constructs. N-terminal (amino acids [aa] 63-288), GID-1 (aa 277 to 545), and C-terminal (aa 429 to 713) fragments were isolated from a stage VI oocyte cDNA library as described previously (16). These cDNAs were subcloned into pCS2MT in frame with an N-terminal six-Myc epitope tag. Full-length (FL) Xaxin was assembled into CS2MT by using restriction fragments of partial cDNA clones as well as PCR products, and the complete sequence was confirmed by DNA sequencing. The deletion constructs GID 2-6, Delta GID (deletion of aa 324 to 504), and Delta RGS (deletion of aa 80 to 290) were cloned in frame following the Myc tag of pCS2MT, using PCR products generated from the FL Xaxin template. Delta DIX (deletion of aa 778 to 842) was created by restriction digest of FL Xaxin in pCS2MT. A GID-1 construct lacking the Myc epitope tag had activity similar to that of the Myc-tagged construct.

Xenopus embryo and oocyte expression. Stage VI Xenopus oocytes were isolated by collagenase treatment (44) and were injected with mRNA prepared by in vitro transcription (mMessage Machine; Ambion, Austin, Tex.); 10 nl of mRNA (1 to 2 ng/nl) was injected for each construct (unless otherwise specified), and oocytes were incubated for 16 h at 18°C. To analyze the effects of Xaxin constructs on dorsal-ventral pattern formation, Xenopus embryos were injected with 10 nl of mRNA (0.1 to 0.2 ng/nl) into one dorsal or ventral cell of a four-cell embryo, and dorsal axes were assessed at the tadpole stage. For Xaxin coimmunoprecipitation, fertilized eggs were injected with 10 nl of Myc and/or hemagglutinin epitope (HA)-tagged Xaxin (1 ng/nl) and harvested at the blastula stage (stage 8).

GSK-3beta assays. In ovo phosphorylation of tau by GSK-3beta was performed by microinjection of tau protein into oocytes expressing GSK-3beta and Xaxin constructs; after 90 min, oocytes were homogenized and tau phosphorylation was analyzed in Western blots with phosphospecific antibodies as described previously (15). In vitro assays for GSK-3beta were performed as described previously (26). Affinity-purified, recombinant His epitope-tagged GID-2 (GID-2/His protein [see below]) was added to in vitro assays at the concentrations indicated in Fig. 4.

Immunoprecipitation and immunoblotting. Oocytes were homogenized in Triton X-100 lysis buffer (41). Embryos expressing Myc and/or HA-tagged Xaxin were homogenized in a mixture containing 20 mM Tris (pH 7.6), 150 mM NaCl, 0.5% Triton X-100, 1 mM EDTA, 50 mM NaF, 0.5 mM NaVO4, 10 nM microcystin, and Sigma bacterial protease inhibitor cocktail at 1:100. Lysates were immunoprecipitated with anti-Myc epitope antibody 9E10 at approximately 10 µg/ml. After 1 h on ice, immune complexes were collected on anti-mouse immunoglobulin G-coupled protein-A beads (Upstate Biotechnology), washed three times in cold phosphate-buffered saline, and eluted in Laemmli sample buffer. Eluted samples were separated by electrophoresis on sodium dodecyl sulfate-10% polyacrylamide gels and then immunoblotted with GSK-3beta antibodies (0.25 µg/ml; Transduction Laboratories), Myc monoclonal antibody 9E10 (1 µg/ml), or HA antibodies (1:1,000; Amersham) and visualized by enhanced chemiluminescence detection (Amersham).

Yeast two-hybrid assay. FL Xaxin was cloned as a fusion protein with the GAL4 DNA binding domain in pAS2-1 (Clontech). FL Xaxin, Delta DIX Xaxin, and a fragment encoding aa 510 to 777 of Xaxin (AID) were cloned as fusion proteins with the GAL4 transcriptional activation domain in the vector pACT2. Yeast were transformed with these plasmids by using previously described protocols (Clontech). Colony lifts were performed on transformants and were assayed for beta -galactosidase activity to detect interacting proteins. In addition, three Xaxin partial-length cDNAs (Y2H 2, Y2H 6, and Y2H 7; all in pACT2) isolated from a previous yeast two-hybrid screen (16) were analyzed.

Purification of GID. A cDNA encoding aa 320 to 429 (GID-2) of Xaxin was cloned into pET29b (Novagen) in frame with the His epitope tag. This GID-2/His protein was expressed in Escherichia coli BL21/DE3 and purified on nickel-agarose according to standard procedures. Purified GID-2/His was added to a reaction cocktail (final volume = 20 µl) containing recombinant GSK-3beta (25 nM) in GSK-3 assay buffer. This reaction mixture was incubated on ice for 1 h, and then half was assayed for GSK-3beta activity as described above while the other half was incubated with nickel-agarose at 4°C for 1 h with rotation. Following three washes in phosphate-buffered saline Laemmli sample buffer was added; the samples were boiled for 5 min and then subjected to immunoblotting with the anti-GSK-3beta antibody.


