Article |
Address correspondence to Marc W. Kirschner, Dept. of Cell Biology, Harvard Medical School, 240 Longwood Ave., C1-517, Boston, MA 02115. Tel.: (617) 432-2250. Fax: (617) 432-0420. E-mail: marc{at}hms.harvard.edu
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Abstract |
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Key Words: ß-catenin; Tcf; wnt; casein kinase; Xenopus
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Introduction |
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Signaling through the wnt pathway is initiated upon binding of wnts to members of a family of seven transmembrane receptors (frizzled receptors). Through an as yet unknown mechanism, frizzled receptors activate a cytoplasmic protein, dishevelled (dsh), which interacts directly with axin via their DIX domains (Fukui et al., 2000; Julius et al., 2000; Salic et al., 2000). The GSK3 binding protein (GBP) (Yost et al., 1998) is recruited through its interaction with the PDZ domain of dsh and thus brought in the proximity of axin-bound GSK3. Binding of GBP to GSK3 inhibits its kinase activity against ß-catenin, reducing its degradation by the SCF ubiquitin ligase complex and resulting in a increased steady state level of free ß-catenin.
Free ß-catenin interacts with the DNA binding proteins Tcf3 and Lef1 (Behrens et al., 1996; Huber et al., 1996; Molenaar et al., 1996) to form a bipartite transactivator that stimulates the transcription (van de Wetering et al., 1997) of immediate gene targets (for example, siamois [Brannon et al., 1997] and Xnr3 [McKendry et al., 1997] in Xenopus). In the absence of ß-catenin, Tcf3/Lef1 proteins mediate repression when bound to the Groucho family of transcription factor, CREB binding protein (CBP), and CtBP. Interaction between Tcf3 and ß-catenin occurs at the NH2 terminus of Tcf3 and is separate from the Groucho and CtBP binding regions (for review see Barker et al., 2000).
Models of the wnt pathway suggest a role for Tcf3/Lef1 as an unregulated scavenger of free ß-catenin. However, CBP binds and acetylates a lysine in the ß-catenin interaction domain of Tcf3, thereby lowering its affinity for ß-catenin (Waltzer and Bienz, 1998). Some members of the Sox family of HMG box proteins also bind ß-catenin and block its binding to Tcf (Zorn et al., 1999). In Caenorhabditis elegans, components of the mitogen-activated kinase pathway phosphorylate Tcf-bound ß-catenin so as to block nuclear localization (Rocheleau et al., 1999). These studies indicate that the interaction between ß-catenin and Tcf3 is dynamic and that regulating it may play an important role in modulating wnt signaling. We developed recently an in vitro system to examine the cytoplasmic components of the wnt signaling pathway using Xenopus egg extracts (Salic et al., 2000). We have used this system to study the effects of Tcf3 on ß-catenin stability and the interaction between Tcf3 and ß-catenin.
Components of the wnt pathway upstream of Tcf3 regulate ß-catenin stability. However, a clear role for Tcf3 itself in the stabilization of ß-catenin has not been demonstrated. In the present study, we show that Tcf3 inhibits the interaction between ß-catenin and axin/APC and that Tcf3 and ß-catenin interact significantly even in the absence of wnt signaling to modulate ß-catenin turnover. We show that GSK3 and casein kinase (CK) 1 both have direct but opposite effects in regulating the ß-cateninTcf3 interaction. We also find that a significant fraction of Tcf3 is cytoplasmic in both Xenopus embryos and cultured cells, indicating that Tcf3 can act outside the nucleus to regulate ß-catenin degradation. Additionally, we provide evidence that CKI
stimulates the interaction between dsh and GBP. These results suggest two possible mechanisms for the role of CK1
in wnt signaling and provide further evidence that regulated formation of the ß-cateninTcf3 complex and the dshGBP complex play a crucial role in wnt signaling.
