Control of Aurora-A stability through interaction with TPX2

113

Research Article

Control of Aurora-A stability through interaction with TPX2 Maria Giubettini1, Italia A. Asteriti1, Jacopo Scrofani1, Maria De Luca1,2, Catherine Lindon2, Patrizia Lavia1 and Giulia Guarguaglini1,* 1

Institute of Molecular Biology and Pathology, CNR, c/o Sapienza University of Rome, Via degli Apuli 4, 00185, Rome, Italy University of Cambridge, Department of Genetics, Downing Street, Cambridge, UK CB2 3EH

2

*Author for correspondence ([email protected])

Journal of Cell Science

Accepted 18 October 2010 Journal of Cell Science 124, 113-122 © 2011. Published by The Company of Biologists Ltd doi:10.1242/jcs.075457

Summary The Aurora-A kinase has well-established roles in spindle assembly and function and is frequently overexpressed in tumours. Its abundance is cell cycle regulated, with a peak in G2 and M phases, followed by regulated proteolysis at the end of mitosis. The microtubule-binding protein TPX2 plays a major role in regulating the activity and localisation of Aurora-A in mitotic cells. Here, we report a novel regulatory role of TPX2 and show that it protects Aurora-A from degradation both in interphase and in mitosis in human cells. Specifically, Aurora-A levels decrease in G2 and prometaphase cells silenced for TPX2, whereas degradation of Aurora-A is impaired in telophase cells overexpressing the Aurora-A-binding region of TPX2. The decrease in Aurora-A in TPX2-silenced prometaphases requires proteasome activity and the Cdh1 activator of the APC/C ubiquitin ligase. Reintroducing either full-length TPX2, or the Aurora-A-binding region of TPX2, but not a truncated TPX2 mutant lacking the Aurora-A-interaction domain, restores Aurora-A levels in TPX2-silenced prometaphases. The control by TPX2 of Aurora-A stability is independent of its ability to activate Aurora-A and to localise it to the spindle. These results highlight a novel regulatory level impinging on Aurora-A and provide further evidence for the central role of TPX2 in regulation of Aurora-A. Key words: Aurora-A, mitosis, protein degradation, TPX2

Introduction The Aurora-A kinase (officially known as Serine/threonine-protein kinase 6) is an important regulator of cell division and acts in several aspects of spindle formation and function (for reviews, see Barr and Gergely, 2007; Vader and Lens, 2008). The levels of Aurora-A are abnormally high in many tumour types, and altered abundance is thought to be of relevance for oncogenic transformation (for reviews, see Gautschi et al., 2008; Vader and Lens, 2008). Indeed, Aurora-A has recently been proposed as a potential target in anti-cancer therapy; inhibitors of its activity have been synthesised, some of which are currently being tested in clinical trials (for reviews, see Gautschi et al., 2008; Pérez de Castro et al., 2008; Karthigeyan et al., 2010). In recent years, several factors have been shown to interact with Aurora-A and to modulate its activity (for a review, see Carmena et al., 2009), among which the microtubule (MT)-binding protein TPX2 (‘targeting protein for Xklp2’) has a prominent role (Gruss and Vernos, 2004). The mechanism through which TPX2 activates Aurora-A has been extensively characterised: TPX2 binding induces a conformational change in Aurora-A, modifying the position of a key residue (Thr288) in the three-dimensional structure of the kinase and rendering it inaccessible to the PP1 phosphatase. Thus, TPX2 binding stabilises Thr288-phosphorylated Aurora-A, which represents the active form of the kinase (Bayliss et al., 2003; Eyers et al., 2003; Tsai et al., 2003). TPX2 is also required to target Aurora-A to the spindle MTs (Kufer et al., 2002; De Luca et al., 2006); a truncated form of TPX2 lacking the first 33 amino acids and unable to bind to Aurora-A cannot restore Aurora-A localisation to MTs in a TPX2-deficient background (Bird and Hyman, 2008). Despite of the well-documented effects of TPX2 on Aurora-A,

