Structural and kinetic investigation into the roles of scaffolding and phosphopriming on GSK3 pathway insulation

Abstract

The kinase GSK3 is key regulator of multiple signaling pathways, including differentiation, energy homeostasis, and development. Its involvement in many distinct pathways causes the potential for crosstalk, where the upstream signal from one pathway inappropriately activates the downstream response of another. To phosphorylate specific substrates, GSK3 recognizes pre-phosphorylated, or ‘phosphoprimed’ sequence motifs on substrate proteins. Phosphopriming ensures that GSK3 substrates have already been acted upon by upstream kinases within specific signaling pathways, making GSK3 activity dependent on pathway activation. Using structural and quantitative biochemical studies, we found that substrate phosphopriming has large effects on GSK3 catalysis that do not cause detectable changes in crystal structures. These results indicate that subtle or transient structural changes in and around the GSK3 active site may play a crucial role in directing GSK3 towards specific substrates. Alongside phosphopriming, scaffold proteins coordinate GSK3 with specific substates, promoting phosphorylation. In the Wnt signaling pathway, the scaffold protein Axin specifies GSK3 to interact with the Wnt substrate β-catenin, preventing GSK3 crosstalk into other pathways. We sought to investigate if Axin also insulates GSK3 from external signals. Using quantitative biochemical analysis and cell culture studies, we found that Axin prevents GSK3 phosphorylation at Ser9, a crucial step of both Insulin and Growth factor signaling. In doing so, Axin protects GSK3 from insulin and growth factor pathways, preventing crosstalk. To determine if other GSK3-dependent pathways use the same insulation mechanism as Axin, we used interactome labeling to find scaffolds that compete with Axin for GSK3 binding. We discovered that the Hedgehog scaffold SUFU competes with Axin to coordinate GSK3, and that SUFU can be displaced by Axin in vivo. Taken together, these findings resolve how phosphopriming enables GSK3 catalysis, uncovers how Axin insulates GSK3 from other pathways, and characterizes developmental scaffold proteins compete for the same GSK3 binding interface to define distinct subpopulations.