Review The TACC : TACC-ling dynamics and function

Isabel Peset1 and Isabelle Vernos1,2

1 Cell and Developmental Biology Program, Centre for Genomic Regulation (CRG), University Pompeu Fabra (UPF), Dr Aiguader 88, Barcelona 08003, Spain 2 Institucio´ Catalana de Recerca i Estudis Avanc¸ats (ICREA), Passeig Lluis Companys 23, 08010 Barcelona, Spain

A major quest in cell biology is to understand the Transforming acidic coiled-coil (TACC) proteins emerged molecular mechanisms underlying the high plasticity initially as a group of proteins implicated in . The of the microtubule network at different stages of the first member of the TACC family to be discovered was , and during and after differentiation. Initial identified in a search of genomic regions that are amplified reports described the centrosomal localization of in breast cancer. It was named transforming acidic coiled- proteins possessing transforming acidic coiled-coil coil 1 (TACC1) because of its highly acidic nature, the (TACC) domains. This discovery prompted several presence of a predicted coiled-coil domain at its C terminus groups to examine the role of TACC proteins during cell (now known as the TACC domain), and its ability to division, leading to indications that they are important promote cellular transformation [6]. TACC proteins are players in this complex process in different organisms. present in different organisms, ranging from yeasts to Here, we review the current understanding of the role of mammals. There is only one TACC in the nema- TACC proteins in the regulation of microtubule tode Caenorhabditis elegans (TAC-1), in Drosophila mel- dynamics, and we highlight the complexity of centro- anogaster (D-TACC), in Xenopus laevis (Maskin), and some function.

Introduction Cell proliferation and differentiation require dramatic Abbreviations rearrangements of the cytoskeleton that rely on the highly AINT: ARNT interacting protein dynamic nature of the cytoskeletal components. Microtu- AKAP350: A kinase (PRKA) anchor protein bules are dynamic filaments with fundamental roles in Alp7: Altered growth polarity 7 eukaryotic cell organization and function. During cell Ark1: aurora-related kinase ARNT: aryl hydrocarbon nuclear translocator protein division, they form the bipolar spindle, which segregates AZU-1: anti-zuai-1 the into the two daughter cells. CBP: calcium-binding protein show prolonged states of polymerization and depolymer- CPEB: cytoplasmic polyadenylation element binding protein ization that interconvert stochastically, exhibiting fre- DCLK: doublecortin-like kinase quent transitions between growing and shrinking ECTACC: endothelial cell TACC E1F4E: eukaryotic initiation factor 4E phases, a property called ‘dynamic instability’ [1]. In the ERIC: -induced cDNA cell, multiple factors modulate this property by acting FOG-1: Friend of Gatal positively or negatively on the nucleation, elongation or GAS41: glioma amplified sequence 41 destabilization of microtubules [1–3]. The relative activity GCN5L2: general control of amino-acid synthesis 5-like 2 g-TURC: g-tubulin related complex of all these factors determines the steady-state length and HEAT: huntingtin, elongation factor 3, A subunit of protein stability of microtubules, in addition to their organization, phosphatase 2A and TOR1 and it is largely dictated by global and local phosphoryl- INI-1: SWI/SNF core subunit ation–dephosphorylation reactions [2,3]. In addition, other Ipl1: Increase-in-ploidy 1 types of factors that have microtubule-severing and - ISREC: Swiss Institute for Experimental Cancer Research KIF2C: kinesin family member 2C anchoring activities also influence the microtubule net- LIS1: Lissencephaly-1 work. The main microtubule-organizing centre (MTOC) LSM7: U6 small nuclear NRA associated of animal cells, the centrosome, acts as a platform upon MBD2: methyl-CpG binding domain protein 2 which the different factors and activities accumulate in a Mial: melanoma inhibitory activity 1 regulated manner. It therefore exerts a tight local and Mps1: MonoPolar Spindle 1 NDEL1 and NUDEL: nude nuclear distribution E homolog temporal control on the number, distribution and polarity (A. nidulans)-like 1 pCAF, p300/CBP-associated factor of microtubules [4,5]. SmG: snRNP Sm protein G TTK: TTK protein kinase Zyg-8: ZYGote defective Corresponding authors: Peset, I. ([email protected]); Zyg-9: ZYGote defective Vernos, I. ([email protected]).

