|
|
|
|
|
|
|
||
© The Rockefeller University Press,
0021-9525/2000//691 $5.00
The Journal of Cell Biology, Volume 148, Number 4,
, 2000 691-702
Original Article |
Interaction among Gsk-3, Gbp, Axin, and APC in Xenopus Axis Specification
kimelman{at}u.washington.edu
Glycogen synthase kinase 3 (GSK-3) is a constitutively active kinase that negatively regulates its substrates, one of which is β-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 β-catenin form a complex that promotes the GSK-3–mediated phosphorylation and subsequent degradation of β-catenin. Adenomatous polyposis coli (APC) joins the complex and downregulates β-catenin in mammalian cells, but its role in Xenopus is less clear. In contrast, GBP, which is required for axis formation in Xenopus, binds and inhibits GSK-3. We show here that GSK-3 binding protein (GBP) inhibits GSK-3, in part, by preventing Axin from binding GSK-3. Similarly, we present evidence that a dominant-negative GSK-3 mutant, which causes the same effects as GBP, keeps endogenous GSK-3 from binding to Axin. We show that GBP also functions by preventing the GSK-3–mediated phosphorylation of a protein substrate without eliminating its catalytic activity. Finally, we show that the previously demonstrated axis-inducing property of overexpressed APC is attributable to its ability to stabilize cytoplasmic β-catenin levels, demonstrating that APC is impinging upon the canonical Wnt pathway in this model system. These results contribute to our growing understanding of how GSK-3 regulation in the early embryo leads to regional differences in β-catenin levels and establishment of the dorsal axis.
Key Words: Wnt pathway • dorsal/ventral • β-catenin
© 2000 The Rockefeller University Press
 |
Introduction |
|---|
 TopAbstract Introduction Materials and MethodsResultsDiscussionReferences |
|---|
A large body of evidence has implicated the Wnt pathway in the establishment of the early dorsal signaling center in Xenopus (for reviews see Harland and Gerhart 1997; Heasman 1997; Moon and Kimelman 1998). In response to sperm entry, a microtubule array is established that causes a rotation of a thin layer of cortical cytoplasm towards the side opposite sperm entry (Elinson and Rowning 1988). Cortical rotation leads to the movement of a transplantable dorsalizing activity from the vegetal pole of the egg to the future dorsal side of the embryo (Fujisue et al. 1993; Kikkawa et al. 1996; Sakai 1996; Rowning et al. 1997). Positive effectors of the Wnt pathway, when overexpressed ventrally, mimic this endogenous dorsalizing activity (Moon and Kimelman 1998). However, the role of more upstream members of the pathway, Wnt itself and Dishevelled, is still unclear. Dominant-negative versions of these proteins do not affect axis formation (Hoppler et al. 1996; Sokol 1996), but it may not be possible to introduce these constructs early enough to affect endogenous axis formation. Two recent findings leave open the possibility that these upstream components of the pathway may play a role. First, Dishevelled has been shown recently to be enriched dorsally in one-cell embryos, and ectopic GFP-tagged Dishevelled is transported along the microtubule array during cortical rotation (Miller et al. 1999). Second, a maternal Wnt, Wnt-11, has been shown recently to be asymetrically distributed at the protein level as a result of asymmetric polyadenylation, which is dependent on cortical rotation (Schroeder et al. 1999).
Numerous studies indicate that the dorsal determinant functions to inhibit GSK-3 activity. A kinase dead GSK-3 acts as a dominant-negative, duplicating the axis when expressed ventrally (Dominguez et al. 1995; He et al. 1995; Pierce and Kimelman 1995), and a β-catenin mutant that lacks the GSK-3 phosphorylation sites necessary for its degradation is a more potent axis inducer than the wild-type protein (Yost et al. 1996). β-Catenin is required for axis formation (Heasman et al. 1994) and is enriched dorsally by the two-cell stage in a manner dependent on cortical rotation (Larabell et al. 1997). The dorsal accumulation of β-catenin activates transcription of dorsal-specific genes such as siamois (Brannon et al. 1997) and Xnr-3 (McKendry et al. 1997). Finally, the embryonic cytoplasm containing the dorsalizing activity can cause nuclear accumulation of β-catenin and induce expression of siamois and Xnr3 (Darras et al. 1997; Marikawa et al. 1997).
With β-catenin established as the direct regulator of gene transcription downstream of Wnt signaling, and GSK-3 established as the direct regulator of cytoplasmic β-catenin levels, attention has shifted to the question of how GSK-3 itself is regulated in the early embryo. Two novel families of GSK-3 binding proteins (GBP) have been identified, and both clearly have been shown to regulate GSK-3 function, although in opposite ways. The first of these families of GSK-3 binding proteins includes Xenopus GBP and the mammalian FRATs (Jonkers et al. 1997; Yost et al. 1998). GBP is required for the formation of the endogenous Xenopus axis, and both GBP and FRAT2 have axis-inducing activity when ectopically expressed in Xenopus (Yost et al. 1998). Ectopic GBP stabilizes β-catenin levels in Xenopus (Yost et al. 1998), and FRAT1 elevates the level of cytosolic β-catenin in NIH3T3 cells (Yuan et al. 1999). GBP inhibits the ability of GSK-3 to phosphorylate a protein substrate, tau, in an in vivo assay, suggesting that GBP inhibits the kinase function of GSK-3 (Yost et al. 1998). The presence of mammalian homologues, and the fact that FRAT1 was cloned as a cooperating oncogene which confers a selective advantage to Myc- and Pim1-expressing tumors (Jonkers et al. 1997), suggest that this family of GSK-3 inhibitors is important in processes besides Xenopus axis formation. At present, GBP is the most upstream component shown to be required for specification of the endogenous dorsal axis, though whether it plays a role in all Wnt-mediated signaling events is an open question. It is possible that GBP activates a unique maternal Wnt-related intracellular pathway, independent of Wnt ligand.
