Mechanistic analysis of scaffold contributions to specificity and insulation in Wnt signaling

Abstract

Glycogen Synthase Kinase 3β (GSK3β) plays a pivotal role in a large number of signaling pathways. To avoid cross-pathway interference, signaling pathways that rely on GSK3β must have regulatory features that allow signals to be passed through GSK3β to the correct downstream substrates without activating other pathways. Scaffold proteins, binding platforms that assemble and regulate signaling pathways, may provide a mechanism to direct shared signaling components to specific downstream targets. In Wnt signaling, the scaffold protein Axin is thought to serve as a control point, coordinating the phosphorylation of the Wnt substrate β- catenin by GSK3β. We tested the long-standing hypothesis that Axin directs GSK3β activity in the Wnt pathway by accelerating GSK3β phosphorylation of the substrate β-catenin. We measured reaction rates in a minimal reconstituted in vitro system and found that, surprisingly, Axin produced only modest increases in β-catenin phosphorylation rates. Instead, Axin increases the specificity of GSK3β for β-catenin by suppressing competing kinase reactions. By suppressing the competition, Axin can produce >10-fold increases in the β-catenin phosphorylation rate when competing substrates are present. Acting through Axin, Wnt signals could regulate just the pool of GSK3β that is bound to Axin, allowing Wnt signals to be sent without affecting other signaling pathways. Axin may also play a role in protecting Wnt- associated GSK3β from being affected by signals from other pathways. We found that binding to Axin protects GSK3β from phosphorylation at Ser9, a key regulatory event in insulin and other signaling pathways. These mechanisms allow the scaffold to create a pool of GSK3β that preferentially interacts with β-catenin and can be independently regulated through Axin without affecting, or being affected by, other signaling pathways.