The TACC proteins: TACC-ling microtubule dynamics and centrosome function

Review

The TACC proteins: TACC-ling microtubule dynamics and centrosome 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 molecular mechanisms underlying the high plasticity of the microtubule network at different stages of the cell cycle, and during and after differentiation. Initial reports described the centrosomal localization of proteins possessing transforming acidic coiled-coil (TACC) domains. This discovery prompted several groups to examine the role of TACC proteins during cell division, leading to indications that they are important players in this complex process in different organisms. Here, we review the current understanding of the role of TACC proteins in the regulation of microtubule dynamics, and we highlight the complexity of centrosome function. Introduction Cell proliferation and differentiation require dramatic rearrangements of the cytoskeleton that rely on the highly dynamic nature of the cytoskeletal components. Microtubules are dynamic filaments with fundamental roles in eukaryotic cell organization and function. During cell division, they form the bipolar spindle, which segregates the chromosomes into the two daughter cells. Microtubules show prolonged states of polymerization and depolymerization that interconvert stochastically, exhibiting frequent transitions between growing and shrinking phases, a property called ‘dynamic instability’ [1]. In the cell, multiple factors modulate this property by acting positively or negatively on the nucleation, elongation or destabilization of microtubules [1–3]. The relative activity of all these factors determines the steady-state length and stability of microtubules, in addition to their organization, and it is largely dictated by global and local phosphorylation–dephosphorylation reactions [2,3]. In addition, other types of factors that have microtubule-severing and anchoring activities also influence the microtubule network. The main microtubule-organizing centre (MTOC) of animal cells, the centrosome, acts as a platform upon which the different factors and activities accumulate in a regulated manner. It therefore exerts a tight local and temporal control on the number, distribution and polarity of microtubules [4,5]. Corresponding authors: Peset, I. ([email protected]); Vernos, I. ([email protected]).

Transforming acidic coiled-coil (TACC) proteins emerged initially as a group of proteins implicated in cancer. The first member of the TACC family to be discovered was identified in a search of genomic regions that are amplified in breast cancer. It was named transforming acidic coiledcoil 1 (TACC1) because of its highly acidic nature, the presence of a predicted coiled-coil domain at its C terminus (now known as the TACC domain), and its ability to promote cellular transformation [6]. TACC proteins are present in different organisms, ranging from yeasts to mammals. There is only one TACC protein in the nematode Caenorhabditis elegans (TAC-1), in Drosophila melanogaster (D-TACC), in Xenopus laevis (Maskin), and

Abbreviations AINT: ARNT interacting protein AKAP350: A kinase (PRKA) anchor protein Alp7: Altered growth polarity 7 Ark1: aurora-related kinase ARNT: aryl hydrocarbon nuclear translocator protein AZU-1: anti-zuai-1 CBP: calcium-binding protein CPEB: cytoplasmic polyadenylation element binding protein DCLK: doublecortin-like kinase ECTACC: endothelial cell TACC E1F4E: eukaryotic initiation factor 4E ERIC: erythropoietin-induced cDNA FOG-1: Friend of Gatal GAS41: glioma amplified sequence 41 GCN5L2: general control of amino-acid synthesis 5-like 2 g-TURC: g-tubulin related complex HEAT: huntingtin, elongation factor 3, A subunit of protein phosphatase 2A and TOR1 INI-1: SWI/SNF core subunit Ipl1: Increase-in-ploidy 1 ISREC: Swiss Institute for Experimental Cancer Research KIF2C: kinesin family member 2C LIS1: Lissencephaly-1 LSM7: U6 small nuclear NRA associated MBD2: methyl-CpG binding domain protein 2 Mial: melanoma inhibitory activity 1 Mps1: MonoPolar Spindle 1 NDEL1 and NUDEL: nude nuclear distribution gene E homolog (A. nidulans)-like 1 pCAF, p300/CBP-associated factor SmG: snRNP Sm protein G TTK: TTK protein kinase Zyg-8: ZYGote defective Zyg-9: ZYGote defective