    RESULTS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

The GSK-3beta binding domain of axin potently inhibits GSK-3beta . Since GSK-3beta appears to be an important target of lithium action, we were interested in identifying endogenous proteins that might also regulate GSK-3beta activity. Thus, we identified Xaxin in a yeast two-hybrid screen using GSK-3beta as bait (16), similar to the work of others identifying chick and mammalian axins (1, 11, 20, 21, 43). The interaction between axin and GSK-3beta raises the possibility that axin regulates GSK-3beta activity directly. While axin has been shown to act as a protein scaffold to bring the substrate beta -catenin within proximity to GSK-3beta , direct regulation of GSK-3beta enzymatic activity by axin has not been reported. Therefore, we used the tau phosphorylation assay to examine the activity of GSK-3beta in Xenopus oocytes in the presence of FL Xaxin or axin deletion mutants (15). In this assay, phosphorylation of tau, which is detected by Western blotting with the phosphospecific tau antibody PHF-1 (9, 36), is completely dependent on expression of GSK-3beta , as shown previously (15) and in Fig. 1B (compare lanes 1 and 2). Furthermore, GSK-3beta -dependent tau phosphorylation in oocytes occurs at the same sites (serines 396 and 404) as those phosphorylated by GSK-3beta in vitro and with similar, rapid kinetics, indicating that PHF-1 immunoreactivity reflects GSK-3beta activity, as described previously (15). Thus, GSK-3beta was expressed in oocytes together with Myc-tagged FL Xaxin or with the N-terminal GID or C-terminal fragments shown in Fig. 1A. Purified tau protein was then microinjected, and phosphorylation of tau was measured by immunoblotting with PHF-1 or with antibodies that detect all forms of tau.


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  		FIG. 1.   The axin GID inhibits GSK-3beta activity. (A) Schematic of Myc-tagged constructs used in coimmunoprecipitation and activity assays. N-terminal (N-term; aa 63 to 288), GID-1 (aa 277 to 545), C-terminal (C-term; aa 429 to 713), and FL Xaxin (aa 1 to 842) constructs were expressed in oocytes. Shading differentiates the RGS domain (grey), GID (black), and DIX domain (striped). Results from panels B and C for each construct are summarized at the right "Binding" refers to coimmunoprecipitation of GSK-3beta with Myc-tagged axin; "Activity" refers to GSK-3beta -dependent tau phosphorylation; "act" and "inh" stand for active and inhibited GSK-3beta , respectively. (B) Tau phosphorylation in oocytes expressing GSK-3beta (lanes 2 to 6), C-terminal (lane 3), GID (lane 4), and N-terminal (lane 5) fragments, and FL Xaxin (lane 6). Top, immunoblot of oocyte lysates, using an antibody specific for phosphorylated tau (tau-P; lanes 1 to 6), middle, immunoblot with antibodies that recognize both phosphorylated and unphosphorylated tau (tau; lanes 1 to 6); bottom, immunoblot with an antibody to GSK-3beta (lanes 1 to 5). (C) The axin GID binds GSK-3beta in oocytes. Oocytes were injected with GSK-3beta mRNA alone (lane 1) or with RNA encoding Myc-tagged C-terminal (lane 2), GID (lane 3), and N-terminal (lane 4) fragments of axin and FL Xaxin (lane 5). After 16 h, Myc-tagged axin fragments were immunoprecipitated with anti-Myc antibodies and immunoblotted with the anti-GSK-3beta antibody. GSK-3beta coimmunoprecipitates only with the GID (lane 3) and FL Xaxin (lane 5). Approximately equal amounts of axin or axin fragments were present in the immunoprecipitates (data not shown). IgG, immunoglobulin G.

Surprisingly, expression of the GID led to virtually complete inhibition of GSK-3beta -mediated tau phosphorylation (Fig. 1B, lane 4). This is evident from loss of PHF-1 immunoreactivity as well as an increase in the electrophoretic mobility of tau protein (seen with phosphorylation-independent tau antibodies). In contrast, FL Xaxin (lane 6) had no discernible effect on tau phosphorylation. Inhibition of GSK-3beta activity was not due to changes in the level of GSK-3beta protein, as demonstrated by Western blotting for GSK-3beta (Fig. 1B, lower panel, lanes 1 to 5). Coexpression with either the N- or C-terminal fragment (or with unrelated mRNAs) had no effect on GSK-3beta activity (lanes 3 and 5). Both GID and FL Xaxin bound to GSK-3beta , as detected by immunoprecipitation of Myc-tagged axin constructs and Western blotting with a GSK-3beta antibody (Fig. 1C, lanes 3 and 5); neither N- nor C-terminal fragments of axin bound to GSK-3beta (Fig. 1C, lanes 2 and 4).

