AURKA destruction is decoupled from its activity at mitotic exit but suppresses interphase activity

Activity of AURKA is controlled through multiple mechanisms including phosphorylation, ubiquitin-mediated degradation, and allosteric interaction with TPX2. Activity peaks at mitosis before AURKA is degraded during mitotic exit in a process strictly dependent on APC/C coactivator FZR1. We used FZR1 knockout cells (FZR1KO) and a novel FRET-based AURKA biosensor to investigate how activity is regulated in absence of destruction. We found that AURKA activity in FZR1KO cells dropped at mitotic exit as rapidly as in parental cells, despite absence of destruction. Unexpectedly, TPX2 was degraded normally in FZR1KO cells. Overexpression of an N-terminal TPX2 fragment sufficient for AURKA binding, but not degraded at mitotic exit, caused delay in AURKA inactivation. We conclude that AURKA inactivation in mitotic exit is determined not by its own degradation but by degradation of TPX2 and therefore dependent on CDC20 rather than FZR1. The biosensor revealed that FZR1 instead suppresses AURKA activity in interphase and is critically required for assembly of the interphase mitochondrial network after mitosis.


Introduction
Aurora kinase A (AURKA) is a major mitotic kinase required for multiple steps in mitosis, including centrosome maturation, microtubule nucleation and organisation into a bipolar spindle and mitotic checkpoint function [1,2]. Recent studies of the effects of acute inhibition of AURKA during mitosis have concluded that it plays an important role during mitotic exit in regulating assembly of the anaphase spindle upon which sister chromatids are segregated [3][4][5]. AURKA also has a number of non-mitotic roles that include disassembly of the primary cilium, regulation of myc family transcription factors and response to replication stress [6][7][8][9][10]. We and others recently reported that AURKA constitutively regulates mitochondrial morphology and function [11,12] in addition to a previously reported role in promoting mitochondrial fission in mitosis [13].
Widespread reports in the literature of interphase activity of AURKA, and of its activation through a number of interacting partners [14], can be explained by the unique allosteric properties of this kinase. Either autophosphorylation of the T-loop [15][16][17] or allosteric interactors [18][19][20][21] contribute to the active conformation of AURKA. The most prominent of these allosteric activators is TPX2, which controls AURKA localization, activation and stability on the mitotic spindle during mitosis [16,22,23]. Interaction of TPX2, whilst protecting the autophosphorylated site (pT288 in hsAURKA) from access by PP1 phosphatase [16], also acts to stabilize the T-loop with or without its phosphorylation. Consequently, phosphatase-mediated reversal of T288 phosphorylation may not be sufficient to eliminate activity of AURKA.
In somatic cells, both AURKA protein and kinase activity are low or undetectable through much of the cell cycle, rising sharply in G2 phase, most prominently on the duplicated centrosomes and then on the bipolar spindle. pT288 staining is almost entirely confined to the centrosomes, in line with the idea that autophosphorylation is just one route to activation of AURKA. Indeed a conformational sensor of AURKA detects active kinase on the spindle, and in the cytoplasm, both during and after mitosis [24]. Mitotic destruction of AURKA begins late in anaphase following assembly of the spindle midzone and at the time of spindle pole disassembly, in a manner that depends on the Cdh1 co-activator (hereafter referred to as FZR1) of the Anaphase Promoting Complex/Cyclosome (APC/C) [25][26][27][28].
The APC/C is responsible for destruction of dozens of substrates during mitotic exit and coordinates mitotic exit with the processes of chromosome segregation and cytokinesis through the concerted regulation of its coactivators [29]. APC/C-CDC20 targets cyclin B and securin during metaphase to drive chromosome segregation and mitotic exit. After anaphase onset a switch in specificity of APC/C-CDC20 and/or activation of APC/C-FZR1 degrades all the remaining substrates including Aurora kinases. Like most APC/C substrates, AURKA destruction is efficiently accomplished by APC/C-FZR1 in a variety of different assays. Unlike most substrates, AURKA and AURKB show strict dependence on FZR1 for their destruction in cellbased assays [28,30,31]. Neither the molecular basis, nor the functional significance of this specificity in AURK targeting, is understood.
