Department of Neurologic Surgery, Mayo Clinic, Jacksonville, FL 32224, USA.
Department of Cellular & Molecular Physiology, Yale School of Medicine, New Haven, CT 06520, USA; Systems Biology Institute, Yale University, West Haven, CT 06516, USA.
Cell Rep. 2021 Jul 20;36(3):109416. doi: 10.1016/j.celrep.2021.109416.
Advances in genetic code expansion have enabled the production of proteins containing site-specific, authentic post-translational modifications. Here, we use a recoded bacterial strain with an expanded genetic code to encode phosphoserine into a human kinase protein. We directly encode phosphoserine into WNK1 (with-no-lysine [K] 1) or WNK4 kinases at multiple, distinct sites, which produced activated, phosphorylated WNK that phosphorylated and activated SPAK/OSR kinases, thereby synthetically activating this human kinase network in recoded bacteria. We used this approach to identify biochemical properties of WNK kinases, a motif for SPAK substrates, and small-molecule kinase inhibitors for phosphorylated SPAK. We show that the kinase inhibitors modulate SPAK substrates in cells, alter cell volume, and reduce migration of glioblastoma cells. Our work establishes a protein-engineering platform technology that demonstrates that synthetically active WNK kinase networks can accurately model cellular systems and can be used more broadly to target networks of phosphorylated proteins for research and discovery.
遗传密码扩展的进展使得能够生产含有特定位置、真实翻译后修饰的蛋白质。在这里，我们使用一种经过重新编码的具有扩展遗传密码的细菌菌株，将磷酸丝氨酸编码到人类激酶蛋白中。我们直接将磷酸丝氨酸编码到多个不同位置的 WNK1（无赖氨酸 [K] 1）或 WNK4 激酶中，产生了激活的、磷酸化的 WNK，磷酸化并激活 SPAK/OSR 激酶，从而在重新编码的细菌中人工激活这种人类激酶网络。我们使用这种方法来鉴定 WNK 激酶的生化特性、SPAK 底物的基序以及磷酸化 SPAK 的小分子激酶抑制剂。我们表明，激酶抑制剂在细胞中调节 SPAK 底物，改变细胞体积，并减少神经胶质瘤细胞的迁移。我们的工作建立了一种蛋白质工程平台技术，证明了人工活性 WNK 激酶网络可以准确模拟细胞系统，并可以更广泛地用于针对磷酸化蛋白质网络进行研究和发现。
