The TBC1D31/praja2 complex controls primary ciliogenesis through PKA‐directed OFD1 ubiquitylation
2021; Springer Nature; Volume: 40; Issue: 10 Linguagem: Inglês
10.15252/embj.2020106503
ISSN1460-2075
AutoresEmanuela Senatore, Francesco Chiuso, Laura Rinaldi, Daniela Intartaglia, Rossella Delle Donne, Emilia Pedone, Bruno Catalanotti, Luciano Pirone, Bianca Fiorillo, Federica Moraca, Giuliana Giamundo, Giovanni Scala, Andrea Raffeiner, Omar Torres‐Quesada, Eduard Stefan, Marcel Kwiatkowski, Alienke van Pijkeren, Manuela Morleo, Brunella Franco, Corrado Garbi, Iván Conte, Antonio Feliciello,
Tópico(s)Renal and related cancers
ResumoArticle2 May 2021Open Access Source DataTransparent process The TBC1D31/praja2 complex controls primary ciliogenesis through PKA-directed OFD1 ubiquitylation Emanuela Senatore Department of Molecular Medicine and Medical Biotechnologies, University Federico II, Naples, Italy Search for more papers by this author Francesco Chiuso Department of Molecular Medicine and Medical Biotechnologies, University Federico II, Naples, Italy Search for more papers by this author Laura Rinaldi Department of Molecular Medicine and Medical Biotechnologies, University Federico II, Naples, Italy Search for more papers by this author Daniela Intartaglia Telethon Institute of Genetics and Medicine, Pozzuoli, Italy Search for more papers by this author Rossella Delle Donne Department of Molecular Medicine and Medical Biotechnologies, University Federico II, Naples, Italy Search for more papers by this author Emilia Pedone Institute of Biostructures and Bioimaging, CNR, Naples, Italy Search for more papers by this author Bruno Catalanotti Department of Pharmacy, University Federico II, Naples, Italy Search for more papers by this author Luciano Pirone Institute of Biostructures and Bioimaging, CNR, Naples, Italy Search for more papers by this author Bianca Fiorillo Department of Pharmacy, University Federico II, Naples, Italy Search for more papers by this author Federica Moraca Department of Pharmacy, University Federico II, Naples, Italy Net4Science srl, University "Magna Græcia", Catanzaro, Italy Search for more papers by this author Giuliana Giamundo Telethon Institute of Genetics and Medicine, Pozzuoli, Italy Search for more papers by this author Giovanni Scala Department of Biology, University Federico II, Naples, Italy Search for more papers by this author Andrea Raffeiner Institute of Biochemistry, University of Innsbruck, Innsbruck, Austria Center for Molecular Biosciences, University of Innsbruck, Innsbruck, Austria Search for more papers by this author Omar Torres-Quesada Institute of Biochemistry, University of Innsbruck, Innsbruck, Austria Center for Molecular Biosciences, University of Innsbruck, Innsbruck, Austria Tyrolean Cancer Research Institute, Innsbruck, Austria Search for more papers by this author Eduard Stefan Institute of Biochemistry, University of Innsbruck, Innsbruck, Austria Center for Molecular Biosciences, University of Innsbruck, Innsbruck, Austria Tyrolean Cancer Research Institute, Innsbruck, Austria Search for more papers by this author Marcel Kwiatkowski Institute of Biochemistry, University of Innsbruck, Innsbruck, Austria Search for more papers by this author Alienke van Pijkeren Institute of Biochemistry, University of Innsbruck, Innsbruck, Austria Search for more papers by this author Manuela Morleo orcid.org/0000-0002-7553-3245 Telethon Institute of Genetics and Medicine, Pozzuoli, Italy Search for more papers by this author Brunella Franco orcid.org/0000-0001-5588-4569 Telethon Institute of Genetics and Medicine, Pozzuoli, Italy Department of Translational Medical Science, University Federico II, Naples, Italy Search for more papers by this author Corrado Garbi Department of Molecular Medicine and Medical Biotechnologies, University Federico II, Naples, Italy Search for more papers by this author Ivan Conte orcid.org/0000-0002-8968-9021 Telethon Institute of Genetics and Medicine, Pozzuoli, Italy Department of Biology, University Federico II, Naples, Italy Search for more papers by this