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EB1-Binding–Myomegalin Protein Complex Promotes Centrosomal Microtubules Functions

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EB1-Binding–Myomegalin Protein Complex Promotes Centrosomal Microtubules Functions EB1-binding–myomegalin protein complex promotes PNAS PLUS centrosomal microtubules functions Habib Bougueninaa, Danièle Salauna,1, Aurélie Mangona,1, Leslie Mullerb, Emilie Baudeleta, Luc Camoina, Taro Tachibanac, Sarah Cianféranib, Stéphane Audeberta, Pascal Verdier-Pinarda,2, and Ali Badachea,2 aCentre de Recherche en Cancérologie de Marseille (CRCM), INSERM, Institut Paoli-Calmettes, CNRS, Aix-Marseille Université,13009 Marseille, France; bLaboratoire de Spectrométrie de Masse BioOrganique, CNRS, Institut Pluridisciplinaire Hubert Curien UMR 7178, Université de Strasbourg, 67087 Strasbourg, France; and cDepartment of Bioengineering, Graduate School of Engineering, Osaka City University, Osaka 558-8585, Japan Edited by Andrew J. Holland, Johns Hopkins University Medical School, Baltimore, MD, and accepted by Editorial Board Member Tak W. Mak October 28, 2017 (received for review April 6, 2017) Control of microtubule dynamics underlies several fundamental investigating the mechanism through which the ErbB2 receptor processes such as cell polarity, cell division, and cell motility. To gain tyrosine kinase controls breast tumor cell motility, we character- insights into the mechanisms that control microtubule dynamics ized a signaling pathway whereby the ErbB2 effector Memo con- during cell motility, we investigated the interactome of the microtu- trols the relocalization to the plasma membrane of a protein bule plus-end–binding protein end-binding 1 (EB1). Via molecular complex comprising APC and ACF7 that captures and stabilizes mapping and cross-linking mass spectrometry we identified and char- MT plus-ends at the cell periphery (10–12), thereby regulating acterized a large complex associating a specific isoform of myomega- directed cell motility (13, 14). To further characterize the mech- “ ” lin termed SMYLE (for short myomegalin-like EB1 binding protein), anisms underlying MT capture, we aimed to define the MT plus- the PKA scaffolding protein AKAP9, and the pericentrosomal protein end–associated protein network by characterizing the EB1 inter- CDK5RAP2. SMYLE was associated through an evolutionarily con- actome. We identified an EB1-interacting isoform of myomegalin served N-terminal domain with AKAP9, which in turn was anchored that assembled a macromolecular complex at the centrosome and at the centrosome via CDK5RAP2. SMYLE connected the pericentro- defined its function in microtubule assembly, directed cell motility, somal complex to the microtubule-nucleating complex (γ-TuRC) via Galectin-3–binding protein. SMYLE associated with nascent centroso- and orientation of cell division. mal microtubules to promote microtubule assembly and acetylation. Results Disruption of SMYLE interaction with EB1 or AKAP9 prevented mi- The EB1-Associated Network Includes MTOC-Associated Proteins. crotubule nucleation and their stabilization at the leading edge of β migrating cells. In addition, SMYLE depletion led to defective astral Upon addition of heregulin (HRG), SKBr3 cells extend wide, flat protrusions populated by MTs that extend from the microtubules and abnormal orientation of the mitotic spindle and centrosomal area to the cell periphery. For further insights into triggered G1 cell-cycle arrest, which might be due to defective cen- MT plus-end complexes involved in MT extension and mainte- trosome integrity. As a consequence, SMYLE loss of function had a nance within cell protrusions, we investigated the EB1 protein- profound impact on tumor cell motility and proliferation, suggesting interaction network via affinity purification mass spectrometry. that SMYLE might be an important player in tumor progression. microtubules | centrosome | mitotic spindle | cell motility Significance he regulation of the microtubule (MT) network is central to Microtubule dynamics is tightly regulated during fundamental Tmany fundamental biological processes, including cell po- biological processes such as mitosis, thereby representing a major larization, cell motility, and mitosis. MTs are polarized tubulin target for anticancer therapies. To better understand the molec- polymers that assemble from MT organizing centers (MTOCs) at ular mechanisms underlying the organization of the microtubule network, we systematically investigated proteins interacting with centrosomal and noncentrosomal sites and grow radially until EB1, a major regulator of microtubules dynamics. We identified a reaching stabilizing structures. Because MT nucleation is a ki- specific isoform of myomegalin, which we termed “SMYLE,” that netically unfavorable process, in cells it often occurs from tem- γ γ assembles a macromolecular complex