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Results |
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Tcf3 and axin/APC compete for ß-catenin binding
Phosphorylation and subsequent degradation of ß-catenin requires its association with both APC and axin. Since binding of Tcf3 to ß-catenin blocks its degradation, we tested if this is due to blocking binding of ß-catenin to axin and/or APC. As shown in Fig. 2 A, purified his-tagged Tcf3 but not his-tagged NTcf3 blocked binding of ß-catenin to axin beads. However, Tcf3 had no effect on GSK3 binding to axin (unpublished data). A purified 100-kD fragment of APC (APCm3) that spans the axin and ß-catenin binding sites stimulates binding of ß-catenin to axin beads (Fig. 2 B). In the presence of 100 nM APCm3, addition of up to 1 µM Tcf3 protein had no detectable effect on the binding of ß-catenin to axin beads. Furthermore, binding of ß-catenin to Tcf3 beads (Fig. 2 C) is effectively inhibited in the presence of 1 µM APCm3. Consistent with a dominant effect of APC in Xenopus extracts, a majority of the soluble ß-catenin is in anti-APC immunoprecipitates (Salic et al., 2000). In extracts, 1 µM of added Tcf3 released radiolabeled ß-catenin from APC immunoprecipitates (Fig. 2 D). As a control, 1 µM
NTcf3 had no detectable effect on ß-catenin binding to endogenous APC. These results indicate that Tcf3 competes with both axin and APC for ß-catenin binding. The failure of Tcf3 to block ß-catenin binding to axin in the presence of APCm3 highlights the strong effect of APC in driving the degradation of free ß-catenin by promoting its binding to axin and in inhibiting competing reactions driven by Tcf3. In contrast, the ability of Tcf3 to compete effectively with endogenous APC for ß-catenin probably reflects the fact that most of ß-catenin is not in a complex with axin/APC, since cellular axin levels are very low (1020 picomolar; unpublished data).
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Since cat449/645 below 2 µM blocks the interaction between ß-catenin and Tcf3 without affecting the interaction between ß-catenin and axin and APC, we tested its effect on the rate of ß-catenin degradation. Addition of 200 nM MBP-cat449/645 to Xenopus extracts accelerated the rate of ß-catenin degradation by 50% (Fig. 3 E). Similarly, when injected into Xenopus embryos cat449/645 stimulated the degradation of coinjected ß-cateninluciferase by
70% (Fig. 3 F). Paradoxically, high concentrations (>2 µM) of cat449/645 inhibited ß-catenin degradation (unpublished data), probably reflecting the ability of high concentrations of this mutant to compete with wild-type ß-catenin for binding to APC (Fig. 3 D). However, in the concentration range of cat449/645 where the ß-cateninAPC interaction is not affected, the mutant blocks the Tcfß-catenin binding and stimulates ß-catenin turnover.
The NH2-terminal regions of Tcf3 and Tcf4 contain a conserved ß-catenin binding domain (Molenaar et al., 1996). We wanted to determine if a peptide corresponding to the NH2-terminal region of Tcf4 could similarly disrupt the interaction between ß-catenin and Tcf3. A human Tcf4 NH2-terminal peptide blocked the binding of ß-catenin to Tcf3 beads (Fig. 4 A). As for cat449/645, the Tcf4 NH2-terminal peptide had no effect on the interaction between ß-catenin and axin (Fig. 4 B) or on the interaction between ß-catenin and APC (Fig. 2 H). Addition of the Tcf4 NH2-terminal peptide to Xenopus extracts stimulated the degradation of ß-catenin by 65% (Fig. 4 C), further demonstrating that Tcf proteins are normally involved in modulating ß-catenin degradation. A control peptide had no effect on the degradation of ß-catenin (unpublished data).
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We examined the interaction of GSK3 and CK1 with Tcf3. Radiolabeled GSK3 and CK1
both significantly bound Tcf3 beads (Fig. 6, C and D). Furthermore, the binding sites of GSK3 and CK1
on Tcf3 appear to be distinct from one another, since GSK3 binding cannot be competed with excess cold CK1
. Conversely, the binding of CK1
to Tcf3 beads is not abolished when incubated with excess cold GSK3. As with GSK3, CK1
can phosphorylate recombinant Tcf3 in vitro. CK1
can enhance the phosphorylation of Tcf3 in an in vitro kinase assay above the level due to that of contaminating GSK3 (Fig. 6 E). Furthermore, the enhanced Tcf3 phosphorylation by CK1
is partially inhibited when CKI-7 (IC50, 1030 µM; purchased from Seikagaku Corporation), a selective inhibitor of CK1 activity (Chijiwa et al., 1989), is included in the kinase reaction. The association between Tcf3 and GKS3/CK1
represents bona fide in vivo interactions, since both endogenous Xenopus GSK3 and CK1e immunoprecipitated with an anti-myc antibody from extracts made from embryos injected with myc6-xTcf3 mRNA (Fig. 6 F). Although Tcf3 binds GSK3, it does not inhibit the activity of GSK3 against axin (Fig. 1 D).
