Interaction among GSK-3, GBP, axin, and APC in Xenopus axis specification

Abstract

Glycogen synthase kinase 3 (GSK-3) is a constitutively active kinase that negatively regulates its substrates, one of which is beta-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 beta-catenin form a complex that promotes the GSK-3-mediated phosphorylation and subsequent degradation of beta-catenin. Adenomatous polyposis coli (APC) joins the complex and downregulates beta-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 beta-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 beta-catenin levels and establishment of the dorsal axis.

Figure 3

GBP does not inhibit Xgsk-3 phosphorylation of a peptide substrate. Embryos were injected with RNA encoding Xgsk-3-myc together with control RNA or GBP-FLAG RNA. After 3 h, proteins were extracted and immunoprecipitated with anti-FLAG (Xgsk-3 + GBP), anti-myc (Xgsk-3 + control), or both (uninjected) antibodies. The kinase activity of immune complexes was measured by phosphorus-32 incorporation into the GSK-3–specific substrate prephosphorylated CREB peptide (p-CREB; dark bars). The nonphosphorylated CREB peptide (CREB; light bars) is not a GSK-3 substrate and was used as a control. The activity of duplicate immune complexes is shown.

Figure 4

Dominant-negative Xgsk-3 binds Axin. Embryos were injected at the two- to eight-cell stage with 1 ng Axin-myc, 0.5 ng Xgsk-3-FLAG, and 0.5 ng dnXgsk-3-FLAG in the animal pole. Embryo extracts were precipitated 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). Lane numbers in the right panel refer to the same injections as shown above corresponding lane numbers in the left panel.

Figure 7

Model for GSK-3 regulation in the early Xenopus embryo. (a) On the ventral side of the embryo, GSK-3 is part of a functional degradation complex that includes APC and Axin. When in this complex, GSK-3 phosphorylates β-catenin, targeting it for degradation via the proteosome pathway. Under these conditions, dorsal genes are repressed. (b) On the dorsal side of the embryo, GSK-3 is excluded from the Axin–APC complex by GBP. In addition, GBP may prevent GSK-3 from phosphorylating its normal substrates by blocking access to the active site. β-Catenin accumulates and activates the transcription of dorsal genes. (c) The kinase-deficient dnXgsk-3 functions by binding Axin, thus, keeping endogenous Xgsk-3 from the degradation complex. As in the situation with GBP, β-catenin accumulates and activates the transcription of dorsal genes.