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

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Review in the fission yeast Schizosaccharomyces pombe (Alp7 also known as Mia1p); by contrast, mammals have three such proteins (TACC1, TACC2 [also known as AZU-1 and ECTACC] and TACC3 [also known as AINT and ERIC1]) [7–11]. Alternative splicing further increases the complexity of the TACC protein family in mammals and flies [12–16]. The three human genes encoding TACC proteins are all in genomic regions that are rearranged in certain cancers, and their expression is altered in cancers from different tissues. TACC1 and TACC2 are located in chromosomes 8p11 and 10q26, respectively, two regions that are implicated in breast cancer and other tumors [6], and TACC3 maps to 4p16, within a translocation breakpoint region associated with the disease multiple myeloma [17].

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Although TACC1 was originally found to be upregulated in breast cancer [6], subsequent studies found that its expression is reduced in ovarian and breast cancer tissues [18,19]. TACC3 is also upregulated in several cancer cell lines, including lung cancer [17,20]; but, again, it was reported as being absent or reduced in ovarian and thyroid cancer tissues [21]. Initially, it was suggested that the TACC2 splice variant AZU-1 is a tumor suppressor in breast cancer. However, the lack of any tumor phenotype in Tacc2-knockout mice did not support this idea [22]. It therefore appears that these proteins can be upregulated or downregulated in different types of cancer or, surprisingly, even in the same type [14,18–25]; as such, their putative involvement in cancer development and/or progression 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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Review Almost at the same time as the identification of TACC1 in humans, Maskin was identified and extensively characterized as a factor involved in the regulation of mRNA translation during maturation of Xenopus oocytes [26]. Other TACC family members have also been implicated in various events related to gene regulation, including the regulation of translation, RNA maturation and gene expression (Figure 1, Table 1) [13,25,27–31]. However, to date, no major common role has emerged for TACC proteins in these processes. By contrast, a major breakthrough came with the identification of D-TACC as a Drosophila microtubule-associated and centrosomal protein required for centrosome activity and microtubule assembly during mitosis [12]. Since then, the idea that TACC proteins have a role in regulating microtubule assembly has gained solid support through various studies performed in different experimental systems. In the light of these data, we review here our current understanding of

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the role of TACC proteins at the centrosome, and we discuss some of the issues that still remain to be addressed. The TACC proteins The TACC domain is the signature of this protein family. This coiled-coil domain is found at the C terminus of all the family members, which have otherwise very diverse N-terminal domains (Figure 1) [7,16]. The TACC domain shows a high level of conservation throughout evolution, and the shorter member of the family, C. elegans TAC-1, consists of basically one TACC domain [8–10]. Together, this suggests that the TACC domain carries most of the common functional properties of this family of proteins. The temporal and tissue-specific expression patterns of the three mammalian TACC proteins have been more extensively studied. TACC1 can be detected in several 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.

