The trimeric coiled‐coil HSBP 1 protein promotes WASH complex assembly at centrosomes
2018; Springer Nature; Volume: 37; Issue: 13 Linguagem: Inglês
10.15252/embj.201797706
ISSN1460-2075
AutoresSai Prasanna Visweshwaran, Peter A. Thomason, Raphaël Guérois, Sophie Vacher, Evgeny V. Denisov, Л. А. Таширева, Maria E. Lomakina, Christine Lazennec‐Schurdevin, Goran Lakisic, Sérgio Lilla, Nicolas Molinié, Véronique Henriot, Yves Mechulam, Antonina Y. Alexandrova, Н. В. Чердынцева, Ivan Bièche, Emmanuelle Schmitt, Robert H. Insall, Alexis Gautreau,
Tópico(s)DNA Repair Mechanisms
ResumoArticle29 May 2018free access Source DataTransparent process The trimeric coiled-coil HSBP1 protein promotes WASH complex assembly at centrosomes Sai P Visweshwaran Ecole Polytechnique, CNRS UMR7654, Université Paris-Saclay, Palaiseau, France Search for more papers by this author Peter A Thomason Beatson Institute for Cancer Research, Bearsden, UK Search for more papers by this author Raphael Guerois orcid.org/0000-0001-5294-2858 Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Université Paris-Saclay, Gif-sur-Yvette, France Search for more papers by this author Sophie Vacher Pharmacogenomics Unit, Department of Genetics, Institut Curie, Paris, France Search for more papers by this author Evgeny V Denisov orcid.org/0000-0003-2923-9755 Laboratory of Molecular Oncology and Immunology, Cancer Research Institute, Tomsk National Research Medical Center, Tomsk, Russia Laboratory for Translational Cellular and Molecular Biomedicine, Tomsk State University, Tomsk, Russia Search for more papers by this author Lubov A Tashireva Department of General and Molecular Pathology, Cancer Research Institute, Tomsk National Research Medical Center, Tomsk, Russia Search for more papers by this author Maria E Lomakina Institute of Carcinogenesis, N.N. Blokhin Cancer Research Center, Moscow, Russia Search for more papers by this author Christine Lazennec-Schurdevin Ecole Polytechnique, CNRS UMR7654, Université Paris-Saclay, Palaiseau, France Search for more papers by this author Goran Lakisic Ecole Polytechnique, CNRS UMR7654, Université Paris-Saclay, Palaiseau, France Search for more papers by this author Sergio Lilla Beatson Institute for Cancer Research, Bearsden, UK Search for more papers by this author Nicolas Molinie Ecole Polytechnique, CNRS UMR7654, Université Paris-Saclay, Palaiseau, France Search for more papers by this author Veronique Henriot Ecole Polytechnique, CNRS UMR7654, Université Paris-Saclay, Palaiseau, France Search for more papers by this author Yves Mechulam Ecole Polytechnique, CNRS UMR7654, Université Paris-Saclay, Palaiseau, France Search for more papers by this author Antonina Y Alexandrova orcid.org/0000-0001-5071-2290 Institute of Carcinogenesis, N.N. Blokhin Cancer Research Center, Moscow, Russia Search for more papers by this author Nadezhda V Cherdyntseva Laboratory of Molecular Oncology and Immunology, Cancer Research Institute, Tomsk National Research Medical Center, Tomsk, Russia Search for more papers by this author Ivan Bièche Pharmacogenomics Unit, Department of Genetics, Institut Curie, Paris, France Search for more papers by this author Emmanuelle Schmitt Ecole Polytechnique, CNRS UMR7654, Université Paris-Saclay, Palaiseau, France Search for more papers by this author Robert H Insall Beatson Institute for Cancer Research, Bearsden, UK Search for more papers by this author Alexis Gautreau Corresponding Author [email protected] orcid.org/0000-0002-2369-4362 Ecole Polytechnique, CNRS UMR7654, Université Paris-Saclay, Palaiseau, France School of Biological and Medical Physics, Moscow Institute of Physics and Technology, Dolgoprudny, Russia Search for more papers by this author Sai P Visweshwaran Ecole Polytechnique, CNRS UMR7654, Université Paris-Saclay, Palaiseau, France Search for more papers by this author Peter A Thomason Beatson Institute for Cancer Research, Bearsden, UK Search for more papers by this author Raphael Guerois orcid.org/0000-0001-5294-2858 Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Université Paris-Saclay, Gif-sur-Yvette, France Search for more papers by this author Sophie Vacher Pharmacogenomics Unit, Department of Genetics, Institut Curie, Paris, France Search for more papers by this author