The GID of axin activates Wnt signaling. These data indicate that the binding of the Xaxin GID to GSK-3beta inhibits GSK-3beta enzymatic activity toward the exogenous substrate tau protein. Although endogenous GSK-3beta is not present at levels sufficient to detect with the tau assay, inhibition of GSK-3beta is known to cause stabilization of cytoplasmic beta -catenin (2, 33), and this serves as a widely used assay for downstream activation of Wnt signaling; inhibition of endogenous GSK-3beta activity also causes stabilization of beta -catenin in Xenopus embryos and oocytes (15, 49, 50). Therefore, beta -catenin protein levels were assessed in Xenopus oocytes injected with either FL Xaxin mRNA or mRNA encoding the N-terminal, GID, or C-terminal domain. Expression of the GID (Fig. 2A, lanes 4 and 7), but not FL Xaxin (Fig. 2A, lane 8) or the N- or C-terminal domain (Fig. 2A, lanes 3 and 5), resulted in accumulation of beta -catenin, similar to the effect of lithium (Fig. 2A, lane 2), a direct inhibitor of GSK-3beta (26, 45). This observation, taken together with the inhibition of tau phosphorylation shown in Fig. 1, suggests that the GID activates downstream Wnt signaling through inhibition of GSK-3beta . (We also find that the axin GID, but not FL Xaxin, strongly activates a LEF-1-luciferase promoter in 293T cells [data not shown].)


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  		FIG. 2.   The axin GID activates Wnt signaling and dorsalizes Xenopus embryos. (A) Accumulation of beta -catenin protein. Cytoplasmic extracts from oocytes expressing beta -catenin (lanes 1 to 8) and incubated in 20 mM LiCl (lane 2) or coexpressing C-terminal (lane 3), GID (lane 4), or N-terminal (lane 5) axin fragments were immunoblotted with a beta -catenin antibody (30). LiCl treatment (lane 2) and the GID (lane 4 and 7) cause accumulation of cytoplasmic beta -catenin. (B) Axis duplication in Xenopus tadpoles by the axin GID. Representative samples are shown at stage 40 (top right) or stage 30 (bottom right) with complete dorsal-anterior axis duplication after injection of 100 pg of axin GID mRNA into one ventral cell of a four-cell embryo. Original axis (arrow) and secondary axis (arrowhead) are indicated. UNIN, uninjected; GID, embryos injected with GID mRNA. (C) Dose-dependent axis duplication by the axin GID. GID mRNA was injected as above at the doses indicated, and axis duplication was scored in tadpoles. Presence of cement gland and eyes was scored as complete axis (solid bars); partial duplications of the trunk and/or heads lacking eyes or cement gland were scored as partial axes (open bars). FL Xaxin mRNA strongly ventralizes embryos when expressed in dorsal blastomeres (data not shown), as described for mouse axin (51).

Activation of Wnt signaling in vivo has been shown by numerous laboratories to lead to axis duplication in Xenopus (14, 31, 33) and mouse (40) embryos. Thus, if inhibition of GSK-3beta by the Xaxin GID activates downstream Wnt signaling, as suggested by the beta -catenin stabilization, then expression of the GID on the ventral side of early Xenopus embryos should also lead to axis duplication, similar to the ventral expression of Wnts or dominant negative GSK-3beta . mRNA encoding FL Xaxin or the GID was microinjected into either ventral or dorsal blastomeres of four-cell Xenopus embryos, which were then cultured until the tadpole stage, and the frequency of ectopic dorsal axes was scored. Ventral injection of GID mRNA caused a high frequency of secondary axis formation (Fig. 2B), with complete axes (including eyes and cement gland) in up to 65% of injected embryos (Fig. 2C). Ectopic axes were detected when as little as 100 pg of mRNA per embryo was microinjected. Dorsal injection of the GID mRNA had no effect on axial development (not shown). Conversely, FL Xaxin caused ventralization when expressed in dorsal blastomeres, as described for mouse axin (51), and induced ectopic cement glands when expressed ventrally, as described for overexpression of GSK-3beta (22). In addition, injection of mRNAs encoding the N- or C-terminal domain had no obvious effect on axial development (not shown). These observations demonstrate that the GID activates downstream Wnt signaling in Xenopus embryos, most likely through inhibition of GSK-3beta and consequent stabilization of beta -catenin.

