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The Centrosomal Adaptor TACC3 and the Microtubule Polymerase chTOG Interact via Defined C-terminal Subdomains in an Aurora-A Kinase-independent Manner*

  • Harish C. Thakur
    Footnotes
    Affiliations
    Institut für Biochemie und Molekularbiologie II, Medizinische Fakultät der Heinrich-Heine-Universität, D-40225 Düsseldorf, Germany
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  • Madhurendra Singh
    Affiliations
    Institut für Biochemie und Molekularbiologie II, Medizinische Fakultät der Heinrich-Heine-Universität, D-40225 Düsseldorf, Germany
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  • Luitgard Nagel-Steger
    Affiliations
    Institut für Physikalische Biologie, Heinrich-Heine-Universität, 40225 Düsseldorf, Germany

    ICS-6, Strukturbiochemie, Forschungszentrum, 52425 Jülich, Germany
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  • Jana Kremer
    Affiliations
    Institut für Biochemie und Molekularbiologie II, Medizinische Fakultät der Heinrich-Heine-Universität, D-40225 Düsseldorf, Germany
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  • Daniel Prumbaum
    Affiliations
    Max-Planck-Institut für Molekulare Physiologie, 44227 Dortmund, Germany
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  • Eyad Kalawy Fansa
    Footnotes
    Affiliations
    Institut für Biochemie und Molekularbiologie II, Medizinische Fakultät der Heinrich-Heine-Universität, D-40225 Düsseldorf, Germany
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  • Hakima Ezzahoini
    Affiliations
    Institut für Biochemie und Molekularbiologie II, Medizinische Fakultät der Heinrich-Heine-Universität, D-40225 Düsseldorf, Germany
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  • Kazem Nouri
    Affiliations
    Institut für Biochemie und Molekularbiologie II, Medizinische Fakultät der Heinrich-Heine-Universität, D-40225 Düsseldorf, Germany
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  • Lothar Gremer
    Affiliations
    Institut für Physikalische Biologie, Heinrich-Heine-Universität, 40225 Düsseldorf, Germany

    ICS-6, Strukturbiochemie, Forschungszentrum, 52425 Jülich, Germany
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  • André Abts
    Affiliations
    Institut für Biochemie, Heinrich-Heine-Universität, 40225 Düsseldorf, Germany
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  • Lutz Schmitt
    Affiliations
    Institut für Biochemie, Heinrich-Heine-Universität, 40225 Düsseldorf, Germany
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  • Stefan Raunser
    Affiliations
    Max-Planck-Institut für Molekulare Physiologie, 44227 Dortmund, Germany
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  • Mohammad R. Ahmadian
    Footnotes
    Affiliations
    Institut für Biochemie und Molekularbiologie II, Medizinische Fakultät der Heinrich-Heine-Universität, D-40225 Düsseldorf, Germany
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  • Roland P. Piekorz
    Correspondence
    To whom correspondence should be addressed: Institut für Biochemie und Molekularbiologie II, Universitätsklinikum der Heinrich-Heine-Universität, D-40225 Düsseldorf, Germany. Tel.: 49-211-81-12739; Fax: 49-211-81-12726
    Affiliations
    Institut für Biochemie und Molekularbiologie II, Medizinische Fakultät der Heinrich-Heine-Universität, D-40225 Düsseldorf, Germany
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  • Author Footnotes
    * This work was supported by a fellowship of the NRW (North Rhine-Westphalia) graduate school “BioStruct; Biological Structures in Molecular Medicine and Biotechnology” (to H. C. T.), the Deutsche Forschungsgemeinschaft (SFB 728/TP A5;to R. P. P.), the research commission of the medical faculty of the Heinrich-Heine-University (grants to R. P. P. and M. R. A.), the strategic research fund of the Heinrich-Heine-University (grants to R. P. P. and M. R. A.), and the International Graduate School of Protein Science and Technology (iGRASP; to K. N. and M. R. A.).
    This article contains supplemental Table S1 and Figs. S1–S11.
    1 Present address: Wellcome Trust Centre for Cell Biology, The University of Edinburgh, Edinburgh EH9 3JR, Scotland, United Kingdom.