Effects of Tcf3 phosphorylation on its activity
These experiments establish that both GSK3 and CK1 can bind and phosphorylate Tcf3 and suggest a possible role for both GSK3 and CK1
in modulating Tcf3 activity. We therefore tested if these kinases affect the interaction of Tcf3 with ß-catenin. Tcf3 can inhibit the degradation of ß-catenin (Fig. 1, A and B). CK1
and Tcf3 act synergistically to inhibit ß-catenin degradation: as shown in Fig. 7 A, concentrations of CK1
and Tcf3 that by themselves are not inhibitory together cause significant inhibition of ß-catenin degradation. In addition, CKI-7 blocks the effect of Tcf3 in a dose-dependent manner in Xenopus extracts (Fig. 7 B). As expected, 100 µM CKI-7 (even in the presence of 10 nM Tcf3) actually accelerates the rate of degradation of ß-catenin in extracts compared with a buffer control. Thus, CK1
activity is required for inhibition of ß-catenin degradation by Tcf3. The simplest model consistent with these experimental results is that phosphorylation of Tcf3 by CK1
promotes the interaction of Tcf3 with ß-catenin. As shown in Fig. 7 C, preincubation of Tcf3 beads with CK1
increases their affinity for ß-catenin nearly fourfold compared with untreated beads. Furthermore, although preincubating Tcf3 beads with GSK3 had very little effect on ß-catenin binding, GSK3 abolished the effect of CK1
. One prediction from these experiments is that blocking GSK3 activity would further potentiate the effect of CK1
. As expected, GBP acts synergistically with CK1
to inhibit ß-catenin degradation (Fig. 7 D). Failure of GSK3 to inhibit the effects of Tcf3 in our earlier experiments (Fig. 1 B) was probably due to the high concentration of Tcf3 (1 µM) used. In fact, the effect of adding Tcf3 at concentrations closer to its IC50 for ß-catenin degradation (100 nM) can be reversed by the addition of 1 µM GSK3 (Fig. 1 C).
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Role of CK1 in mediating dsh activity
Given the fact that CK1 has been shown to bind axin (Sakanaka et al., 1999) and dsh (Peters et al., 1999), it appears that CK1
like GSK3 simultaneously affects many components of the wnt pathway. Dsh acts synergistically with CK1
in Xenopus embryos (Fig. 8 A). Embryos injected ventrally at the 48-cell stage with low doses of Dsh (100 pg) or CK1
(50 pg) RNA develop normally (100%, n = 30), whereas injection of embryos with both Dsh and CK1
resulted in axis duplication (68%, n = 50). Dsh acts synergistically with CK1
to inhibit ß-catenin degradation in Xenopus extracts (Fig. 8 B), consistent with the in vivo results. Preliminary experiments indicate that both GSK3 and CK1
can bind the PDZ domain of dsh (unpublished data). Since GBP binds the PDZ domain of dsh, we tested whether CK1
or GSK3 can alter the affinity of dsh for GBP. CK1
stimulates the binding of radiolabeled dsh to GBP in Xenopus extracts four- to fivefold greater than in buffer- or GSK3-treated extracts (Fig. 8 C). This result indicates that in addition to promoting the association between Tcf3 and ß-catenin, CK1
stimulates the binding of GBP to dsh. Interestingly, in the absence of Xenopus extracts CK1
has no effect on binding of dsh to GBP, suggesting that extracts contain an activity that mediates the effect of CK1
on the dshGBP interaction.
