Department of Structural Biology, Stanford University School of Medicine, Stanford, CA 94035, USA.
Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA 94035, USA.
Sci Signal. 2024 Sep 3;17(852):eado0881. doi: 10.1126/scisignal.ado0881.
Glycogen synthase kinase 3β (GSK-3β) targets specific signaling pathways in response to distinct upstream signals. We used structural and functional studies to dissect how an upstream phosphorylation step primes the Wnt signaling component β-catenin for phosphorylation by GSK-3β and how scaffolding interactions contribute to this reaction. Our crystal structure of GSK-3β bound to a phosphoprimed β-catenin peptide confirmed the expected binding mode of the phosphoprimed residue adjacent to the catalytic site. An aspartate phosphomimic in the priming site of β-catenin adopted an indistinguishable structure but reacted approximately 1000-fold slower than the native phosphoprimed substrate. This result suggests that substrate positioning alone is not sufficient for catalysis and that native phosphopriming interactions are necessary. We also obtained a structure of GSK-3β with an extended peptide from the scaffold protein Axin that bound with greater affinity than that of previously crystallized Axin fragments. This structure neither revealed additional contacts that produce the higher affinity nor explained how substrate interactions in the GSK-3β active site are modulated by remote Axin binding. Together, our findings suggest that phosphopriming and scaffolding produce small conformational changes or allosteric effects, not captured in the crystal structures, that activate GSK-3β and facilitate β-catenin phosphorylation. These results highlight limitations in our ability to predict catalytic activity from structure and have potential implications for the role of natural phosphomimic mutations in kinase regulation and phosphosite evolution.
糖原合酶激酶 3β（GSK-3β）针对特定的信号通路，以响应不同的上游信号。我们使用结构和功能研究来剖析上游磷酸化步骤如何使 Wnt 信号成分β-连环蛋白（β-catenin）为 GSK-3β的磷酸化做好准备，以及支架相互作用如何对此反应做出贡献。我们结合了结构和功能研究来剖析上游磷酸化步骤如何使 Wnt 信号成分β-连环蛋白（β-catenin）为 GSK-3β的磷酸化做好准备，以及支架相互作用如何对此反应做出贡献。我们结合了结构和功能研究来剖析上游磷酸化步骤如何使 Wnt 信号成分β-连环蛋白（β-catenin）为 GSK-3β的磷酸化做好准备，以及支架相互作用如何对此反应做出贡献。我们结合了结构和功能研究来剖析上游磷酸化步骤如何使 Wnt 信号成分β-连环蛋白（β-catenin）为 GSK-3β的磷酸化做好准备，以及支架相互作用如何对此反应做出贡献。我们的研究结果表明，底物定位本身不足以进行催化，并且需要天然的磷酸化启动相互作用。我们还获得了与支架蛋白 Axin 的延伸肽结合的 GSK-3β结构，其结合亲和力大于先前结晶的 Axin 片段。该结构既没有揭示产生更高亲和力的其他接触点，也没有解释 Axin 结合如何调节 GSK-3β 活性位点中的底物相互作用。总之，我们的研究结果表明，磷酸化启动和支架作用产生了小的构象变化或变构效应，这些效应在晶体结构中无法捕捉到，从而激活了 GSK-3β 并促进了β-连环蛋白的磷酸化。这些结果突出了我们从结构预测催化活性的能力的局限性，并可能对天然磷酸模拟突变在激酶调节和磷酸化位点进化中的作用产生影响。