functional studies in Xenopus extracts (Brunet et al., 2004; Tsai and Zheng, 2005; Sardon et al., 2008) and human cells (Bird and Hyman, 2008) have not yet provided a complete understanding of the actual role of the complex in spindle assembly and mitotic control. At the end of mitosis, the abundance of Aurora-A is downregulated through APC/C–Cdh1-dependent proteasomemediated proteolysis (Taguchi et al., 2002; Lindon and Pines, 2004); this downregulation is important for the organisation of the anaphase spindle (Floyd et al., 2008). The molecular determinants of Aurora-A degradation have been characterised: a canonical destruction box (D-box) in the C-terminal region and a novel motif in the N-terminus (A-box) are required for APC/C–Cdh1-dependent destruction of human Aurora-A. The phosphorylation state of a serine residue (Ser51) in the A-box modulates degradation of Aurora-A as mutants mimicking constitutive phosphorylation of this site cannot be degraded by the APC/C–Cdh1 (Crane et al., 2004). Recent studies suggest that the PP2A phosphatase is responsible for dephosphorylation of Ser51 (Horn et al., 2007). Abnormal phosphorylation of Ser51 has been observed in head and neck cancer, suggesting a link between control of the stability of Aurora-A and tumorigenesis (Kitajima et al., 2007). Interestingly, addition of the Aurora-A-binding region of TPX2 to Xenopus oocyte extracts impairs APC/C–Cdh1-dependent degradation of Aurora-A (Sardon et al., 2008). Over the past few years, we contributed to the demonstration that both Plk1 (at centrosomes) and TPX2 (at MTs) regulate the localisation of Aurora-A in mitotic cells (De Luca et al., 2006). We now report that TPX2 is also required for regulation of the stability of Aurora-A protein in human cells: we show that Aurora-A protein

114

Journal of Cell Science 124 (1)

levels decrease in cells lacking TPX2, in a proteasome- and Cdh1dependent manner, and that the interaction between Aurora-A and TPX2 is required for protecting Aurora-A from degradation. This novel mechanism of Aurora-A regulation is relevant to the kinetics of accumulation and disappearance of Aurora-A during the cell cycle and hence for the proper execution and exit from mitosis; in addition, it might indicate poorly explored routes to increased kinase abundance in tumours. Results The abundance of Aurora-A decreases in TPX2-silenced prometaphases

Journal of Cell Science

In previous experiments, we noticed that the Aurora-A signal decreased in cells silenced for TPX2 by RNA interference (RNAi) (TPX2i) compared with that of controls, possibly hinting at a novel level of control exerted by TPX2 on the abundance of Aurora-A protein. That prompted us to design a series of experiments to substantiate that observation. First, we quantified the Aurora-A-

specific immunofluorescence (IF) signal in U2OS single mitotic cells with or without TPX2 after RNAi. Given that TPX2-defective cells reach prometaphase – the stage at which Aurora-A levels are highest – but cannot progress any further (Garret et al., 2002; Gruss et al., 2002; De Luca et al., 2006), we limited our analysis to prometaphase cells from control (GL2i) and TPX2i cultures (examples are shown in supplementary material Fig. S1). Placing selections in different areas of the cells (Fig. 1A), we observed that (i) the intensity of Aurora-A signal at spindle poles was lower in TPX2i compared with control cells, and this was not associated with a decrease in MT density at poles; (ii) there was no concomitant increase in the Aurora-A cytoplasmic fraction; consistent with this, (iii) the total amount of Aurora-A was reduced by ~50% compared with control prometaphases. Comparable results were obtained using independent TPX2-targeting small-interfering RNA (siRNA) oligonucleotides: Fig. 1A shows results obtained with TPX2144, used throughout the paper; results obtained with TPX2168, previously characterised by Bird and Hyman (Bird and

Fig. 1. The levels of Aurora-A decrease in TPX2-silenced prometaphases. (A)The Aurora-A IF signals at poles and microtubules (POLES/MTs), in the mitotic cytoplasm (CYT) and in the whole cell (TOT) from control and TPX2i prometaphases (24 and 27 cells, respectively, from two experiments) were measured (arbitrary units, a.u.). Selections are shown in the schematisation. Black diamonds represent single cells. Poles and cytoplasmic values are mean pixel intensity; for the ‘total’, absolute values are shown. **P60%) cells were in G2 and M phases and cyclin B1 levels were highest (Fig. 3A, left panels). IF analysis (Fig. 3A, right panels) confirmed that TPX2-positive cells are already present at the time of release from thymidine arrest, with an increased signal intensity thereafter. By contrast, Aurora-A was barely detectable at the time of release,