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Review only at the beginning of development, after which it becomes dramatically downregulated [6,32]. TACC2 is also widely expressed, showing the highest levels in heart and muscle [32,33]. In mice, expression was detected at all developmental stages [13]. By contrast, TACC3 is expressed in relatively few adult tissues, but it shows elevated levels in testis and ovary, and in the hematopoietic lineages [17,32,34]. During mouse development, TACC3 is present in all the embryonic stages and particularly in proliferating tissues [15,32,35]. These data suggest that TACC3 has a role during cell division, in particular during development. Indeed, TACC3-deficient mice show embryonic lethality, associated with a greatly reduced cell number, widespread apoptotic cell death, and mitotic defects [36,37]. Interestingly, this phenotype was partially rescued in mice that had reduced levels of the tumor suppressor protein p53 [36]. However, to date, no clear picture has emerged to describe the molecular mechanism linking p53 activity and TACC3. Intracellular localization of TACC proteins Little information is available concerning the cellular localization of TACC family members in interphase, although studies have revealed that some of them – the three human members and Maskin – are nuclear [38,39]. It is during cell division that TACC proteins show their most characteristic localization – within the centrosome (Figure 2, Box 1) [8–12,38,40–43]. In humans, the three family members show slightly different distribution patterns. TACC2 is strongly associated with the centrosome throughout the cell cycle, whereas TACC1 and TACC3 only localize to the centrosome during mitosis – TACC1 weakly, and TACC3 covering a larger area [38]. These differences in localizations suggest that the three human TACC proteins have non-overlapping functions. The centrosomal localization of TACC proteins is highly dynamic. However, given that microtubules do not modulate the rapid exchange between centrosomal and cytoplasmic pools in C. elegans, and considering that the centrosomal localization of TAC-1, D-TACC and human TACC proteins is insensitive to microtubule-depolymerizing drugs, this class of proteins can be considered as core components of the centrosome [8,9,12,38]. The characteristic centrosomal targeting of TACC proteins relies on their conserved TACC domain. Indeed, this domain alone was shown to localize strongly to the spindle poles and to the centre of centrosomal asters [12,42]. Recently, it was demonstrated that two residues (L229 and M581) in the TACC domain of C. elegans TAC-1 are important for targeting TAC-1 to the centrosome [44]. It is unclear at the moment whether these residues are important for structural reasons or for protein–protein interactions. However, the localization of TACC proteins is not restricted to the centrosome, and most of them also associate with microtubules during cell division to various extents (Figure 2). Human TACC3 shows only restricted association with spindle microtubules, whereas Maskin and C. elegans TAC-1 localize all along spindle microtubules, and D-TACC associates with both spindle and astral microtubules (Figure 2a,b) [8,12,38,40,42,45–47]. These 382

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microtubule localization patterns are therefore specific for each protein and are determined by sequences outside the TACC domain. It is interesting to note that truncated proteins lacking the TACC domain do not localize to centrosomes or microtubules in Drosophila or Xenopus [12,42]. Interestingly, some data suggest that TACC proteins bind to the ends of microtubules. On the one hand, the high degree of accumulation to the spindle poles suggests that TACC proteins bind to microtubule minus-ends. In fact, this localization was directly observed for D-TACC in Drosophila embryos, in which microtubule minus-ends can be distinguished from the centrosomal aster at the spindle poles (Figure 2a) [12]. Consistently, Maskin localizes to the centre of taxol-induced asters in Xenopus egg extracts [42]. On the other hand, green fluorescent protein (GFP)-labeled D-TACC proteins were visualized in living Drosophila embryos as dots moving towards and away from the centrosome, presumably associated to shrinking or growing microtubule plus-ends [48]. Immunolocalization studies also suggest that TACC proteins are associated with microtubule plus-ends in the vicinity of the chromosomes [8,9,11,12,48]. The mechanism underlying the preferential microtubule end localization of TACC proteins is still unclear. In vitro studies did not reveal any preference for binding of Maskin to either the plusends or the minus-ends [40], suggesting that other proteins mediate these end localizations. Function(s) of the TACC proteins during cell division To date, all the phenotypes described for situations in which the expression of TACC proteins is altered are related to defects in microtubule stability. In C. elegans, TAC-1-depleted embryos show defects in pronuclear migration, shorter spindles and defective spindle elongation in anaphase. They also have shorter astral microtubules and, as a consequence, spindle-positioning defects. Interestingly, microtubules do form in the cytoplasm of TAC-1-depleted embryos, suggesting that TAC-1 is required for microtubule assembly only at the centrosome. Consistently, the recovery of fluorescent tubulin at the centrosome after photobleaching is slower in TAC-1depleted embryos [8–10]. In Drosophila, d-tacc mutants are female-sterile and have failures of pronuclear fusion. The majority of embryos appear to be arrested in the first mitotic division, and those that develop have abnormally short centrosomal microtubules at all stages of the cell cycle and eventually die as a consequence of the accumulation of mitotic defects [12]. In Xenopus, spindles assembled in egg extracts depleted of Maskin have reduced size and microtubule content, and the centrosomes nucleate fewer and shorter microtubules (Figure 2) [40–42]. In HeLa cells, depletion of TACC1 does not affect the cell cycle, although multipolar spindles form and the cells also show proliferation defects [18]. By contrast, the silencing of TACC3 results in partially destabilized microtubules, spindles with reduced microtubule content, and defects in chromosome alignment, in addition to a high mitotic index due to mitotic arrest [46,49]. Consistently, increasing the concentration of D-TACC and Maskin results in the accumulation of these proteins at the spindle poles and an increase in spindle microtubule