A 25-aa sequence of Xaxin is sufficient to bind and inhibit GSK-3beta in vivo. To identify the domain of axin necessary for GSK-3beta binding, multiple Myc-tagged GID deletion constructs were expressed in Xenopus oocytes along with GSK-3beta (Fig. 3A). Tau protein was then microinjected, and immunoblotting was performed with phosphorylation-specific tau antibodies. A parallel group of oocytes expressing GSK-3beta and GID mutants were lysed, and GID proteins were immunoprecipitated with the Myc antibody. GSK-3beta was detected in GID immunoprecipitates with GSK-3beta antibodies. Constructs containing aa 380 to 404 of Xaxin (GID-1, -2, -4, -5, and -6) bound to GSK-3beta and inhibited GSK-3beta mediated tau phosphorylation (Fig. 3A). GID-3, which lacks this 25-residue sequence, did not bind to GSK-3beta and had no effect on GSK-3beta activity. These data show that both the binding and inhibitory activities of the GID reside within a 25-aa sequence that is well conserved between Xenopus, chick, mouse, and human axins (Fig. 3B). The sequence does not contain obvious sequence similarity to GBP, a recently described protein that also binds and inhibits GSK-3beta (49).


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  		FIG. 3.   A 25-residue sequence of axin is sufficient for binding and inhibition of GSK-3beta . (A) Schematic of Myc-tagged GID constructs. GID-1 (aa 277 to 545), GID-2 (aa 320 to 429), GID-3 (aa 320 to 375), GID-4 (aa 350 to 429), GID-5 (aa 380 to 429), and GID-6 (aa 380 to 404) were coexpressed in oocytes with GSK-3beta . GSK-3beta binding to axin-GID fragments was assessed by coimmunoprecipitation, and GSK-3beta activity was measured by tau phosphorylation (as in Fig. 1). (B) GID-6 (Xenopus GID) corresponds to a highly conserved region of axin shared between Xenopus, chick (cAxin), mouse (mAxin), and human (hAxin) sequences.

The GID of axin binds but does not inhibit GSK-3beta in vitro. Interestingly, the GID and FL Xaxin have fundamentally different activities in vivo. Ikeda et al., though, recently reported that a region containing the GSK-3beta and beta -catenin interaction domains of rat axin (aa 289 to 506) promoted GSK-3beta -mediated phosphorylation of beta -catenin in vitro (20). Since this sequence is similar to the GID constructs that inhibit GSK-3beta in vivo (Fig. 1B), we investigated whether the GID from Xaxin inhibits GSK-3beta activity in vitro, using GID-2/His protein purified from E. coli. GSK-3beta (25 nM) was incubated with GID-2/His (up to 200-fold molar excess), and each mixture was then assayed for protein kinase activity or, in parallel, for protein-protein interaction by purification on nickel-agarose followed by immunoblotting with GSK-3beta antibodies. GSK-3beta bound specifically to GID-2/His (Fig. 4A, lanes 4 to 6). However, GID-2/His had no significant effect on GSK-3beta -mediated phosphorylation of the GS-2 peptide derived from glycogen synthase (Fig. 4B) and also did not inhibit tau phosphorylation even when GID-2/His was present at a 200-fold molar excess. Taking these findings together with the results of Ikeda et al. (20), we conclude that the GID binds directly to GSK-3beta but does not inhibit its activity in vitro, in contrast to the robust inhibition seen in oocytes and embryos. This observation indicates that an additional factor (or factors) present in vivo is required to inhibit GSK-3beta bound to the axin GID.


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  		FIG. 4.   The GID binds but does not inhibit GSK-3beta in vitro. (A) In vitro binding. Purified, recombinant GSK-3beta (lane 1) was incubated with purified GID-2/His and bound to nickel-agarose. Eluted samples were then immunoblotted with GSK-3beta antibodies. Lanes: 1, GSK-3beta protein prior to column binding; 2, minimal GSK-3beta binding to nickel-agarose in the absence of GID-2/His; 3, GID-2/His without GSK-3beta ; 4 to 6, copurification of GSK-3beta with increasing amounts of GID-2/His. (B) GID-2/His does not inhibit GSK-3beta in vitro. GSK-3beta (25 nM) was incubated with multiple concentrations of GID protein (0.5 nM to 5.0 µM), and phosphorylation of the GS-2 peptide was assayed. Phosphorylation of tau protein was also not inhibited in vitro by up to 5 µM GID-2/His (not shown).

Identification of an AID. Using the yeast two-hybrid assay, we investigated whether axin can bind to itself, to other members of the Wnt pathway, or to the Galpha q subunit of heterotrimeric G proteins. FL Xaxin was cloned into the bait vector (as a fusion with the GAL4 DNA binding domain) and transformed into yeast with FL Xaxin, various axin fragments, or other genes as indicated below in the target vector (as fusion proteins with the GAL4 activation domain [Fig. 5A]). Interaction was assessed in a filter assay for beta -galactosidase activity (6, 10). Axin did not interact with disheveled, C-terminal fragments of Xenopus frizzled 3 and 7 or Galpha q. However, FL Xaxin interacted strongly with GSK-3beta (as expected), as well as with itself. A number of axin deletion constructs (Fig. 5A) were then used to define a domain of axin between aa 510 to 777 that is sufficient to mediate self-interaction. Axin also interacts with itself in Xenopus embryos, as detected by coimmunoprecipitation of Myc-tagged and HA-tagged axin (Fig. 5B). Thus, axin appears to interact with a number of proteins, including itself, APC, GSK-3beta , and beta -catenin. A recent report also showed that axin interacts with itself in two-hybrid assays and in coimmunoprecipitations; however, that work identified a distinct interaction domain lying within the DIX domain (19).