    2 Supported by Bundesministerium für Bildung und Forschung (NGFNplus program Grant 01GS08100).
Open AccessPublished:November 22, 2013DOI:https://doi.org/10.1074/jbc.M113.532333
      The cancer-associated, centrosomal adaptor protein TACC3 (transforming acidic coiled-coil 3) and its direct effector, the microtubule polymerase chTOG (colonic and hepatic tumor overexpressed gene), play a crucial function in centrosome-driven mitotic spindle assembly. It is unclear how TACC3 interacts with chTOG. Here, we show that the C-terminal TACC domain of TACC3 and a C-terminal fragment adjacent to the TOG domains of chTOG mediate the interaction between these two proteins. Interestingly, the TACC domain consists of two functionally distinct subdomains, CC1 (amino acids (aa) 414–530) and CC2 (aa 530–630). Whereas CC1 is responsible for the interaction with chTOG, CC2 performs an intradomain interaction with the central repeat region of TACC3, thereby masking the TACC domain before effector binding. Contrary to previous findings, our data clearly demonstrate that Aurora-A kinase does not regulate TACC3-chTOG complex formation, indicating that Aurora-A solely functions as a recruitment factor for the TACC3-chTOG complex to centrosomes and proximal mitotic spindles. We identified with CC1 and CC2, two functionally diverse modules within the TACC domain of TACC3 that modulate and mediate, respectively, TACC3 interaction with chTOG required for spindle assembly and microtubule dynamics during mitotic cell division.

      Introduction

      The centrosome represents the main microtubule (MT)
      The abbreviations used are:
      MT
      microtubule
      aa
      amino acid
      SEC
      size exclusion chromatography
      aSEC
      analytical SEC
      CBB
      Coomassie Brilliant Blue
      CC
      coiled-coil
      chTOG
      colonic and hepatic tumor overexpressed gene
      ITC
      isothermal titration calorimetry
      7R
      serine-proline-glutamate-rich repeat region
      TACC
      transforming acidic coiled-coil
      XMAP215
      Xenopus microtubule associated protein 215 kDa
      ARNT
      aryl hydrocarbon receptor nuclear translocator.
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      ,
      • Lauffart B.
      • Howell S.J.
      • Tasch J.E.
      • Cowell J.K.
      • Still I.H.
      Interaction of the transforming acidic coiled-coil 1 (TACC1) protein with ch-TOG and GAS41/NuBI1 suggests multiple TACC1-containing protein complexes in human cells.
      ,
      • Sato M.
      • Vardy L.
      • Angel Garcia M.
      • Koonrugsa N.
      • Toda T.
      Interdependency of fission yeast Alp14/TOG and coiled coil protein Alp7 in microtubule localization and bipolar spindle formation.
      ), thereby targeting them to spindle poles. In contrast, the functional role of the N-terminal part of TACC3 outside of the TACC domain is rather undefined besides being a substrate for Aurora-A-mediated phosphorylation that is required for centrosomal and proximal spindle localization of TACC3 (
      • Peset I.
      • Vernos I.
      The TACC proteins. TACC-ling microtubule dynamics and centrosome function.
      ).
      From the analysis of X. laevis TACC3, it has been proposed that the N-terminal part masks the TACC domain and thereby inhibits its function (
      • Albee A.J.
      • Tao W.
      • Wiese C.
      Phosphorylation of maskin by Aurora-A is regulated by RanGTP and importin β.
      ,
      • Albee A.J.
      • Wiese C.
      Xenopus TACC3/maskin is not required for microtubule stability but is required for anchoring microtubules at the centrosome.
      ). Aurora-A mediated phosphorylation of TACC3 was implicated to “unmask” and thereby expose the TACC domain to intermolecular interaction with XMAP215 (
      • Peset I.
      • Seiler J.
      • Sardon T.
      • Bejarano L.A.
      • Rybina S.
      • Vernos I.
      Function and regulation of Maskin, a TACC family protein, in microtubule growth during mitosis.
      ,
      • Albee A.J.
      • Wiese C.
      Xenopus TACC3/maskin is not required for microtubule stability but is required for anchoring microtubules at the centrosome.