Evgeny V Denisov orcid.org/0000-0003-2923-9755 Laboratory of Molecular Oncology and Immunology, Cancer Research Institute, Tomsk National Research Medical Center, Tomsk, Russia Laboratory for Translational Cellular and Molecular Biomedicine, Tomsk State University, Tomsk, Russia Search for more papers by this author Lubov A Tashireva Department of General and Molecular Pathology, Cancer Research Institute, Tomsk National Research Medical Center, Tomsk, Russia Search for more papers by this author Maria E Lomakina Institute of Carcinogenesis, N.N. Blokhin Cancer Research Center, Moscow, Russia Search for more papers by this author Christine Lazennec-Schurdevin Ecole Polytechnique, CNRS UMR7654, Université Paris-Saclay, Palaiseau, France Search for more papers by this author Goran Lakisic Ecole Polytechnique, CNRS UMR7654, Université Paris-Saclay, Palaiseau, France Search for more papers by this author Sergio Lilla Beatson Institute for Cancer Research, Bearsden, UK Search for more papers by this author Nicolas Molinie Ecole Polytechnique, CNRS UMR7654, Université Paris-Saclay, Palaiseau, France Search for more papers by this author Veronique Henriot Ecole Polytechnique, CNRS UMR7654, Université Paris-Saclay, Palaiseau, France Search for more papers by this author Yves Mechulam Ecole Polytechnique, CNRS UMR7654, Université Paris-Saclay, Palaiseau, France Search for more papers by this author Antonina Y Alexandrova orcid.org/0000-0001-5071-2290 Institute of Carcinogenesis, N.N. Blokhin Cancer Research Center, Moscow, Russia Search for more papers by this author Nadezhda V Cherdyntseva Laboratory of Molecular Oncology and Immunology, Cancer Research Institute, Tomsk National Research Medical Center, Tomsk, Russia Search for more papers by this author Ivan Bièche Pharmacogenomics Unit, Department of Genetics, Institut Curie, Paris, France Search for more papers by this author Emmanuelle Schmitt Ecole Polytechnique, CNRS UMR7654, Université Paris-Saclay, Palaiseau, France Search for more papers by this author Robert H Insall Beatson Institute for Cancer Research, Bearsden, UK Search for more papers by this author Alexis Gautreau Corresponding Author [email protected] orcid.org/0000-0002-2369-4362 Ecole Polytechnique, CNRS UMR7654, Université Paris-Saclay, Palaiseau, France School of Biological and Medical Physics, Moscow Institute of Physics and Technology, Dolgoprudny, Russia Search for more papers by this author Author Information Sai P Visweshwaran1, Peter A Thomason2, Raphael Guerois3, Sophie Vacher4, Evgeny V Denisov5,6, Lubov A Tashireva7, Maria E Lomakina8, Christine Lazennec-Schurdevin1, Goran Lakisic1, Sergio Lilla2, Nicolas Molinie1, Veronique Henriot1, Yves Mechulam1, Antonina Y Alexandrova8, Nadezhda V Cherdyntseva5, Ivan Bièche4, Emmanuelle Schmitt1, Robert H Insall2 and Alexis Gautreau *,1,9 1Ecole Polytechnique, CNRS UMR7654, Université Paris-Saclay, Palaiseau, France 2Beatson Institute for Cancer Research, Bearsden, UK 3Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Université Paris-Saclay, Gif-sur-Yvette, France 4Pharmacogenomics Unit, Department of Genetics, Institut Curie, Paris, France 5Laboratory of Molecular Oncology and Immunology, Cancer Research Institute, Tomsk National Research Medical Center, Tomsk, Russia 6Laboratory for Translational Cellular and Molecular Biomedicine, Tomsk State University, Tomsk, Russia 7Department of General and Molecular Pathology, Cancer Research Institute, Tomsk National Research Medical Center, Tomsk, Russia 8Institute of Carcinogenesis, N.N. Blokhin Cancer Research Center, Moscow, Russia 9School of Biological and Medical Physics, Moscow Institute of Physics and Technology, Dolgoprudny, Russia *Corresponding author. Tel: +33 169334870; E-mail: [email protected] EMBO J (2018)37:e97706https://doi.org/10.15252/embj.201797706 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract The Arp2/3 complex generates branched actin networks that exert pushing forces onto different cellular membranes. WASH complexes activate Arp2/3 complexes at the surface of endosomes and thereby fission transport intermediates containing endocytosed receptors, such as α5β1 integrins. How WASH complexes are assembled in the cell is unknown. Here, we identify the small coiled-coil protein