Ancestral resurrection reveals mechanisms of kinase regulatory evolution

Abstract Protein kinases are crucial to coordinate cellular decisions and therefore their activities are strictly regulated. We used ancestral resurrection to uncover a mechanism underlying the evolution of kinase control within the ERK family of Mitogen Activated Protein Kinases (MAPKs). Kinase activities switched from high to low intrinsic autophosphorylation at the transition from the ancestors of ERKs1-5 and ERKs1-2. A shortening of the loop between β3-αC and a mutation in the gatekeeper residue drove this transition. Molecular dynamics simulations suggested that the change in the β3-αC loop length affected kinase cis-autophosphorylation by altering the positioning of catalytic residues and by allowing greater flexibility in the L16 kinase loop. This latter effect likely synergizes with the known role of gatekeeper mutations in facilitating domain closure and thus kinase activation, providing a rationale for the synergy between the two evolutionary mutations. Our results shed light on the evolutionary mechanisms that led to tight regulation of a central kinase in development and disease.