associated with the cen- plates such as the -tubulin ring complex ( -TuRC), a multiprotein trosome, the major microtubule-organizing center in cells, and complex in which proteins of the γ-tubulin complex organize γ also connected to the microtubule nucleating complex. SMYLE -tubulin into a ring (1). Nucleation is regulated by factors that promoted microtubule assembly from the centrosome and sub- γ α β attach the -TuRCs to the MTOCs (2). Dimers of - tubulin are sequent stabilization of microtubules at the cell periphery. This then processively assembled into MTs through a highly dynamic had consequences on cell motility, mitosis, and cell-cycle pro- process, with MTs constantly alternating between phases of gression, suggesting that SMYLE might be an important player growth and shortening while exploring the cytoplasmic space (3). in tumor progression. MT stability is regulated by MT-associated proteins that bind to the MT lattice; stable MTs often carry posttranslational modifi- Author contributions: A.B. designed research; H.B., D.S., A.M., L.M., E.B., L.C., S.A., and cations, such as detyrosination and acetylation (4). Recent studies P.V.-P. performed research; T.T. contributed new reagents/analytic tools; H.B., D.S., A.M., L.M., L.C., S.C., S.A., P.V.-P., and A.B. analyzed data; and H.B., P.V.-P., and A.B. wrote indicate that tubulin acetylation contributes to regulating micro- the paper. tubule architecture and mechanics (5–7). MT dynamics are con- The authors declare no conflict of interest. trolled by MT plus-end tracking proteins (+TIPs), a family of This article is a PNAS Direct Submission. A.J.H. is a guest editor invited by the unrelated proteins, including EB1/2/3, CLASP1/2, CLIP170, the Editorial Board. tumor suppressor APC, and the spectraplakin ACF7, that form Published under the PNAS license. comet-shaped structures at MT distal ends (8). End-binding 1 1D.S. and A.M. contributed equally to this work. (EB1) is considered a major binding hub for CAP-Gly domain or 2 + To whom correspondence may be addressed. Email: [email protected] or SxIP motif-containing TIPs forming a complex protein- [email protected]. interaction network that was proposed to regulate MT dynamics This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. CELL BIOLOGY and interactions with peripheral stabilizing structures (9). While 1073/pnas.1705682114/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1705682114 PNAS | Published online November 21, 2017 | E10687–E10696 Downloaded by guest on September 29, 2021 SKBr3 cells stably expressing moderate amounts of EB1-GFP dem mass spectrometry (Fig. S1D) revealed that the 1,116-aa-long displayed typical EB1 tip tracking (Fig. S1 A–C). GFP-trap myomegalin-like protein identified here (UniProt accession no. pulldown from EB1-GFP cell lysates followed by label-free mass E9PL24) is clearly distinct from the originally identified 2,346-aa spectrometry analysis revealed a group of proteins that robustly myomegalin (UniProt accession no. Q5VU43; Human Genome associated with EB1 (Fig. 1A and Dataset S1). Of note, HRG Organization Gene Nomenclature Committee: phosphodiesterase treatment did not significantly modify the EB1 interactome 4D-interacting protein, PDE4DIP) (15). It has a specific 373-aa (Dataset S1). Many proteins contained the typical SxIP motifs N-terminal sequence lacking the CM1 motif, aligns with PDE4DIP known to contribute to EB1 binding, suggesting that they are di- from residues 374–1,102, lacks the phosphodiesterase 4D-binding rect EB1 interactors. We identified EB3, known to dimerize with region, and has a unique 14-aa C-terminal sequence (Fig. 1B and EB1, and several known MT plus-end–binding proteins, such as Fig. S1D). The protein, which we termed “SMYLE” (for short KIF2C/MCAK, dystonin, and NAV1. Unexpectedly, among the myomegalin-like EB1-binding protein) to prevent confusion with best interactors we identified a set of proteins previously known to the original long isoform, differs from the previously described be associated with MTOCs, in particular the cAMP-dependent Golgi-associated EB–MMG at its C terminus (16) but is identical PKA scaffold AKAP9 (also called “AKAP350” or “AKAP450”), to the Golgi-associated protein (MMG8) identified by Wang et al. regulatory and catalytic subunits of PKA, the pericentrosomal (17). Extensive analysis of SMYLE sequences across distant spe- matrix protein CDK5RAP2, and a specific isoform of PDE4DIP/ cies through homologous sequence alignments, secondary struc- myomegalin. Sequence analysis of the peptides identified by tan- ture prediction, and computation of conservation scores revealed CD2AP SPAG5 A Syntabulin* B PDE4DIP PDE4D binding TAO Kinase 2 * CM1 PKP3 Dystonin* 1001 1670 1 2346 SMYLE-specific KIF2C* SMYLE JUP CLASP2 * C-terminus GAS2L1* Smy SxIP EB3 H APC * 1 1116 Coiled-coil (ANNIE) chTOG GlobDoms/Disordered
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