0962-8924/$ – see front matter ß 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.tcb.2008.06.005 Available online 23 July 2008 379 Review Trends in Cell Biology Vol.18 No.8 in the fission yeast Schizosaccharomyces pombe (Alp7 Although TACC1 was originally found to be upregulated also known as Mia1p); by contrast, mammals have three in breast cancer [6], subsequent studies found that its such proteins (TACC1, TACC2 [also known as AZU-1 and expression is reduced in ovarian and breast cancer tissues ECTACC] and TACC3 [also known as AINT and ERIC1]) [18,19]. TACC3 is also upregulated in several cancer cell [7–11]. Alternative splicing further increases the comp- lines, including lung cancer [17,20]; but, again, it was lexity of the TACC protein family in mammals and flies reported as being absent or reduced in ovarian and thyroid [12–16]. cancer tissues [21]. Initially, it was suggested that the The three human encoding TACC proteins are all TACC2 splice variant AZU-1 is a tumor suppressor in in genomic regions that are rearranged in certain , breast cancer. However, the lack of any tumor phenotype and their expression is altered in cancers from different in Tacc2-knockout mice did not support this idea [22].It tissues. TACC1 and TACC2 are located in chromosomes therefore appears that these proteins can be upregulated 8p11 and 10q26, respectively, two regions that are impli- or downregulated in different types of cancer or, surpris- cated in breast cancer and other tumors [6], and TACC3 ingly, even in the same type [14,18–25]; as such, their maps to 4p16, within a translocation breakpoint region putative involvement in cancer development and/or pro- associated with the disease multiple myeloma [17]. gression is unclear.

Figure 1. The TACC family of proteins: structural organization and regions of interaction with binding partners. The figure shows alignment of the key structural features, and the position of domains that interact with binding partners (underlined regions). TACC proteins have the conserved coiled-coil TACC domain at their C terminus (blue box). In addition, some members have highly acidic, imperfect repeats of 33 amino acids (termed SPD repeats [28] owing to their specific amino acid composition [pale-blue boxes]) or a Ser–Pro Azu-1 motif (SPAZ) [24] (dark-grey boxes). Yellow lines indicate the position of nuclear localization signals (NLSs). The conserved consensus sequences for AurA phosphorylation are shown as orange bars. The conserved Ser residue is highlighted in orange, and additional consensus sites in Maskin are indicated in grey. The position of the Leu residue, which is important for the C. elegans TAC-1–Zyg-9 interaction, is shown with a white line [44]. For the sake of simplicity, only TACC family proteins that have mapped interactions are shown.

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Almost at the same time as the identification of the role of TACC proteins at the centrosome, and we TACC1 in humans, Maskin was identified and extensively discuss some of the issues that still remain to be addressed. characterized as a factor involved in the regulation of mRNA translation during maturation of Xenopus oocytes The TACC proteins [26]. Other TACC family members have also been impli- The TACC domain is the signature of this protein family. cated in various events related to gene regulation, in- This coiled-coil domain is found at the C terminus of all cluding the regulation of translation, RNA maturation the family members, which have otherwise very diverse and (Figure 1, Table 1) [13,25,27–31]. N-terminal domains (Figure 1) [7,16]. The TACC domain However, to date, no major common role has emerged shows a high level of conservation throughout evolution, for TACC proteins in these processes. By contrast, a major and the shorter member of the family, C. elegans TAC-1, breakthrough came with the identification of D-TACC as a consists of basically one TACC domain [8–10]. Together, Drosophila microtubule-associated and centrosomal this suggests that the TACC domain carries most of protein required for centrosome activity and microtubule the common functional properties of this family of assembly during mitosis [12]. Since then, the idea that proteins. TACC proteins have a role in regulating microtubule The temporal and tissue-specific expression patterns of assembly has gained solid support through various studies the three mammalian TACC proteins have been more performed in different experimental systems. In the light extensively studied. TACC1 can be detected in several of these data, we review here our current understanding of adult tissues, but relatively high levels of expression occur

Table 1. Partners of TACC proteins, and the putative functions of their interactions

The table summarizes all the interreactions described in the literature for some TACC proteins. Proteins involved in MT dynamics and centrosomal functions are indicated in red; proteins involved in RNA regulation are indicated in green; proteins involved in gene regulation are indicated in blue and proteins involved in nucleo-cytoplasmic transport are indicated in black. Abbreviations: aa, amino acids; AKAP350, A kinase (PRKA) CPC, chromosomal passenger complex; CRC, chromatin-remodeling complex; CT, centrosome; HAT, histone acetyltransferase; IF, immunofluorescence; IP, immunoprecipitation; MT, microtubule; n.d., not described; PCM, pericentriolar material; PD, pulldown; Y2H, Yeast two hybrid.

