Redox Biology 28 (2020) 101318 Contents lists available at ScienceDirect Redox Biology journal homepage: www.elsevier.com/locate/redox Research Paper Covalent Aurora A regulation by the metabolic integrator coenzyme A T Yugo Tsuchiyaa,#, Dominic P. Byrneb,#, Selena G. Burgessc,#, Jenny Bormannd, Jovana Bakovića, Yueyang Huanga, Alexander Zhyvoloupa, Bess Yi Kun Yua, Sew Peak-Chewe, Trang Tranf, Fiona Bellanyf, Alethea B. Taborf, AW Edith Chang, Lalitha Guruprasadh, Oleg Garifulini, Valeriy Filonenkoi, Matthias Vonderachj, Samantha Ferriesj, Claire E. Eyersb,j, John Carrolld,1, ∗∗ ∗∗∗ ∗ Mark Skehele, Richard Baylissc, , Patrick A. Eyersb, , Ivan Gouta,i, a Department of Structural and Molecular Biology, University College London, London, WC1E 6BT, UK b Department of Biochemistry, Institute of Integrative Biology, University of Liverpool, Liverpool, L69 7ZB, UK c School of Molecular and Cellular Biology, Astbury Centre for Structural and Molecular Biology, University of Leeds, Leeds, LS2 9JT, UK d Department of Cell and Developmental Biology, University College London, London, WC1E 6BT, UK e MRC Laboratory of Molecular Biology, Cambridge Biomedical Campus, Cambridge, CB2 0QH, UK f Department of Chemistry, University College London, London, WC1E 6BT, UK g Wolfson Institute for Biomedical Research, University College London, London, WC1E 6BT, UK h School of Chemistry, University of Hyderabad, Hyderabad, 500 046, India i Department of Cell Signaling, Institute of Molecular Biology and Genetics, Kyiv 143, Ukraine j Centre for Proteome Research, Department of Biochemistry, Institute of Integrative Biology, University of Liverpool, Liverpool, L69 7ZB, UK 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 bythe 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 byoxidative 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 Cysresidues. 1. Introduction distinct sub-families composed of Aurora A and Aurora B and C [5]. Aurora kinases contain a conserved C-terminal kinase domain, which Aurora kinases are Ser/Thr kinases that play well-documented roles phosphorylates substrates with a minimal R-X-S/T consensus motif in eukaryotes, where they control meiosis, mitosis and cell division [1]. [6,7]. The Aurora kinase domain is itself regulated by a critical au- Aurora kinases differ in relative expression levels, stability and sub- tophosphorylation event in the activation loop [8–11], which is asso- cellular localization, the latter linked to single amino acid differences ciated with catalytic activation and timely execution of different phases that dramatically affect biological distribution and function [2–4]. In of mitosis [12–15] and meiosis [16]. During the cell cycle, Aurora A has vertebrates, the Aurora family of protein kinases are grouped into two canonical roles in centrosome duplication, spindle bipolarity, ∗ Corresponding author. Department of Structural and Molecular Biology, University College London, London, WC1E 6BT, UK. ∗∗ Corresponding author. ∗∗∗ Corresponding author. E-mail addresses: [email protected] (R. Bayliss), [email protected] (P.A. Eyers), [email protected] (I. Gout). # These authors contributed equally to this study. 1 Current address: Development and Stem Cell Program, Monash Biomedicine Discovery Institute, Monash University, Melbourne, Victoria, Australia. https://doi.org/10.1016/j.redox.2019.101318 Received 10 April 2019; Received in revised form 14 August 2019; Accepted 1 September 2019 Available online 05 September 2019 2213-2317/ © 2019 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/BY/4.0/). Y. Tsuchiya, et al. Redox Biology 28 (2020) 101318 chromosome segregation and spindle checkpoint maintenance [17], CoA. Intracellular levels of CoA and its thioester-derivatives are tightly which together contribute to fundamental processes such as cen- regulated by a variety of stimuli, including hormones, nutrients, me- trosomal and genomic stability and likely reflect the long-recognised tabolites and stresses [43,44]. The main rate-limiting enzyme in CoA oncogenic properties associated with overexpressed Aurora A [18]. biosynthesis is pantothenate kinase which initiates the biosynthetic Interestingly, Aurora A has also recently been implicated in the control pathway [45]. Furthermore, the activity of the last enzyme in the of energy production in cancer cells through mitochondrial targeting pathway, CoA synthase, is regulated by extracellular stimuli and stress [19]. In contrast, Aurora B is a chromosomal passenger protein required response [46–49]. for the spindle assembly checkpoint and rate-limiting for cytokinesis in CoA and its thioester derivatives are strategically positioned at the cells [20]. Aurora C is most highly expressed in germ cells, where it can crossroads of cellular anabolic and catabolic pathways, controlling the functionally replace Auora B as a chromosome passenger protein [1]. Krebs cycle, ketogenesis, the biosynthesis of proteins, lipids and cho- Aurora A biology is controlled via targeted subcellular localization lesterol, oxidation of fatty acids and degradation of amino acids. In mediated through the formation of dynamic protein complexes with addition, they are also involved in the regulation of gene expression and non-catalytic binding partners such as TPX2 [9,10,21–23], TACC3 [24] cellular metabolism via post-translational modifications, such as pro- and NMYC [25], which control distinct Aurora A signaling outputs. tein acetylation, butyrylation, malonylation and succinylation. Indeed, Aurora A/TPX2 holoenzyme complexes co-localise at the polar Abnormal biosynthesis and homeostasis of CoA and its derivatives is end of spindle microtubules, where Aurora A is maintained in dynamic associated with various human pathologies, including neurodegenera- pools alongside the mitoitc kinesin Eg5, which is localised by a C- tion, cancer, metabolic disorders and cardiac hypertrophy [44]. terminal motif in TPX2 [22,26,27]. Allosteric Aurora A activation on Protein CoAlation is a recently described post-translational mod- the spindle is thought to occur by binding to microtubule-associated ification (PTM) that involves covalent modification of cysteine residues proteins (which are also Aurora A substrates) such as TPX2 and TACC3 by CoA [50]. New research tools and methodologies have revealed [28]. Mechanistically, Aurora A activation requires autopho- protein CoAlation as a widespread and reversible PTM involved in sphorylation of Thr 288 (and possibly Thr 287), which drives the kinase cellular redox regulation [51]. To date, more than one thousand pro- into the most catalytically-competent conformation [3,9,29]. Interest- teins have been found to be CoAlated in a variety of prokaryotic and ingly, the TPX2 complex also protects Aurora A from Thr 288 depho- eukaryotic cells [50,52]. Protein CoAlation occurs at a low level in sphorylation by PP1 and PP2A phosphatases in vitro [3,9], although exponentialy growing cells, but is strongly induced in reponse to var- accumulating evidence suggests that the PP6 Ser/Thr phosphatase is ious oxidising agents, including H2O2, diamide, menadione and t-butyl rate-limiting for mitotic dephosphorylation at this site in cells [27]. In hydroperoxide. Protein CoAlation alters the molecular mass, charge, addition to TPX2, Aurora A is targeted to core protein co-factors, in- and activity of modified proteins, and is thought to protect them from cluding the centrosomal protein Cep192, the mitotic entry factor BORA irreversible over-oxidation. and the transcription factor NMYC [30], whose interaction with a The presence of exposed cysteine residues on the surface of proteins specific inactive Aurora A conformation is important for controlling provides a simple, sometimes reversible, mechanism for post-transla- NMYC stability in cells, and the basis for new approaches