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  		FIG. 5.   AID. (A) FL Xaxin fused to the GAL4 activation domain was cotransformed into S. cerevisiae with the constructs above fused to the GAL4 DNA binding domain. After growth on selective medium, colonies were assayed for expression of beta -galactosidase to identify clones which harbored interacting proteins. FL Xaxin, Delta DIX, Y2H4, and AID constructs all interacted strongly with FL Xaxin. Y2H6 and Y2H7 had no significant interaction with FL Xaxin while retaining the ability to interact with GSK-3beta (16). +, positive interaction; -, no interaction. (B) Myc-tagged FL Xaxin was coexpressed with HA-tagged FL Xaxin in embryos. Samples were immunoprecipitated (IP) with the anti-Myc antibody and immunoblotted with anti-HA antibodies. Top, immunoprecipitates; bottom, embryo lysates prior to immunoprecipitation.

Axin complex formation and Wnt signaling. (i) Axin deletion mutants bind and inhibit GSK-3beta . Because of the inhibitory activity of the Xenopus GID, we have investigated whether other Xaxin deletion mutants might have similar activity. Delta GID lacks aa 324 to 504, removing both the GSK-3beta and beta -catenin binding sites. Delta RGS lacks the RGS domain, which binds the APC protein, and is similar to the mouse Delta RGS mutant described previously (51). Delta DIX lacks the last 64 aa, removing the disheveled homology domain.

Delta RGS bound to GSK-3beta (Fig. 6A) and inhibited GSK-3beta -mediated tau phosphorylation in a dose-dependent manner (Fig. 6B, lanes 6 to 13). Furthermore, inhibition by a fixed concentration of Delta RGS is overcome by increased levels of GSK-3beta (lanes 6 to 9). This inhibition was similar to the effect of the GID (Fig. 1 and 2) as well as lithium (15) and is a likely explanation for the dorsalizing activity of the mouse Delta RGS mutant (51), which is also seen for Xenopus Delta RGS (Fig. 7). The Delta DIX mutant also bound to GSK-3beta and partially inhibited GSK-3beta . This observation suggests that the presence of the RGS and DIX domains, or the proteins that bind to them, is critical for GSK-3beta activity and normal axis formation. Finally, the Delta GID construct did not bind GSK-3beta and had no discernible effect on GSK-3beta activity in the tau assay (Fig. 6A). These observations indicate that the GID is both necessary and sufficient for the in vivo binding and inhibition of GSK-3beta by the axin mutants.


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  		FIG. 6.   Deletion of RGS, GID, or DIX domains. (A) Myc epitope-tagged Delta RGS (deletion of aa 80 to 290), Delta GID (deletion of aa 324 to 504), and Delta DIX (deletion of aa 778 to 842) proteins were expressed in oocytes along with Xenopus GSK-3beta . Interaction with GSK-3beta was assessed by immunoprecipitation with the Myc antibody followed by immunoblotting with anti-GSK-3beta (as in Fig. 1C); GSK-3beta activity was assayed by tau phosphorylation (as in Fig. 1B). Data for FL Xaxin and GID-2 are from Fig. 1 and 3, respectively. (B) Delta RGS inhibits GSK-3beta -mediated tau phosphorylation in a dose-rependent manner. GSK-3beta phosphorylates tau, as assessed by a decrease in electrophoretic mobility (lane 2, 20 ng of RNA; lane 3, 2 ng; lane 4, 1 ng; lane 5, 0.4 ng), and this is inhibited by coexpression of Delta RGS (20 ng; lanes 6 to 9). Delta RGS also inhibits GSK-3beta (2 ng) in a dose-dependent manner (lane 10, 20 ng of Delta RGS mRNA; lane 11, 2 ng; lane 12, 1 ng; lane 13, 0.4 ng). Note that the highest level of GSK-3beta expression overcomes the inhibition by Delta RGS (lane 6).



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  		FIG. 7.   Delta GID reverses dorsalization by Delta RGS. (A) Schematic of proposed complementation of Delta RGS by Delta GID utilizing the AID. The GID lacks the AID. (B) Axis duplication. Delta RGS and GID cause complete axis duplication, with eyes and cement glands present. Coexpression of Delta GID with Delta RGS (Delta RGS+Delta GID) reverses axis duplication, while coexpression of Delta GID with GID (GID+Delta GID) does not. Delta GID alone causes infrequent ectopic posterior axes. (C) Scoring for complete and partial secondary axes. Filled bars represent complete secondary axes (including eyes and cement glands); open bars represent partial secondary axis, including head but lacking eyes or cement gland. The few secondary axes in Delta GID-injected embryos showed posterior duplications without head formation. Delta GID did not affect the level of Delta RGS expression as detected by immunoblotting (data not shown).