Given the complexity of AURKA activity during the cell cycle, we investigated what the destruction of AURKA contributes to its inactivation during mitotic exit using novel tools in the form of a CRISPR/Cas9 FZR1 knockout cell line we have recently generated and a FRET-based AURKA activity biosensor. We conclude from our studies that FZR1-mediated destruction of AURKA plays no role in the timing of its inactivation during mitotic exit but is critical to suppress interphase activity and function of the kinase. We test this idea by demonstrating that reestablishment of mitochondrial connectivity after mitosis requires suppression of activity of undegraded AURKA.

Results
Although AURKA activity has been described to peak in M phase there has been no detailed characterization of how AURKA activity varies through mitosis. In particular the question of how AURKA activity is regulated during mitotic exit, given that AURKA also has anaphase functions [32,33], and the importance of its ongoing destruction for attenuation of activity, remain unresolved. We first used a commercially available antibody against the activated T-loop of Aurora kinases (pT288 in AURKA) that confirms by immunoblot the peak of active AURKA at mitosis in extracts from synchronised U2OS cells ( Figure 1A). We then used the same antibody to look by immunofluorescence (IF) in fixed mitotic cells, marking centrosomes with γ-tubulin staining. All phospho-epitope signal on the centrosomes and spindle poles was abolished by treatment with AURKA inhibitor MLN8237, whereas the signal at the midbody (where the same antibody recognizes pT232 of AURKB) was insensitive to 100 nM MLN8237, indicating that the centrosomal pT288 signal is specific to AURKA as expected ( Figure 1B, Supplementary Figure   S1) [34,35]. We quantified fluorescence intensity associated with pT288 at different stages of mitosis scored according to DNA morphology. We found that pT288 was evident at the centrosomes from G2 phase onwards. Following nuclear envelope breakdown (NEB), prometaphase (PM) cells showed a strong increase in centrosome-and spindle pole-associated pT288 signal, peaking at metaphase. pT288 signal remained high at anaphase before starting to fall significantly in telophase cells ( Figure 1C, D). We concluded that inactivation of AURKA through dephosphorylation of its T-loop occurs at around the time of onset of AURKA destruction, which occurs about 10 minutes after anaphase onset in human cells [28]. Immunoblot analysis of extracts from cells synchronized through mitotic exit using a spindle assembly checkpoint (SAC) arrest/release protocol confirmed that pT288 levels and total AURKA both fall rapidly during forced mitotic exit ( Figure 1E).
Since the kinetics of dephosphorylation and destruction of AURKA in mitotic exit were not identical ( Figure 1E), we investigated whether AURKA destruction contributes to the fall in kinase activity at mitotic exit. Mitotic AURKA destruction is critically dependent on the FZR1 co-activator of APC/C, so we used a FZR1 knockout (FZR1 KO ) in U2OS cells generated by CRISPR/Cas9 (Supplementary Figure S2) to study AURKA inactivation in the absence of its destruction at mitotic exit. In FZR1 KO cells, AURKA-Venus protein levels measured in single cell degradation assays remained constant through mitotic exit, compared to the parental U2OS cell line ( Figure   2A), and as previously reported using siRNA-mediated suppression of FZR1 [28]. We then compared loss of pT288 staining in mitotic exit between parental and FZR1 KO cells using the assays established in Figure 1. We found by immunoblot that loss of pT288 signal was identical in both cell lines during synchronized mitotic exit, despite strong stabilization of the AURKA signal in FZR1 KO cells ( Figure 2B). AURKB was also stabilized but less so, consistent with slower degradation of AURKB [36]. We note that degradation of endogenous TPX2 during mitotic exit, like inactivation of AURKA, was insensitive to FZR1 knockout, and appeared more complete in FZR1 KO cells, where CDC20 levels persisted compared to the parental cells ( Figure 2B). TPX2 was first proposed as a substrate of the Cdh1/FZR1-activated version of APC/C [37] but our results suggest that in an unperturbed mitotic exit TPX2 -like cyclin B1 (CCNB1) -is a substrate for CDC20 ahead of FZR1 activation. We then fixed parental U2OS cells and FZR1 KO cells and processed them for quantitative IF analysis of pT288-AURKA and total AURKA staining at spindle poles at metaphase and telophase. In this analysis AURKA persisted on spindle poles during mitotic exit in FZR1 KO cells compared to parental U2OS, but with no corresponding persistence in pT288 label ( Figure 2C). In both cell lines, the presence of active AURKA, as measured by T-loop phosphorylation of the kinase, was strongly reduced after onset of mitotic exit independent of the level of protein remaining. We confirmed that the persistence of AURKA on spindle poles is most likely a direct effect of AURKA non-degradation, since wild-type AURKA-Venus, but not a nondegradable version, is rapidly lost from spindle poles in unperturbed mitotic exit (Supplementary Figure S2).