The second of these GSK-3 binding protein families includes Axin (Zeng et al. 1997) and the related proteins Axil (Yamamoto et al. 1998) and Conductin (Behrens et al. 1998). Axin functions as a scaffolding protein that directly binds both GSK-3 and its substrate β-catenin (Hart et al. 1998; Ikeda et al. 1998; Kishida et al. 1998; Nakamura et al. 1998; Sakanaka et al. 1998) and greatly enhances GSK-3's ability to phosphorylate β-catenin (Hart et al. 1998; Ikeda et al. 1998), leading to its degradation (Aberle et al. 1997; Hart et al. 1998). Axin mutations in the mouse embryo lead to axis duplication, and overexpression of Axin on the dorsal side of the Xenopus embryo abolishes the endogenous axis (Zeng et al. 1997). In Drosophila, D-Axin is required for the negative regulation of Wg signaling (Hamada et al. 1999; Willert et al. 1999), d-axin mutant clones contain elevated levels of the Drosophila homologue of β-catenin, Arm (Hamada et al. 1999), and D-Axin interacts with Arm and Zeste-white 3 (Willert et al. 1999). Axin is a maternal protein, present throughout development (Zeng et al. 1997; Hamada et al. 1999; Hedgepeth et al. 1999) and found throughout adult mouse tissues (Zeng et al. 1997). The ubiquitous expression of Axin, and its presence in both vertebrates and invertebrates, suggests that it plays a role in a broad range of GSK-3–regulated processes.
Axin functions as part of a multiprotein complex that also includes the APC tumor suppressor protein (for reviews see Polakis 1997; Bienz 1999). APC directly binds Axin and β-catenin in a protein complex that includes GSK-3 (Rubinfeld et al. 1996; Hart et al. 1998; Kishida et al. 1998). Cell culture experiments have implicated APC in the downregulation of β-catenin (Munemitsu et al. 1995; Hayashi et al. 1997; Hart et al. 1998), although studies in Xenopus have suggested that APC has an alternative role, activating dorsal axis formation in a pathway requiring β-catenin but independent of β-catenin stabilization (Vleminckx et al. 1997). Thus, a picture has begun to emerge wherein multiple proteins with opposing effects converge on GSK-3 to regulate its activity and, therefore, cytoplasmic β-catenin levels. The next question is how are these various inputs on GSK-3 activity integrated to regulate the level of β-catenin available for transcription of target genes? We sought to build on our previous work showing that GBP inhibits GSK-3 by determining how this inhibition occurs, and specifically, how GBP opposes the activity of Axin. To gain further insight into the relationship between Axin and GSK-3, we investigated the mechanism of action of the dominant-negative Xenopus GSK-3 (dnXgsk-3). Finally, we sought to clarify the role of APC in Xenopus axis formation by determining the effects of overexpressed APC on β-catenin levels in the embryo.
We show here, both in vivo and in vitro, that GSK-3 cannot bind GBP and Axin simultaneously, leading to the model that GBP functions in part to prevent GSK-3 binding to the Axin/APC/β-catenin complex. We show that dnXgsk-3 binds Axin in vivo, and propose that it functions to induce an ectopic axis in a manner analogous to GBP by keeping endogenous Xenopus GSK-3 (Xgsk-3) from binding to Axin. Additionally, we demonstrate that GBP inhibits Xgsk-3–mediated phosphorylation of protein substrates without eliminating the kinase activity of Xgsk-3. Finally, we show that the APC constructs that duplicate the axis in Xenopus also stabilize β-catenin, providing an explanation for this surprising result. These results provide a framework for understanding how positive and negative regulators affect GSK-3 activity and, subsequently, β-catenin levels.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Embryos and Microinjection
Embryos were obtained as previously described (Newport and Kirschner 1982). UV ventralization of embryos was performed as described (Gerhart et al. 1989). Embryos were microinjected (Moon and Christian 1989) with RNA synthesized from CS2+ (Turner and Weintraub 1994) -derived constructs linearized with NotI or Asp718 using the mMessage mMachine kit (Ambion) according to the manufacturer's instructions.
Production of Proteins
rAxin (298-506) (Ikeda et al. 1998) was produced as an MBP fusion in Escherichia coli (MBP-Axin). Recombinant MBP and MBP-Axin proteins were purified over amylose resin (New England Biolabs) and eluted with maltose according to the manufacturer's directions. GSK-3 was produced as a glutathione-S-transferase fusion in E. coli (GST-GSK-3) (Ikeda et al. 1998), purified over glutathione resin (Pharmacia Biotech), and eluted with 20 mM glutathione. Kinase activity was confirmed using the peptide phosphorylation assay as described (Wang et al. 1994). 35S-labeled proteins were produced in Promega TNT coupled reticulocyte lysate systems according to the manufacturer's directions.
Immunoprecipitation, Western Blotting, and Pull-down Assays
Immunoprecipitations and Western blotting were performed as described (Yost et al. 1998), except for experiments involving detection of endogenous APC. In this case, embryo lysates, prepared as described (Yost et al. 1998), were electrophoresed on 4–12% gradient gels (Novex) and transferred to PVDF in Tris-glycine buffer lacking SDS and methanol. Endogenous APC was immunoprecipitated and detected with anti-APC2 antibody (Rubinfeld et al. 1993; provided by P. Polakis). Anti-MBP antibody was purchased from New England Biolabs, anti-GST antibody was purchased from Amersham Pharmacia Biotech, and anti-HA antibody was purchased from Santa Cruz Biotechnology. Extinction coefficients for MBP, MBP-Axin, and GST-GSK-3 were calculated using ExPASy ProtParam and protein concentrations were determined by UV absorbance at 280 nm. 4 µg of GST-GSK-3 was incubated at room temperature with the specified molar excess concentrations of MBP and MBP-Axin for 10 min with 15 µg RNaseH in binding buffer (20 mM Tris, pH 7.4, 200 mM NaCl). After this short incubation, 15 µg BSA and 9 µl of the [35S]GBP translation mix were added and the volume was increased to 1,500 µl with binding buffer. Proteins were nutated at 4°C for 1 h. 50 µl of prerinsed glutathione resin was added to each reaction and proteins were nutated an additional hour at 4°C. The resin was spun down for 1 min at 1,000 rpm and washed four times in 1 ml PBS, 0.5% NP-40. Sample buffer was added directly to the beads, and proteins were run on a 15% polyacrylamide gel. Alternatively, 4 µg of MBP or MBP-Axin was incubated with 10 µg BSA and 7 µl of the [35S]GBP ± [35S]GSK-3 translation mix, in a final volume of 1 ml binding buffer. Proteins were collected with 50 µl of prerinsed amylose resin.
