Covalent Aurora A regulation by the metabolic integrator coenzyme A

Abstract

Aurora A kinase is a master mitotic regulator whose functions are controlled by several regulatory interactions and post-translational modifications. It is frequently dysregulated in cancer, making Aurora A inhibition a very attractive antitumor target. However, recently uncovered links between Aurora A, cellular metabolism and redox regulation are not well understood. In this study, we report a novel mechanism of Aurora A regulation in the cellular response to oxidative stress through CoAlation. A combination of biochemical, biophysical, crystallographic and cell biology approaches revealed a new and, to our knowledge, unique mode of Aurora A inhibition by CoA, involving selective binding of the ADP moiety of CoA to the ATP binding pocket and covalent modification of Cys290 in the activation loop by the thiol group of the pantetheine tail. We provide evidence that covalent CoA modification (CoAlation) of Aurora A is specific, and that it can be induced by oxidative stress in human cells. Oxidising agents, such as diamide, hydrogen peroxide and menadione were found to induce Thr 288 phosphorylation and DTT-dependent dimerization of Aurora A. Moreover, microinjection of CoA into fertilized mouse embryos disrupts bipolar spindle formation and the alignment of chromosomes, consistent with Aurora A inhibition.Altogether, our data reveal CoA as a new, rather selective, inhibitor of Aurora A, which locks this kinase in an inactive state via a “dual anchor” mechanism of inhibition that might also operate in cellular response to oxidative stress. Finally and most importantly, we believe that these novel findings provide a new rationale for developing effective and irreversible inhibitors of Aurora A, and perhaps other protein kinases containing appropriately conserved Cys residues.

Are redox changes a critical switch for mitotic progression?

“…It was found that CoA was able to form a disulfide adduct (CoAlation) on Cys-290 within the AURKA activation segment, near the conserved Thr-288 autophosphorylation/activation site. 4 A crystal structure of an AURKA kinase domain CoAlated on Cys-290 and phosphorylated at Thr-288 revealed a monomeric kinase domain with the adenosine diphosphate (ADP) moiety of CoA occupying the adenosine triphosphate (ATP) binding pocket, consistent with an inhibitory effect. CoAlation as well as oxidation of Cys-290 to sulfenic acid were found to be stimulated in mammalian cells artificially treated with oxidizing agents, suggesting a possible link between increased protein thiol oxidation during mitosis and AURKA regulation.…”

“…CoAlation as well as oxidation of Cys-290 to sulfenic acid were found to be stimulated in mammalian cells artificially treated with oxidizing agents, suggesting a possible link between increased protein thiol oxidation during mitosis and AURKA regulation. [3][4][5] Treatment of cells with oxidizing agents has been shown to stimulate AURKA phosphorylation at Thr-288. 4,6 Yet, paradoxically, in biochemical assays using purified protein, oxidative modification of AURKA by CoAlation or sulfenylation of Cys-290 potently inhibited kinase activity.…”

“…[3][4][5] Treatment of cells with oxidizing agents has been shown to stimulate AURKA phosphorylation at Thr-288. 4,6 Yet, paradoxically, in biochemical assays using purified protein, oxidative modification of AURKA by CoAlation or sulfenylation of Cys-290 potently inhibited kinase activity. 4,5 To help resolve this paradox, we recently showed that disulfide modifications of Cys-290 can directly promote AURKA activation by Thr-288 autophosphorylation in a dimerizationdependent manner.…”

“…4,6 Yet, paradoxically, in biochemical assays using purified protein, oxidative modification of AURKA by CoAlation or sulfenylation of Cys-290 potently inhibited kinase activity. 4,5 To help resolve this paradox, we recently showed that disulfide modifications of Cys-290 can directly promote AURKA activation by Thr-288 autophosphorylation in a dimerizationdependent manner. 7 We determined a crystal structure of the AURKA kinase domain covalently modified by the arseniccontaining buffering agent cacodylate, revealing Cys-247 and Cys-290 as redox-sensitive residues within the kinase domain that could allosterically regulate its conformation.…”

“…Indeed, our crystal structure of a fully CoAlated but unphosphorylated AURKA kinase domain revealed a unique activation segment-swapped dimer with the CoA adduct of each monomer bound within the active site of the opposing monomer, in stark contrast to the previously reported monomeric structure of a CoAlated but phosphorylated and inhibited AURKA kinase domain. 4 We next crystallized a mixture of CoAlated and non-CoAlated AURKA kinase domains. That structure showed an activation segment-swapped dimer with clear electron density for a Cys-290-Cys-290 disulfide bond formed between the two monomers in a subpopulation of the molecules in the crystal, suggesting that CoAlation may promote the formation of AURKA disulfide dimers.…”