with the earliest detectable signals found around centrosomes in isolated cells 6 hours after release (arrowed in Fig. 3A), indicating that the earliest pool of newly synthesised Aurora-A is recruited promptly at centrosomes. Twelve hours after release, Aurora-A signals accumulated both around centrosomes and within nuclei (Fig. 3A); high TPX2 levels were detected in these same cells. We wondered whether TPX2 inactivation would also influence AuroraA accumulation in interphase cells during G2 (i.e. adherent cells with an intact nuclear envelope examined at 12 hours after thymidine release). Both WB and IF analyses showed that inactivation of TPX2 by RNAi hinders the accumulation of AuroraA (Fig. 3B), while affecting neither cell cycle progression (60.1±4.1% G2–M cells in GL2i cultures and 65.7±0.7% in TPX2i by FACS analysis) nor the levels of cyclin B1 (Fig. 3B). Thus, TPX2 exerts a protective effect against Aurora-A degradation already in interphase and contributes to its accumulation during G2 phase.

Fig. 3. The levels of Aurora-A in interphase are sensitive to the presence of TPX2. (A)Kinetics of accumulation of Aurora-A and TPX2 during progression from G1–S to G2 determined by WB and IF analysis (indicated are the hours after release from a treatment with thymidine for 24 hours). Cyclin B1 was used as a marker of cell cycle progression in WB. More than 1000 cells for each condition were counted in the IF analysis (graph on the left; shown are the means ± s.d.), in at least two independent experiments. Arrows in the IF panels indicate examples of Aurora-A-positive centrosomes. Scale bar: 10m. (B)Cultures were analysed after 12 hours of release from a treatment with thymidine for 24 hours. Shown is the efficiency of TPX2 RNAi by IF (~1000 cells were counted for each condition, in two experiments). Inactivation of TPX2 impairs the accumulation of Aurora-A in G2-enriched cultures, as assessed by counting Aurora-A-positive cells on IF coverslips (left; ~1000 cells were counted for each condition, in two experiments; shown are the means ± s.d.) and by WB (right).

TPX2 controls Aurora-A stability

the localisation and stability of Aurora-A by quantifying both total and pole-associated Aurora-A IF signals, as done for Fig. 1 (Fig. 4B). Expression of full-length TPX2 (TPX2res) restored normal levels of Aurora-A compared with TPX2-silenced cells expressing empty vector (GFP lanes), both at poles and MTs (POLES/MTs) and in the whole cell. By contrast, TPX2res/43 failed to stabilise either Aurora-A population. Importantly, the N-terminal TPX2 fragment (TPX2/1–43) was sufficient to obtain a recovery of the overall Aurora-A abundance, although it was unable to restore the signal at spindle MTs. These data indicate that the TPX2 region of interaction with Aurora-A is necessary and sufficient to mediate the stabilising effect of TPX2. TPX2 differentially regulates Aurora-A activity, localisation and stability

The findings that the first 43 amino acids of TPX2 are sufficient to restore normal levels of Aurora-A, but not MT localisation, in a TPX2-deficient background (Fig. 4) suggest that the mechanistic

Journal of Cell Science

of TPX2 are required for localisation of Aurora-A to spindle MTs in human cells (Bird and Hyman, 2008). We wanted to establish whether the formation of the Aurora-A–TPX2 complex is required for control of Aurora-A stability. To address that question, we used constructs encoding fluorescently tagged TPX2-deleted versions (Fig. 4A; for details, see the Materials and Methods section). Fulllength TPX2 was encoded by pEGFP-TPX2res, expressing an RNAi-resistant transcript (Fig. 4A). That construct was compared with pEGFP-TPX2res/43, also RNAi resistant, encoding a TPX2 version lacking the first 43 amino acids and thus unable to bind to Aurora-A (Bird and Hyman, 2008), and with pEYFP-TPX2/1–43, coding for the first 43 amino acids of TPX2 only and not including the siRNA target sequence. In a TPX2-silenced background, exogenously expressed TPX2res and TPX2res/43 did both accumulate at spindle poles, as expected, whereas TPX2/1–43, which lacks the MT-binding regions of TPX2, was distributed diffusely in the mitotic cytoplasm (Fig. 4A). We then were in a position to investigate whether these TPX2 mutants would affect