CREB Peptide Kinase Assay
Immunoprecipitates were washed as usual in NP-40 lysis buffer and were washed additionally with 50 mM Tris, pH 8.0, plus 1 M NaCl, and finally in 50 mM Tris, pH 8.0. Kinase reactions were carried out essentially as described (Wang et al. 1994) in a 30-µl reaction volume for 20 min at 30°C. Levels of Xgsk-3-myc immunoprecipitated alone or with GBP were quantified using densitometry and phosphorus-32 incorporation was normalized to Xgsk-3 protein levels. CREB peptide and the prephosphorylated p-CREB peptide were synthesized by Genosys.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
|
GBP Does Not Inhibit the Ability of Xgsk-3 to Phosphorylate a Peptide Substrate
The demonstration that GBP and Axin cannot bind GSK-3 simultaneously suggests that GBP might inhibit GSK-3 by removing it from the Axin complex or by preventing GSK-3 from associating with Axin. Since we previously demonstrated that GBP inhibits the in vivo phosphorylation of tau (Yost et al. 1998), a protein not thought to be involved in Wnt signaling, we wanted to examine whether GBP might also be able to inhibit GSK-3 by binding and inactivating the catalytic site. We used a modification of a published assay for GSK-3 activity that measures the ability of GSK-3 to phosphorylate the peptide substrate P-CREB, which contains a GSK-3 consensus phosphorylation site, in comparison to the negative control peptide, CREB (Wang et al. 1994). Embryos were injected with RNA encoding Xgsk-3-myc together with control RNA or GBP-FLAG (Yost et al. 1998) RNA and, after 3 h, proteins were extracted and immunoprecipitated. Anti-FLAG antibodies were used to isolate Xgsk-3 bound to GBP when both were injected; anti-myc antibodies were used to isolate Xgsk-3 when it was injected with a control RNA; and uninjected embryos were immunoprecipitated with both antibodies to measure background. The immunoprecipitates were incubated with 
-[32P]ATP in kinase buffer containing P-CREB or CREB, and the incorporated radioactivity was quantified. Western blotting showed that GBP and Xgsk-3 were both expressed and efficiently immunoprecipitated, and remained associated throughout the assay (data not shown). As shown in Fig. 3, GBP does not affect the ability of Xgsk-3 to phosphorylate this peptide substrate. This shows that GBP can inhibit GSK-3 in a way that does not inactivate its catalytic activity.
|
|
Since we were interested in understanding this potentially novel means of β-catenin regulation by APC, we repeated the ectopic expression experiments with APC. Like Vleminckx et al. 1997, we observed that ectopic full-length Xenopus APC (XAPCFL) has axis-inducing activity (Fig. 5 b). Whereas the previous study measured the ability of XAPCFL to induce a dorsal axis when ectopically expressed on the ventral side of the embryo, we measured the ability of XAPCFL to induce an axis in embryos whose endogenous axis has been ablated by UV light. Whereas the latter assay (the UV rescue assay) is more easily quantified than secondary axis formation, the two assays measure the same process.
|
We next wished to determine if the observed truncated protein products we observed when we injected XAPCFL might be generated during processing of the embryos for immunoprecipitation or if they were present in the intact cells. Lysate from uninjected embryos was immunoprecipitated with antibody to APC (Rubinfeld et al. 1993) in parallel with lysate from embryos injected with XAPCFL, which was immunoprecipitated with antibody to the myc epitope. A single band of endogenous APC was detected in the uninjected sample (Fig. 5 d, lane 1), whereas injected siblings produced a variety of faster migrating species in addition to full-length myc-tagged APC (Fig. 5 d, lane 3). Blotting the XAPCFL-injected sample with the anti-APC2 antibody shows that this antibody is capable of recognizing the larger of the truncated products (Fig. 5 d, lane 5). Because this antibody was generated against only the central third of APC, it would not be expected to recognize truncations that contain only the NH2-terminal third or less of APC. Because endogenous APC appeared undegraded, the lysis and immunoprecipitation protocols are not responsible for the observed truncated XAPCFL products. Importantly, this indicates that these truncated products are actually present in the embryo before lysis.
Next, we wished to reexamine the results obtained by Vleminckx et al. 1997 in a β-catenin stabilization experiment, in which an assay we developed (Yost et al. 1996) was used to claim that β-catenin levels were unchanged by injection of full-length XAPC RNA into Xenopus embryos. Low levels of ectopically expressed myc-tagged β-catenin are used to measure the rate of β-catenin degradation in this assay, which is very sensitive to the dose of injected β-catenin-myc (Yost et al. 1996). At the dose of β-catenin-myc used by Vleminckx et al. 1997 (1 ng), we were concerned that it was not possible to measure the degradation of the ectopically expressed β-catenin since it overwhelms the endogenous degradation machinery. To test this, embryos were injected with a range of doses of β-catenin-myc from 10 pg to 500 pg, with or without 1 ng of GBP-myc to stabilize β-catenin levels, and with a control RNA (GFP) to equalize the mass of RNA injected for each treatment (Fig. 6 a). When 50 pg or less of β-catenin-myc was used, there was an increase in the levels of β-catenin protein when GBP-myc was coinjected with β-catenin-myc. Above this dose, however, β-catenin levels were identical in the presence and absence of GBP. Therefore, the previous measurement of β-catenin stability by Vleminckx et al. 1997 would not have given a meaningful result. To reexamine whether ectopic XAPCFL stabilizes β-catenin, embryos were injected with two doses of XAPCFL RNA along with 50 pg β-catenin-myc RNA. The high dose of XAPCFL caused significant axis rescue in UV-irradiated embryos, whereas the low dose did not (not shown). Correspondingly, the high dose of XAPCFL stabilized β-catenin, whereas the low dose did not (Fig. 6 b).