381 Review Trends in Cell Biology Vol.18 No.8 only at the beginning of development, after which it microtubule localization patterns are therefore specific becomes dramatically downregulated [6,32]. TACC2 is also for each protein and are determined by sequences outside widely expressed, showing the highest levels in heart and the TACC domain. It is interesting to note that truncated muscle [32,33]. In mice, expression was detected at all proteins lacking the TACC domain do not localize to cen- developmental stages [13]. By contrast, TACC3 is trosomes or microtubules in Drosophila or Xenopus [12,42]. expressed in relatively few adult tissues, but it shows Interestingly, some data suggest that TACC proteins elevated levels in testis and ovary, and in the hematopoie- bind to the ends of microtubules. On the one hand, the high tic lineages [17,32,34]. During mouse development, TACC3 degree of accumulation to the spindle poles suggests that is present in all the embryonic stages and particularly in TACC proteins bind to microtubule minus-ends. In fact, proliferating tissues [15,32,35]. These data suggest that this localization was directly observed for D-TACC in TACC3 has a role during cell division, in particular during Drosophila embryos, in which microtubule minus-ends development. Indeed, TACC3-deficient mice show embryo- can be distinguished from the centrosomal aster at the nic lethality, associated with a greatly reduced cell num- spindle poles (Figure 2a) [12]. Consistently, Maskin loca- ber, widespread apoptotic cell death, and mitotic defects lizes to the centre of taxol-induced asters in Xenopus egg [36,37]. Interestingly, this phenotype was partially rescued extracts [42]. On the other hand, green fluorescent protein in mice that had reduced levels of the tumor suppressor (GFP)-labeled D-TACC proteins were visualized in living protein p53 [36]. However, to date, no clear picture has Drosophila embryos as dots moving towards and away emerged to describe the molecular mechanism linking p53 from the centrosome, presumably associated to shrinking activity and TACC3. or growing microtubule plus-ends [48]. Immunolocaliza- tion studies also suggest that TACC proteins are associ- Intracellular localization of TACC proteins ated with microtubule plus-ends in the vicinity of the Little information is available concerning the cellular chromosomes [8,9,11,12,48]. The mechanism underlying localization of TACC family members in interphase, the preferential microtubule end localization of TACC although studies have revealed that some of them – the proteins is still unclear. In vitro studies did not reveal three human members and Maskin – are nuclear [38,39].It any preference for binding of Maskin to either the plus- is during cell division that TACC proteins show their most ends or the minus-ends [40], suggesting that other proteins characteristic localization – within the centrosome mediate these end localizations. (Figure 2, Box 1) [8–12,38,40–43]. In humans, the three family members show slightly different distribution pat- Function(s) of the TACC proteins during cell division terns. TACC2 is strongly associated with the centrosome To date, all the phenotypes described for situations in throughout the cell cycle, whereas TACC1 and TACC3 only which the expression of TACC proteins is altered are localize to the centrosome during mitosis – TACC1 weakly, related to defects in microtubule stability. In C. elegans, and TACC3 covering a larger area [38]. These differences TAC-1-depleted embryos show defects in pronuclear in localizations suggest that the three human TACC migration, shorter spindles and defective spindle proteins have non-overlapping functions. elongation in anaphase. They also have shorter astral The centrosomal localization of TACC proteins is highly microtubules and, as a consequence, spindle-positioning dynamic. However, given that microtubules do not modu- defects. Interestingly, microtubules do form in the cyto- late the rapid exchange between centrosomal and cyto- plasm of TAC-1-depleted embryos, suggesting that TAC-1 plasmic pools in C. elegans, and considering that the is required for microtubule assembly only at the centro- centrosomal localization of TAC-1, D-TACC and human some. Consistently, the recovery of fluorescent tubulin at TACC proteins is insensitive to microtubule-depolymeriz- the centrosome after photobleaching is slower in TAC-1- ing drugs, this class of proteins can be considered as core depleted embryos [8–10].InDrosophila, d-tacc mutants components of the centrosome [8,9,12,38]. are female-sterile and have failures of pronuclear fusion. The characteristic centrosomal targeting of TACC The majority of embryos appear to be arrested in the first proteins relies on their conserved TACC domain. Indeed, mitotic division, and those that develop have abnormally this domain alone was shown to localize strongly to the short centrosomal microtubules at all stages of the cell spindle poles and to the centre of centrosomal asters cycle and eventually die as a consequence of the accumu- [12,42]. Recently, it was demonstrated that two residues lation of mitotic defects [12].InXenopus, spindles (L229 and M581) in the TACC domain of C. elegans TAC-1 assembled in egg extracts depleted of Maskin have are important for targeting TAC-1 to the centrosome [44]. reduced size and microtubule content, and the centro- It is unclear at the moment whether these residues are somes nucleate fewer and shorter microtubules important for structural reasons or for protein–protein (Figure 2) [40–42]. In HeLa cells, depletion of TACC1 does interactions. not affect the cell cycle, although multipolar spindles form However, the localization of TACC proteins is not and the cells also show proliferation defects [18].Bycon- restricted to the centrosome, and most of them also associ- trast, the silencing of TACC3 results in partially destabi- ate with microtubules during cell division to various lized microtubules, spindles with reduced microtubule extents (Figure 2). Human TACC3 shows only restricted content, anddefects inchromosome alignment, in addition association with spindle microtubules, whereas Maskin to a high mitotic index due to mitotic arrest [46,49]. and C. elegans TAC-1 localize all along spindle microtu- Consistently, increasing the concentration of D-TACC bules, and D-TACC associates with both spindle and astral and Maskin results in the accumulation of these proteins microtubules (Figure 2a,b) [8,12,38,40,42,45–47]. These at the spindle poles and an increase in spindle microtubule

