Redox priming promotes Aurora A activation during mitosis

Abstract

Cell cycle-dependent redox changes can mediate transient covalent modifications of cysteine thiols to modulate the activities of regulatory kinases and phosphatases. Our previously reported finding that protein cysteine oxidation is increased during mitosis relative to other cell cycle phases suggests that redox modifications could play prominent roles in regulating mitotic processes. The Aurora family of kinases and their downstream targets are key components of the cellular machinery that ensures the proper execution of mitosis and the accurate segregation of chromosomes to daughter cells. In this study, x-ray crystal structures of the Aurora A kinase domain delineate redox-sensitive cysteine residues that, upon covalent modification, can allosterically regulate kinase activity and oligomerization state. We showed in both Xenopus laevis egg extracts and mammalian cells that a conserved cysteine residue within the Aurora A activation loop is crucial for Aurora A activation by autophosphorylation. We further showed that covalent disulfide adducts of this residue promote autophosphorylation of the Aurora A kinase domain. These findings reveal a potential mechanistic link between Aurora A activation and changes in the intracellular redox state during mitosis and provide insights into how novel small-molecule inhibitors may be developed to target specific subpopulations of Aurora A.

Fig. 1.. Crystallization of the Aurora A kinase domain with cacodylate buffer identifies two conserved redox-active cysteines and an activation segment-swapped dimer.

(A) Left, the crystal structure of the Aurora A kinase domain in an active conformation, shown in grey ribbons representation, adopts the canonical kinase domain fold with N-terminal and C-terminal lobes. The ATP binding pocket containing bound AMP-PNP, shown in stick representation (with carbons colored dark grey, oxygens red, and phosphorus orange) is located within the active site cleft in between the two lobes. Side chains of Cys²⁴⁷, Cys²⁹⁰, and phosphorylated Thr²⁸⁸ are also shown in stick representation. Right, the crystal structure of the Aurora A kinase domain obtained with cacodylate buffer, shown in blue ribbons representation, depicting covalent modification of Cys²⁴⁷ and Cys²⁹⁰ (shown in stick representation with cyan carbons, overlayed with transparent space filled rendering) and a large displacement of the activation segment (shown in orange). TPX2 residues 7–20 are shown in yellow. (B) The crystal structure of the activation segment-swapped dimer of the cacodylate-modified Aurora kinase domain. Monomers are colored blue and purple and shown as ribbons representations, with a cartoon indicating the relative orientation of each monomer in the upper right of the structure. The activation segment is orange. Symmetry-related kinase domain monomers within the crystal structure can be seen in a dimeric arrangement with their activation segments exchanged between the monomers at the dimer interface. The monomers are oriented with their N-terminal lobes pointing in near-orthogonal directions, as indicated by the dotted axis lines in the cartoon representation.

Fig. 2.. Cacodylate-modification of Cys²⁴⁷ in the Aurora A kinase domain induces an inactive conformation, suggesting that compounds that selectively modify Cys²⁴⁷ may provide novel Aurora A inhibitors.

(A) Superposition of the active sites in the cacodylate-modified Aurora A kinase domain (blue and cyan) with the corresponding unmodified structure (grey and dark grey) shows spatial overlap between Phe²⁷⁵ in the active DFG-in conformation (dark grey, in stick representation) and the dimethyl arsenic adduct of the cacodylate-modified Cys²⁴⁷ (cyan and purple in stick representation, overlayed with transparent space filled rendering). AMP-PNP in the active structure (stick representation with dark grey carbons) spatially overlaps with Phe²⁷⁵ in the inactive cacodylate-modified DFG-out structure (cyan), and the side chain of Glu¹⁸¹ is also displaced in the cacodylate-modified structure (cyan) relative to the unmodified structure (dark grey). (B) The displacement of the Glu¹⁸¹ side chain expands the ATP-binding pocket, allowing an oxidized DTT molecule to occupy this space in the cacodylate-modified Aurora A structure. Superposition of the previous structure of the Aurora A kinase domain in complex with a 5-aminopyrimidinyl quinazoline inhibitor (beige and yellow; PDB code 2C6E) shows how this expanded ATP-binding pocket accommodates existing Aurora A-selective inhibitors. (C) Schematic of the experiment in which an Aurora A kinase domain construct containing a single cysteine (Cys²⁴⁷) was used for a mass spectrometry-based high throughput tethering screen of 880 disulfide-containing compounds (listed in data file S1) to identify additional covalent modifiers of Cys²⁴⁷. Each compound was incubated separately with Aurora A kinase domain, to allow thiol-disulfide exchange with the Cys²⁴⁷ side chain thiol in the presence of ß-mercaptoethanol, a non-specific disulfide reducing agent. Stable covalent labeling of Cys²⁴⁷ under these mildly reducing conditions requires additional stabilizing contacts between the particular compound and residues in Aurora A in close proximity to Cys²⁴⁷. These stable disulfide adducts were then detected by an increased total mass of the protein using mass spectrometry (data file S1).