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
GBP Does Not Inhibit GSK-3 Kinase Activity
Since the inhibition of tau phosphorylation requires GBP binding to Xgsk-3 (Yost et al. 1998), we hypothesized that GBP could bind to GSK-3 in a manner that inactivates its catalytic cleft. This mechanism has been shown for the interaction between Cdk2, which shares a high degree of sequence homology with GSK-3, and the Kip/Cip family of CDK inhibitors, which includes p21Cip1,WAF-1, p27Kip1, and p57Kip2. When p27Kip1 binds to Cdk2, it causes large structural changes in the NH2 terminus and catalytic cleft, which eliminates ATP binding and kinase activity (Russo et al. 1996). Using a peptide phosphorylation assay, we find that Xgsk-3 kinase activity is not inhibited by GBP binding. Additionally, FRAT1 does not affect the ability of GSK-3 to phosphorylate a peptide substrate (Yuan et al. 1999). Taken together, the results from the tau assay and the peptide assay suggest that GBP binds to GSK-3 in a manner that does not inhibit the catalytic activity of the active site, unlike the case for p27Kip1 binding to Cdk2. GBP might sterically block access of protein substrates to the active site of GSK-3, and we are currently attempting to map the residues of Xgsk-3 that are important for GBP binding to Xgsk-3 to determine if GBP might bind in the region of Xgsk-3's active site. These results also show that this widely used assay of GSK-3 activity in some important cases may not accurately reflect the extent of GSK-3 functional inhibition.
The Dominant-negative Xgsk-3 Binds Axin
Mutation of a lysine residue, conserved in all kinases, in the ATP binding domain of GSK-3 creates a kinase-deficient protein that acts as a dominant-negative mutant in Xenopus. Overexpression of this mutant on the ventral side of embryos results in the formation of a second body axis (Dominguez et al. 1995; He et al. 1995; Pierce and Kimelman 1995) by locally preventing the degradation of β-catenin (Yost et al. 1996; Larabell et al. 1997). It was previously assumed that dnXgsk-3 functions by competing with endogenous Xgsk-3 for substrates or regulatory molecules. The demonstration that Axin binds GSK-3 and β-catenin and promotes GSK-3 phosphorylation of β-catenin suggested that Axin might be the target of the dnXgsk-3. However, Ikeda et al. 1998 showed that a kinase-deficient mammalian GSK-3 analogous to ours does not bind Axin in cell culture, and other workers have found that similar GSK-3 mutants do not act as dominant-negatives in the Wnt pathway in mammalian cells (Woodgett, J., personal communication). A different GSK-3 kinase mutant, GSK-3Y>F also coimmunoprecipitates less Axin than wild-type GSK-3 in mammalian cells (Yuan et al. 1999). Furthermore, Akt, which inhibits GSK-3 by phosphorylating it, reduces the amount of Axin coimmunoprecipitated by GSK-3 (Yuan et al. 1999). However, we show here that the dominant-negative Xgsk-3 and wild-type Xgsk-3 bind Axin equally well in vivo. This is consistent with a model in which overexpressed kinase-deficient Xgsk-3 acts as a dominant- negative either by displacing endogenous Xgsk-3 already bound to Axin or by preventing endogenous Xgsk-3 from binding Axin as new complexes form. In either case, endogenous Xgsk-3 is prevented from phosphorylating β-catenin by the dominant-negative Xgsk-3 because it is prevented from associating with Axin. In mammalian cells, the lack of a dominant-negative activity for kinase-deficient GSK-3 can be attributed to its inability to bind Axin. It is unclear why a more severe mutation in a mammalian GSK-3 than that of Ikeda et al. 1998 apparently retains its ability to bind Axin in vitro (Sakanaka et al. 1998). However, it was not addressed whether this mutant acted as a dominant-negative.
Overexpressed APC Induces Axis Formation by Stabilizing β-Catenin
APC appears to be an important regulator of β-catenin levels and, as such, has been implicated in cancer progression and in development (for reviews see Polakis 1997; Bienz 1999). Evidence from tissue culture suggests that APC functions in conjunction with GSK-3 and Axin to downregulate β-catenin levels, and that GSK-3 phosphorylates both APC and β-catenin as a prerequisite for β-catenin degradation (Rubinfeld et al. 1996). Data obtained from a study overexpressing APC in Xenopus, however, suggested that the role of APC in frog embryos might be different. Vleminckx et al. 1997 showed that APC could induce an ectopic axis and induce expression of siamois, apparently without stabilizing β-catenin, though cytoplasmic β-catenin was required. The authors concluded that Xenopus APC has a signaling role independent of β-catenin regulation.
Studies in C. elegans have shown that Wnt signaling is also involved in establishing cell polarity in the EMS cell division (Rocheleau et al. 1997; Thorpe et al. 1997). Downstream components of the Wnt pathway, however, including POP-1 (TCF), WRM-1 (β-catenin), and APR-1 (APC), may be used differently in worms than in other organisms (for review see Han 1997). For example, WRM-1 is required for excluding POP-1 from the nucleus of the E cell, allowing that cell to develop as an endoderm (Lin et al. 1998). In vertebrates, in contrast, β-catenin interacts with Tcf-1 in the nucleus to activate transcription. Furthermore, RNAi loss-of-function experiments have shown that apr-1(RNAi) and wrm-1(RNAi) C. elegans embryos have similar defects in E blastoderm development, suggesting that APR-1 positively regulates WRM-1 (Rocheleau et al. 1997).
