Phosphoregulation, in which the addition of a negatively charged phosphate group modulates protein activity, enables dynamic cellular responses. To understand how new phosphoregulation might be acquired, we mutationally scanned the surface of a prototypical yeast kinase (Kss1) to identify potential regulatory sites. The data revealed a set of spatially distributed “hotspots” that might have coevolved with the active site and preferentially modulated kinase activity. By engineering simple consensus phosphorylation sites at these hotspots, we rewired cell signaling in yeast. Using the same approach with a homolog yeast mitogen-activated protein kinase, Hog1, we introduced new phosphoregulation that modified its localization and signaling dynamics. Beyond revealing potential use in synthetic biology, our findings suggest that the identified hotspots contribute to the diversity of natural allosteric regulatory mechanisms in the eukaryotic kinome and, given that some are mutated in cancers, understanding these hotspots may have clinical relevance to human disease.

磷酸化（其中添加带负电荷的磷酸基团可调节蛋白质活性）可实现动态细胞反应。为了了解如何获得新的磷酸化，我们对原型酵母激酶（Kss1）的表面进行了突变扫描，以发现潜在的调控位点。数据揭示了一组空间分布的“热点”，这些热点可能与活性位点和优先调节的激酶活性共同进化。通过在这些热点处设计简单的共有磷酸化位点，我们重新连接了酵母中的细胞信号传导。使用与同源酵母促细胞分裂素激活的蛋白激酶Hog1相同的方法，我们引入了新的磷酸化作用，从而改变了其定位和信号传导动力学。除了揭示合成生物学的潜在用途外，