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Figure 2. Localization and function of TACC proteins at the centrosome. (a) Localization of different members of the TACC family of proteins. Immunofluorescence images of a one-cell stage C.elegans embryo (courtesy of P. Go¨ nczy, Swiss Institute for Experimental Cancer Research, Ch. Des Boveresses 155, Case postale, CH – 1066 Epalinges), a D. melanogaster early embryo (courtesy of J.Raff, Cancer Research UK Senior Group Leader, The Gordon Institute, Tennis Court Road, Cambridge, CB2 1QN) and a X. laevis tissue culture cell, showing the localization of TAC-1, D-TACC and Maskin respectively during anaphase. The upper row shows overlay images with microtubules in green, DNA in blue and the corresponding TACC proteins in red. The lower row shows the distribution of each TACC protein alone. (b) Model for TACC protein function at the spindle pole. (i) Maskin (TACC3) localizes to the spindle poles and along spindle microtubules in Xenopus egg extracts. Maskin is shown in green, microtubules in red, and DNA in blue. Below, the model shows that the targeting of TACC proteins to the spindle pole requires their phosphorylation by Aurora A (AurA), which is active at the centrosome (as indicated by its phosphorylation). Phosphorylated TACC proteins accumulate at the centrosome and recruit ch-TOG/XMAP215. TACC–TOG complexes interact efficiently with nascent microtubules and stabilize them, counteracting the microtubule-destabilizing activity of MCAK at the centrosome. TACC–TOG complexes also associate with microtubule plus- ends. (ii) Spindles assembled in Xenopus egg extracts depleted of Maskin are on average 30% smaller and have reduced microtubule mass. Immunofluorescence with antibodies against Maskin verifies that Maskin is not associated with the spindle under these conditions, confirming the efficiency of the depletion. Below, the model shows that, in the absence of TACC proteins, the recruitment of TOG to the centrosome is not as efficient. As a result, nascent microtubules are not efficiently protected from destabilization by centrosomal MCAK, and therefore fewer microtubules elongate from the centrosome. (iii) Addition of GFP–TD (TACC Domain) to a Maskin-depleted extract rescues the size of the spindle and its microtubule density. GFP–TD localizes to the spindle poles in the same way as Maskin but not along spindle microtubules. Below, the model shows that GFP- TD localizes to the spindle poles and recruits TOG. The TD–TOG complex stabilizes nascent microtubules at the centrosome, protecting them against the destabilizing activity of centrosomal MCAK. TD ensures the loading of TOG to the microtubule plus-end, promoting microtubule growth from the centrosome.

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Box 1. The centrosome Box 2. The ch-TOG/XMAP215 family