author Antonio Feliciello Corresponding Author [email protected] orcid.org/0000-0002-7932-2170 Department of Molecular Medicine and Medical Biotechnologies, University Federico II, Naples, Italy Search for more papers by this author Emanuela Senatore Department of Molecular Medicine and Medical Biotechnologies, University Federico II, Naples, Italy Search for more papers by this author Francesco Chiuso Department of Molecular Medicine and Medical Biotechnologies, University Federico II, Naples, Italy Search for more papers by this author Laura Rinaldi Department of Molecular Medicine and Medical Biotechnologies, University Federico II, Naples, Italy Search for more papers by this author Daniela Intartaglia Telethon Institute of Genetics and Medicine, Pozzuoli, Italy Search for more papers by this author Rossella Delle Donne Department of Molecular Medicine and Medical Biotechnologies, University Federico II, Naples, Italy Search for more papers by this author Emilia Pedone Institute of Biostructures and Bioimaging, CNR, Naples, Italy Search for more papers by this author Bruno Catalanotti Department of Pharmacy, University Federico II, Naples, Italy Search for more papers by this author Luciano Pirone Institute of Biostructures and Bioimaging, CNR, Naples, Italy Search for more papers by this author Bianca Fiorillo Department of Pharmacy, University Federico II, Naples, Italy Search for more papers by this author Federica Moraca Department of Pharmacy, University Federico II, Naples, Italy Net4Science srl, University "Magna Græcia", Catanzaro, Italy Search for more papers by this author Giuliana Giamundo Telethon Institute of Genetics and Medicine, Pozzuoli, Italy Search for more papers by this author Giovanni Scala Department of Biology, University Federico II, Naples, Italy Search for more papers by this author Andrea Raffeiner Institute of Biochemistry, University of Innsbruck, Innsbruck, Austria Center for Molecular Biosciences, University of Innsbruck, Innsbruck, Austria Search for more papers by this author Omar Torres-Quesada Institute of Biochemistry, University of Innsbruck, Innsbruck, Austria Center for Molecular Biosciences, University of Innsbruck, Innsbruck, Austria Tyrolean Cancer Research Institute, Innsbruck, Austria Search for more papers by this author Eduard Stefan Institute of Biochemistry, University of Innsbruck, Innsbruck, Austria Center for Molecular Biosciences, University of Innsbruck, Innsbruck, Austria Tyrolean Cancer Research Institute, Innsbruck, Austria Search for more papers by this author Marcel Kwiatkowski Institute of Biochemistry, University of Innsbruck, Innsbruck, Austria Search for more papers by this author Alienke van Pijkeren Institute of Biochemistry, University of Innsbruck, Innsbruck, Austria Search for more papers by this author Manuela Morleo orcid.org/0000-0002-7553-3245 Telethon Institute of Genetics and Medicine, Pozzuoli, Italy Search for more papers by this author Brunella Franco orcid.org/0000-0001-5588-4569 Telethon Institute of Genetics and Medicine, Pozzuoli, Italy Department of Translational Medical Science, University Federico II, Naples, Italy Search for more papers by this author Corrado Garbi Department of Molecular Medicine and Medical Biotechnologies, University Federico II, Naples, Italy Search for more papers by this author Ivan Conte orcid.org/0000-0002-8968-9021 Telethon Institute of Genetics and Medicine, Pozzuoli, Italy Department of Biology, University Federico II, Naples, Italy Search for more papers by this author Antonio Feliciello Corresponding Author [email protected] orcid.org/0000-0002-7932-2170 Department of Molecular Medicine and Medical Biotechnologies, University Federico II, Naples, Italy Search for more papers by this author Author Information Emanuela Senatore1,†, Francesco Chiuso1,†, Laura Rinaldi1, Daniela Intartaglia2, Rossella Delle Donne1, Emilia Pedone3, Bruno Catalanotti4, Luciano Pirone3, Bianca Fiorillo4, Federica Moraca4,5, Giuliana Giamundo2, Giovanni Scala6, Andrea Raffeiner7,8, Omar Torres-Quesada7,8,9, Eduard Stefan7,8,9, Marcel