(ii) Dorsalization by Delta RGS is rescued by Delta GID. As described above, deletion mutants of axin that bind GSK-3beta inhibit its enzymatic activity in vivo, yet FL Xaxin, which also binds GSK-3beta , does not inhibit its activity. Two general mechanisms could explain this difference. First, deletion of domains such as the RGS or DIX domains could allow axin mutants to become inhibitory in vivo. Second, the presence of these domains could protect GSK-3beta from inhibition, for example, by recruiting additional proteins, such as APC, into the axin complex.

To address this second possibility, we have taken advantage of the AID to reconstitute an axin-GSK-3beta complex in vivo. We coexpressed the inhibitory Delta RGS mutant together with Delta GID, which does not bind GSK-3beta or affect its activity. Interaction between Delta RGS and Delta GID should occur through the AID(s), thus providing RGS and GID binding domains in trans (Fig. 7A). While Delta RGS potently dorsalizes (Fig. 7B and C; 86%, n = 59), embryos expressing both Delta GID and Delta RGS mutants displayed a marked reduction in the frequency and extent of secondary axes (30%, n = 62). Dorsalization by the GID, which lacks the AID, was not rescued by Delta GID (Fig. 7B and C) or by coexpression with an N-terminal fragment that includes the RGS domain (data not shown). Thus, Delta GID specifically rescues the dominant inhibitory effects of Delta RGS. These observations indicate that self-interaction allows recruitment of a cellular factor(s) that prevents inhibition of GSK-3beta (Fig. 8). While these experiments do not rule out the interesting possibility that deletion mutants such as Delta RGS are modified to an inhibitory form in vivo, they nevertheless support the importance of axin complex formation to prevent inhibition of GSK-3beta and to maintain ventral cell fate.


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  		FIG. 8.   Hypothetical model of axin complex formation. In the unstimulated state (top), axin forms a complex with itself (blue), GSK-3beta (green), beta -catenin (red), APC (brown), and protein phosphatase 2A (not shown). In this model, APC (and/or other proteins that bind to the RGS domain) blocks access of a GSK-3beta inhibitor (black box). GSK-3beta remains active, phosphorylating beta -catenin, which is then degraded. Wnt signaling (bottom right) would cause a conformational change in the axin complex (this could be a reversible conformational change or proteolytic cleavage) that now allows access of the GSK-3beta inhibitor. The Delta RGS mutant (bottom left) mimics this conformational change, allowing constitutive access of the GSK-3beta inhibitor. In both cases, inhibition of GSK-3beta leads to stabilization of beta -catenin, which then translocates to the nucleus, binds to LEF-1, and activates transcription of Wnt-responsive genes. Alternative models are discussed in the text.


    DISCUSSION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Our work has focused on understanding the role of axin in the regulation of GSK-3beta activity. To that end, we have defined a 25-aa sequence in axin that binds GSK-3beta and potently inhibits its activity in vivo. We have shown that axin is capable of interacting with itself and have presented data from assays utilizing this self-interaction domain to suggest that a multimeric axin complex is required to maintain GSK-3beta activity in vivo and thus to antagonize Wnt signaling. Furthermore, we have provided an explanation for the in vivo activity of the Delta RGS mutant. These data raise the interesting possibility that axin, in addition to facilitating beta -catenin phosphorylation by GSK-3beta , can also mediate the inhibition of GSK-3beta in response to extracellular signals such as Wnts.

Axin deletion mutants inhibit GSK-3beta and activate Wnt signaling. FL Xaxin and axin deletion mutants containing the GID bind to GSK-3beta (Fig. 1C, 3, and 4). The results of three independent assays suggest that these deletion mutants also inhibit GSK-3beta activity. First, the GID-containing mutants potently block GSK-3beta -mediated tau phosphorylation in oocytes (Fig. 1B). Second, expression of the GID-containing mutants causes accumulation of beta -catenin protein, consistent with inhibition of endogenous GSK-3beta (Fig. 2A). Third, expression of these mutants on the ventral side of Xenopus embryos leads to ectopic axis formation, similar to the effects of Wnts (31), dominant negative GSK-3beta (4, 13, 38), GBP (49), and lithium (24). Clearly, the axin mutants antagonize the activity of GSK-3beta in vivo.