Review

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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 plusends. (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 GFPTD 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) of animal cells [4,5]. Centrosomes are small cellular organelles with diverse morphologies, but they each consist of a pair of centrioles surrounded by pericentriolar material (PCM), an electron-dense material comprising core resident proteins and several nonpermanent structural and regulatory proteins. One characteristic component of the PCM is g-tubulin, which promotes efficient nucleation of microtubules at the centrosome. This imposes a natural polarity on the resulting microtubule network, with microtubule minus-ends focused at the PCM, and microtubule plus-ends reaching out into the surrounding cytoplasm. In addition, centrosomes act as platforms for the recruitment of multiple structural and regulatory proteins. These include various microtubule nucleation factors (e.g. pericentrin and centrosomin), microtubule-stabilizing factors (e.g. ch-TOG/XMAP215 and TACC proteins), and microtubule-destabilizing or -severing factors (e.g. the kinesin-like protein MCAK, and katanin) [59]. The efficiency of microtubule elongation from the centrosome is therefore determined by the relative abundance and activity of all of these factors at any given time [65]. The dynamic changes in centrosome morphology and activity are tightly regulated by several centrosomal kinases, including members of the Aurora, Polo-like kinase (Plk) and Never In Mitosis A (NIMA) [4,5] families. Remarkably, many centrosomal proteins also exist in a soluble, cytoplasmic pool, indicating that centrosomes are highly dynamic organelles [5]. In resting mammalian cells, the centrosome migrates to the cell surface, and one of the centrioles differentiates into the basal body of a cilium [4], which functions as a sensory organelle or as a fluid propeller. The presence of a cilium is transient in proliferating cells in which the activity and number of centrosomes varies in tight coordination with the cell cycle. The primary cilium present in G1 disassembles before the cell progresses into the cell cycle, and the two centrioles duplicate during S phase. During G2, the two newly formed centrosomes undergo a process called maturation, and they integrate the control of entry into M phase with an increase in their microtubule-nucleation capacity. This generates a robust aster of highly dynamic microtubules, which are involved in centrosome separation and spindle assembly. During mitosis, the centrosomes are positioned at each spindle pole and have an important role in determining spindle orientation and the plane of cell division [4].