The centrosome is the main microtubule-organizing centre (MTOC) XMAP215 is the founding member of a large family of microtubule- of animal cells [4,5]. are small cellular organelles with binding proteins. It was originally purified in the late 1980s from diverse morphologies, but they each consist of a pair of centrioles Xenopus egg extracts. It was characterized as a factor that increases surrounded by pericentriolar material (PCM), an electron-dense the elongation rate of microtubules in vitro [66], and was found to material comprising core resident proteins and several non- be related to chTOG (for colonic and hepatic tumor overexpressed permanent structural and regulatory proteins. One characteristic protein), a human protein overexpressed in tumor cells. component of the PCM is g-tubulin, which promotes efficient XMAP215 shows very different effects on microtubule dynamics nucleation of microtubules at the centrosome. This imposes a in vitro compared with other microtubule-associated proteins natural polarity on the resulting microtubule network, with micro- (MAPs) such as tau and MAP2. Indeed, although these brain MAPs tubule minus-ends focused at the PCM, and microtubule plus-ends promote a strong reduction in the frequency of microtubule reaching out into the surrounding cytoplasm. In addition, centro- catastrophe without substantially altering the growth rate, somes act as platforms for the recruitment of multiple structural and XMAP215 stimulates the growth rate of microtubules without regulatory proteins. These include various microtubule nucleation changing the catastrophe frequency. XMAP215 has also been found factors (e.g. pericentrin and centrosomin), microtubule-stabilizing to increase the depolymerization rate and to reduce the frequency of factors (e.g. ch-TOG/XMAP215 and TACC proteins), and microtu- rescues (i.e. switching between depolymerization and polymeriza- bule-destabilizing or -severing factors (e.g. the kinesin-like protein tion phases). As a result, XMAP215 promotes an increase in MCAK, and katanin) [59]. The efficiency of microtubule elongation microtubule length and mass but, in so doing, promotes the from the centrosome is therefore determined by the relative formation of microtubules of a highly dynamic nature, a property abundance and activity of all of these factors at any given time that can be particularly important during M phase. XMAP215 has [65]. The dynamic changes in centrosome morphology and activity also been shown to counteract the activity of the microtubule- are tightly regulated by several centrosomal kinases, including destabilizing motor MCAK [51]. Consistently, all of the phenotypes members of the Aurora, Polo-like kinase (Plk) and Never In Mitosis A associated with the disruption of ch-TOG/XMAP215 family members (NIMA) [4,5] families. Remarkably, many centrosomal proteins also are related to changes in microtubule stability, including decreased exist in a soluble, cytoplasmic pool, indicating that centrosomes are microtubule growth and defects in spindle function [67]. highly dynamic organelles [5]. ch-TOG/XMAP215 family proteins have a C-terminal domain In resting mammalian cells, the centrosome migrates to the cell involved in MT binding and a N-terminal domain consisting of a surface, and one of the centrioles differentiates into the basal body variable number of TOG domains. Each TOG domain contains six of a cilium [4], which functions as a sensory organelle or as a fluid HEAT repeats, which fold into a paddle-like domain, and wrap itself propeller. The presence of a cilium is transient in proliferating cells around one tubulin dimer [68]. Recently, it was proposed that in which the activity and number of centrosomes varies in tight XMAP215 acts as a processive polymerase, catalyzing the addition