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Discussion |
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The inhibitory effect of Tcf3 depends on its ability to bind ß-catenin because an NH2-terminal deletion mutant of Tcf3 that lacks the ß-catenin binding site has no effect on ß-catenin degradation. A ß-catenin COOH-terminal deletion mutant (ß-cateninC2) is regulated in a manner indistinguishable from the wild-type protein except it is no longer sensitive to Tcf3 and is degraded at a faster rate than the full-length protein in Xenopus extracts. Based on experiments with this mutant, we defined a fragment of ß-catenin (cat449/645) that can disrupt the interaction between ß-catenin and Tcf but not the interaction of ß-catenin with axin/APC. Addition of this purified fragment accelerates the rate of ß-catenin degradation in both Xenopus extracts and embryos. Similarly, a peptide encoding the NH2-terminal ß-catenin binding region of Tcf4 (which also blocks the binding of ß-catenin to Tcf3) stimulates ß-catenin degradation. Our attempts to determine whether this peptide has any effect on axis formation have been unsuccessful because injections of this peptide into Xenopus embryos at concentrations sufficient to block Tcf3ß-catenin interaction severely perturbed gastrulation. Although the effects of the Tcf4 peptide and the cat449/645 fragment might seem modest, these results should be viewed in the context of the effects of known regulators of the wnt pathway (for example, axin and dsh), which alter the rate of ß-catenin degradation only three- to fourfold.
Structure-function analysis of ß-catenin (von Kries et al., 2000) and the x-ray crystal structure of its complex with the ß-catenin binding domain of Tcf3 (Graham et al., 2000) indicate that the Tcf/Lef1 binding site of ß-catenin forms a positively charged groove. This interacts with the ß-catenin binding domain of Tcf3 consisting of an extended structure that can be divided into three sites. A comparison of this structure to ß-cateninC2 indicates that this mutant is missing one of the flanking regions and part of the central region involved in Tcfß-catenin interaction.
Our results suggest that cytoplasmic Tcf3 competes for ß-catenin binding with both APC and axin (Fig. 8 A); however, once a trimeric complex consisting of ß-catenin, axin, and APC is formed Tcf3 is unable to compete effectively for ß-catenin binding (Fig. 2 B). By sequestering ß-catenin in the cytoplasm or enhancing its export from the nucleus (Henderson, 2000), APC inhibits signaling through the wnt pathway by further blocking assembly of the ß-cateninTcf3 bipartite complex. Therefore, in the absence of APC the effect of Tcf proteins on wnt signaling would predominate. Loss of APC function would not only result in an increase in the amount of free ß-catenin available to bind Tcf3 but would also allow Tcf3 to compete effectively with axin for ß-catenin binding.
Factors that change the concentration of Tcf3 protein or its affinity for ß-catenin can alter the amount of the ß-cateninTcf complex. Decreases or increases in the ratio of soluble ß-catenin to Tcf3 can modulate the transcription level of target genes, since Tcf3 acts as a transcriptional repressor in the absence of ß-catenin and as a transcriptional activator when bound to ß-catenin (Cavallo et al., 1998; Roose et al., 1998). A model of the wnt pathway in which the level of signaling is continually being modulated (rather than simply being in the "on" or "off" position) is an attractive one. In fact, experiments in which cells have been transfected with axam (Kadoya et al., 2000), an axin binding protein that blocks its interaction with dsh, show a reduced level of ß-catenin. This result indicates that even in the absence of ligand a significant amount of signal modulation occurs in the wnt pathway.
A major mechanism by which the rate of ß-catenin degradation is regulated by a wnt ligand is through its control of GSK3 kinase activity (Fig. 8 B). Previous studies of GSK3 have indicated that it controls nearly every level of ß-catenin degradation: (a) phosphorylation of APC to facilitate its interaction with ß-catenin, (b) phosphorylation of axin to promote its stability and possibly to increase its affinity for ß-catenin, and (c) phosphorylation of ß-catenin so as to allow recognition by the SCF ubiquitin ligase complex. Studies using promoter-based assays to assess the activation state of the wnt pathway have found disparities between the steady-state levels of ß-catenin and the transcriptional activation of reporter constructs. Using a biochemical approach, we have revealed an additional role for GSK3 in the wnt pathway that may account for these discrepancies: GSK3 phosphorylates Tcf3 thereby decreasing its affinity for ß-catenin and antagonizing the activity of CK1. This model helps explain why insulin growth factor (IGF) 1 can enhance the stability of ß-catenin but not transcriptional activation of downstream targets (Playford et al., 2000), whereas addition of the GSK3 inhibitor lithium resulted in a dramatic increase in ß-cateninTcfmediated transcriptional activation. IGF-1 promotes the stability of ß-catenin by stimulating its tyrosine phosphorylation without affecting the activity of GSK3. Increases in ß-catenin levels mediated by IGF-1 may not be sufficient to overcome the inhibitory effect of GSK3 on ß-cateninTcf interaction. It will be interesting to explore the biochemical state of Tcf in early Xenopus development and its contribution to wnt signaling, particularly the role of its interactions with GSK3 and CK1
.