      ). However, the molecular basis/details of the masking/unmasking mechanism of the TACC domain and its interaction with the C terminus of XMAP215 remained enigmatic. Here, we subjected recombinant murine TACC3 and the C-terminal part of the murine XMAP215 homologue chTOG to a deletion and biochemical interaction analysis. We identify within the TACC domain two functionally distinct subdomains, CC1 (aa 414–530) and CC2 (aa 530–630), which are involved in interdomain and intradomain protein interaction, respectively. We demonstrate that TACC3 forms a stable intramolecular complex through the interaction of 7R with CC2 (TACC domain “masked”). Interestingly, the C terminus of chTOG (aa 1806–2032) right hand to the putative MT-interacting TOG6 domain (
      • Hood F.E.
      • Williams S.J.
      • Burgess S.G.
      • Richards M.W.
      • Roth D.
      • Straube A.
      • Pfuhl M.
      • Bayliss R.
      • Royle S.J.
      Coordination of adjacent domains mediates TACC3-ch-TOG-clathrin assembly and mitotic spindle binding.
      ) binds selectively to the CC1 module and thereby disrupts the intramolecular CC2–7R complex, thereby giving rise to the effector-bound state of the TACC domain (TACC domain “unmasked”). Neither intradomain interaction of TACC3 nor its binding to chTOG was affected by Aurora-A kinase. Thus, consecutive intra- and intermolecular protein interactions direct and determine TACC3-chTOG protein complex formation before its Aurora-A-regulated centrosomal and proximal spindle recruitment required for MT growth and mitotic spindle assembly.

      DISCUSSION

      This study provides novel molecular insight into the basis of spindle MT stability and dynamics during mitosis by determining the interaction between the centrosomal adaptor protein TACC3 and the MT polymerase chTOG. The main findings of our work are as follows. 1) The C-terminal TACC domain of TACC3 consists of two functionally distinct modules, CC1 and CC2. 2) CC2 performs an intradomain interaction with the central repeat region (7R), a complex that masks intermolecular interaction of TACC3. 3) chTOG directly binds CC1 via a C-terminal fragment adjacent to N-terminal MT binding TOG domains. 4) Aurora-A kinase, a major regulator of TACC3, does not interfere with TACC3-chTOG complex formation either in vitro or in vivo. 5) Thus, Aurora-A solely acts as a centrosomal/proximal spindle recruitment factor for the TACC3-chTOG complex consistent with previous findings
      • Barros T.P.
      • Kinoshita K.
      • Hyman A.A.
      • Raff J.W.
      Aurora A activates D-TACC-Msps complexes exclusively at centrosomes to stabilize centrosomal microtubules.
      • Kinoshita K.
      • Noetzel T.L.
      • Pelletier L.
      • Mechtler K.
      • Drechsel D.N.
      • Schwager A.
      • Lee M.
      • Raff J.W.
      • Hyman A.A.
      Aurora A phosphorylation of TACC3/maskin is required for centrosome-dependent microtubule assembly in mitosis.
      • LeRoy P.J.
      • Hunter J.J.
      • Hoar K.M.
      • Burke K.E.
      • Shinde V.
      • Ruan J.
      • Bowman D.
      • Galvin K.
      • Ecsedy J.A.
      Localization of human TACC3 to mitotic spindles is mediated by phosphorylation on Ser-558 by Aurora A. A novel pharmacodynamic method for measuring Aurora A activity.
      • Peset I.
      • Seiler J.
      • Sardon T.
      • Bejarano L.A.
      • Rybina S.
      • Vernos I.
      Function and regulation of Maskin, a TACC family protein, in microtubule growth during mitosis.
      .
      Our data argue against the possibility that the evolutionary conserved interaction between TACC3 and chTOG family members, as observed by several groups (
      • Hood F.E.
      • Royle S.J.
      Pulling it together. The mitotic function of TACC3.
      ,
      • Gergely F.
      • Draviam V.M.
      • Raff J.W.
      The ch-TOG/XMAP215 protein is essential for spindle pole organization in human somatic cells.
      ,
      • Albee A.J.
      • Tao W.
      • Wiese C.
      Phosphorylation of maskin by Aurora-A is regulated by RanGTP and importin β.
      ,
      • Albee A.J.
      • Wiese C.
      Xenopus TACC3/maskin is not required for microtubule stability but is required for anchoring microtubules at the centrosome.
      ,
      • Cassimeris L.
      • Morabito J.
      TOGp, the human homolog of XMAP215/Dis1, is required for centrosome integrity, spindle pole organization, and bipolar spindle assembly.