coordination with the cell cycle. The primary cilium present in G1 of 25 tubulin dimers while moving with the assembling microtubule disassembles before the cell progresses into the cell cycle, and the tip. Under some circumstances, XMAP215 can also catalyze the

two centrioles duplicate during S phase. During G2, the two newly reverse reaction, therefore modulating microtubule dynamics [69]. formed centrosomes undergo a process called maturation, and they Although, Drosophila Msps also localizes to the acentrosomal integrate the control of entry into M phase with an increase in their spindle poles during female meiosis [50,70], a universal feature of this microtubule-nucleation capacity. This generates a robust aster of family of proteins is their localization to the centrosome of metazoan highly dynamic microtubules, which are involved in centrosome cells and to the spindle pole bodies of yeast. The centrosomal separation and spindle assembly. During mitosis, the centrosomes localization of XMAP215 is mediated by its C-terminal microtubule- are positioned at each spindle pole and have an important role in binding domain [67]. In some organisms, this domain interacts with determining spindle orientation and the plane of cell division [4]. TACC proteins [11,18]. Given that the localization of Msps is also dependent on AurA in Drosophila [47], these data suggest that the localization of XMAP215 family members to the centrosome relies on length and number, which are effects opposite to those their interaction with members of the TACC family. caused by depletion. A similar phenotype – accumulation at spindle poles and increase in microtubule length and number – arises upon overexpression of human TACC3 The TACC proteins only interact very weakly with but not TACC1 or TACC2 [38,42,48]. Altogether, these polymerized microtubules in vitro, but they do co-pellet data clearly indicate that the TACC proteins have a con- very efficiently with microtubules in Drosophila embryos served function in promoting centrosomal microtubule and Xenopus egg extracts, suggesting that other factor(s) assembly. are involved [12,42,48]. The first clue to shed some light upon this issue was a report describing an interaction How do TACC proteins participate in microtubule between D-TACC and Minispindles (Msps), the Drosophila stabilization? member of the colonic and hepatic tumor-overexpressed Several observations strongly suggest that TACC proteins gene (ch-TOG; also known as XMAP215) family of micro- function not at the level of nucleation of microtubules but, tubule-associated proteins (Box 2) [48]. The functional rather, in the stabilization of microtubules. Experiments relevance of this interaction is underscored by the obser- performed in Xenopus egg extracts have clearly shown that vation that TACC and ch-TOG/XMAP215 proteins have Maskin has no role in centrosomal microtubule nucleation similar localizations. Furthermore, perturbing any of these activity [42].InC. elegans, TAC-1 mutant embryos do not proteins produces similar phenotypes [8,49,50]. Interest- show defects in the distribution of the microtubule-nuclea- ingly, this interaction turned out to be conserved through- tor g-tubulin [8].InDrosophila d-tacc mutant embryos, the out evolution (Table 1) and to be mediated by the TACC localization of g-tubulin and the centrosomal proteins domain (Figure 1) [8,10,11,18,40–42,48]. This is consistent CP190 and CP60 to the centrosome is normal, suggesting with the idea that this domain has a crucial role in promot- that microtubule nucleation at centrosomes is also unaf- ing microtubule assembly, and it also agrees with exper- fected [12]. Therefore, TACC proteins promote microtubule imental data showing that the TACC domain is sufficient growth from the centrosome without altering the nuclea- to rescue the phenotype of Maskin depletion in Xenopus tion of microtubules. egg extracts (Figure 2b) [40,42]. Overexpression studies