HSBP1 as a factor that specifically promotes the assembly of a ternary complex composed of CCDC53, WASH, and FAM21 by dissociating the CCDC53 homotrimeric precursor. HSBP1 operates at the centrosome, which concentrates the building blocks. HSBP1 depletion in human cancer cell lines and in Dictyostelium amoebae phenocopies WASH depletion, suggesting a critical role of the ternary WASH complex for WASH functions. HSBP1 is required for the development of focal adhesions and of cell polarity. These defects impair the migration and invasion of tumor cells. Overexpression of HSBP1 in breast tumors is associated with increased levels of WASH complexes and with poor prognosis for patients. Synopsis WASH activates the Arp2/3 complex at the endosomal surface. To perform its function, WASH needs first to be assembled at centrosomes into a functional trimeric complex by a conserved assembly factor. The small coiled coil protein HSBP1 specifically promotes the assembly of a ternary complex composed of CCDC53, WASH and FAM21. HSBP1 localizes to and operates at centrosomes. HSBP1 depletion in human cancer cells phenocopies WASH depletion, affecting focal adhesions, cell polarity and cancer cell migration. Overexpression of HSBP1 is associated with increased levels of WASH complexes and poor prognosis of breast cancer patients. Introduction Cells use branched actin networks to control their shape, to power cell migration, and to drive membrane remodeling in intracellular traffic (Rotty et al, 2013). The Arp2/3 complex is the stable multiprotein complex that generates branched actin networks. It contains two actin-related proteins, Arp2 and Arp3, and five other subunits that maintain the two actin-related proteins associated. When activated by the WCA domain of so-called nucleation promoting factors (NPFs), the Arp2/3 complex creates an actin branch (Pollard, 2007): It associates with a pre-existing actin filament and nucleates a new filament from its two subunits, Arp2 and Arp3, brought into close contact, thus mimicking a filament end (Rouiller et al, 2008). Such a multiprotein complex can be referred to as a molecular machine to highlight the coordinated work it performs (Alberts, 1998). No function has been ascribed to Arp2 or Arp3 in isolation, outside of the Arp2/3 complex. Nucleation promoting factors activates the Arp2/3 complex at different subcellular locations: WAVE at the lamellipodium edge, where the branched actin network provides the force for membrane protrusion (Rotty et al, 2013), and WASH at the surface of endosomes, where the force generated by the branched actin network contributes to the scission of transport intermediates (Derivery et al, 2009b; Gomez & Billadeau, 2009). These transport intermediates either follow the retrograde route toward the Golgi (Gomez & Billadeau, 2009; Harbour et al, 2010) or recycle internalized receptors to the plasma membrane (Temkin et al, 2011; Piotrowski et al, 2013). The α5β1 integrin is a cargo that takes the two WASH-dependent routes, since it is recycled to the plasma membrane both from endosomes and after a detour through the trans-Golgi network (Zech et al, 2011; Duleh & Welch, 2012; De Franceschi et al, 2015; Shafaq-Zadah et al, 2016; Nagel et al, 2017). WAVE and WASH are both stably associated with four other proteins, which integrate inputs to control WCA exposure as an output (Derivery & Gautreau, 2010b; Rotty et al, 2013). The endosomal recruitment of the WASH complex depends on the cargo-recognition complex of the retromer (Harbour et al, 2012; Jia et al, 2012; Helfer et al, 2013; Gautreau et al, 2014). Formation of branched actin networks thus involves cascades of molecular machines. How cells assemble these molecular machines from neosynthesized subunits is not known in most cases. Indeed, molecular machines are not just simple assemblies driven by the spontaneous association of subunits. The simple stepwise addition of subunits or subcomplexes yields a WAVE complex, where the WCA domain is not properly masked (Innocenti et al, 2004; Derivery et al, 2009a). The reconstitution of a native WAVE complex from recombinant subunits was a tour-de-force, which required a decade of work (Chen et al, 2014). In fact, in the cell, proteasomes exert a quality control and degrade