Scaffold proteins are thought to promote signaling specificity by accelerating reactions between bound kinase and substrate proteins. To test the long-standing hypothesis that the scaffold protein Axin accelerates glycogen synthase kinase 3β (GSK3β)-mediated phosphorylation of β-catenin in the Wnt signaling network, we measured GSK3β reaction rates with multiple substrates in a minimal, biochemically reconstituted system. We observed an unexpectedly small, ∼2-fold Axin-mediated rate increase for the β-catenin reaction when measured in isolation. In contrast, when both β-catenin and non-Wnt pathway substrates are present, Axin accelerates the β-catenin reaction by preventing competition with alternative substrates. At high competitor concentrations, Axin produces >10-fold rate effects. Thus, while Axin alone does not markedly accelerate the β-catenin reaction, in physiological settings where multiple GSK3β substrates are present, Axin may promote signaling specificity by suppressing interactions with competing, non-Wnt pathway targets. This mechanism for scaffold-mediated control of competition enables a shared kinase to perform distinct functions in multiple signaling networks.

支架蛋白被认为通过加速结合激酶和底物蛋白之间的反应来促进信号特异性。为了检验支架蛋白 Axin 加速糖原合酶激酶 3β (GSK3β) 介导的 Wnt 信号网络中 β-连环蛋白磷酸化的长期假设，我们在最小的生化重组系统中测量了 GSK3β 与多种底物的反应速率。我们观察到当单独测量时，β-连环蛋白反应的 Axin 介导的速率增加出乎意料地小，约为 2 倍。相反，当 β-catenin 和非 Wnt 途径底物都存在时，Axin 通过阻止与替代底物的竞争来加速 β-catenin 反应。在高竞争者浓度下，Axin 产生 >10 倍的速率效应。因此，虽然单独的 Axin 不会显着加速 β-catenin 反应，但在存在多种 GSK3β 底物的生理环境中，Axin 可能通过抑制与竞争性非 Wnt 途径靶标的相互作用来促进信号特异性。这种支架介导的竞争控制机制使共享激酶能够在多个信号网络中执行不同的功能。