Our results support a model in which APC acts to downregulate β-catenin in Xenopus. We found that injection of RNA encoding full-length APC (XAPCFL) in Xenopus results in the accumulation of a number of COOH terminally truncated products, presumably because of either incomplete translation or proteolytic cleavage in the embryo. While we (Fig. 5 d) and Vleminckx et al. 1997 did detect a high molecular mass APC product from injected RNA, both studies also observed the presence of truncated species. We also found that a level of XAPCFL, XAPC1, or XAPC4 RNA that rescues axis formation in UV-irradiated embryos also stabilizes β-catenin levels. Thus, we conclude that the APC products expressed from the injected RNA induce an ectopic axis by increasing β-catenin levels, as is observed when Wnt (Larabell et al. 1997), β-catenin (Funayama et al. 1995; Yost et al. 1996), dnXgsk-3 (Yost et al. 1996), or GBP (Yost et al. 1998) are overexpressed in Xenopus embryos. No alternate or parallel pathway needs be invoked to explain the axis-inducing effect of ectopically expressed APC in Xenopus.
Because the various deletion constructs cause axis formation through β-catenin stabilization, we propose that the effects of ectopic XAPCFL are mediated not by the full-length protein, but by the abundant truncated products. We suggest that the ability of ectopic APC to induce an axis in Xenopus is a dominant-negative effect in which the truncated APC products displace the endogenous APC from its normal partners. XAPC4, for example, contains two of the three Axin binding domains identified in APC (Behrens et al. 1998; Hart et al. 1998), and is likely to displace endogenous APC from the Axin–Xgsk-3 complex, preventing it from participating in the degradation of β-catenin. Supporting this interpretation, it has been shown that an APC fragment that binds Conductin stabilizes β-catenin in Neuro2A cells and prevents APC-induced degradation of β-catenin in SW480 cells (Behrens et al. 1998). However, a fragment of human APC similar to XAPC4 reduces cytoplasmic β-catenin levels in SW480 cells (Munemitsu et al. 1995). The different effects of this central part of APC on β-catenin stability may be accounted for by differences in the systems examined. For example, a dominant-negative effect may depend on full-length APC, present in Xenopus but not in SW480 cells, or that more protein may be produced by overexpression in Xenopus than transfection of mammalian cells. Unlike Vleminckx et al. 1997, we also observed axis rescue by XAPC1, which is the most NH2-terminal fragment and lacks the identified Axin binding domains (Fig. 5 a). This fragment contains an NH2-terminal oligomerization domain (Su et al. 1993), which could bind the endogenous APC and block its function, and also a domain of Arm repeats, which might be expected to mediate an interaction between APC and an unidentified partner.
Model for GSK-3 Dorsal–Ventral Regulation in Xenopus
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 (for review see Yost et al. 1997). The experiments presented here advance our understanding of GSK-3 regulation and allow us to propose the following model for Xgsk-3 regulation in the fertilized Xenopus egg. On the ventral side, β-catenin levels are kept low by Xgsk-3–dependent phosphorylation in a complex including Axin and APC (Fig. 7 a). Phosphorylation of Axin and APC by Xgsk-3 may be required to assemble this complex. On the dorsal side, GBP inhibits phosphorylation of β-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 (Fig. 7 b). 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 β-catenin degradation (Fig. 7 c).
|
Note Added in Proof. While this work was under review, a study was published showing that a peptide derived from the GSK-3 binding region of FRAT1 prevents the association of Axin and GSK-3 in agreement with the results presented here (Thomas, G.M., S. Frame, M. Goedert, L. Nathke, P. Polakis, and P. Cohen. 1999. FEBS (Fed. Eur. Biochem. Soc.) Lett. 458:247–251.
|
Acknowledgments |
|---|
This work was supported by grant HD27262 to D. Kimelman; C. Yost, and D. Ferkey were supported by National Institutes of Health training grants T32-GM07270 and T32-HD07183, respectively.
Submitted: 12 April 1999
Revised: 20 December 1999
Accepted: 10 January 2000
G.H. Farr III and D.M. Ferkey contributed equally to this work.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Aberle H. Bauer A. Stappert J. Kispert A. Kemler R. β-Catenin is a target for the ubiquitin-proteasome pathway, EMBO (Eur. Mol. Biol. Organ.) J. 16, 1997 3797–3804.[Medline]
Behrens J. Jerchow B.A. Wurtele M. Grimm J. Asbrand C. Wirtz R. Kuhl M. Wedlich D. Birchmeier W. Functional interaction of an axin homolog, conductin, with β-catenin, APC, and GSK-3β, Science 280, 1998 596–599.
Behrens J. von Kries J.P. Kühl M. Bruhn L. Wedlich D. Grosschedl R. Birchmeier W. Functional interaction of β-catenin with the transcription factor LEF-1, Nature 382, 1996 638–642.[Medline]
Bhanot P. Brink M. Samos C. Hsieh J. Wang Y. Macke J. Andrew D. Nathans J. Nusse R. A new member of the frizzled family from Drosophila functions as a Wingless receptor, Nature 382, 1996 225–230.[Medline]
Bienz M. APCthe plot thickens, Curr. Opin. Genet. Dev 9, 1999 595–603.[Medline]
Brannon M. Gomperts M. Sumoy L. Moon R.T. Kimelman D. A β-catenin/XTcf-3 complex binds to the siamois promoter to regulate specification of the dorsal axis in Xenopus, Genes Dev. 11, 1997 2359–2370.
Dale T.C. Signal transduction by the Wnt family of ligands, Biochem. J 329, 1998 209–223.[Medline]
Darras S. Marikawa Y. Elinson R.P. Lemaire P. Animal and vegetal pole cells of early Xenopus embryos respond differently to maternal dorsal determinantsimplications for the patterning of the organiser, Development 124, 1997 4275–4286.[Abstract]
Dominguez I. Itoh K. Sokol S.Y. Role of glycogen synthase kinase 3β as a negative regulator of dorsoventral axis formation in Xenopus embryos, Proc. Natl. Acad. Sci. USA 92, 1995 8498–8502.