up to 30% of neosynthesized proteins (Schubert et al, 2000). When one subunit of WAVE, WASH or Arp2/3 complexes is depleted, remaining subunits of the same molecular machine are usually degraded by proteasomes (Kunda et al, 2003; Steffen et al, 2006; Derivery et al, 2009a,b; Jia et al, 2010). Conversely, when an exogenous, usually tagged, subunit is overexpressed, the endogenous subunit is degraded, because its partner subunits have been titrated by the more abundant exogenous protein (Derivery et al, 2009a,b). These observations suggest that subunits need to assemble with their partner subunits to reach their native state and become stable (Derivery & Gautreau, 2010b). In the case of the WAVE complex, one subunit, Brk1, forms a homotrimeric precursor, even though only a single Brk1 subunit is present in the native complex (Derivery et al, 2008; Linkner et al, 2011). Brk1 turns over more rapidly than the WAVE complex, suggesting that the two Brk1 molecules that should remain after dissociation of the trimeric precursor are also degraded (Derivery & Gautreau, 2010b; Wu et al, 2012). Large molecular machines, like proteasomes, require many factors for their assembly (Sahara et al, 2014; Budenholzer et al, 2017). Assembly factors transiently associate with one or several subunits, but eventually dissociate before the machine is completed. If they were to remain associated, they would be subunits. It is not yet established whether assembly factors are systematically required for the formation of smaller molecular machines, like the Arp2/3 or NPF complexes. So far, an assembly factor was only identified in the case of the WAVE complex: The Nudel protein, which transiently interacts with two subcomplexes, is critical to maintain WAVE complex levels and thus to form lamellipodia (Wu et al, 2012). Assembly factors offer a means to control the levels of assembled complexes. For example, starvation induces the expression of proteasome assembly factors and hence favors the assembly of new proteasomes to promote the degradation of old proteins, thereby allowing biosynthesis of new proteins in amino acid-restricted conditions (Rousseau & Bertolotti, 2016). The Arp2/3 and the WAVE complexes are overexpressed in a variety of cancers (Molinie & Gautreau, 2018). This overexpression is usually associated with a high-grade, lymph node invasion and poor prognosis for patients (Semba et al, 2006; Iwaya et al, 2007). Since most subunits of these molecular machines are only stable within the whole complex, it means that invasive tumor cells managed to assemble more of these machines, but the mechanism involved is not known. The WASH complex, which allows focal delivery of metalloproteases and integrin recycling, is critical for tumor cell invasion (Zech et al, 2011; Monteiro et al, 2013), but whether its expression is deregulated in tumors is not known. Here, we identify the first assembly factor of the WASH complex, HSBP1, and characterize how it promotes WASH assembly. We found that HSBP1 is overexpressed in breast cancer and that its overexpression is associated with increased levels of WASH complex and poor survival of patients. Results Identification of HSBP1 as an assembly factor of the WASH complex To look for putative assembly factors of the WASH complex, we derived stable 293 cell lines expressing tagged subunits of the WASH complex. We noticed a small molecular weight protein in the immunoprecipitates of CCDC53, but not of WASH, nor of Strumpellin (Fig 1A). We identified this protein as HSBP1 by mass spectrometry. The gene encoding this small protein of 76 amino acids (8.5 kDa of predicted mass) was cloned and transiently co-expressed with each subunit of the WASH complex in 293T cells. Indeed, out of the five subunits of the WASH complex, CCDC53, WASH, Strumpellin, SWIP, and FAM21, only CCDC53 interacted with HSBP1 (Fig 1B). HSBP1 is thus a possible assembly factor, because it binds to a single subunit, and not to the whole WASH complex. Figure 1. HSBP1 is a specific partner of the CCDC53 subunit of the WASH complex Identification of HSBP1. 