117

Fig. 4. TPX2 binding protects Aurora-A from degradation in prometaphase. (A)Left: maps of the TPX2 constructs; asterisk (*): mutation conferring resistance to RNAi. The WB shows expression of mutants, but not wild-type GFP–TPX2, in TPX2i cultures (GFP row); endogenous TPX2 is also shown (TPX2 row). Note that the TPX2/1–43 panel (separated by a vertical black line) has been artificially aligned to the other signals, while migrating ahead of the other constructs. The mitotic localisation of the exogenous proteins (green) is shown; DNA is in blue in the merged images. (B)Aurora-A IF signals from cultures transfected with the indicated combinations of plasmids and siRNA oligonucleotides. Aurora-A signals at poles (‘POLES/MTs’) and in the whole cell (TOTAL) from GL2i or TPX2i prometaphases were measured (arbitrary units, a.u.). Areas used for selections are as in Fig. 1. Black diamonds represent single cells. ‘POLES/MTs’ values are mean pixel intensity; for the ‘TOTAL’, absolute values are shown. A representative experiment is shown; the same trend was confirmed in two additional experiments. **P0.1, Mann–Whitney test). Images show a typical transfected cell for each condition. Scale bars: 10m.

Journal of Cell Science

TPX2 controls Aurora-A stability roles of TPX2 in regulating the stabilisation and localisation of Aurora-A are independent. To address this question directly, we took the approach of experimentally altering each one of these effects individually and analysing the other one. First, we stabilised Aurora-A levels in TPX2-silenced prometaphase cells by inhibiting proteasome activity by treatment with MG132 (Fig. 5A): under these conditions, Aurora-A became more abundant in the cytoplasm yet failed to associate with MTs (Aurora-A signals at spindle poles and throughout mitotic cells are quantified in supplementary material Fig. S4). Next, we prevented the localisation of Aurora-A along the spindle by treating cultures with nocodazole, so as to inhibit MT assembly, in a TPX2-proficient background (Fig. 5B). This yielded Aurora-A dispersal in the cytoplasm, yet the overall abundance of Aurora-A remained similar to that seen in prometaphases with properly assembled MTs. These results demonstrate unambiguously that TPX2 regulates the MT localisation and stability of Aurora-A through two independent mechanisms; furthermore, Aurora-A protection from proteasomedependent degradation is not conferred through MT binding of the Aurora-A–TPX2 complex. Given that the first 43 amino acids of TPX2 that regulate the overall abundance of Aurora-A (Fig. 4B) also regulate its kinase activity (Bayliss et al., 2003), we asked whether catalytic activation per se confers stability to Aurora-A. To test this, we compared wildtype (wt) and catalytically inactive [K162R, kinase-dead (KD)] (Meraldi and Nigg, 2001; Meraldi et al., 2002) Aurora-A versions in U2OS cells. Both constructs were myc-tagged and showed a comparable transfection efficiency. We found that exogenous wildtype and KD proteins are expressed at comparable levels in both WB (Fig. 5C, myc row) and IF assays (Fig. 5D), both in interphase and mitotic cells. This yielded a comparable overall abundance of the Aurora-A protein, with a similar cell cycle phase distribution, as revealed by cyclin B1 levels (Fig. 5C) in the cell populations transfected with either construct. These assays indicate that the loss of catalytic activity per se does not decrease the stability of AuroraA, suggesting therefore that the stabilising role of TPX2 is not necessarily dependent on its ability to activate the kinase. Downregulation of TPX2 in telophase is required to clear Aurora-A in cells exiting mitosis

Proteasome- and Cdh1-dependent downregulation of Aurora-A physiologically occurs at exit from mitosis (Taguchi et al., 2002; Lindon and Pines, 2004). Based on the data obtained at this point, we surmised that the protective effect of TPX2 on Aurora-A would need to be lost or attenuated at that time. TPX2 is itself a substrate of degradation at mitotic exit (Stewart and Fang, 2005), and single studies of TPX2 and Aurora-A indicate that both proteins disappear within 2–4 hours of release from nocodazole-dependent prometaphase arrest in HeLa cells – that is, a time corresponding approximately to telophase (Lindon and Pines, 2004; Stewart and Fang, 2005; Floyd et al., 2008). To gain insight into their dependence for degradation, we first characterised their timing of downregulation in U2OS cultures exiting mitosis. U2OS cells were pre-synchronised by thymidine followed by nocodazole treatment during thymidine release; mitoses were collected at round-up by shake-off, replated to terminate mitosis and analysed at intervals of 30 minutes until re-entry in the next G1 phase (for details, see Ciciarello et al., 2010): this showed that the levels of TPX2 indeed begin to decrease before Aurora-A (Fig. 6A). In order to evaluate whether that event is causally required for Aurora-A degradation, we analysed the endogenous levels of Aurora-A in U2OS cells that

