👤 Denise M Ferkey

Glycogen synthase kinase-3 beta (GSK-3) is a key downstream target of Wnt signaling and is regulated by its interactions with activating and inhibitory proteins. We and others have shown that GSK-3 activity toward non-primed substrates is regulated in part through a competition between its activating (Axin) and inhibitory (GBP/FRAT) binding partners. Here we use a reverse two-hybrid screen to identify mutations in GSK-3 that alter binding to GBP and Axin. We find that these mutations overlap and propose that GBP and Axin compete for binding to the same region of GSK-3. We use these mutations to examine the ability of GSK-3 to block eye development in Xenopus embryos and suggest that GSK-3 regulates eye development through a non-Wnt pathway. Show less

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. Show less