Fig. 3.. Crystal structures of Aurora A kinase domain modified with disulfide adducts on C290 show a catalytically active dimer facilitating autophosphorylation in trans.

(A and B) Structures of the Aurora A kinase domain modified with compounds 7–80 (A) and 8–34 (B) at Cys²⁹⁰ show an activation segment-swapped dimer, in which the monomers are oriented with their N-terminal lobes pointing in the same direction, as indicated by the dotted axis lines in the cartoon representation in the center bottom. Monomers are colored blue and purple and shown as ribbons representations. The activation segment is orange. This dimer configuration is distinct from the near orthogonal arrangement seen in the cacodylate-modified structure (Fig. 1B). (C) Superposition of the active site regions of the 7–80 modified Aurora A structure with the Akt kinase domain (dark green and chartreuse) in complex with AMP-PNP and a GSK3ß substrate peptide (beige and yellow) from PDB code 1O6L. The active site of one monomer of the 7–80 modified Aurora A is shown in blue and cyan, with the Thr²⁸⁸/Cys²⁹⁰ activation loop from the other monomer shown in purple and magenta. Sulfur atoms of Cys²⁹⁰ are colored bright green. (D and E) Structure of the 7–80-modified structure, superimposed and contrasted with each of the monomers from a structure of a fully reduced and unphosphorylated Aurora A kinase domain in a similar dimer configuration (PDB code 4C3P). Large differences in the positioning of the phopsho acceptor residue (Thr²⁸⁸) and of the catalytic base (Asp²⁵⁶) in the 4C3P structure (emphasized with black outlines) can be seen between the 7–80-modified and the unmodified structures.

Fig. 4.. The activation loop cysteine is crucial for Aurora A activation by autophosphorylation.

(A) Sequence alignment of human Aurora A (residues 246–293) with X. laevis Aurora A (residues 253–300). The highlighted amino acid residues denote residues mutated in the constructs used in the assays described in the remainder of this figure (B to D). (B to D) Xenopus egg extracts were used for xAurora A activation assays to examine the requirement for the activation loop cysteine (Cys²⁹⁷) of xAurora A in autophosphorylation. Endogenous xAurora A was depleted from Xenopus egg extracts using an immobilized xAurora A-binding fragment of xCEP192. Wild-type and mutant xAurora A constructs were then added to this depleted extract and assayed for activation by addition of either sperm nuclei as a source of centrosomes (B) or by addition of an antibody to xAurora A (C). In (D), xAurora A activation was also assayed using undepleted Xenopus egg extract and selective activation of exogenous wild-type and mutant FLAG-tagged xAurora A constructs using an antibody to FLAG. To the right of each experimental schematic, total and autophosphorylated (pThr²⁹⁵) Aurora A was detected by Western blotting. Blots in (B and D) are representative of 5 independent experiments, and the blots in (C) are representative of 3 independent experiments.

Fig. 5.. The activation loop cysteine in Aurora A is critical for autophosphorylation in mammalian cells.

(A) Experimental schematic of the creation of stable HeLa cell lines by incorporating a doxycycline-inducible shRNA against endogenous Aurora A and transfected with or without shRNA-resistant, FLAG-tagged, wild-type and mutant Aurora A constructs driven by a native Aurora A promoter fragment. Following induction of shAurora A, the cells were nocodazole arrested. (B) Western blotting to assess Aurora A autophosphorylation at pThr²⁸⁸ in the nocodazole-arrested lysates described in (A). Total Aurora A and β-tubulin were blotted for reference. Blots are representative of 2 independent experiments.

Fig. 6.. Disulfide modification of Aurora A is involved in its activation by autophosphorylation of its activation loop.