      ), requires the complete TACC domain. By analyzing the TACC3-chTOG protein complex, we define CC1 as an chTOG interacting domain. Moreover, we show that the deletion mutant TACC3-ΔCC1, in contrast to TACC3-ΔCC2, fails to co-immunoprecipitate/interact with chTOG in vivo (Fig. 5). Our findings are consistent with recent work of Hood et al. (
      • Hood F.E.
      • Williams S.J.
      • Burgess S.G.
      • Richards M.W.
      • Roth D.
      • Straube A.
      • Pfuhl M.
      • Bayliss R.
      • Royle S.J.
      Coordination of adjacent domains mediates TACC3-ch-TOG-clathrin assembly and mitotic spindle binding.
      ) that has analyzed the interaction of human TACC3 and chTOG isoforms using a deletion mapping approach. The authors narrowed down the corresponding human CC1 domain to a short region of 12 amino acids (aa 673–684) that appears to be sufficient for chTOG binding and chTOG localization on spindle MTs in vivo (
      • Hood F.E.
      • Williams S.J.
      • Burgess S.G.
      • Richards M.W.
      • Roth D.
      • Straube A.
      • Pfuhl M.
      • Bayliss R.
      • Royle S.J.
      Coordination of adjacent domains mediates TACC3-ch-TOG-clathrin assembly and mitotic spindle binding.
      ). Interestingly, centrosomal localization of chTOG was apparently reduced but still detectable, further indicating that chTOG may be recruited to centrosomes via both TACC3-dependent and -independent mechanisms.
      As indicated in our model (Fig. 9), the mutually exclusive intradomain 7R-CC2 and interdomain CC1-chTOG interactions, respectively, provide novel functional insight into the subdomain selectivity and directionality of TACC3-chTOG complex formation. Our findings obtained by ITC analysis (Figs. 3E and 4E) are of particular relevance by providing clear insights into differential binding affinities for a strong chTOG-Cterm-CC1 interaction versus a weak 7R-CC2 interaction. Accordingly, we propose that chTOG binding to CC1 results in a conformational change of the CC2 subdomain, which is in turn released from its intramolecular complex with 7R and hence unmasks both CC2 and the central repeat region of TACC3. As a consequence, not only CC2, but also 7R may become available for further interactions with other downstream binding partners. However, in the latter case, no protein is currently known that binds to the central repeat domain of TACC3 despite the presence of bona fide PXXP binding motifs known to interact with SH3 domain-containing proteins in intracellular signaling processes. This is different for the TACC domain that has been identified by yeast two hybrid-based screening as well as pulldown and immunoprecipitation assays as major binding partner for various, functionally rather diverse proteins. These include factors involved in cortical neurogenesis (Cep192, DOCK7) (
      • Xie Z.
      • Moy L.Y.
      • Sanada K.
      • Zhou Y.
      • Buchman J.J.
      • Tsai L.-H.
      Cep120 and TACCs control interkinetic nuclear migration and the neural progenitor pool.
      ,
      • Yang Y.T.
      • Wang C.L.
      • Van Aelst L.
      DOCK7 interacts with TACC3 to regulate interkinetic nuclear migration and cortical neurogenesis.
      ), hematopoietic development (FOG-1) (
      • Garriga-Canut M.
      • Orkin S.H.
      Transforming acidic coiled-coil protein 3 (TACC3) controls friend of GATA-1 (FOG-1) subcellular localization and regulates the association between GATA-1 and FOG-1 during hematopoiesis.
      ), hypoxia response and gene expression (ARNT) (
      • Partch C.L.
      • Gardner K.H.
      Coactivators necessary for transcriptional output of the hypoxia inducible factor, HIF, are directly recruited by ARNT PAS-B.
      ), transcriptional regulation (MBD2) (
      • Angrisano T.
      • Lembo F.
      • Pero R.
      • Natale F.
      • Fusco A.
      • Avvedimento V.E.
      • Bruni C.B.
      • Chiariotti L.
      TACC3 mediates the association of MBD2 with histone acetyltransferases and relieves transcriptional repression of methylated promoters.
      ), and regulation of mTOR signaling (TSC2) (
      • Gómez-Baldó L.
      • Schmidt S.
      • Maxwell C.A.
      • Bonifaci N.
      • Gabaldón T.
      • Vidalain P.O.
      • Senapedis W.
      • Kletke A.
      • Rosing M.
      • Barnekow A.
      • Rottapel R.
      • Capellá G.