A number of AURKA binding partners have been shown to affect the activity of AURKA, some independently of phosphorylation on T288, via distinct effects on its conformational dynamics. Therefore, we used a diffusible kinase biosensor, based on the well-established design of a fluorescent protein FRET pair separated by a phospho-threonine binding domain and specific phosphorylation motif [38], to provide a more comprehensive readout of AURKA activity. An AURKA-directed version of this biosensor was created using the T210 motif from the T-loop of Plk1 as a known target of AURKA activity [39,40]. Phosphorylation of the T210 motif in the biosensor causes loss of FRET in mitotic cells, measured as an increase in CFP/YFP emission ratio of approximately 10%, in a manner dependent on its phosphorylation site and sensitive to specific inhibitors of AURKA ( Figure 3A-B, Supplementary Figure S3). Some sensitivity to inhibitors of AURKB at higher doses indicated that the biosensor might not be completely specific to AURKA. (Supplementary Figure S3). This finding was not unexpected for a diffusible biosensor, since some of the specificity in substrate phosphorylation by Aurora kinases is proposed to reside in the colocalization of kinase with substrates [3,35]. We transfected this biosensor into U2OS and FZR1 KO cells to measure Aurora kinase activity at mitosis and during mitotic exit. FRET measurements in mitotic cells were in agreement with the analysis of pT288 staining shown in Figure 2, in showing that biosensor activity started to increase in late G2, peaking after NEB and decaying during mitotic exit. We found that the increase in activity measured at mitotic entry was identical in individual U2OS and FZR1 KO cells in silico synchronized to NEB ( Figure 3D): peak activity showed a small but not significant increase in FZR1 KO cells. In silico synchronization to anaphase onset (Supplementary Figure S3) revealed that the timing of Aurora kinase inactivation was identical. Moreover, if we normalized FRET signals to the anaphase onset value, the inactivation curves were directly superimposable ( Figure   3E). Therefore, mitotic Aurora kinase inactivation is independent of FZR1. FRET measurements returned to pre-mitotic levels at about 1 hour after anaphase onset, indicating complete inactivation of the mitotic pool of Aurora kinases. We observed, however, that biosensor activity starts to increase again gradually in G1 in FZR1 KO cells compared to parental U2OS cells and is significantly increased at 160 minutes after NEB ( Figure 3D). We concluded that destruction of AURKA does not influence its inactivation during mitotic exit but may be important to prevent reactivation early in the cell cycle.
How can we explain the timing of AURKA inactivation at mitotic exit? We have previously shown that interaction with TPX2 stabilizes AURKA against APC/C-FZR1-mediated degradation [23], and from this concluded that loss of interaction with TPX2 contributes to the timing of AURKA degradation in mitotic exit. Our finding here that AURKA degradation plays no role in its inactivation led us to evaluate whether AURKA inactivation also could depend on TPX2 degradation. Since transient overexpression of full-length TPX2 inhibited mitotic progression in our hands, we used an N-terminal fragment of TPX2 (amino acids 1-43) known to be sufficient for binding to and activating AURKA [16]. We overexpressed TPX2(1-43)-CFP in U2OS and FZR1 KO cells and monitored pT288-AURKA during mitotic exit ( Figure 4). We found that persistence of TPX2(1-43) through mitotic exit stabilized AURKA protein during mitotic exit in U2OS cells ( Figure 4A,B) as previously described [23]. As expected, TPX2(1-43) showed no effect on AURKA levels in FZR1 KO cells where the protein is not degraded ( Figure 4C,D). Importantly, we found a marked effect of TPX2  in stabilizing the pT288-AURKA signal during mitotic exit in both parental U2OS cells and FZR1 KO cells ( Figure 4A-D). We concluded that loss of interaction with TPX2 is the rate-limiting step in inactivation of AURKA in mitotic exit. We confirmed that inhibition of the candidate phosphatase, PP1, after mitotic exit also stabilized pT288 signal ( Figure 4E), consistent with the conclusion that de-activation rather than destruction controls AURKA activity in mitotic exit.
