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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
* 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).
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.
. Numerical and structural abnormalities of centrosomes are associated with aneuploidy, chromosomal instability and transformation, developmental defects, apoptotic cell death, and cell cycle arrest through induction of premature senescence
A crucial regulator of TACC3 is the mitotic kinase Aurora-A that phosphorylates TACC3 (pTACC3) and thereby determines its differential centrosomal/proximal spindle (pTACC3) versus distal spindle MT (TACC3) localization during (pro)metaphase
). Family members, which comprise XMAP215 in Xenopus laevis, Msps in Drosophila melanogaster, and chTOG/CKAP5 (cytoskeleton associated protein 5) in Homo sapiens, are identified by the presence of several “TOG” domains involved in MT binding.
TACC proteins are structurally characterized by a rather variable N-terminal region of which the approximately first 100 residues are uniquely conserved among vertebrate TACC3 isoforms. Further features include a central serine-proline-glutamate-rich repeat region (
), 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 (
). 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 (
) 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.
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
), 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. (
) 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 (
). 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) (
). Consistently, CC2 may be involved not only in intradomain but also in intermolecular protein interactions, whereas CC1 may only undergo intermolecular effector binding.
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 (
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 (
). 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 (
) 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) (
). 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 (
). 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) (
). 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 (
). 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 (
). 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 (
). 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) (
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.
Thakur, H. C., (2012) Biochemical and biophysical characterization of the centrosomal protein TACC3. Doctoral dissertation, Faculty of Mathematics and Natural Sciences of the Heinrich Heine University, Düsseldorf, Germany,