      • Vidal M.
      • Astrinidis A.
      • Piekorz R.P.
      • Pujana M.A.
      TACC3-TSC2 maintains nuclear envelope structure and controls cell division.
      ). Interestingly, FOG-1 and ARNT have been proposed to bind to a region containing the last 20 residues of the CC2 subdomain (
      • Partch C.L.
      • Gardner K.H.
      Coactivators necessary for transcriptional output of the hypoxia inducible factor, HIF, are directly recruited by ARNT PAS-B.
      ,
      • Simpson R.J.
      • Yi Lee S.H.
      • Bartle N.
      • Sum E.Y.
      • Visvader J.E.
      • Matthews J.M.
      • Mackay J.P.
      • Crossley M.
      A classic zinc finger from friend of GATA mediates an interaction with the coiled-coil of transforming acidic coiled-coil 3.
      ). Consistently, CC2 may be involved not only in intradomain but also in intermolecular protein interactions, whereas CC1 may only undergo intermolecular effector binding.
      Figure thumbnail gr9
      FIGURE 9Integrative model for the domain specificity and directionality of TACC3-chTOG complex formation. We propose that the intradomain (7R-CC2; TACC3 masked) and intermolecular (CC1-chTOG; TACC3 unmasked) binding states of TACC3 are mutually exclusive, thereby defining a directionality of TACC3 regulated adaptor function toward chTOG binding and function. TACC3 can be phosphorylated by Aurora-A kinase in both binding states, indicating that Aurora-A acts as a centrosomal recruitment factor but is not involved in exposing the TACC domain for intermolecular interaction with chTOG or TACC3-chTOG complex formation. Moreover, based on biophysical characterization of murine TACC3 (; Ref.
      • Thakur H.C.
      • Singh M.
      • Nagel-Steger L.
      • Prumbaum D.
      • Fansa E.K.
      • Gremer L.
      • Ezzahoini H.
      • Abts A.
      • Schmitt L.
      • Raunser S.
      • Ahmadian M.R.
      • Piekorz R.P.
      Role of centrosomal adaptor proteins of the TACC family in the regulation of microtubule dynamics during mitotic cell division.
      ) we conclude that TACC3 displays an dimeric to oligomeric state. Thr., thrombin.
      Aurora-A-mediated phosphorylation of TACC3 seems not to interfere with TACC3 intradomain and TACC3-chTOG interdomain interactions under in vitro conditions (Fig. 8). Accordingly, in vivo, TACC3-chTOG interaction and centrosomal colocalization was still detectable in HeLa cells that have been subjected to treatment with the Aurora-A kinase inhibitor MLN8237 (supplemental Figs. S8 and S9). These findings also contradict the previous model proposing that Aurora-A-mediated phosphorylation of X. laevis TACC3 triggers unmasking of the TACC domain and thereby exposes it for intermolecular interaction (i.e. XMAP215 binding) and centrosomal targeting (
      • Peset I.
      • Seiler J.
      • Sardon T.
      • Bejarano L.A.
      • Rybina S.
      • Vernos I.
      Function and regulation of Maskin, a TACC family protein, in microtubule growth during mitosis.
      ,
      • Albee A.J.
      • Wiese C.
      Xenopus TACC3/maskin is not required for microtubule stability but is required for anchoring microtubules at the centrosome.
      ). In fact, Aurora-A-mediated phosphorylation of TACC3 seems to be solely required for targeting of the TACC3-chTOG complex to centrosomes and spindle MTs (
      • Peset I.
      • Vernos I.
      The TACC proteins. TACC-ling microtubule dynamics and centrosome function.
      ,
      • Kinoshita K.
      • Noetzel T.L.
      • Pelletier L.
      • Mechtler K.
      • Drechsel D.N.
      • Schwager A.
      • Lee M.
      • Raff J.W.
      • Hyman A.A.
      Aurora A phosphorylation of TACC3/maskin is required for centrosome-dependent microtubule assembly in mitosis.
      ). In the latter case, pTACC-chTOG interacts with another key effector in mitotic spindle assembly, the clathrin heavy chain, thereby cross-linking and stabilizing MT bundles (
      • Hood F.E.
      • Royle S.J.
      Pulling it together. The mitotic function of TACC3.
      ,
      • Booth D.G.
      • Hood F.E.
      • Prior I.A.