293 stable cell lines expressing PC-mCherry tagged subunits of the WASH complex were subjected to PC immunoprecipitations. Immunoprecipitates were resolved by SDS–PAGE and stained with colloidal Coomassie. *indicates the precipitated bait. °indicates the position of a faint protein partner, which appears to co-immunoprecipitate with CCDC53, but not with the other WASH subunits. This protein was identified as HSBP1 by mass spectrometry. M, molecular weight markers in kDa. 293T cells were transiently transfected with plasmids expressing FLAG-HSBP1 and PC-GFP tagged subunits of the WASH complex as indicated. GFP precipitation of WASH complex subunits confirms the specific co-immunoprecipitation of HSBP1 with the CCDC53 subunit. His-tagged full-length CCDC53 and HSBP1 were purified from Escherichia coli. Purity was assessed by Coomassie staining. SEC-MALS analysis of CCDC53, HSBP1 or of a complex of the two proteins. CCDC53 and HSBP1 proteins are both trimeric. When mixed, His-tagged CCDC53 and untagged HSBP1 spontaneously form a heterotrimer that contains a single molecule of the CCDC53 subunit of the WASH complex. Source data are available online for this figure. Source Data for Figure 1 [embj201797706-sup-0006-SDataFig1.pdf] Download figure Download PowerPoint To examine whether HSBP1 directly binds to CCDC53, we produced and purified these two recombinant proteins in Escherichia coli (Fig 1C). HSBP1 was previously crystallized, and the HSBP1 core is formed by a trimeric coiled coil (Liu et al, 2009). HSBP1 was, however, described as a hexamer, because the asymmetric unit of the crystal contains two trimeric coiled coils associated end-to-end. In this structure, the two trimers differ by the conformation of a bulky residue in their coiled coil, phenylalanine 27 (F27; Appendix Fig S1A). The end-to-end association of two HSBP1 trimers is a crystal-induced contact, because we found that HSBP1 in solution behaves as a homotrimer, when its mass is evaluated by size exclusion chromatography coupled to multi-angle light scattering (SEC-MALS; Fig 1D). The structure of CCDC53 was not previously characterized. We found by SEC-MALS that free CCDC53 also behaves as a homotrimer (Fig 1D). The situation is reminiscent of the distantly related Brk1 subunit of the WAVE complex (Jia et al, 2010): Free Brk1 exists as a homotrimer, even though a single subunit of Brk1 is present in the WAVE complex (Derivery et al, 2008). When CCDC53 and HSBP1 were mixed, we detected a new peak by SEC-MALS, which surprisingly corresponded to a mixed heterotrimer, containing two HSBP1 for a single CCDC53 (Fig 1D). So not only HSBP1 directly binds to CCDC53, but also this interaction remodels the quaternary structure of each component. Trimeric HSBP1 displays a distorted coiled coil. This distortion most likely arises from the accommodation of the bulky F27, which creates a steric hindrance (Appendix Fig S1A). Such a bulky residue at the d position of a coiled-coil heptad is highly unusual in trimeric coiled coils (Woolfson, 2005). However, F27 is strictly conserved in all HSBP1 homologs (Appendix Fig S1B). To better understand the reaction occurring between HSBP1 and CCDC53, we modeled the structure of free CCDC53 according to the crystal structure of free Brk1 (Linkner et al, 2011). CCDC53, whose name stands for coiled-coil domain-containing protein 53, can be modeled as a trimeric coiled-coil protein, like Brk1 (Fig 2A). Then, we used the two previous structural models of HSBP1 and CCDC53 to model the mixed HSBP1-CCDC53 heterotrimer. In heptads 4 and 5 of the mixed heterotrimer, we noticed that the single strand of CCDC53 brings two optimal electrostatic couples with the two strands contributed by HSBP1 (Fig 2B). These salt bridges formed between residues E(e)-K/R(g') of heptads were identified as the most stabilizing motifs in experimental studies of model trimeric coiled coils (Roy & Case, 2009). The residues involved are strongly conserved in the sequence alignments of both HSBP1 and CCDC53 (Appendix Fig S1B). These two stabilizing salt bridges probably contribute to the spontaneous assembly of the mixed HSBP1-CCDC53 heterotrimer. Figure 2. Structural model of the CCDC53-HSBP1 heterotrimer Ribbon representation of trimeric coiled-coil assemblies formed by HSBP1, CCDC53, or the mixed heterotrimer. The HSBP1 homotrimer comes from its X-ray structure (PDB code 3CI9). The CCDC53 homotrimer is modeled based on the X-ray structure of