in Xenopus axis formation by determining the effects of overexpressed APC on b-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 bind- ing to the Axin/APC/b-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 bind- ing to Axin. Additionally, we demonstrate that GBP in- hibits Xgsk-3–mediated phosphorylation of protein sub- strates without eliminating the kinase activity of Xgsk-3. Finally, we show that the APC constructs that duplicate the axis in Xenopus also stabilize b-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, b-cate- nin levels. Materials and Methods RNA Expression Constructs The FLAG-epitope–tagged GBP mutant that does not bind Xgsk-3 (BP139), myc-epitope–tagged Xgsk-3 (XG134), and FLAG-epitope– tagged wild-type GBP (BP20) have been described previously (Yost et al., 1998). Axin-myc (Zeng et al., 1997), b-catenin-myc (XBC40; Yost et al., 1996), and XAPCFL, XAPC1, and XAPC4 (Vleminckx et al., 1997) also have been described. Xgsk-3-FLAG (XG140) was constructed by cloning the Xgsk-3 fragment from XG134 into CS2 1 FLAG. GBP-myc (BP25) was constructed by cloning the GBP fragment from BP20 into CS2 1 MT (Turner and Weintraub, 1994), and GBP-HA was constructed by cloning this fragment into CS2 1 HA, a derivative of CS21 (Turner and Wein- traub, 1994) containing an HA epitope (details available upon request). BP139-HA was constructed by cloning the mutant GBP from BP139 into CS2 1 HA, and an HA-epitope–tagged b-catenin was constructed by sub- cloning b-catenin from XBC40 into CS2 1 HA to generate XBC-HA. A FLAG-epitope–tagged dominant-negative Xgsk-3 (XG210) was con- structed as follows: the Xgsk-3–containing BamHI fragment from XG21 (Pierce and Kimelman, 1995) was cloned into CS21 to make XG114. The HindIII-EcoRV fragment of XG140 was replaced with the same fragment from XG114 to generate XG210. Embryos and Microinjection Embryos were obtained as previously described (Newport and Kirschner, 1982). UV ventralization of embryos was performed as described (Ger- hart et al., 1989). Embryos were microinjected (Moon and Christian, 1989) with RNA synthesized from CS21 (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 pro- teins 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 endoge- nous APC. In this case, embryo lysates, prepared as described (Yost et al., 1998), were electrophoresed on 4–12% gradient gels (Novex) and trans- ferred to PVDF in Tris-glycine buffer lacking SDS and methanol. Endoge- nous APC was immunoprecipitated and detected with anti-APC2 anti- body (Rubinfeld et al., 1993; provided by P. Polakis). Anti-MBP antibody was purchased from New England Biolabs, anti-GST antibody was pur- chased 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 Prot- Param and protein concentrations were determined by UV absorbance at 280 nm. 4 mg 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 mg RNaseH in binding buffer (20 mM Tris, pH 7.4, 200 mM NaCl). After this short incubation, 15 mg BSA and 9 ml of the [35S]GBP transla- tion mix were added and the volume was increased to 1,500 ml with bind- ing buffer. Proteins were nutated at 48C for 1 h. 50 ml of prerinsed glu- tathione resin was added to each reaction and proteins were nutated an additional hour at 48C. 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 mg of MBP or MBP-Axin was incubated with 10 mg BSA and 7 ml of the [35S]GBP 6 [35S]GSK-3 translation mix, in a final volume of 1 ml binding buffer. Proteins were collected with 50 ml 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 de- scribed (Wang et al., 1994) in a 30-ml reaction volume for 20 min at 308C. Levels of Xgsk-3-myc immunoprecipitated alone or with GBP were quan- tified using densitometry and phosphorus-32 incorporation was normal- ized to Xgsk-3 protein levels. CREB peptide and the prephosphorylated p-CREB peptide were synthesized by Genosys. Results GSK-3 Cannot Bind GBP and Axin Simultaneously The observations that Axin binds GSK-3 and b-catenin to promote the phosphorylation of b-catenin by GSK-3 (Hart et al., 1998; Ikeda et al., 1998; Yamamoto et al., 1998), and that GBP binds and inhibits GSK-3 (Yost et al., 1998), led us to ask if GBP might function by preventing GSK-3 from binding Axin. Coimmunoprecipitations and in vitro pull-down assays were performed to address this question. In the first set of experiments, we asked if the presence of an excess of GBP could reduce the amount of Axin coimmunoprecipitated with Xgsk-3, when the levels of the latter two were kept constant. RNA constructs encoding FLAG-epitope–tagged Xgsk-3 (Xgsk-3-FLAG) and myc-epitope–tagged GBP (GBP-myc) and Axin (Axin-myc) (Zeng et al., 1997) were injected into the ani- mal region of two- to eight-cell stage Xenopus embryos. Embryos were lysed after 4–5 h, and immunocomplexes were precipitated with anti-FLAG antibodies. The immu- noprecipitates were analyzed on a blot with both anti- FLAG and anti-myc antibodies. In preliminary experi- ments, injection of a combination of 50 pg of Xgsk-3 RNA and 1 ng of Axin RNA was shown to result in the produc- tion of an excess of Axin protein relative to Xgsk-3 (data not shown). Coinjection of increasing amounts of GBP RNA with this mixture of Xgsk-3 and Axin RNAs resulted in a decrease of Axin coimmunoprecipitated with Xgsk-3 (Fig. 1 a, lanes 2–6), though both were produced at the same level in all samples. To demonstrate that the reduc- tion in the amount of Axin bound to Xgsk-3 is specifically Farr et al. Interaction among Intracellular Wnt Factors 693