XMAP215 is the founding member of a large family of microtubulebinding proteins. It was originally purified in the late 1980s from Xenopus egg extracts. It was characterized as a factor that increases the elongation rate of microtubules in vitro [66], and was found to be related to chTOG (for colonic and hepatic tumor overexpressed protein), a human protein overexpressed in tumor cells. XMAP215 shows very different effects on microtubule dynamics in vitro compared with other microtubule-associated proteins (MAPs) such as tau and MAP2. Indeed, although these brain MAPs promote a strong reduction in the frequency of microtubule catastrophe without substantially altering the growth rate, XMAP215 stimulates the growth rate of microtubules without changing the catastrophe frequency. XMAP215 has also been found to increase the depolymerization rate and to reduce the frequency of rescues (i.e. switching between depolymerization and polymerization phases). As a result, XMAP215 promotes an increase in microtubule length and mass but, in so doing, promotes the formation of microtubules of a highly dynamic nature, a property that can be particularly important during M phase. XMAP215 has also been shown to counteract the activity of the microtubuledestabilizing motor MCAK [51]. Consistently, all of the phenotypes associated with the disruption of ch-TOG/XMAP215 family members are related to changes in microtubule stability, including decreased microtubule growth and defects in spindle function [67]. ch-TOG/XMAP215 family proteins have a C-terminal domain involved in MT binding and a N-terminal domain consisting of a variable number of TOG domains. Each TOG domain contains six HEAT repeats, which fold into a paddle-like domain, and wrap itself around one tubulin dimer [68]. Recently, it was proposed that XMAP215 acts as a processive polymerase, catalyzing the addition of 25 tubulin dimers while moving with the assembling microtubule tip. Under some circumstances, XMAP215 can also catalyze the reverse reaction, therefore modulating microtubule dynamics [69]. Although, Drosophila Msps also localizes to the acentrosomal spindle poles during female meiosis [50,70], a universal feature of this family of proteins is their localization to the centrosome of metazoan cells and to the spindle pole bodies of yeast. The centrosomal localization of XMAP215 is mediated by its C-terminal microtubulebinding domain [67]. In some organisms, this domain interacts with 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 their interaction with members of the TACC family.

length and number, which are effects opposite to those caused by depletion. A similar phenotype – accumulation at spindle poles and increase in microtubule length and number – arises upon overexpression of human TACC3 but not TACC1 or TACC2 [38,42,48]. Altogether, these data clearly indicate that the TACC proteins have a conserved function in promoting centrosomal microtubule assembly. How do TACC proteins participate in microtubule stabilization? Several observations strongly suggest that TACC proteins function not at the level of nucleation of microtubules but, rather, in the stabilization of microtubules. Experiments performed in Xenopus egg extracts have clearly shown that Maskin has no role in centrosomal microtubule nucleation activity [42]. In C. elegans, TAC-1 mutant embryos do not show defects in the distribution of the microtubule-nucleator g-tubulin [8]. In Drosophila d-tacc mutant embryos, the localization of g-tubulin and the centrosomal proteins CP190 and CP60 to the centrosome is normal, suggesting that microtubule nucleation at centrosomes is also unaffected [12]. Therefore, TACC proteins promote microtubule growth from the centrosome without altering the nucleation of microtubules. 384

The TACC proteins only interact very weakly with polymerized microtubules in vitro, but they do co-pellet very efficiently with microtubules in Drosophila embryos and Xenopus egg extracts, suggesting that other factor(s) are involved [12,42,48]. The first clue to shed some light upon this issue was a report describing an interaction between D-TACC and Minispindles (Msps), the Drosophila member of the colonic and hepatic tumor-overexpressed gene (ch-TOG; also known as XMAP215) family of microtubule-associated proteins (Box 2) [48]. The functional relevance of this interaction is underscored by the observation that TACC and ch-TOG/XMAP215 proteins have similar localizations. Furthermore, perturbing any of these proteins produces similar phenotypes [8,49,50]. Interestingly, this interaction turned out to be conserved throughout evolution (Table 1) and to be mediated by the TACC domain (Figure 1) [8,10,11,18,40–42,48]. This is consistent with the idea that this domain has a crucial role in promoting microtubule assembly, and it also agrees with experimental data showing that the TACC domain is sufficient to rescue the phenotype of Maskin depletion in Xenopus egg extracts (Figure 2b) [40,42]. Overexpression studies