Our initial experiments indicated that GSK3 does not readily reverse the inhibitory effect of Tcf3 at high concentration (1 µM), but subsequent experiments revealed that GSK3 reverses the effect of Tcf3 at lower concentrations that are close to the IC50 of Tcf3 for ß-catenin degradation. These conflicting results demonstrate the subtleties of these experiments and the hazards inherent in overexpression studies and highlight the advantages of using a biochemical system to study the wnt pathway. The concentrations of individual components can be manipulated so as to reveal regulations that might otherwise be overlooked in experiments involving overexpression of components by transfection of DNA into cultured cells or injection of RNAs into embryos. CK1 is both necessary and sufficient for wnt signaling (Peters et al., 1999; Sakanaka et al., 1999). Our results suggest that one of the roles of CK1
in wnt signaling is to phosphorylate Tcf3 thereby increasing its affinity for ß-catenin. In Xenopus extracts, CK1
and Tcf3 synergize to inhibit ß-catenin degradation. Studies with the CK1
inhibitor CKI-7 (Fig. 6 C) indicate that CK1
regulates the formation of the ß-cateninTcf3 complex: CKI-7 blocks the inhibitory effect of Tcf3 on ß-catenin degradation and therefore increases the rate of ß-catenin degradation above basal levels. The mechanism by which GSK3 and CK1
oppose each other's actions is not known. Intriguingly, our analysis of the crystal structure of ß-catenin complexed with the ß-catenin binding domain of Tcf3 (Graham et al., 2000) indicates that one of the three ß-catenin binding regions of Tcf3 (amino acids 4964 of Xenopus Tcf3) contains a conserved and overlapping phosphorylation site for both GSK3 and CK1
(which is also present in other members of the Tcf/LEF-1 family that bind ß-catenin). This may indicate that binding and/or phosphorylation of Tcf3 by one kinase may preclude the binding and/or phosphorylation of Tcf3 by the other kinase.
The interaction between ß-catenin and Tcf appears to be regulated in multiple ways. In Drosophila, the CBP acetylates a lysine in the ß-catenin binding domain of dTcf, thereby lowering its affinity for ß-catenin (Waltzer and Bienz, 1998). However, in vertebrates CBP has been shown to bind to the COOH-terminal region of ß-catenin to activate Tcf-mediated gene transcription (Takemaru and Moon, 2000). Certain Sox proteins have been shown to interact with ß-catenin and block its interaction with Tcf proteins (Zorn et al., 1999). ICAT, a ß-catenin interacting protein inhibits the interaction of ß-catenin with Tcf4 and represses ß-cateninTcf4mediated gene transcription (Tago et al., 2000). In C. elegans, the LIT-1 protein phosphorylates POP-1, a Tcf/LEF-related protein, to downregulate its activity (Rocheleau et al., 1999). Interestingly, phosphorylation of POP-1 requires an active LIT-1 kinase complex containing WRM-1, a ß-cateninlike homologue belonging to a divergent wnt pathway. Finally, the bipartite ß-cateninTcf complex can directly bind Smad4 to stimulate expression of a target gene, twin (Nishita et al., 2000). Each of these interactions could also modulate ß-catenin stability, leading to pleiotropic and often synergistic effects in wnt signaling. Similarly to GSK3, CK1 appears to regulate the wnt pathway at more than one level. CK1
stimulates the binding of dsh to GBP in Xenopus extracts. Although CK1e can directly bind dsh, the mechanism by which CK1
promotes dshGBP interaction is not clear, since this effect of CK1
was only detected in the presence of Xenopus extracts. Interestingly, the amount of dsh bound to GBP in the presence of Xenopus extracts was much less than the amount of dsh bound to GBP in the absence of extracts. Dsh is heavily phosphorylated in Xenopus extracts (unpublished data), which possibly inhibits its binding to GBP. Addition of CK1
to Xenopus extracts increases the binding of dsh to GBP to levels comparable to that seen in the absence of extract. These results suggest the existence of an as yet unidentified factor(s) that functions to inhibit the binding of GBP to dsh; one role of CK1
may be to counteract the activity of this factor.
