Gavagan, M. | Search bioRxiv

bioRxiv2022

The Axin scaffold protects the kinase GSK3β from cross-pathway inhibition

Multiple signaling pathways regulate the kinase GSK3{beta} by inhibitory phosphorylation at Ser9, which then occupies the GSK3{beta} priming pocket and blocks substrate binding. Since this mechanism should affect GSK3{beta} activity towards all primed substrates, it is unclear why Ser9 phosphorylation does not affect other GSK3{beta}-dependent pathways, such as Wnt signaling. We used biochemical reconstitution and cell culture assays to evaluate how Wnt-associated GSK3{beta} is insulated from cross-activation by other signals. We found that the Wnt-specific scaffold protein Axin allosterically protects GSK3{beta} from phosphorylation at Ser9 by upstream kinases, which prevents accumulation of pS9-GSK3{beta} in the Axin-GSK3{beta} complex. Scaffold proteins that protect bound proteins from alternative pathway reactions could provide a general mechanism to insulate signaling pathways from improper crosstalk.

biochemistry↗

bioRxiv2019

The Wnt pathway scaffold protein Axin promotes signaling specificity by suppressing competing kinase reactions

GSK3{beta} is a multifunctional kinase that phosphorylates {beta}-catenin in the Wnt signaling network and also acts on other protein targets in response to distinct cellular signals. To test the long-standing hypothesis that the scaffold protein Axin specifically accelerates {beta}-catenin phosphorylation, we measured GSK3{beta} reaction rates with multiple substrates in a minimal, biochemically-reconstituted system. We observed an unexpectedly small, ~2-fold Axin-mediated rate increase for the {beta}-catenin reaction. The much larger effects reported previously may have arisen because Axin can rescue GSK3{beta} from an inactive state that occurs only under highly specific conditions. Surprisingly, Axin significantly slows the reaction of GSK3{beta} with CREB, a non-Wnt pathway substrate. When both {beta}-catenin and CREB are present, Axin accelerates the {beta}-catenin reaction by preventing competition with CREB. Thus, while Axin alone does not markedly accelerate the {beta}-catenin reaction, in physiological settings where multiple GSK3{beta} substrates are present, Axin can promote signaling specificity by suppressing interactions with competing, non-Wnt pathway targets.

biochemistry↗