Review have also provided some additional insights into the functional role of this domain. In HeLa cells, the overexpression of any of the three TACC domains results in the formation of highly ordered, cytoplasmic polymers that interact with bundled microtubules but not with tubulin oligomers [38]. In Drosophila embryos, overexpression of the C-terminal part of D-TACC results in the formation of microtubule asters in the cytoplasm – but only if Msps is also present [48]. All these data support the idea that the function of the TACC domain in promoting microtubule assembly is highly dependent on its interaction with ch-TOG/ XMAP215. So what is the underlying mechanism promoting the assembly of microtubules? Experiments performed in different systems have shown that reducing TACC protein levels impairs the correct localization of ch-TOG/XMAP125 to the centrosome [8–11,41,48,50]. One exception concerns TACC3 in HeLa cells, but this might have been due to incomplete TACC3 depletion or because any of the other TACC proteins were compensating for the lack of TACC3 by targeting ch-TOG to the centrosome [49]. In any case, increasing the concentration of TACC proteins results in an increase in the recruitment of ch-TOG/XMAP215 to the spindle poles [38,42,48]. By contrast, ch-TOG/XMAP215 is not required for the localization of TACC proteins to the centrosome [11,44,49]. Although all of these data strongly support the idea that TACC proteins are required for the efficient recruitment of ch-TOG/XMAP215 proteins to the centrosome, it is still unclear whether this targeting function is sufficient to explain the function of TACC proteins. It is also possible that a functional relationship exists between TACC proteins and ch-TOG/XMAP215. In this context, it is interesting to recall that Msps is required for the formation of microtubule asters in Drosophila embryo extracts containing the C-terminal part of D-TACC [48]. Although there are 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, and this complex possesses a higher affinity for microtubules than do each protein on its own [41,42]. TACC proteins might therefore promote a conformational change in ch-TOG/XMAP215 that renders the molecule more efficient for microtubule binding. Interestingly, under these conditions, microtubules are more resistant to depolymerization by a destabilizing factor, the mitotic centromereassociated kinesin MCAK (a kinesin-13 also known as KIF2C) [41,42]. The model that emerges is one in which TACC proteins recruit ch-TOG/XMAP215 to the centrosome and enhances its microtubule binding and stabilizing activity. This counteracts the destabilizing activity of MCAK and thereby promotes microtubule growth from the centrosome (Figure 2) [41,42,51]. Regulation of TACC proteins by Aurora A Another conserved partner of TACC proteins is the Ser– Thr kinase Aurora A (AurA–STK6) (Table 1, Box 3) [18,41,42,45,47,52]. In vitro pull-down experiments have shown that Maskin interacts directly with AurA [42]. Moreover, TACC proteins are good substrates for this

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Box 3. The Aurora kinase family The Aurora kinase family is an evolutionarily conserved family of serine–threonine kinases. Although there is only one Aurora kinase in yeasts (Ipl1p in S. cerevisiae, and Ark1 in S. pombe), metazoans have three Aurora kinases: Aurora A (also known as STK6), Aurora B (STK12) and Aurora C (STK13). Each of these proteins shows its highest levels and activity during the G2 and M phases of the cell cycle. The initial discovery of the Aurora mutation in Drosophila implicated the Aurora protein in spindle assembly, but extensive studies have shown that these kinases have more functions [71]. AurA and AurB have been more extensively studied. During cell division, they have non-overlapping roles related to their distinctive localizations. AurB is a chromosomal passenger protein required for phosphorylation of histone H3, chromosome bi-orientation, the spindle assembly checkpoint, and cytokinesis. The centrosomal AurA, by contrast, has emerged as a major regulator of centrosome activity, participating in centrosome maturation and separation, and in spindle assembly. In addition, AurA has been implicated in entry to M phase, and in mRNA translation, cilia disassembly, and asymmetric cell division [72]. One clear function of AurA is the recruitment and regulation of proteins at the centrosome, including centrosomin, g-TURC and TACC proteins [71]. Relatively few substrates of the AurA kinase have been identified, but several are spindle-assembly factors, such as TACC proteins and NDEL1. Although AurA kinase can selfactivate by autophosphorylation, several activators have been reported. One of them is TPX2, a RanGTP-regulated factor involved in spindle assembly. TPX2 also targets AurA to spindle microtubules [73,74]. In humans, the three Aurora kinases are overexpressed in a variety of human cancers and are believed to have multiple roles in the development and progression of cancer. Moreover, the gene encoding the Aurora A kinase (AURKA) maps to the chromosome region 20q13, which is frequently amplified in many human cancers. The overexpression of AURKA induces centrosome amplification and aneuploidy. It also confers resistance to taxol-dependent apoptosis in cancer cells. In this context, it is also interesting to note that AurA interacts with and inactivates the tumor suppressor p53, and that it also interacts with the breast cancer susceptibility gene BRCA1 and colocalizes with it at the centrosome. Given that the overexpression of AurA has been shown to cause tumorigenic transformation of human and rodent cells in vitro and in vivo [72], it has been proposed that AurA acts as an oncogene.