Elinson R.P. Rowning B. A transient array of parallel microtubules in frog eggspotential tracks for a cytoplasmic rotation that specifies the dorso-ventral axis, Dev. Biol. 128, 1988 185–197.[Medline]
Fujisue M. Kobayakawa Y. Yamana K. Occurrence of dorsal axis-inducing activity around the vegetal pole of an uncleaved Xenopus egg and displacement to the equatorial region by cortical rotation, Development 118, 1993 163–170.[Abstract]
Funayama N. Fagotto F. McCrea P. Gumbiner B.M. Embryonic axis induction by the Armadillo repeat domain of β-cateninevidence for intracellular signaling, J. Cell Biol. 128, 1995 959–968.
Gerhart J.C. Danilchick M. Doniach T. Roberts S. Rowning B. Stewart R. Cortical rotation of the Xenopus eggconsequences for the anteroposterior pattern of embryonic dorsal development, Dev. Suppl. 107, 1989 37–51.
Hamada F. Tomoyasu Y. Takatsu Y. Nakamura M. Nagai S. Suzuki A. Fujita F. Shibuya H. Toyoshima K. Ueno N. Akiyama T. Negative regulation of wingless signaling by D-Axin, a Drosophila homolog of Axin, Science 283, 1999 1739–1742.
Han M. Gut reaction to Wnt signaling in worms, Cell 90, 1997 581–584.[Medline]
Harland R. Gerhart J. Formation and function of Spemann's organizer, Annu. Rev. Cell Dev. Biol. 13, 1997 611–667.[Medline]
Hart M.J. de los Santos R. Albert I.N. Rubinfeld B. Polakis P. Downregulation of β-catenin by human Axin and its association with the APC tumor suppressor, β-catenin and GSK-3β, Curr. Biol. 8, 1998 573–581.[Medline]
Hayashi S. Rubinfeld B. Souza B. Polakis P. Wieschaus E. Levine A.J. A Drosophila homolog of the tumor suppressor gene adenomatous polyposis coli down-regulates β-catenin but its zygotic expression is not essential for the regulation of Armadillo, Proc. Natl. Acad. Sci. USA 94, 1997 242–247.
He X. Saint-Jeannet J.-P. Woodgett J.R. Varmus H.E. Dawid I. Glycogen synthase kinase-3 and dorsoventral patterning in Xenopus embryos, Nature 374, 1995 617–622.[Medline]
He X. Saint-Jeannet J.P. Wang Y. Nathans J. Dawid I. Varmus H. A member of the Frizzled protein family mediating axis induction by Wnt-5A, Science 275, 1997 1652–1654.
Heasman J. Patterning the Xenopus blastula, Development 124, 1997 4179–4191.[Abstract]
Heasman J. Crawford A. Goldstone K. Garner-Hamrick P. Gumbiner B. McCrea P. Kintner C. Noro C.Y. Wylie C. Overexpression of cadherins and underexpression of β-catenin inhibit dorsal mesoderm induction in early Xenopus embryos, Cell 79, 1994 791–803.[Medline]
Hedgepeth C.M. Deardorff M.A. Klein P.S. Xenopus axin interacts with glycogen synthase kinase-3β and is expressed in the anterior midbrain, Mech. Dev 80, 1999 147–151.[Medline]
Hoppler S. Brown J.D. Moon R.T. Expression of a dominant negative Wnt blocks induction of MyoD in Xenopus embryos, Genes Dev. 10, 1996 2805–2817.
Ikeda S. Kishida S. Yamamoto H. Murai H. Koyama S. Kikuchi A. Axin, a negative regulator of the Wnt signaling pathway, forms a complex with GSK-3β and β-catenin and promotes GSK-3β-dependent phosphorylation of β-catenin, EMBO (Eur. Mol. Biol. Organ.) J. 17, 1998 1371–1384.[Medline]
Itoh K. Krupnik V.E. Sokol S.Y. Axis determination in Xenopus involves biochemical interactions of axin, glycogen synthase kinase 3 and β-catenin, Curr. Biol. 8, 1998 591–594.[Medline]
Jonkers J. Korswagen H.C. Acton D. Breuer M. Berns A. Activation of a novel proto-oncogene, Frat1, contributes to progression of mouse T-cell lymphomas, EMBO (Eur. Mol. Biol. Organ.) J. 16, 1997 441–450.[Medline]
Kikkawa M. Takano K. Shinagawa A. Location and behavior of dorsal determinants during first cell cycle in Xenopus eggs, Development 122, 1996 3687–3696.[Abstract]
Kishida S. Yamamoto H. Ikeda S. Kishida M. Sakamoto I. Koyama S. Kikuchi A. Axin, a negative regulator of the wnt signaling pathway, directly interacts with adenomatous polyposis coli and regulates the stabilization of β-catenin, J. Biol. Chem. 273, 1998 10823–10826.
Kishida S. Yamamoto H. Hino S. Ikeda S. Kishida M. Kikuchi A. DIX domains of Dvl and axin are necessary for protein interactions and their ability to regulate β-catenin stability, Mol. Cell. Biol 19, 1999 4414–4422.
Larabell C.A. Torres M. Rowning B.A. Yost C. Miller J.R. Wu M. Kimelman D. Moon R.T. Establishment of the dorso-ventral axis in Xenopus embryos is presaged by early asymmetries in β-catenin that are modulated by the Wnt signaling pathway, J. Cell Biol. 136, 1997 1123–1136.