      • Royle S.J.
      A TACC3/ch-TOG/clathrin complex stabilises kinetochore fibres by inter-microtubule bridging.
      ,
      • Hood F.E.
      • Williams S.J.
      • Burgess S.G.
      • Richards M.W.
      • Roth D.
      • Straube A.
      • Pfuhl M.
      • Bayliss R.
      • Royle S.J.
      Coordination of adjacent domains mediates TACC3-ch-TOG-clathrin assembly and mitotic spindle binding.
      ).
      Based on this study a sequential function of TACC3-chTOG effector complexes in the course of mitosis can be proposed. TACC3 interacts with the C terminus of chTOG thereby targeting it in an Aurora-A-dependent manner to spindle poles. On the other hand, the evolutionary conserved N terminus of chTOG likely comprises MT-stabilizing activity as demonstrated for XMAP215. In particular, XMAP215/chTOG proteins contain a variable number of TOG domains that bind to αβ-tubulin heterodimers, load them as MT polymerase (
      • Brouhard G.J.
      • Stear J.H.
      • Noetzel T.L.
      • Al-Bassam J.
      • Kinoshita K.
      • Harrison S.C.
      • Howard J.
      • Hyman A.A.
      XMAP215 is a processive microtubule polymerase.
      ) to the plus ends of MTs, and thereby inhibit “MT catastrophes.” In contrast, the C-terminal part of XMAP215 (and likely also chTOG) suppresses MT growth by promoting MT catastrophes (
      • Popov A.V.
      • Pozniakovsky A.
      • Arnal I.
      • Antony C.
      • Ashford A.J.
      • Kinoshita K.
      • Tournebize R.
      • Hyman A.A.
      • Karsenti E.
      XMAP215 regulates microtubule dynamics through two distinct domains.
      ). Therefore, the engagement of chTOG-Cterm by the CC1 subdomain of TACC3 during G2/M transition and metaphase might be a vital step in shifting the equilibrium toward MT polymerization. Upon mitotic exit, Cdh1 and ubiquitin-dependent degradation of TACC3 (
      • Jeng J.C.
      • Lin Y.M.
      • Lin C.H.
      • Shih H.M.
      Cdh1 controls the stability of TACC3.
      ) then “disengages” the MT catastrophe promoting activity of the C terminus of XMAP215/chTOG. As a consequence, a shift of the equilibrium occurs toward MT “shrinkage” and disassembly of the spindle apparatus. Thus, TACC3 family members may function as “engagement factors” for the C terminus of XMAP215/chTOG to ensure a dynamic balance between MT rescue and catastrophe during the course of mitosis.
      Besides a better molecular understanding regarding the mechanism and directionality of TACC3-chTOG interaction, we furthermore obtained novel insight into the unusual biophysical properties of TACC3. Analysis by aSEC (e.g. Fig. 1) clearly demonstrated that TACC3 displays a higher oligomeric mass and/or an elongated rod-like structure, obviously due to the presence of the coiled-coil containing TACC domain that elutes inherently at an apparent molecular mass of ∼630 kDa (Fig. 3B). Moreover, endogenous TACC3 or FLAG-tagged TACC3 from transfected eukaryotic cells behaves on aSEC comparable to purified TACC3 (data not shown). These findings are in accordance with the observation that TACC isoforms overexpressed in HeLa cells form in a TACC domain-dependent manner punctuate-like structures resembling cytoplasmic polymers (data not shown) (
      • Gergely F.
      • Karlsson C.
      • Still I.
      • Cowell J.
      • Kilmartin J.
      • Raff J.W.
      The TACC domain identifies a family of centrosomal proteins that can interact with microtubules.
      ). Employing further analytical methods including multiangle light scattering and analytical ultracentrifugation allowed us to conclude that TACC3 is characterized by a oligomeric (i.e. dimeric to hexameric) structure and a highly extended shape (supplemental Fig. 11, B and C, and supplemental Table S1). These findings are consistent with data from electron microscopic analysis where TACC3 depicts an elongated, fiber-like appearance (
      • Thakur H.C.
      • Singh M.
      • Nagel-Steger L.
      • Prumbaum D.
      • Fansa E.K.
      • Gremer L.
      • Ezzahoini H.
      • Abts A.
      • Schmitt L.
      • Raunser S.
      • Ahmadian M.R.
      • Piekorz R.P.