kinase in vitro, and most of them have one or more sequences that conform to a consensus motif for phosphorylation by AurA. In all cases, these sites are located outside the TACC domain (Figure 1, Table 1) [41,42,45,47,52,53]. One of them is conserved in several TACC orthologs, indicating that it is functionally important [45,52,53]. There is strong experimental support in several systems (i.e. nematode, fly, frog and human cells) indicating that phosphorylation has a role in TACC function at the centrosome [8,41,42,47,53]. Experiments with purified proteins have shown that Maskin can bind simultaneously to AurA and XMAP215, suggesting that the binding sites for these two proteins do not overlap (Figure 1) [42]. Moreover, phosphorylation does not appear to have any positive or negative influence on these interactions. Indeed, in Xenopus egg extract, XMAP215 is pulled down as efficiently by phosphorylated wild type Maskin as it is by an unphosphorylatable, mutated Maskin protein [41]. Finally, AurA does not enhance the microtubule binding affinity achieved by the Maskin–XMAP215 complex in comparison with Maskin or XMAP215 proteins in isolation [41]. All of these data suggest that phosphorylation does not regulate the 385

Review interaction or functionality of TACC–XMAP215 complexes. It also seems unlikely that phosphorylation induces a change in the oligomerization state of Maskin, because Maskin and unphosphorylatable mutants of Maskin are found at the same position on sucrose gradients of various protein combinations [41]. What then does phosphorylation regulate? One possibility is that it regulates the turnover of the protein at the centrosome, favoring the retention of the phosphorylated form. Another possibility is that a direct interaction with the AurA is involved, resulting in the phosphorylation of the TACC protein at the centrosome. In fact, in Drosophila, phosphorylation of D-TACC seems to be required not for its localization to the centrosome but, rather, to enable it to load at the microtubule minus-ends [53]. However, the inhibition of AurA kinase activity by a small molecule results in TACC3 failing to localize to centrosomes in human cells [45], suggesting that kinase activity rather than the protein itself is required. In any case, various data support the idea that AurA regulates the centrosomal recruitment of TACC. Immunofluorescence studies performed with an antibody against the phosphorylated form of TACC3 have shown that phosphorylated TACC proteins are mostly found at the centrosome in HeLa cells and Drosophila embryos [41,53,54]. Consistent with this, a non-phosphorylatable mutant of TACC3 does not localize efficiently to the centrosome in transfected HeLa cells or in Xenopus egg extracts [41,53,54]. In Drosophila embryos, a similar, although less pronounced, effect is observed for the localization of a non-phosphorylatable mutant of D-TACC [53]. In further agreement, TACC proteins do not localize to the centrosome in AurA mutants in C. elegans or Drosophila, and there is a strong reduction in the association of Maskin with spindles assembled in Xenopus egg extracts depleted of AurA [8,42,47]. However, these observations cannot exclude the possibility that these mislocalizations are attributable to indirect effects on either centrosome content or functionality due to the absence of this important centrosomal kinase. Overall, AurA phosphorylation of TACC proteins appears to contribute substantially to their recruitment to or accumulation at the centrosome, and, as a consequence, to the recruitment of ch-TOG/XMAP215 (Figure 2) [41]. The precise molecular mechanism involved remains to be elucidated. In this context, it is noteworthy that alternative pathways to target TACC proteins to the centrosome have been described recently. Indeed, the centrosomal localization of D-TACC relies on its interaction with motif 1 of the Drosophila protein centrosomin [55], and the dualspecificity kinase TTK is essential for the centrosomal localization of human TACC2 [56]. Recently, it was also reported that the centrosomal protein NDEL1 (also known as NUDEL), another substrate of AurA, is required for the targeting of TACC3 to the centrosome in human cells [57]. Interestingly, NDEL1 is a binding partner of LIS1, a protein that participates in the regulation of cytoplasmic dynein function and microtubule organization during cell division and neuronal migration [58]. NDEL1 is also related to microtubule-remodeling mechanisms through the recruitment of the microtubule-severing factor katanin [59]. 386