Furthermore, we have identified a 25-aa sequence in axin that, in vivo, is sufficient to mediate the binding and inhibition of GSK-3beta . This 25-aa sequence is well conserved between axin homologues but shows no similarity with other GSK-3beta -interacting proteins such as GBP or the alpha subunit of pyruvate dehydrogenase (18). All of the GID-containing mutants that bind to GSK-3beta result in in vivo inhibition, which suggests that GID binding to GSK-3beta is required for inhibition. GSK-3beta lacking up to 62 residues from the N terminus or 132 residues from the C terminus still binds to axin, suggesting that axin binds within the catalytic domain of GSK-3beta (data not shown). In addition, this N-terminal deletion inactivates GSK-3beta but does not affect axin binding. Thus, kinase activity is not required for axin binding, similar to the results of Sakanaka et al. for an inactive GSK-3beta mutant (43). In contrast, Ikeda et al. have reported that mutants in the ATP binding (K85M) and tyrosine phosphorylation (Y216F) sites of GSK-3beta do not bind rat axin in COS cells (20). It is not clear, therefore, whether GSK-3beta activity is required for axin binding in all cell types.

Axin mutants are not in vitro inhibitors of GSK-3beta activity. In vitro, aa 289 to 506 (a region containing the GSK-3beta and beta -catenin interaction domains) of rat axin promotes GSK-3beta -mediated phosphorylation of beta -catenin (20). Consistent with this, we find that GID-2 protein binds to GSK-3beta but does not inhibit the activity of the enzyme toward either GS-2 peptide or tau in in vitro assays (Fig. 4B and data not shown) even when the GID is in considerable molar excess. This observation suggests that an additional factor (or factors) is required in vivo to inhibit GSK-3beta bound to the GID. In SW480 cells, a Delta RGS mutant of human axin facilitates the turnover of beta -catenin more effectively than full-length human axin (11). This is in contrast to the effects of GID-1 and Delta RGS mutants in Xenopus, which potently inhibit GSK-3beta activity toward multiple substrates (Fig. 1B and 2A) and cause axis duplication (Fig. 2C and 7B) (51). This discrepancy could be explained by regulatory factors present in oocytes and embryos that are absent in SW480 cells. In fact, SW480 cells have an abnormal karyotype, express several mutated genes including the tumor suppressor p53 and APC genes as well as an activated Ki-ras gene (8), and have extremely high basal levels of beta -catenin protein. This raises the possibility that factors mutated or deleted in SW480 cells are required for the proper regulation of GSK-3beta activity in vivo.

Several possible mechanisms may explain why axin mutants, but not full-length protein, inhibit GSK-3beta in vivo. The axin-GID could inhibit GSK-3beta activity by blocking access to substrate. However, fragments of axin that bind both GSK-3beta and beta -catenin (e.g., aa 320 to 502 [Fig. 3A]) still inhibit GSK-3beta activity. In addition, full-length axin also binds GSK-3beta and does not inhibit its activity. Thus, inhibition cannot be explained by limited access to substrate. Alternatively, axin mutants could be modified in vivo to become GSK-3beta inhibitors or could alter the subcellular distribution of GSK-3beta . While these explanations cannot be ruled out, the ability of the Delta GID construct to rescue normal development in embryos expressing Delta RGS suggests another mechanism, as described below.

Axin self-interaction and complex formation. An alternative explanation for inhibition of GSK-3beta by mutated but not full-length axin is that axin complex formation is essential to maintain the activity of GSK-3beta bound to axin and to ensure normal dorsal-ventral development in the embryo (Fig. 8). Thus far, APC, beta -catenin, and GSK-3beta have been shown to bind to an axin complex in vivo (1, 11, 16, 20, 21, 43). In addition, we show here that axin interacts with itself through a region lying between the beta -catenin binding site and the DIX domain. A recent report also observed axin self-interaction (but involving a region within the DIX domain) as well as interaction with protein phosphatase 2A (19). Thus, we propose that mutations that disrupt axin complex formation will lead to in vivo inhibition of GSK-3beta (Fig. 8). For example, the Delta RGS mutant, a potent in vivo inhibitor of GSK-3beta (Fig. 6B), does not bind APC. The effect of Delta RGS may thus be functionally equivalent to a loss of APC, which results in marked accumulation of beta -catenin protein in colonic epithelia, presumably due to inhibition of GSK-3beta -mediated phosphorylation of beta -catenin (42). Recruitment of APC (and/or other factors) to Delta RGS by coexpression with the Delta GID construct should then rescue GSK-3beta activity and normal ventral axis formation, as we observe (Fig. 7B). This rescue requires the AID domain in addition to the RGS domain, since an N-terminal fragment containing the RGS domain but lacking the AID does not rescue activity (data not shown) and fragments containing the GID but not the RGS or self-interaction domains are not rescued by Delta GID. Therefore, we propose that axin complex formation, involving both homomeric and heteromeric interactions, is required to maintain GSK-3beta activity in vivo. An interesting additional possibility is that axin deletion mutants such as GID and Delta RGS are converted in vivo to forms that directly inhibit GSK-3beta , but this in vivo modification or the inhibition of GSK-3beta by the modified axin mutant is blocked by proteins that interact with the RGS domain. However, this mechanism would still be consistent with the requirement for the RGS domain in an axin complex to prevent in vivo inhibition of GSK-3beta .