Brk1, the distantly related subunit in the WAVE complex. The CCDC53-HSBP1 heterotrimer with 1:2 stoichiometry is modeled based on the two previous structures. Focus on the coiled-coil heptads of the CCDC53-HSBP1 heterotrimer. Residues buried in the trimer core of each heptad (labeled from h1 to h5) and residues that form salt bridges are shown as sticks. The E-K/R salt bridges that were shown in model systems to stabilize trimeric coiled coils are highlighted by a green asterisk in heptads 4 and 5. Download figure Download PowerPoint To evaluate the potential role of HSBP1 in the assembly of the WASH complex, we isolated MDA-MB-231 clones stably depleted of HSBP1 and analyzed their content in WASH complex subunits. Upon HSBP1 knock-down, steady state levels of CCDC53 and WASH, but not those of FAM21 and Strumpellin, were significantly decreased (Fig 3A). The effect of HSBP1 depletion was the same in HeLa cells upon transient transfection of siRNAs (Appendix Fig S2). The subunits of stable multiprotein complexes usually depend on each other for their stability. For the WASH complex, the depletion of a "large" subunit, such as Strumpellin, SWIP, or FAM21, induces the degradation of all remaining subunits, whereas the depletion of a "small" subunit, such as WASH or CCDC53, induces the degradation of the other small subunit, leaving intact levels of large subunits (Jia et al, 2010). Upon HSBP1 depletion, the co-dependent CCDC53 and WASH subunits are thus specifically downregulated. Figure 3. HSBP1 promotes the assembly of a ternary complex containing CCDC53, WASH, and FAM21 MDA-MB-231 clones stably transfected with plasmids expressing control or HSBP1 targeting shRNAs were obtained. HSBP1-depleted cells (shHSBP1) display significant downregulation of the CCDC53 and WASH subunits compared to control cells (shCtrl). Mean ± s.e.m. of densitometric signals; three independent experiments; ANOVA, *P < 0.05, **P < 0.01. Cytosolic extracts from control or HSBP1-depleted cells were fractionated by ultracentrifugation in sucrose gradients, and fractions were analyzed by Western blot with the indicated antibodies. Three color-coded pools containing CCDC53 were detected in control cells, whereas only the higher molecular weight pool was detected in HSBP1-depleted cells. CCDC53 immunoprecipitations of the three pools from control cells were analyzed by Western blot using the indicated antibodies. IgG refers to the control immunoprecipitation performed with non immune IgG. Structural model of WASH complex assembly. HSBP1 promotes WASH complex assembly by dissociating the CCDC53 homotrimeric precursor and delivering a single CCDC53 molecule to the ternary complex composed of CCDC53, WASH, and FAM21. Based on the analogous structure of the WAVE complex, a new heterotrimeric coiled coil within WASH complexes, where CCDC53, WASH, and FAM21 contribute one strand each is proposed. Source data are available online for this figure. Source Data for Figure 3 [embj201797706-sup-0007-SDataFig3.pdf] Download figure Download PowerPoint To characterize WASH subcomplexes, we fractionated cytosolic extracts by ultracentrifugation in sucrose gradients and analyzed the distribution of subunits in each fraction by Western blot (Fig 3B). In control MDA-MB-231 cells, we detected three distinct pools of WASH complex subunits. The low pool P1 was detected at 3.0 S, an intermediate pool P2 was detected at 8.8 S, and a high pool P3 was detected at 12.5 S, which corresponds to the previously determined sedimentation coefficient of the pentameric WASH complex (Derivery et al, 2009b). The P1 pool corresponds to the complex of CCDC53 with HSBP1, as determined by immunoprecipitation (Fig 3C). The P2 pool corresponds to a ternary complex containing CCDC53, WASH, and FAM21. The distribution of FAM21 in P2 is much more extended than the ones of WASH and CCDC53, suggesting that only part of FAM21 is associated with WASH and CCDC53 in the ternary complex. When HSBP1 is depleted, P1 and P2 pools of CCDC53, but surprisingly not the P3 pool, are absent. WASH is also depleted from the P2 ternary complex, but not FAM21, in line with its extended distribution. These distributions suggest that HSBP1 promotes the conversion of the CCDC53-HSBP1 heterotrimer into the CCDC53-WASH-FAM21 