(A) Western blotting for Aurora A autophosphorylation at pThr²⁸⁸ to assess xAurora A activation in Xenopus egg extracts supplemented with demembranated sperm nuclei and exposed to DTT (or buffer, control) for the indicated times. Blots are representative of 4 independent experiments. (B) Schematic for Aurora A kinase domain constructs CoAlated on Cys²⁹⁰ and crystallized in the presence of AMP-PNP. (C) Structure of a wild type Aurora A kinase domain construct CoAlated on Cys²⁹⁰ shows an activation segment-swapped dimer. Monomers are colored blue and purple and shown as ribbons representations, with a cartoon shown above the structure. The activation segments (orange and red) are swapped between the monomers. The CoA adduct of one monomer is bound in the ATP-binding of pocket of the opposing monomer. No electron density was observed for the TPX2 fragment fused to the N-terminus of the Aurora A kinase domain construct used to determine this structure. (D) Structure of a single-cysteine human Aurora A kinase domain construct CoAlated on Cys²⁹⁰ and crystallized in complex with AMP-PNP shows an activation segment-swapped dimer with monomers oriented with their N-terminal lobes pointing in near orthogonal directions (dotted axis lines in the cartoon representation shown in the upper right of the panel), in contrast to the monomer orientation shown in panel C. Monomers are colored blue and purple and shown as ribbons representations. The activation segment is orange. (E) The CoAlated Aurora A kinase domain dimer from (D), with the activation segments colored orange and the DFG phenylalanine in cyan, is shown superimposed on the cacodylate-modified dimer, colored yellow/gold with activation segments colored green. Both structures show a catalytically inactive DFG-out activation segment conformation. (F) A 2mF_o-DF_c map contoured at 0.5σ shows electron density consistent with a subpopulation of the Aurora A kinase domain molecules in the crystal containing a Cys²⁹⁰-Cys²⁹⁰ symmetric disulfide.

Fig. 7.. Crystal structure of a disulfide-linked Aurora A kinase domain homodimer.

(A) The structure shows an activation segment-swapped dimer with the disulfide bond formed between Cys²⁹⁰ in each monomer (with sulfur atoms colored green) at the center of the dimer interface. Molecules are shown in cartoon representation with the monomers colored blue and magenta, and with a cartoon representation shown above the structure indicating the relative orientations of the monomers. (B) The cacodylate-modified Aurora A kinase domain dimer in an identical orientation as in (A), shows a similar overall dimer configuration but with conformational differences in the activation segments [green in (B) vs orange in (A)]. (C) Monomers of Aurora A from (A) and (B) are shown superimposed. The DFG-in active conformation of a monomer of the Aurora A kinase domain disulfide homodimer (Phe shown in stick representation, colored cyan) contrasts with the inactive DFG-out conformation of the cacodylate-modified kinase domain (colored yellow), and also with the inactive DFG-out conformation of the CoAlated disulfide-linked dimer structure (Fig. 6E).

Fig. 8.. Disulfide-mediated dimerization of Aurora A promotes autophosphorylation.

(A) Catalytically active Aurora A kinase domain constructs treated with the disulfide-promoting Ellman’s reagent and incubated with ATP show robust Thr²⁸⁸ autophosphorylation as assayed by Western blotting. Phosphorylation of Thr²⁸⁸ is detected in the bands corresponding to dimers for both the wild type and the C247V + C319V mutant construct. Inclusion of DTT in the kinase assay abrogates Thr²⁸⁸ phosphorylation. Blots are representative of 2 independent experiments. (B) Upper panel, purification scheme for disulfide-linked heterodimers containing an MBP-tagged kinase dead and untagged catalytically active Aurora A kinase domain. Lower panel, following incubation with ATP, the samples were analyzed by SDFS-PAGE under non-reducing or reducing conditions, and probed for autophosphorylation by Western blotting. Blots are representative of 8 independent experiments. (C) Proposed model of redox and dimerization-dependent activation of Aurora A. We posit that increased protein cysteine oxidation during mitosis results in increased levels of disulfide modifications of proteins, such as CoAlation of Aurora A. Clustering of Aurora A molecules upon centrosomal recruitment promotes dimerization and thiol-disulfide exchange between kinase domains to form a disulfide homodimer that facilitates autophosphorylation. Resolution of the disulfide homodimer releases activated (pThr²⁸⁸) Aurora A monomers.