If FZR1-mediated destruction of AURKA is not required for its inactivation at mitotic exit, then what is it for? We noticed a small but significant increase in accumulation of Aurora kinase biosensor activity following mitotic exit in FZR1 KO cells ( Figure 3D). Using both AURKA and AURKB biosensors [41] and pT288 staining, we examined Aurora kinase activity in cells arrested at the G1/S boundary by double thymidine block. The AURKA biosensor revealed increased Aurora kinase activity in FZR1 KO cells that was abolished by treatment with MLN8237 ( Figure 5A), but insensitive to a low dose of AZD1152 inhibitor sufficient to abolish AURKB-specific activity (Supplementary Figure S3). Since the AURKB sensor is insensitive to MLN8237 at the dose used (Supplementary Figure S4), we concluded that there is increased AURKA activity in interphase in FZR1 KO cells. Consistent with this conclusion, we found that pT288 stained centrosomes in interphase FZR1 KO cells but not parental U2OS ( Figure 5B).
Next, we explored how FZR1 affects known AURKA functions in interphase. We and others have previously described a role for AURKA, at physiologically relevant levels of expression, in promoting mitochondrial fission during interphase [11,12], an observation thought to be significant to cellular metabolism given the intimate link between mitochondrial morphology and function [42]. We hypothesized therefore that one consequence of increased interphase AURKA activity should be increased mitochondrial fragmentation in FZR1 KO cells, and indeed mitochondrial fragmentation following FZR1 siRNA treatment of HeLa cells has previously been described [43]. We compared the mitochondrial networks in parental U2OS and FZR1 KO cells arrested at the G1/S boundary, when the mitochondria tend to form large interconnected mitochondrial networks [44,45], and found the mitochondrial network to be highly fragmented in FZR1 KO cells in a manner dependent on AURKA activity ( Figure 5C-F). We measured reduced mean mitochondrial lengths in FZR1 KO cells ( Figure 5E), and classified mitochondria according to morphological subtypes, to find a significant increase in the percentage of fragmented globules and significantly reduced tubular morphology in FZR1 KO cells ( Figure 5F). Horn and co-authors had identified the fission factor dynamin-related protein-1 (DRP1) as the target of FZR1 required for its effect on mitochondrial morphology [43]. Here we found that inhibition of AURKA activity completely rescued mitochondrial length and morphology in FZR1 KO cells ( Figure 5E,F), consistent with data showing AURKA to be an upstream regulator of DRP1 [11,13]. Treatment of FZR1 KO cells with an AURKB-specific inhibitor had no effect on mitochondrial length (Supplementary Figure S4). Moreover we found no alteration in DRP1 levels in FZR1 KO cells ( Figure 5G) although, in agreement with [43], we observed loss of DRP1 during mitotic exit in parental cells ( Figure 5H) and suggest that it could be a substrate for APC/C-CDC20 as well as APC/C-FZR1, or another ubiquitin ligase. We concluded that APC/C-FZR1 regulates mitochondrial dynamics by preventing AURKA reactivation in interphase.
To further test our hypothesis that mitochondrial fragmentation is a direct response to undegraded AURKA, we tested if the introduction of nondegradable (nd, Δ32-66) and wild-type (WT) versions of AURKA would show differential effects on mitochondrial dynamics ( Figure 6).