ternary complex. To confirm the role of HSBP1 in promoting WASH assembly, we derived a stable MDA-MB-231 cell line that overexpresses tagged WASH. The increased WASH levels were associated with increased levels of CCDC53, but not of FAM21 and Strumpellin (Fig EV1A). When tagged WASH was overexpressed, the endogenous WASH was downregulated, as observed previously (Derivery et al, 2009b), suggesting that the levels of partner subunits control the total amount of WASH. In this experiment, WASH was tagged with both FLAG and HaloTag sequences. The HaloTag covalently reacts with haloalkane reactive ligands. A chemical compound called HaloPROTAC3 induces the specific degradation of HaloTagged proteins through the recruitment of the VHL E3 ubiquitin ligase (Buckley et al, 2015). When cells expressing HaloTagged WASH were incubated with 1 μM HaloPROTAC3 for 24 h, levels of tagged WASH and CCDC53 significantly decreased, while endogenous WASH reappeared (Fig EV1A). Click here to expand this figure. Figure EV1. HSBP1 is required for CCDC53 assembly into the WASH complex The stable MDA-MB-231 cell line expressing FLAG-HaloTag-WASH (FHT-WASH) was analyzed by Western blot. Overexpression of tagged WASH induces upregulation of CCDC53, whereas FAM21 and Strumpellin levels are not affected. Tagged WASH replaces the endogenous WASH in the WASH complex. Stable MDA-MB-231 cells expressing FHT-WASH were treated for 24 h with HaloPROTAC3, a small molecule that degrades HaloTag tagged proteins (Buckley et al, 2015). FHT-WASH degradation induced CCDC53 downregulation and reappearance of endogenous WASH. FAM21 and Strumpellin levels were not affected. HaloPROTAC3 wash-out for the indicated time allows to monitor subunit buildup around FHT-WASH by FLAG immunoprecipitations. Upon HSBP1 depletion, the association of WASH with CCDC53 is delayed. Source data are available online for this figure. Download figure Download PowerPoint To monitor the assembly of the WASH complex, we washed out the HaloPROTAC3 compound to resume expression of HaloTagged WASH and to follow its assembly into WASH complexes by immunoprecipitation. Using this procedure, we were able to detect the buildup of WASH and associated subunits over several hours (Fig EV1B). Upon HSBP1 depletion, HaloTagged WASH associated with a reduced amount of CCDC53, but with normal amounts of FAM21 and Strumpellin, in line with the disappearance of the ternary complex (P2), but the maintenance of the pentameric complex (P3), seen in sucrose gradients. To conceive better the sequence of events in WASH complex assembly, we then built a structural model of the pentameric WASH complex, based on the crystal structure of the analogous WAVE complex (Chen et al, 2010). In this model, WASH, CCDC53, and FAM21 interact through a heterotrimeric coiled coil (Fig 3D). It should be stated that the model of the WASH complex is not as robust as the one of CCDC53-HSBP1 heterotrimer, because apart from a predicted coiled-coil segment in its N-terminus, the structure of FAM21 cannot be accurately predicted. This model suggests, however, a molecular scenario where CCDC53 must be dissociated from a precursor homotrimeric form to contribute a single subunit to the ternary complex. HSBP1, which dissociates the free homotrimer of CCDC53, would deliver this single CCDC53 to WASH and FAM21. Phenotype associated with HSBP1 depletion in MDA-MB-231 cells Because WASH is involved in recycling α5β1 integrins from intracellular compartments to the plasma membrane, we checked the localization of this particular integrin in the stable MDA-MB-231 clones depleted or not of HSBP1. We found that α5β1 focal adhesions from HSBP1-depleted cells were strongly reduced in numbers and slightly reduced in size (Fig 4A and B). HSBP1-depleted cells display decreased levels of α5β1 integrins at their surface, as measured by FACS, and decreased total levels of β1, as measured by densitometry of Western blots (Fig 4C and D). As expected, these defects were associated with reduced adhesion to fibronectin, the major ligand of the α5β1 integrin, and to collagen type I (Appendix Fig S3). Similar defects were previously documented in WASH-depleted cells (Zech et al, 2011; Duleh & Welch, 201
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