Comparison with the mouse fused alleles. Two alleles of fused, Futg1 and FuKb, that cause a recessive, embryonic lethal phenotype, with duplication of axial structures, as well as neurological and cardiac abnormalities have been described (37, 51). Interestingly, both alleles produce mRNAs that could encode truncated axin proteins. Futg1 contains a 600-kb insertion that replaces exon 2 (removing the RGS domain), eliminating the major 3.9-kb transcript, but Futg1 homozygotes express a 3.0-kb mRNA that contains exons 3 through 10 (37, 51). Fukb produces two RNA species, one with a deletion in exon 7 and the other with a premature stop predicted to encode the N-terminal 637 residues (46). If translated, both alleles would encode the GID but not sequences that may be required to maintain GSK-3beta activity. These truncated forms of axin would resemble axin deletion mutants, such as Delta RGS, which inhibit GSK-3beta activity, as shown here. Thus, the axis duplication phenotype seen with these alleles could in principle arise from the expression of inhibitory forms of axin rather than the absence of the protein. It will therefore be important to test whether Futg1 and Fukb mice express GID-containing proteins and to define the phenotype of axin gene knockouts in mice and Xenopus.

Regulation of Wnt signaling through inhibition of GSK-3beta . The data presented here suggest that the endogenous axin complex may mediate inhibition of GSK-3beta in response to extracellular signals, such as Wnts, in addition to its proposed role as an antagonist of Wnt signaling. This additional role for the axin complex can be explained if basally, the complex serves as a scaffold to facilitate GSK-3beta mediated phosphorylation of beta -catenin (20), but in response to Wnts, a conformational change in the axin complex allows GSK-3beta to be inhibited (Fig. 8). One candidate for the proposed endogenous inhibitor may be GBP, since it is expressed during early development, can inhibit GSK-3beta activity in vivo, and is required for dorsal axis formation (49). This speculative but testable hypothesis predicts that full-length axin would prevent GBP, or another inhibitory molecule, from binding to and inhibiting GSK-3beta in vivo, but a Wnt-dependent change in the conformation of the axin complex would then allow access to the inhibitor.

Alternative GSK-3beta inhibitors mimic lithium action. We have shown previously that lithium is a direct inhibitor of GSK-3beta activity and proposed that this may explain the mechanism by which lithium alters cell fate in diverse organisms (26). This hypothesis has subsequently been confirmed in vivo by several laboratories (5, 15, 17, 27-29, 35, 45, 47). Nevertheless, it remains possible that lithium alters cell fate by acting through a different target and that inhibition of GSK-3beta by lithium is a remarkable coincidence. One way to test this alternative hypothesis is to determine the effects of other inhibitors of GSK-3beta on cell fate. The data presented here, as well as the effects of GBP (49), show that alternative GSK-3beta inhibitors lead to axis duplication in Xenopus in a manner similar to lithium. (Dominant negative GSK-3beta also mimics lithium in embryos [4, 13, 38] but does not inhibit the enzymatic activity of GSK-3beta [25a].) Together with the observation that lithium phenocopies null mutations in Dictyostelium GSK-3beta , these data argue strongly that the effect of lithium on development is through inhibition of GSK-3beta .

Conclusions. In summary, we have defined the GID of Xaxin to within 25 aa and have shown that in vivo it is a potent inhibitor of GSK-3beta and an activator of Wnt signaling. We have identified a novel AID. Furthermore, we have shown that formation of an axin regulatory complex is critical for both normal axis formation and GSK-3beta activity. Axin mutants containing both substrate and enzyme binding sites but lacking other domains (such as the APC binding domain) inhibit GSK-3beta activity, suggesting that axin may act as more than a scaffolding protein and may regulate GSK-3beta activity directly in response to Wnt signaling.


    ACKNOWLEDGMENTS

We owe many thanks to Leslee Conrad and Jie Zhang for excellent technical assistance. We are also grateful to David Kimelman, Barry Gumbiner, Peter Davies, and Virginia Lee for providing reagents. We thank Tom Kadesch for reading the manuscript and making helpful comments. Many helpful comments were also made by Betsy Wilder, Steve Liebhaber, Hui-Chuan Huang, Dan Kessler, Morrie Birnbaum, and Mitch Lazar.

This work was supported in part by grants from the National Institute of Mental Health and the EJLB Foundation. P.S.K. is an assistant investigator in the Howard Hughes Medical Institute.


    FOOTNOTES

* Corresponding author. Mailing address: Howard Hughes Medical Institute, University of Pennsylvania School of Medicine, 415 Curie Blvd., Philadelphia, PA 19104-6148. Phone: (215) 898-2179. Fax: (215) 573-4320. E-mail: pklein{at}hhmi.upenn.edu.


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