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Clearly, additional work is needed to address some key questions and to understand how TACC proteins function to promote the assembly of microtubules. Conclusions TACC proteins have recently emerged as important players in the complex process of regulating microtubule dynamics during cell division. Although it is now clearly established that they have a major role at the centrosome – promoting microtubule elongation together with ch-TOG/ XMAP215 proteins – the molecular mechanism underlying their activity is still unclear. Solid data support the fact that they interact with, and are substrates of, the kinase AurA; but, again, although phosphorylation is essential for their localization to and function at the centrosome, the molecular mechanism involved is far from being understood. In fact, it is noteworthy that TACC proteins also function in pathways that do not involve the centrosome. Indeed, D-TACC and Msps both localize at the acentrosomal poles of Drosophila female meiotic spindles, and both are required for maintaining the bipolarity of these spindles [50]. In addition, some data from frog and fission yeast support the idea that TACC proteins participate in the RanGTP-dependent spindle assembly pathway [39,40,60]. Furthermore, in the fission yeast S. pombe, Alp7 functions in the maintenance of microtubule organization during interphase [61]. It is therefore very likely that TACC proteins have a more general role in microtubule stabilization than currently appreciated. Finally, it is still unclear whether the quite distinct roles of TACC proteins in different aspects of gene regulation and in microtubule assembly are connected in some way. In this context, it is noteworthy that recent studies have revealed that different classes of RNAs are associated with the mitotic spindle [62,63]. Interestingly, both Maskin and AurA are involved in the regulation of mRNA translation in Xenopus oocytes [26]. It is quite apparent that much remains to be done to understand more clearly how TACC proteins function. In a wider context, it will be interesting to elucidate the functional connections between their roles in microtubule stabilization and in RNA control. In addition, future work should reveal whether their role is only restricted to events occurring during cell division. As a final remark, it should be noted that the study of TACC proteins is starting to offer a variety of promising applications related to cancer therapies. Recently, it was shown that monitoring phospho-TACC3 is an efficient way of evaluating the effectiveness of AurA inhibitors that are promising anti-cancer drugs, some of which have recently entered clinical trials [45]. The observation that TACC3 depletion sensitizes cells to paclitaxel-induced cell death also suggests that TACC3 itself is a promising target for the treatment of the tumors resistant to this widely used therapy [64]. Finally, TACC3 is also emerging as a good prognostic marker for some cancers [20]. Acknowledgements We thank all members of the Vernos Laboratory, especially Luis Bejarano, Teresa Sardon and Roser Pinyol, for critically reading the manuscript and providing helpful comments. We thank P. Go¨nczy (ISREC, CH) and J. Raff (The Gurdon Institute, UK) for the kind gift of

Review the immunofluorescence images of C. elegans and D. melanogaster embryos, respectively. We also thank Christoph Spinzig for creative suggestions. Work in the I.V. laboratory is supported by the CRG, the European Union MRTN/CT 2004 512348 and the Spanish Ministry grants BFU2006–04694 and CSD2006–0023.

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