Computational and Systems Biology Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, United States; Gerstner Sloan Kettering Graduate School, Memorial Sloan Kettering Cancer Center, New York, United States
Computational and Systems Biology Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, United States
Julie M Behr
Computational and Systems Biology Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, United States; Tri-Institutional Program in Computational Biology and Medicine, Weill Cornell Medical College, New York, United States
David D Thomas
Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, United States
Many eukaryotic protein kinases are activated by phosphorylation on a specific conserved residue in the regulatory activation loop, a post-translational modification thought to stabilize the active DFG-In state of the catalytic domain. Here we use a battery of spectroscopic methods that track different catalytic elements of the kinase domain to show that the ~100 fold activation of the mitotic kinase Aurora A (AurA) by phosphorylation occurs without a population shift from the DFG-Out to the DFG-In state, and that the activation loop of the activated kinase remains highly dynamic. Instead, molecular dynamics simulations and electron paramagnetic resonance experiments show that phosphorylation triggers a switch within the DFG-In subpopulation from an autoinhibited DFG-In substate to an active DFG-In substate, leading to catalytic activation. This mechanism raises new questions about the functional role of the DFG-Out state in protein kinases.