Both Tcf and ß-catenin are required for axis formation in Xenopus development. ß-catenin is stabilized on the dorsal side of the embryo during early development, and when zygotic transcription starts at the midblastula transition, ß-catenin translocates into the nuclei of dorsal cells. How ß-catenin stabilization and nuclear translocation on the dorsal side takes place is still unclear, although both processes are perhaps essential for axis formation. Regulation of ß-catenin stability by Tcf might play an important role in these processes.
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Materials and methods |
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Immunoprecipitation of myc6-Tcf3
Both cells of 2-cell embryos were injected with myc6-Tcf3 RNA (500 pg/blastomere) and homogenized at stage 7.5 by resuspending the embryos three times with a clipped P-200 pipet tip (cut so that the opening was slightly smaller than the diameter of an embryo). The lysate (100 µl) was incubated with 25 µl of anti-myc antibody-coupled beads (Santa Cruz Biotechnology, Inc.) or control beads for 2 h at 4°C. Beads were washed and the samples eluted (see Binding assay). The eluted samples were subjected to SDS-PAGE followed by Western blotting using an anti-myc antibody (9E10; Santa Cruz Biotechnology, Inc.), an anti-GSK3ß antibody (Transduction Laboratory), and an anti-CK1 antibody (Transduction Laboratory).
Tcf fractionation
Egg and embryonic lysates were prepared as described above. Supernatants were obtained by centrifuging the lysates for 2 h at 100,000 g at 4°C. Equivalent volumes of lysate and supernatant were subjected to SDS-PAGE followed by Western blotting using either an anti-myc antibody (9E10; Santa Cruz Biotechnology, Inc.) or an anti-Tcf monoclonal antibody (Exalpha Biological). Nuclear and cytoplasmic extracts were prepared from cultured 293 cells as described previously (Heintz and Stillman, 1988) with modifications. Cells were washed in a cold hypotonic buffer (lysis buffer; 25 mM Hepes, pH 7.7, 5 mM CH3COOK, 0.5 mM MgCl2, 1 mM DTT, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 10 µg/ml pepstatin, and 100 µM PMSF) and disrupted with 25 strokes in a dounce homogenizer. Nuclei were pelleted at 4,000 rpm for 5 min in an Eppendorf centrifuge. The supernatant was removed, recentrifuged at 4,000 rpm for 5 min, and transferred to a new tube. The nuclear pellet was washed twice with PBS and resuspended in lysis buffer with a volume equivalent to the supernatant fraction. SDS-PAGE and Western blotting was performed on the nuclear and cytoplasmic fractions using an antitopoisomerase II monoclonal antibody (StressGen Biotechnologies) and an anti-Tcf monoclonal antibody (Exalpha Biological).
mRNA synthesis, translation in extracts, and degradation assays
Capped mRNAs were synthesized in vitro from linearized plasmid DNA templates, purified using RNAeasy spin columns (QIAGEN), and resuspended in water to 0.20.5 mg/ml. For experiments requiring translations, fresh extracts prepared as described above were supplemented with placental ribonuclease inhibitor (Promega) (1:100 ratio), mixed with mRNA to a final concentration of 50100 ng/µL, and incubated for 12 h at room temperature. Translations were terminated by the addition of cycloheximide (100 µg/mL) and were either used immediately in degradation assays or snap-frozen in liquid nitrogen and stored at -80°C.
