Ancient Origins of Allosteric Activation in a Ser/Thr Kinase

Despite a myriad of cellular events being regulated by allostery, evolution of this process is largely unexplored territory. Here, we use Ancestral Sequence Reconstruction (ASR) to resurrect ancestors of two colocalizing proteins, Aurora A kinase and its allosteric activator TPX2, to experimentally characterize the evolutionary path of allosteric activation. Autophosphorylation of the activation loop is the most ancient activation mechanism; it is fully developed in the oldest kinase ancestor and remains stable over one billion years of evolution. As the microtubule-associated protein TPX2 appeared, efficient kinase binding to TPX2 evolved likely due to increased fitness by virtue of colocalization. Subsequently, TPX2-mediated allosteric kinase regulation gradually evolved. Surprisingly, evolution of this regulation is encoded in the kinase and did not arise by a dominating mechanism of coevolution.