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

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.

The multifaceted allosteric regulation of Aurora kinase A

“…These results were corroborated using an independent experimental method, double electron–electron resonance (DEER) spectroscopy, in which the fluorescent dyes are replaced by paramagnetic spin probes. The results of the DEER experiments were fully consistent with the FRET experiments [ 88 ], confirming that the activation loop of phosphorylated AurA adopts a range of different conformations, as observed in the unphosphorylated kinase.…”

“…( B ) Schematics of the dye-labeling scheme used to track the position of the activation loop by FRET (top), and (bottom) the distribution of inter-dye distances determined by time-resolved FRET for inactive AurA (dashed line), AurA activated by Tpx2 (pink), and AurA activated by phosphorylation on T288 (dark purple-shaded area). The data are taken from Ruff et al [ 88 ]. ( C ) Structure of the probable DFG-Out state adopted by AurA in solution (purple).…”

“…In contrast, Tpx2 binding was observed to cause a pronounced switch to the active state. A second study used time-resolved FRET to map the distribution of conformations sampled by the activation loop with and without phosphorylation on T288 [ 88 ]. The results showed that the activation loop of both unphosphorylated and phosphorylated AurA samples a range of conformations in solution that span the DFG-Out and DFG-In states ( Figure 3B , compare dashed and solid purple lines in the FRET distance distribution).…”

“…These surprising results raised the question of how phosphorylation on its own activates AurA so strongly, without causing a population shift to the DFG-In state. Reasoning that the large DFG-Out subpopulation of phosphorylated AurA might be obscuring an activating structural change in the DFG-In subpopulation in their ensemble experiments, Ruff et al [ 88 ] used a DFG-In-selective inhibitor, SNS-314, to trap AurA in the DFG-In state. DEER experiments on these samples revealed that phosphorylation on T288 indeed triggers a structural change within the DFG-In state, confirming that phosphorylation operates by altering the structure and dynamics of the DFG-In subpopulation ( Figure 4A ).…”

“… ( A ) Schematic of the spin-probe labeling scheme (top) and measured spin-spin distances (bottom) from double electron–electron resonance experiments on phosphorylated AurA (darker solid line and shading) and unphosphorylated AurA (lighter dashed line). Data are taken from Ruff et al [ 88 ]. The inhibitor SNS-314 was used to isolate the DFG-In state.…”