384 Review Trends in Cell Biology Vol.18 No.8 have also provided some additional insights into the func- Box 3. The Aurora kinase family tional role of this domain. In HeLa cells, the overexpression of any of the three TACC domains results in the formation The Aurora kinase family is an evolutionarily conserved family of serine–threonine kinases. Although there is only one Aurora kinase of highly ordered, cytoplasmic polymers that interact with in yeasts (Ipl1p in S. cerevisiae, and Ark1 in S. pombe), metazoans bundled microtubules but not with tubulin oligomers [38]. have three Aurora kinases: Aurora A (also known as STK6), Aurora B In Drosophila embryos, overexpression of the C-terminal (STK12) and Aurora C (STK13). Each of these proteins shows its part of D-TACC results in the formation of microtubule highest levels and activity during the G2 and M phases of the cell asters in the cytoplasm – but only if Msps is also present cycle. The initial discovery of the Aurora mutation in Drosophila implicated the Aurora protein in spindle assembly, but extensive [48]. All these data support the idea that the function of the studies have shown that these kinases have more functions [71]. TACC domain in promoting microtubule assembly is AurA and AurB have been more extensively studied. During cell highly dependent on its interaction with ch-TOG/ division, they have non-overlapping roles related to their distinctive XMAP215. localizations. AurB is a chromosomal passenger protein required for So what is the underlying mechanism promoting the phosphorylation of histone H3, bi-orientation, the spindle assembly checkpoint, and cytokinesis. The centrosomal assembly of microtubules? Experiments performed in AurA, by contrast, has emerged as a major regulator of centrosome different systems have shown that reducing TACC protein activity, participating in centrosome maturation and separation, and levels impairs the correct localization of ch-TOG/XMAP125 in spindle assembly. In addition, AurA has been implicated in entry to the centrosome [8–11,41,48,50]. One exception concerns to M phase, and in mRNA translation, cilia disassembly, and asymmetric cell division [72]. TACC3 in HeLa cells, but this might have been due to One clear function of AurA is the recruitment and regulation of incomplete TACC3 depletion or because any of the other proteins at the centrosome, including centrosomin, g-TURC and TACC proteins were compensating for the lack of TACC3 TACC proteins [71]. Relatively few substrates of the AurA kinase by targeting ch-TOG to the centrosome [49]. In any case, have been identified, but several are spindle-assembly factors, such increasing the concentration of TACC proteins results in as TACC proteins and NDEL1. Although AurA kinase can self- activate by autophosphorylation, several activators have been an increase in the recruitment of ch-TOG/XMAP215 to the reported. One of them is TPX2, a RanGTP-regulated factor involved spindle poles [38,42,48]. By contrast, ch-TOG/XMAP215 is in spindle assembly. TPX2 also targets AurA to spindle microtubules not required for the localization of TACC proteins to the [73,74]. centrosome [11,44,49]. In humans, the three Aurora kinases are overexpressed in a Although all of these data strongly support the idea that variety of human cancers and are believed to have multiple roles in the development and progression of cancer. Moreover, the gene TACC proteins are required for the efficient recruitment of encoding the (AURKA) maps to the chromosome ch-TOG/XMAP215 proteins to the centrosome, it is still region 20q13, which is frequently amplified in many human cancers. unclear whether this targeting function is sufficient to The overexpression of AURKA induces centrosome amplification explain the function of TACC proteins. It is also possible and aneuploidy. It also confers resistance to taxol-dependent that a functional relationship exists between TACC apoptosis in cancer cells. In this context, it is also interesting to note that AurA interacts with and inactivates the tumor suppressor proteins and ch-TOG/XMAP215. In this context, it is inter- p53, and that it also interacts with the breast cancer susceptibility esting to recall that Msps is required for the formation of gene BRCA1 and colocalizes with it at the centrosome. Given that microtubule asters in Drosophila embryo extracts contain- the overexpression of AurA has been shown to cause tumorigenic ing the C-terminal part of D-TACC [48]. Although there are transformation of human and rodent cells in vitro and in vivo [72],it has been proposed that AurA acts as an oncogene. only a few clues concerning the mechanism involved at the molecular level, gel filtration experiments have shown that XMAP215 and Maskin form a one-to-one complex in vitro, kinase in vitro, and most of them have one or more and this complex possesses a higher affinity for microtu- sequences that conform to a consensus motif for phos- bules than do each protein on its own [41,42]. TACC phorylation by AurA. In all cases, these sites are located proteins might therefore promote a conformational change outside the TACC domain (Figure 1, Table 1) in ch-TOG/XMAP215 that renders the molecule more effi- [41,42,45,47,52,53]. One of them is conserved in several cient for microtubule binding. Interestingly, under these TACC orthologs, indicating that it is functionally import- conditions, microtubules are more resistant to depolymer- ant [45,52,53]. There is strong experimental support in ization by a destabilizing factor, the mitotic centromere- several systems (i.e. nematode, fly, frog and human cells) associated kinesin MCAK (a kinesin-13 also known as indicating that phosphorylation has a role in TACC func- KIF2C) [41,42]. tion at the centrosome [8,41,42,47,53]. The model that emerges is one in which TACC proteins Experiments with purified proteins have shown that recruit ch-TOG/XMAP215 to the centrosome and enhances Maskin can bind simultaneously to AurA and XMAP215, its microtubule binding and stabilizing activity. This coun- suggesting that the binding sites for these two proteins do teracts the destabilizing activity of MCAK and thereby not overlap (Figure 1) [42]. Moreover, phosphorylation promotes microtubule growth from the centrosome does not appear to have any positive or negative influence (Figure 2) [41,42,51]. on these interactions. Indeed, in Xenopus egg extract, XMAP215 is pulled down as efficiently by phosphorylated Regulation of TACC proteins by Aurora A wild type Maskin as it is by an unphosphorylatable, Another conserved partner of TACC proteins is the Ser– mutated Maskin protein [41]. Finally, AurA does not Thr kinase Aurora A (AurA–STK6) (Table 1, Box 3) enhance the microtubule binding affinity achieved by [18,41,42,45,47,52]. In vitro pull-down experiments have the Maskin–XMAP215 complex in comparison with shown that Maskin interacts directly with AurA [42]. Maskin or XMAP215 proteins in isolation [41]. All of these Moreover, TACC proteins are good substrates for this data suggest that phosphorylation does not regulate the