Laurent M.N. Blitz I.L. Hashimoto C. Rothbächer U. Cho K.W. The Xenopus homeobox gene twin mediates Wnt induction of goosecoid in establishment of Spemann's organizer, Development 124, 1997 4905–4916.[Abstract]
Li L. Yuan H. Weaver C. Mao J. Farr G.H. III Sussman D.J. Jonkers J. Kimelman D. Wu D. Axin and Frat-1 interact with Dvl and GSK, bridging Dvl to GSK in Wnt-mediated regulation of LEF-1, EMBO (Eur. Mol. Biol. Organ.) J. 18, 1999 4233–4240.[Medline]
Lin R. Hill R.J. Priess J.R. POP-1 and anterior-posterior fate decisions in C. elegans embryos, Cell 92, 1998 229–239.[Medline]
Marikawa Y. Li Y. Elinson R.P. Dorsal determinants in the Xenopus egg are firmly associated with the vegetal cortex and behave like activators of the Wnt pathway, Dev. Biol. 191, 1997 69–79.[Medline]
McKendry R. Hsu S.C. Harland R.M. Grosschedl R. LEF-1/TCF proteins mediate Wnt-inducible transcription from the Xenopus nodal-related 3 promoter, Dev. Biol. 192, 1997 420–431.[Medline]
McMahon A.P. Moon R.T. Ectopic expression of the proto-oncogene int-1 in Xenopus embryos leads to duplication of the embryonic axis, Cell 58, 1989 1075–1084.[Medline]
Miller J.R. Moon R.T. Signal transduction through β-catenin and specification of cell fate during embryogenesis, Genes Dev. 10, 1996 2527–2539.
Miller J.R. Rowning B.A. Larabell C.A. Yang-Snyder J.A. Bates R.L. Moon R.T. Establishment of the dorsal–ventral axis in Xenopus embryos coincides with the dorsal enrichment of dishevelled that is dependent on cortical rotation, J. Cell Biol 146, 1999 427–437.
Molenaar M. van de Wetering M. Oosterwegel M. Peterson-Maduro J. Godsave S. Korinek V. Roose J. Destrée O. Clevers H. XTcf-3 transcription factor mediates β-catenin-induced axis formation in Xenopus embryos, Cell 86, 1996 391–399.[Medline]
Moon R.T. Christian J.L. Microinjection and expression of synthetic mRNAs in Xenopus embryos, Technique 1, 1989 76–89.
Moon R.T. Kimelman D. From cortical rotation to organizer gene expressiontoward a molecular explanation of axis specification in Xenopus, Bioessays 20, 1998 536–545.[Medline]
Munemitsu S. Albert I. Souza B. Rubinfeld B. Polakis P. Regulation of intracellular β-catenin levels by adenomatous polyposis coli (APC) tumor-suppressor protein, Proc. Natl. Acad. Sci. USA 92, 1995 3046–3050.
Nakamura T. Hamada F. Ishidate T. Anai K. Kawahara K. Toyoshima K. Akiyama T. Axin, an inhibitor of the Wnt signalling pathway, interacts with β-catenin, GSK-3β and APC and reduces the β-catenin level, Genes Cells 3, 1998 395–403.[Abstract]
Newport J. Kirschner M.W. A major developmental transition in early Xenopus embryos. I. Characterization and timing of cellular changes at the midblastula stage, Cell 30, 1982 675–686.[Medline]
Noordermeer J. Klingensmith J. Perrimon N. Nusse R. dishevelled and armadillo act in the Wingless signalling pathway in Drosophila, Nature 367, 1994 80–83.[Medline]
Orford K. Crockett C. Jensen J.P. Weissman A.M. Byers S.W. Serine phosphorylation–regulated ubiquitination and degradation of β-catenin, J. Biol. Chem 272, 1997 24735–24738.
Pierce S.B. Kimelman D. Regulation of Spemann organizer formation by the intracellular kinase Xgsk-3, Development 121, 1995 755–765.[Abstract]
Polakis P. The adenomatous polyposis coli (APC) tumor suppressor, Biochim. Biophys. Acta 1332, 1997 F127–F147.[Medline]
Rocheleau C.E. Downs W.D. Lin R. Wittmann C. Bei Y. Cha Y.H. Ali M. Priess J.R. Mello C.C. Wnt signaling and an APC-related gene specify endoderm in early C. elegans embryos, Cell 90, 1997 707–716.[Medline]
Rothbacher U. Laurent M.N. Blitz I.L. Watabe T. Marsh J.L. Cho K.W.Y. Functional conservation of the Wnt signaling pathway revealed by ectopic expression of Drosophila dishevelled in Xenopus, Dev. Biol. 170, 1995 717–721.[Medline]
Rowning B.A. Wells J. Wu M. Gerhart J.C. Moon R.T. Larabell C.A. Microtubule-mediated transport of organelles and localization of β-catenin to the future dorsal side of Xenopus eggs, Proc. Natl. Acad. Sci. USA 94, 1997 1224–1229.
Rubinfeld B. Souza B. Albert I. Müller O. Chamberlain S.H. Masiarz F.R. Munemitsu S. Polakis P. Association of the APC gene product with β-catenin, Science 262, 1993 1731–1734.
Rubinfeld B. Albert I. Porfiri E. Fiol C. Munemitsu S. Polakis P. Binding of GSK-3β to the APC-β-catenin complex and regulation of complex assembly, Science 272, 1996 1023–1026.[Abstract]
Russo A. Jeffery P.D. Patten A.K. Massagué J. Pavletich N.P. Crystal structure of the p27Kip1 cyclin-dependent-kinase inhibitor bound to the cyclin A-Cdk2 complex, Nature 382, 1996 325–331.[Medline]
Sakai M. The vegetal determinants required for the Spemann organizer move equatorially during the first cell cycle, Development 122, 1996 2207–2214.[Abstract]
Sakanaka C. Weiss J.H. Williams L.T. Bridging of β-catenin and glycogen synthase kinase-3β by Axin and inhibition of β-catenin mediated transcription, Proc. Natl. Acad. Sci. USA 95, 1998 3020–3023.