119

terminated mitosis with overexpressed TPX2/1–43 (Fig. 6B): that fragment, which contains the region interacting with Aurora-A, as described above, is not ubiquitylated in vitro (Stewart and Fang, 2005) and lacks the KEN-box sequence required for degradation of TPX2 at the end of mitosis. We presynchronised transfected cultures by thymidine, as above, accumulated cells in prometaphase in the presence of monastrol after thymidine release, then released prometaphase-arrested cells in drug-free medium to terminate mitosis and analysed Aurora-A by IF during completion of mitosis. The levels of Aurora-A decreased to an almost undetectable level in telophase cells compared with prometaphase and metaphase cells, when either vector-transfected (Fig. 6B) or non-transfected (data not shown). In sharp contrast with this, Aurora-A signals showed no significant variations in TPX2/1–43-transfected prometaphase, metaphase and telophase cells (Fig. 6B), indicating that forced expression of the Aurora-A-interacting region of TPX2 at mitotic exit prevents degradation of Aurora-A. These results are consistent with the notion that degradation of TPX2 is a prerequisite to convey Aurora-A towards proteolysis when cells complete mitosis. Discussion TPX2 is required for Aurora-A regulated abundance in early mitosis

The levels of Aurora-A are regulated through the cell cycle, with a peak of abundance in early mitotic stages, which reflects cellcycle-dependent transcriptional activation taking place in G2 phase and proteasome- and APC/C-mediated proteolysis at the end of mitosis (reviewed in Vader and Lens, 2008). In this study, we show that inactivating the Aurora-A regulator TPX2 by RNAi yields a premature decrease in the levels of Aurora-A in prometaphase – that is, when they are normally due to reach highest abundance – and that this decrease is proteasome dependent. These findings suggest that the abundance of Aurora-A does not simply increase steadily during cell cycle progression but that it is actually subjected to a dynamic turnover resulting from the balance between increased synthesis and degradation, which is counteracted in the presence of TPX2. The protective effect of TPX2 on Aurora-A is not reciprocal because TPX2 stability is not altered in the absence of Aurora-A; in line with this observation, accumulation of TPX2 precedes that of Aurora-A during progression from S phase to mitosis in a normal cell cycle. TPX2 stabilises nuclear Aurora-A during G2 progression

We have found that the stabilising effect of TPX2 on Aurora-A begins in G2 phase. TPX2 localises to the nucleus during that phase, where a large fraction of Aurora-A also colocalises. AuroraA fails to accumulate in nuclei of TPX2-silenced cells, revealing that TPX2 is required for the nuclear accumulation of Aurora-A just before mitotic entry. Actually, the earliest Aurora-A signals detected during interphase progression are first seen at centrosomes, which at this point are devoid of TPX2. This suggests that centrosomal Aurora-A (which accounts for 1 in 50 to 1 in 100 of the entire intracellular pool of Aurora-A, based on measurements of IF signals) does not require TPX2-dependent stabilisation. It is possible that one or more centrosomal interactors of Aurora-A (e.g. Ajuba, PAK1 or HEF1) (for a review, see Carmena et al., 2009) exert a local stabilising effect or, non-mutually exclusively, that active stabilisation is more crucial to nuclear than to centrosomal Aurora-A accumulation owing to their significant differential abundance; furthermore, the centrosomally localised proteasome

Journal of Cell Science 124 (1)

Journal of Cell Science

120

Fig. 6. The disappearance of TPX2 at the exit from mitosis contributes to the degradation of Aurora-A. (A)The abundance of TPX2 and Aurora-A was followed by WB in cultures synchronously exiting mitosis (protocol schematisation is shown above; time intervals are not represented to scale; MSO: mitotic shake-off; NOC, nocodazole). The minutes elapsed after the replating of shaken-off prometaphases are indicated. (B)Aurora-A levels (TOTAL) were measured in prometaphase–metaphase cells, and telophase cells, overexpressing either GFP alone or EYFP-TPX2/1–43 (between 30 and 50 cells per condition were used for the analysis, from two experiments; a.u., arbitrary units). Black diamonds represent single cells. **P