We used stable RPE1-FRT/TO cell lines expressing either AURKA-Venus-WT or AURKA-Venusnd under tetracycline control. Induction of AURKA-Venus-WT caused increased fragmentation in unsynchronized cells as previously described [12], but AURKA-Venus-nd had a greater effect ( Figure 6A,B). Importantly, this mitochondrial phenotype was independent of any effect of AURKA on microtubule dynamics that might contribute to subcellular distribution of mitochondria (Supplementary Figure S5). We then tracked individual cells through mitotic exit in the presence of MitoTracker ® . Treatment with MLN8237 interfered with mitochondrial fragmentation at mitosis as expected from the published findings [13] ( Figure 6C). In the presence of overexpressed AURKA-Venus we found that mitochondria were excessively fragmented as cells progressed out of mitosis into G1 phase, and that this effect was exacerbated in cells expressing AURKA-Venusnd compared to WT ( Figure 6D). We concluded that undegraded AURKA is able to inhibit reassembly of the interphase mitochondrial network, and that AURKA is a direct target of FZR1 in regulating mitochondrial dynamics.

Discussion
AURKA activity increases in preparation for mitosis in parallel with the protein level, and both progressively drop from anaphase onwards. This has led to the expectation that destruction contributes to regulating AURKA activity at mitotic exit [28,33]. We observed, however, that loss of AURKA activity proceeds faster than destruction of the protein. Lack of the APC/C co-activator FZR1 stabilizes AURKA levels but does not affect the timing of AURKA inactivation during mitotic exit, as measured using either pT288 reactivity or a novel FRET biosensor for AURKA activity that we describe in this study. Therefore, AURKA destruction is not required for timing of its inactivation in late mitosis.
Instead we find that FZR1 suppresses AURKA-dependent activity of the FRET biosensor in interphase. In the absence of FZR1 we speculate that the timing of reactivation of AURKA depends on the changing balance of AURKA activators and phosphatases during G1 phase.
Although a low level of pT288 is detectable at centrosomes by IF in FZR1 KO cells, since there are routes to generating active AURKA that do not depend on autophosphorylation (for example under interaction with Nucleophosmin/B23 or TACC3 [46,47], it is likely that basal AURKA activity in interphase is dependent on AURKA levels in the cell, and is calibrated in each cell cycle by mitotic destruction of the AURKA pool. Such multiple 'opportunities' for AURKA activation may underlie the strong association of AURKA overexpression with cancer, particularly where co-overexpressed with an activating partner such as TPX2 [48], and underscores the importance of ubiquitin-mediated destruction of AURKA in suppressing excess AURKA activity in interphase. We investigated further the route to inactivation of the mitotic pool of AURKA. We found as expected that pT288 dephosphorylation during mitotic exit could be triggered through loss of interaction with TPX2. TPX2 has been shown to protect AURKA from negative regulation by PP1 [15][16][17]49]. Indeed, whereas PP6 has been shown to act on the AURKA-TPX2 complex to limit AURKA activity in spindle assembly, PP1 can act only on 'free' AURKA [50]. We found that overexpression of the TPX2 (1-43) peptide that is sufficient to activate AURKA, and which is not degraded at mitotic exit, delayed the timing of kinase inactivation.
Our data indicate that the 'release' of AURKA to PP1-mediated inactivation could depend on APC/C-CDC20-mediated destruction of TPX2. We cannot exclude that ubiquitination of TPX2 would be enough to abrogate interaction, or that loss of interaction would be an indirect consequence of APC/C-CDC20 activity. However, our idea is consistent with the early degradation of TPX2 that we observe, and studies indicating TPX2 as a major substrate for APC/C at mitotic exit [51,52] and numerous observations that amplification of TPX2 is the ratelimiting event for increased AURKA activity associated with tumorigenesis [48,53]. One interesting conclusion to be drawn from our model is that although AURKA destruction during mitotic exit is a FZR1-dependent event, AURKA inactivation during mitotic exit is dependent on CDC20, along with all of the other known critical events of mitotic exit. This helps to explain why mitotic exit occurs essentially normally in absence of FZR1. Indeed, FZR1 is not expressed during early embryonic cell cycles, and AURKA activity there is regulated by activation and inactivation, instead of periodic proteolysis. Throughout development AURKA destruction therefore remains 'decoupled' from mitosis in a manner that contrasts with most other substrates of APC/C-FZR1, that can also be targeted by APC/C-CDC20 [36]. We infer that the unusual relationship between APC/C and Aurora kinases preserves a pool of potential Aurora kinase activity with critical interphase functions, regulatable through proteostasis.
