A dynamic mechanism for allosteric activation of Aurora kinase A by activation loop phosphorylation.

Emily F Ruff,Steven K Albanese,Julie M Behr,Soreen Cyphers,Andrew R Thompson,Eric W Lake,Sonya M Hanson,David D Thomas,Joseph M Muretta,Nicholas M Levinson,John D Chodera

doi:10.7554/elife.32766

Emily F Ruff, Steven K Albanese + Show 9 more

Open Access

https://doi.org/10.7554/elife.32766

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Abstract

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.

Highlights

Stringent regulatory control of protein kinases is critically important for the integrity of cellular signal transduction
We recently showed that activation of Aurora A (AurA) by Tpx2 is driven by a population shift from a DFGOut to the DFG-In state (Cyphers et al, 2017)
The majority of eukaryotic protein kinases are activated by phosphorylation on the activation loop at a site equivalent to T288 in AurA (Johnson et al, 1996)

Summary

Introduction

Stringent regulatory control of protein kinases is critically important for the integrity of cellular signal transduction. The catalytic activity of protein kinases is regulated by finely-tuned allosteric mechanisms that reversibly switch the kinase domain between active and inactive conformational states (Huse and Kuriyan, 2002). X-ray structures show that ionic interactions between the phosphate moiety and a surrounding pocket of basic residues stabilize the activation loop in a conserved active conformation (Knighton et al, 1991; Yamaguchi and Hendrickson, 1996; Steichen et al, 2012). In this active state, a catalytic asp-phe-gly (DFG) motif at the N-terminal end of the activation loop adopts an active ‘DFG-In’ conformation, with the aspartate residue of the DFG motif pointing into the active site to coordinate Mg-ATP, and the C-terminal segment of the activation loop positioned to bind peptide substrates.

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