      Role of centrosomal adaptor proteins of the TACC family in the regulation of microtubule dynamics during mitotic cell division.
      ). Another abnormality of murine TACC3 represents its migration in SDS-PAGE gels at 120–130 kDa (Fig. 1C) as compared with its theoretical molecular mass of 70.5 kDa. Interestingly, this unusual “gel shifting” is not based on the presence of the coiled-coil containing TACC domain (data not shown) but is rather caused by the central repeat region (supplemental Fig. S4, B versus A). As proof, deletion of the 7R domain restored normal gel migration of TACC3 (Fig. 3C and supplemental Fig. S4B). Of note, abnormal SDS-PAGE migration of acidic proteins can be caused by an altered binding of surfactants (like SDS) (
      • Shi Y.
      • Mowery R.A.
      • Ashley J.
      • Hentz M.
      • Ramirez A.J.
      • Bilgicer B.
      • Slunt-Brown H.
      • Borchelt D.R.
      • Shaw B.F.
      Abnormal SDS-PAGE migration of cytosolic proteins can identify domains and mechanisms that control surfactant binding.
      ), a possibility that remains to be clarified for TACC3 and in particular the 7R domain.
      Biological and pathobiological roles of TACC3 are underlined by several observations. TACC3 deficiency leads to severe growth retardation and embryonic lethality (
      • Piekorz R.P.
      • Hoffmeyer A.
      • Duntsch C.D.
      • McKay C.
      • Nakajima H.
      • Sexl V.
      • Snyder L.
      • Rehg J.
      • Ihle J.N.
      The centrosomal protein TACC3 is essential for hematopoietic stem cell function and genetically interfaces with p53-regulated apoptosis.
      ,
      • Yao R.
      • Natsume Y.
      • Noda T.
      TACC3 is required for the proper mitosis of sclerotome mesenchymal cells during formation of the axial skeleton.
      ). This is in line with the anti-proliferative and cell cycle arrest/senescence-inducing impact of shRNA-mediated gene silencing of TACC3 (
      • Schmidt S.
      • Schneider L.
      • Essmann F.
      • Cirstea I.C.
      • Kuck F.
      • Kletke A.
      • Jänicke R.U.
      • Wiek C.
      • Hanenberg H.
      • Ahmadian M.R.
      • Schulze-Osthoff K.
      • Nürnberg B.
      • Piekorz R.P.
      The centrosomal protein TACC3 controls paclitaxel sensitivity by modulating a premature senescence program.
      ,
      • Schneider L.
      • Essmann F.
      • Kletke A.
      • Rio P.
      • Hanenberg H.
      • Schulze-Osthoff K.
      • Nürnberg B.
      • Piekorz R.P.
      TACC3 depletion sensitizes to paclitaxel-induced cell death and overrides p21WAF-mediated cell cycle arrest.
      ). Moreover, it could be shown that TACC3 depletion sensitizes cells to the apoptotic and senescence-inducing effects of mitotic spindle poisons. Accordingly, inducible gene disruption of TACC3 in vivo in the p53−/− sarcolymphoma model is highly effective in causing apoptotic tumor regression (
      • Yao R.
      • Natsume Y.
      • Saiki Y.
      • Shioya H.
      • Takeuchi K.
      • Yamori T.
      • Toki H.
      • Aoki I.
      • Saga T.
      • Noda T.
      Disruption of Tacc3 function leads to in vivo tumor regression.
      ). Interestingly, besides quantitative deregulation of gene expression of TACC isoforms in several tumor types, TACC1 and TACC3 point mutants have been identified in melanoma and ovarian cancer patients (
      • Hodis E.
      • Watson I.R.
      • Kryukov G.V.
      • Arold S.T.
      • Imielinski M.
      • Theurillat J.P.
      • Nickerson E.
      • Auclair D.
      • Li L.
      • Place C.
      • Dicara D.
      • Ramos A.H.
      • Lawrence M.S.
      • Cibulskis K.
      • Sivachenko A.
      • Voet D.
      • Saksena G.
      • Stransky N.
      • Onofrio R.C.
      • Winckler W.
      • Ardlie K.
      • Wagle N.
      • Wargo J.
      • Chong K.
      • Morton D.L.
      • Stemke-Hale K.
      • Chen G.
      • Noble M.
      • Meyerson M.
      • Ladbury J.E.