So, what are the FZR1-sensitive AURKA-dependent events of interphase? Previous studies have shown that AURKA activity acts as an upstream regulator of mitochondrial morphology through RALA-dependent and -independent pathways [11,13]. Mitochondria are highly dynamic organelles that undergo constant fission and fusion to modulate connectivity of the mitochondrial network, allowing the cell to respond to metabolic demands and maintain a healthy mitochondrial network. Increased rates of fission occur in preparation for mitosis so that mitochondrial fragments can be equally distributed between daughter cells. Here we show that the presence of non-degraded AURKA delays reassembly of the mitochondrial network after cell division. Whilst there is increasing evidence that the dynamic state of mitochondria contributes to the overall metabolic state of the cell, our understanding of how this impacts progression in the cell cycle remains limited [44,45]. Our study provides new evidence that APC/C-FZR1 control of AURKA activity in interphase is a critical parameter in regulation of mitochondrial dynamics.
The non-targeted AURKB FRET biosensor was a kind gift from Michael Lampson [41].
The AURKA-directed FRET biosensor was created as followed: We modified a FRET construct backbone containing mTurquoise and mVenus sequences in the pECFP-C1 Clontech vector (construct "F36" [55], a kind gift from Carsten Schultz). First, mVenus was replaced by YPet sequence using KpnI and BamHI restriction sites. The sequence of the FHA2 domain from ScRad53 was amplified by PCR and inserted between AgeI and MluI sites, introducing HindIII and EcoRI sites upstream of MluI. Finally, complementary oligonucleotides encoding the phosphorylation site Thr210 of Plk1 were annealed and inserted between EcoRI and MluI sites.
Cells were transfected using electroporation with Neon Transfection System (Invitrogen™) using the following parameters: pulse voltage 1500 V, pulse width 10 ms, and 2 pulses total on the transfection device according to the manufacturer's protocol.
After 30 min on ice, the lysate was centrifuged at 14,000 rpm (4°C) for 10 min. For immunoblotting, an equal amount of protein (20 µg) were loaded into SDS-PAGE 4-12% pre-cast gradient gels. Proteins were transferred to Immobilon-P or Immobilon-FL membranes using the

Immunofluorescence analysis
Cells were seeded at 2 x 10 4 onto glass coverslips and then fixed with cold 100% methanol (−20°C), permeabilized with 0.5% Triton X-100 in PBS, and incubated in 2% bovine serum albumin (BSA), 0.2% Triton X-100 in PBS (blocking buffer) for 1 h at room temperature. Cells were incubated overnight with primary antibodies (rabbit phospho-AURKA Thr288, 1:50; mouse anti-γ-tubulin GTU-88, Sigma-Aldrich; 1:1000) diluted in blocking buffer at 4°C, then washed three times with blocking buffer and incubated with secondary antibodies at 1:1000 dilution. Alexa Fluor 488 anti-mouse and Alexa Fluor 568 anti-rabbit (Thermo Fisher Scientific) were used as the secondary antibodies. DNA was stained with DAPI. Coverslips were mounted with Prolong Gold antifade reagent. Epifluorescent stacks were acquired using 500 nm step with 2×2 bin using appropriate filter sets and 40X NA 1.3 oil objective. The best in-focus images were selected and integrated intensities were measured by ImageJ (http://rsb.info.nih.gov/ij/; National Institutes of Health, Bethesda, MD).

Mitochondrial imaging and analysis
Cells were seeded at 2 × collected with 2x2 bin applied, exported as tiff files and analysed using MicroP [12,56].

Timelapse imaging and FRET quantification
Cells were imaged in L-15 medium with 10% FBS at 37°C using an automated epifluorescence imaging platform composed of Olympus IX83 motorized inverted microscope, Spectra-X multi-    Figure 1 and are presented as dot plots for total AURKA (C) and pT288-AURKA (D) in both U2OS and FZR1 KO . All values were normalized to the mean value from control metaphase cells. ns, non-significant; ** p < 0.001; *** p < 0.0001, Student's t-test. C, n ≥ 11; D, n ≥ 23. Bars, 10 µm.