385 Review Trends in Cell Biology Vol.18 No.8 interaction or functionality of TACC–XMAP215 com- Clearly, additional work is needed to address some key plexes. It also seems unlikely that phosphorylation induces questions and to understand how TACC proteins function a change in the oligomerization state of Maskin, because to promote the assembly of microtubules. Maskin and unphosphorylatable mutants of Maskin are found at the same position on sucrose gradients of various Conclusions protein combinations [41]. TACC proteins have recently emerged as important What then does phosphorylation regulate? One possib- players in the complex process of regulating microtubule ility is that it regulates the turnover of the protein at the dynamics during cell division. Although it is now clearly centrosome, favoring the retention of the phosphorylated established that they have a major role at the centrosome – form. Another possibility is that a direct interaction with promoting microtubule elongation together with ch-TOG/ the AurA is involved, resulting in the phosphorylation of XMAP215 proteins – the molecular mechanism underlying the TACC protein at the centrosome. In fact, in Droso- their activity is still unclear. Solid data support the fact phila, phosphorylation of D-TACC seems to be required that they interact with, and are substrates of, the kinase not for its localization to the centrosome but, rather, to AurA; but, again, although phosphorylation is essential for enable it to load at the microtubule minus-ends [53]. their localization to and function at the centrosome, the However, the inhibition of AurA kinase activity by a small molecular mechanism involved is far from being under- molecule results in TACC3 failing to localize to centro- stood. In fact, it is noteworthy that TACC proteins also somes in human cells [45], suggesting that kinase activity function in pathways that do not involve the centrosome. rather than the protein itself is required. In any case, Indeed, D-TACC and Msps both localize at the acentroso- various data support the idea that AurA regulates the mal poles of Drosophila female meiotic spindles, and both centrosomal recruitment of TACC. Immunofluorescence are required for maintaining the bipolarity of these spin- studies performed with an antibody against the phos- dles [50]. In addition, some data from frog and fission yeast phorylated form of TACC3 have shown that phosphory- support the idea that TACC proteins participate in the lated TACC proteins are mostly found at the centrosome in RanGTP-dependent spindle assembly pathway [39,40,60]. HeLa cells and Drosophila embryos [41,53,54]. Consistent Furthermore, in the fission yeast S. pombe, Alp7 functions with this, a non-phosphorylatable mutant of TACC3 does in the maintenance of microtubule organization during not localize efficiently to the centrosome in transfected interphase [61]. It is therefore very likely that TACC HeLa cells or in Xenopus egg extracts [41,53,54].InDro- proteins have a more general role in microtubule stabiliz- sophila embryos, a similar, although less pronounced, ation than currently appreciated. effect is observed for the localization of a non-phosphor- Finally, it is still unclear whether the quite distinct roles ylatable mutant of D-TACC [53]. In further agreement, of TACC proteins in different aspects of gene regulation TACC proteins do not localize to the centrosome in AurA and in microtubule assembly are connected in some way. In mutants in C. elegans or Drosophila,andthereisastrong this context, it is noteworthy that recent studies have reduction in the association of Maskin with spindles revealed that different classes of RNAs are associated with assembled in Xenopus egg extracts depleted of AurA the mitotic spindle [62,63]. Interestingly, both Maskin and [8,42,47]. However, these observations cannot exclude AurA are involved in the regulation of mRNA translation the possibility that these mislocalizations are attributable in Xenopus oocytes [26]. to indirect effects on either centrosome content or func- It is quite apparent that much remains to be done to tionality due to the absence of this important centrosomal understand more clearly how TACC proteins function. In a kinase. Overall, AurA phosphorylation of TACC proteins wider context, it will be interesting to elucidate the func- appears to contribute substantially to their recruitment to tional connections between their roles in microtubule or accumulation at the centrosome, and, as a consequence, stabilization and in RNA control. In addition, future work to the recruitment of ch-TOG/XMAP215 (Figure 2) [41]. should reveal whether their role is only restricted to events The precise molecular mechanism involved remains to be occurring during cell division. elucidated. In this context, it is noteworthy that alterna- As a final remark, it should be noted that the study of tive pathways to target TACC proteins to the centrosome TACC proteins is starting to offer a variety of promising have been described recently. Indeed, the centrosomal applications related to cancer therapies. Recently, it was localization of D-TACC relies on its interaction with motif shown that monitoring phospho-TACC3 is an efficient way 1oftheDrosophila protein centrosomin [55],andthedual- of evaluating the effectiveness of AurA inhibitors that are specificity kinase TTK is essential for the centrosomal promising anti-cancer drugs, some of which have recently localization of human TACC2 [56]. Recently, it was also entered clinical trials [45]. The observation that TACC3 reported that the centrosomal protein NDEL1 (also known depletion sensitizes cells to paclitaxel-induced cell death as NUDEL), another substrate of AurA, is required for the also suggests that TACC3 itself is a promising target for targeting of TACC3 to the centrosome in human cells [57]. the treatment of the tumors resistant to this widely used Interestingly, NDEL1 is a binding partner of LIS1, a therapy [64]. Finally, TACC3 is also emerging as a good protein that participates in the regulation of cytoplasmic prognostic marker for some cancers [20]. dynein function and microtubule organization during cell division and neuronal migration [58].NDEL1isalso Acknowledgements related to microtubule-remodeling mechanisms through We thank all members of the Vernos Laboratory, especially Luis Bejarano, Teresa Sardon and Roser Pinyol, for critically reading the the recruitment of the microtubule-severing factor kata- manuscript and providing helpful comments. We thank P. Go¨nczy nin [59]. (ISREC, CH) and J. Raff (The Gurdon Institute, UK) for the kind gift of

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