Degradation assays
For degradation assays, 68 µL of Xenopus egg extracts were supplemented with 0.1 µL cycloheximide (10 mg/mL), 0.2 µL energy mix, 0.2 µL purified bovine ubiquitin (14 mg/mL), and 0.10.3 µL [35S]methionine-labeled ß-catenin. The reactions were incubated at room temperature for 3 h, and 1-µL aliquots were removed at 0, 1, and 3 h for analysis by SDS-PAGE and autoradiography.
Plasmids and recombinant proteins
Xenopus Tcf3, NTcf3 (Molenaar et al., 1996), dsh, and ß-catenin were subcloned by PCR in the pCS2+ vector. ß-catenin deletion mutants were constructed by PCR in pCS2+ and verified by in vitro translation of a protein of the appropriate size. Xenopus GBP and CK1
were cloned from a stage 14 Xenopus plasmid library. MBP-GBP, MBP-cat449/645 (amino acids 449645 of Xenopus ß-catenin), and MBP-CKI
were expressed in bacteria. His-tagged Xenopus Tcf3 (his6-Tcf3), his6-
N Tcf3 (lacking amino acids 131 of Xenopus Tcf3), his6-APCm3 (amino acids 13422075 of Xenopus APC), and his6-GSK3 were produced in baculovirus-infected Sf9 cells.
Kinase reactions
Kinase reactions were performed for 30 min at room temperature in kinase buffer (20 mM Hepes, pH 7.5, 300 mM NaCl, 10 mM MgCl2, 2 mM DTT, 0.2% Tween-20, 50 µM ATP, and 0.25 µCi/µL [-32P]ATP). For the phosphorylation of Tcf3 beads by CK1
and GSK3, Tcf3 beads (5 µl) were incubated with 1 µM GSK3 or MBP-CK1
in a 50-µl reaction containing 20 mM Hepes, pH 7.5, 300 mM NaCl, 10 mM MgCl2, 2 mM DTT, 0.2% Tween-20, and 2 mM ATP. Reactions were performed for 1 h at room temperature with rotation. Beads were subsequently washed with 3x 1 ml of binding buffer (see below) before addition of ß-catenin.
Binding assays
Binding reactions using MBP fusion proteins were performed with the fusion proteins bound to amylose beads (New England Biolabs, Inc.). Other proteins were immobilized by cross-linking to Ultralink beads (Pierce Chemical Co.). For binding experiments performed in extracts, Xenopus egg extracts were diluted 1:5 in buffer containing 20 mM Hepes, pH 7.5, 300 mM NaCl, 1% Tween-20, 1 mg/mL BSA, and protease inhibitors. Binding to purified or in vitrotranslated proteins ([35S]methionine-labeled using the Promega TNT kit) was performed in the same buffer (2 h at 4°C). After incubation, beads were washed with 3 mL of buffer A (20 mM Hepes, pH 7.5, 300 mM NaCl, 1% Tween-20), 3 mL of buffer B (20 mM Hepes, pH 7.5, 400 mM NaCl, 1% Tween-20), and 3 mL of buffer C (20 mM Hepes, pH 7.5, 50 mM NaCl, 1% Tween-20). Bound proteins were eluted with hot sample buffer and analyzed by SDS-PAGE followed by either autoradiography or Western blotting. Western analysis of GSK3 was performed using a monoclonal antibody obtained from Transductions Laboratories. NH2-terminal Tcf4 peptide was obtained from Zymed Laboratories.
ß-cateninluciferase assays
10 ng of recombinant ß-cateninluciferase protein (1 mg/ml in 20 mM Hepes, pH 7.4, and 50 mM NaCl) purified from baculovirus-infected Sf9 cells was injected into 2-cell stage Xenopus embryos and processed as described previously (Salic et al., 2000).
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Footnotes |
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* Abbreviations used in this paper: APC, adenomatous polyposis coli; CBP, CREB binding protein; CK, casein kinase; dsh, dishevelled; GBP, GSK3 binding protein; GSK, glycogen synthase kinase; IGF, insulin growth factor; MBP, maltose-binding protein; SCF, Skp1/cullin/F-box protein complex.
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Acknowledgments |
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Ethan Lee is a Merck Fellow of the Helen Hay Whitney Foundation.
Submitted: 14 February 2001
Revised: 16 July 2001
Accepted: 30 July 2001
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