Schroeder K.E. Condic M.L. Eisenberg L.M. Yost H.J. Spatially regulated translation in embryosasymmetric expression of maternal Wnt-11 along the dorsal-ventral axis in Xenopus, Dev. Biol 214, 1999 288–297.[Medline]
Siegfried E. Wilder E.L. Perrimon N. Components of wingless signaling in Drosophila, Nature 367, 1994 76–80.[Medline]
Smalley M.J. Sara E. Paterson H. Naylor S. Cook D. Jayatilake H. Fryer L.G. Hutchinson L. Fry M.J. Dale T.C. Interaction of axin and Dvl-2 proteins regulates Dvl-2-stimulated TCF-dependent transcription, EMBO (Eur. Mol. Biol. Organ.) J 18, 1999 2823–2835.[Medline]
Smith W.C. Harland R.M. Injected Xwnt-8 acts early in Xenopus embryos to promote formation of a vegetal dorsalizing center, Cell 67, 1991 753–766.[Medline]
Sokol S. Christian J.L. Moon R.T. Melton D.A. Injected wnt RNA induces a complete body axis in Xenopus embryos, Cell 67, 1991 741–752.[Medline]
Sokol S.Y. Analysis of Dishevelled signalling pathways during Xenopus development, Curr. Biol. 6, 1996 1456–1467.[Medline]
Sokol S.Y. Klingensmith J. Perrimon N. Itoh K. Dorsalizing and neuralizing properties of Xdsh, a maternally expressed Xenopus homolog of dishevelled, Development 121, 1995 1637–1647.[Abstract]
Su L.-K. Vogelstein B. Kinzler K.W. Association of the APC tumor suppressor protein with catenins, Science 262, 1993 1734–1737.
Thorpe C.J. Schlesinger A. Carter J.C. Bowerman B. Wnt signaling polarizes an early C. elegans blastomere to distinguish endoderm from mesoderm, Cell 90, 1997 695–705.[Medline]
Turner D.L. Weintraub H. Expression of achaete-scute homolog 3 in Xenopus embryos converts ectodermal cells to a neural fate, Genes Dev. 8, 1994 1434–1447.
Vleminckx K. Wong E. Guger K. Rubinfled B. Polakis P. Gumbiner B.M. Adenamatous polyposis coli tumor suppressor protein has signaling activity in Xenopus laevis embryos resulting in the induction of an ectopic dorsoanterior axis, J. Cell. Biol. 136, 1997 411–420.
Wagner U. Brownlees J. Irving N.G. Lucas F.R. Salinas P.C. Miller C.C. Overexpression of the mouse dishevelled-1 protein inhibits GSK-3β-mediated phosphorylation of tau in transfected mammalian cells, FEBS (Fed. Eur. Biochem. Soc.) Lett 411, 1997 369–372.
Wang Q.M. Fiol C.J. DePaoli-Roach A.A. Roach P.J. Glycogen synthase kinase-3β is a dual specificity kinase differentially regulated by tyrosine and serine/threonine phosphorylation, J. Biol. Chem. 269, 1994 14566–14574.
Willert K. Logan C.Y. Arora A. Fish M. Nusse R. A Drosophila Axin homolog, Daxin, inhibits Wnt signaling, Development 126, 1999 4165–4173.[Abstract]
Wodarz A. Nusse R. Mechanisms of Wnt signaling in development, Annu. Rev. Cell Dev. Biol. 14, 1998 59–88.[Medline]
Yamamoto H. Kishida S. Uochi T. Ikeda S. Koyama S. Asashima M. Kikuchi A. Axil, a member of the Axin family, interacts with both glycogen synthase kinase 3β and β-catenin and inhibits axis formation of Xenopus embryos, Mol. Cell Biol 18, 1998 2867–2875.
Yanagawa S. van-Leeuwen F. Wodarz A. Klingensmith J. Nusse R. The dishevelled protein is modified by wingless signaling in Drosophila, Genes Dev. 9, 1995 1087–1097.
Yang-Snyder J. Miller J.R. Brown J.D. Lai C. Moon R.T. A frizzled homolog functions in a vertebrate Wnt signaling pathway, Curr. Biol. 6, 1996 302–306.
Yost C. Torres M. Miller J.R. Huang E. Kimelman D. Moon R.T. The axis-inducing activity, stability, and subcellular distribution of β-catenin is regulated in Xenopus embryos by glycogen synthase kinase 3, Genes Dev. 10, 1996 1443–1454.
Yost C. Pierce S.B. Torres M. Moon R.T. Kimelman D. Glycogen synthase kinase-3a component of multiple signaling pathways, Cowin P. Klymkowsky M., Cytoskeletal–Membrane Interactions and Signal Transduction, 1997 73–81 R. G. Landes Co Austin, Texas.
Yost C. Farr G.H. III Pierce S.B. Ferkey D.M. Chen M.M. Kimelman D. GBP, an inhibitor of GSK-3, is implicated in Xenopus development and oncogenesis, Cell 93, 1998 1031–1041.[Medline]
Yuan H. Mao J. Li L. Wu D. Suppression of glycogen synthase kinase activity is not sufficient for leukemia enhancer factor-1 activation, J. Biol. Chem 274, 1999 30419–30423.
Zeng L. Fagotto F. Zhang T. Hsu W. Vasicek T.J. Perry W.L. Lee J.J. Tilghman S.M. Gumbiner B.M. Costantini F. The mouse Fused locus encodes Axin, an inhibitor of the Wnt signaling pathway that regulates embryonic axis formation, Cell 90, 1997 181–192.[Medline]
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Facebook 
Reddit 
Technorati 
Twitter What's this?
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
|
|
1 embryo). A portion of the total lysate shown in lane 6 was precipitated in the absence of anti-FLAG antibody as a negative control (lane 7). Lane numbers in the right panel refer to the same injections as shown above the corresponding lane numbers in the left panel. (b) Prevention of GSK-3–Axin binding by GBP is dependent on GBP/GSK-3 binding. Embryos were injected with Axin-myc and Xgsk-3-FLAG as above along with 4 ng HA-tagged wild-type (GBPwt) or the HA-tagged point mutant that does not bind GSK-3 (GBPmut). Immunoprecipitations were performed as above. No GBP is present in the anti-FLAG immunoprecipitates in lane 4 since this is the GSK-3 binding mutant of GBP. Lane numbers in the right panel refer to the same injections as shown above the corresponding lane numbers in the left panel.
