Figure 1. (a) GBP blocks GSK-3 binding to Axin in vivo. Em- bryos were injected at the two to eight-cell stage in the animal pole with 50 pg Xgsk-3-FLAG and 1 ng Axin-myc along with in- creasing doses of GBP-myc, as indicated. After 4–5 h, proteins were isolated by immunoprecipitation with anti-FLAG antibody and detected by Western blotting (left panel). An aliquot of each sample taken before immunoprecipitation is shown in the right panel (Total Lysates); the level of Xgsk-3 produced is not detect- able in these samples (z1 embryo). A portion of the total lysate shown in lane 6 was precipitated in the absence of anti-FLAG an- tibody as a negative control (lane 7). Lane numbers in the right panel refer to the same injections as shown above the cor- responding lane numbers in the left panel. (b) Prevention of GSK-3–Axin binding by GBP is dependent on GBP/GSK-3 bind- ing. 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 corre- sponding lane numbers in the left panel. dependent on GBP binding to Xgsk-3, the coimmunopre- cipitation experiment was repeated using a high dose of HA-tagged GBP (GBPwt) or an equivalent amount of the HA-tagged GBP mutant that does not bind Xgsk-3 (Yost et al., 1998) (GBPmut). The level of Axin coimmunopre- cipitated by Xgsk-3 was reduced by expression of GBPwt but not by GBPmut (Fig. 1 b). These results indicate that GBP can prevent the association of Axin and Xgsk-3, ei- ther by displacing Axin from Xgsk-3, or by preventing the formation of Axin–Xgsk-3 complexes. In a complementary set of experiments, we asked if GBP could join a GSK-3–Axin complex. For these experi- ments, the GSK-3 binding region of rat Axin, rAxin (298- 506), which binds GSK-3 as well as full-length Axin (Ikeda et al., 1998), was purified as a maltose binding pro- tein fusion (MBP-Axin) and GSK-3 was purified as a glu- tathione-S-transferase fusion (GST-GSK-3b) (Ikeda et al., 1998). GBP was synthesized, sulfur-35–labeled in vitro, and was added to mixtures of GST-GSK-3b and increasing amounts of MBP-Axin or MBP. After an incubation, GST-GSK-3b was collected on glutathione-conjugated Sepharose beads, and the levels of [35S]GBP associated Figure 2. (a) GBP and Axin do not bind GSK-3 at the same time. Bacterially produced GST-GSK-3b and MBP-Axin or MBP were incubated with [35S]methionine-labeled GBP. MBP-Axin or MBP was present in excess over GST-GSK-3b as indicated. Pro- tein complexes were precipitated with glutathione-Sepharose beads and [35S]GBP was detected by SDS-PAGE and autora- diography. MBP-Axin/MBP levels were detected by SDS-PAGE and anti-MBP Western blotting of 0.33% input. IVT, an aliquot of the in vitro translation reaction; 11% of binding reaction input. Neg. control, [35S]GBP incubated with glutathione-Sepharose beads. The level of GST-GSK-3 pulled down in each sample was detected by anti-GST Western blotting. (b) GBP does not bind MBP-Axin directly or through an interaction with GSK-3. Bacte- rially produced MBP-Axin was incubated with [35S]methionine- labeled in vitro translated GBP in the presence or absence of [35S]methionine-labeled Xgsk-3. Protein complexes were precipi- tated with amylose resin, and GBP and Xgsk-3 were detected by SDS-PAGE and autoradiography. IVT, an aliquot of the in vitro translation reaction; 14% of binding reaction input. MBP-Axin/ MBP levels were detected by SDS-PAGE and anti-MBP West- ern blotting of 1% of the input. with GST-GSK-3b were determined by SDS-PAGE and autoradiography. Whereas [35S]GBP did not significantly bind the glutathione resin (Fig. 2 a, lane 2) or GST (data not shown), it effectively bound GST-GSK-3b (Fig. 2 a, lane 3). Inclusion of increasing amounts of MBP-Axin with GST-GSK-3b resulted in less [35S]GBP binding to GST-GSK-3b (Fig. 2 a, lanes 4–7). At a 50-fold molar ex- cess of MBP-Axin to GST-GSK-3b, binding of [35S]GBP was completely eliminated compared with the background (Fig. 2 a, compare lanes 6 and 2). This effect is specifically dependent on MBP-Axin binding to GST-GSK-3b since substituting even a 1,000-fold molar excess of maltose binding protein (MBP) does not significantly affect [35S]GBP binding (Fig. 2 a, lane 11). While these results were consistent with the hypothesis that GSK-3 can be bound to either GBP or Axin, it was also possible that MBP-Axin binds GBP and this interac- tion prevents GBP from interacting with GSK-3. To test this, we examined the ability of MBP-Axin to pull down [35S]GBP. As shown in Fig. 2 b, whereas MBP-Axin effec- The Journal of Cell Biology, Volume 148, 2000 694