      • Davies M.A.
      • Gershenwald J.E.
      • Wagner S.N.
      • Hoon D.S.
      • Schadendorf D.
      • Lander E.S.
      • Gabriel S.B.
      • Getz G.
      • Garraway L.A.
      • Chin L.
      A landscape of driver mutations in melanoma.
      ,
      • Lauffart B.
      • Vaughan M.M.
      • Eddy R.
      • Chervinsky D.
      • DiCioccio R.A.
      • Black J.D.
      • Still I.H.
      Aberrations of TACC1 and TACC3 are associated with ovarian cancer.
      ). Moreover, oncogenic fusions between TACC and fibroblast growth factor receptor genes have been recently described in glioblastoma multiforme and bladder cancer patients (
      • Singh D.
      • Chan J.M.
      • Zoppoli P.
      • Niola F.
      • Sullivan R.
      • Castano A.
      • Liu E.M.
      • Reichel J.
      • Porrati P.
      • Pellegatta S.
      • Qiu K.
      • Gao Z.
      • Ceccarelli M.
      • Riccardi R.
      • Brat D.J.
      • Guha A.
      • Aldape K.
      • Golfinos J.G.
      • Zagzag D.
      • Mikkelsen T.
      • Finocchiaro G.
      • Lasorella A.
      • Rabadan R.
      • Iavarone A.
      Transforming fusions of FGFR and TACC genes in human glioblastoma.
      ,
      • Williams S.V.
      • Hurst C.D.
      • Knowles M.A.
      Oncogenic FGFR3 gene fusions in bladder cancer.
      ). The impact of these structural and tumor-associated alterations on Aurora-A-mediated regulation and function of TACCs is currently unknown and requires a more in-depth molecular understanding of TACC-effector interactions. Irrespective, it is tempting to speculate that these TACC mutants translate through loss-of-function or gain-of-function mechanisms into chromosomal instability and aneuploidy and thereby support cellular transformation (
      • Vitre B.D.
      • Cleveland D.W.
      Centrosomes, chromosome instability (CIN) and aneuploidy.
      ,
      • Holland A.J.
      • Cleveland D.W.
      Losing balance. The origin and impact of aneuploidy in cancer.
      ). Taken all these points into account, TACC3 represents an attractive antitumor target that may be at least indirectly drug-treatable at the level of its interactome. This assumption is supported by the recent identification of small drugs that act as inhibitors of protein-protein interaction and thereby impair the half-life and stability of TACC3 (KSH101), disrupt the TACC3-ARNT complex (KG-548), or inhibit the function of the TACC3-chTOG complex (spindlactone) (
      • Yao R.
      • Kondoh Y.
      • Natsume Y.
      • Yamanaka H.
      • Inoue M.
      • Toki H.
      • Takagi R.
      • Shimizu T.
      • Yamori T.
      • Osada H.
      • Noda T.
      A small compound targeting TACC3 revealed its different spatiotemporal contributions for spindle assembly in cancer cells.
      ,
      • Partch C.L.
      • Gardner K.H.
      Coactivators necessary for transcriptional output of the hypoxia inducible factor, HIF, are directly recruited by ARNT PAS-B.
      ,
      • Guo Y.
      • Partch C.L.
      • Key J.
      • Card P.B.
      • Pashkov V.
      • Patel A.
      • Bruick R.K.
      • Wurdak H.
      • Gardner K.H.
      Regulating the ARNT/TACC3 axis. Multiple approaches to manipulating protein/protein interactions with small molecules.
      ,
      • Wurdak H.
      • Zhu S.
      • Min K.H.
      • Aimone L.
      • Lairson L.L.
      • Watson J.
      • Chopiuk G.
      • Demas J.
      • Charette B.
      • Halder R.
      • Weerapana E.
      • Cravatt B.F.
      • Cline H.T.
      • Peters E.C.
      • Zhang J.
      • Walker J.R.
      • Wu C.
      • Chang J.
      • Tuntland T.
      • Cho C.Y.
      • Schultz P.G.
      A small molecule accelerates neuronal differentiation in the adult rat.
      ).

      Acknowledgments

      We thank Britta Tschapek for help in setting up the multiangle light scattering method and Jürgen Scheller and members of the Institute of Biochemistry and Molecular Biology II for input and fruitful comments during the course of this work and on the manuscript.

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