Kwiatkowski7, Alienke van Pijkeren7, Manuela Morleo2, Brunella Franco2,10, Corrado Garbi1, Ivan Conte2,6 and Antonio Feliciello *,1 1Department of Molecular Medicine and Medical Biotechnologies, University Federico II, Naples, Italy 2Telethon Institute of Genetics and Medicine, Pozzuoli, Italy 3Institute of Biostructures and Bioimaging, CNR, Naples, Italy 4Department of Pharmacy, University Federico II, Naples, Italy 5Net4Science srl, University "Magna Græcia", Catanzaro, Italy 6Department of Biology, University Federico II, Naples, Italy 7Institute of Biochemistry, University of Innsbruck, Innsbruck, Austria 8Center for Molecular Biosciences, University of Innsbruck, Innsbruck, Austria 9Tyrolean Cancer Research Institute, Innsbruck, Austria 10Department of Translational Medical Science, University Federico II, Naples, Italy †These authors contributed equally to this work *Corresponding author. Tel: +39 081 7463615; E-mail: [email protected] EMBO J (2021)40:e106503https://doi.org/10.15252/embj.2020106503 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 primary cilium is a microtubule-based sensory organelle that dynamically links signalling pathways to cell differentiation, growth, and development. Genetic defects of primary cilia are responsible for genetic disorders known as ciliopathies. Orofacial digital type I syndrome (OFDI) is an X-linked congenital ciliopathy caused by mutations in the OFD1 gene and characterized by malformations of the face, oral cavity, digits and, in the majority of cases, polycystic kidney disease. OFD1 plays a key role in cilium biogenesis. However, the impact of signalling pathways and the role of the ubiquitin-proteasome system (UPS) in the control of OFD1 stability remain unknown. Here, we identify a novel complex assembled at centrosomes by TBC1D31, including the E3 ubiquitin ligase praja2, protein kinase A (PKA), and OFD1. We show that TBC1D31 is essential for ciliogenesis. Mechanistically, upon G-protein-coupled receptor (GPCR)-cAMP stimulation, PKA phosphorylates OFD1 at ser735, thus promoting OFD1 proteolysis through the praja2-UPS circuitry. This pathway is essential for ciliogenesis. In addition, a non-phosphorylatable OFD1 mutant dramatically affects cilium morphology and dynamics. Consistent with a role of the TBC1D31/praja2/OFD1 axis in ciliogenesis, alteration of this molecular network impairs ciliogenesis in vivo in Medaka fish, resulting in developmental defects. Our findings reveal a multifunctional transduction unit at the centrosome that links GPCR signalling to ubiquitylation and proteolysis of the ciliopathy protein OFD1, with important implications on cilium biology and development. Derangement of this control mechanism may underpin human genetic disorders. Synopsis Oro-facial digital type I syndrome (OFDI) is an X-linked congenital ciliopathy caused by mutations in the OFD1 gene, encoding a centrosome/basal body and pericentriolar satellite protein. This work identifies a multifunctional centrosomal complex that links G-protein-coupled receptor (GPCR) signalling to ubiquitylation and proteolysis of OFD1 protein and regulates cilium biology and development in vertebrates. Centrosomal TBC1D31 interacts with praja2, PKA and OFD1 PKA phosphorylates OFD1 at Ser375 in response to GPCR-cAMP stimulation Phosphorylation primes OFD1 for praja2-mediated ubiquitylation and proteasomal degradation Expression of a non-phosphorylatable OFD1 mutant affects cilium morphology and dynamics The TBC1D31/praja2/OFD1 pathway is essential for cilium dynamics, sonic hedgehog signalling, and development in Medaka fish Introduction The primary cilium is the principal sensory organelle in most eukaryotic cells and represents an intracellular hub where distinct signalling networks integrate and focus. A variety of receptors, scaffolds, ion channels, adaptor molecules and effector enzymes are localized within the ciliary compartment, playing a central role in development, metabolism, growth and differentiation (Ishikawa & Marshall, 2011; Oh et al, 2015; Hilgendorf et al, 2016; Nachury & Mick, 2019). Under growth-arrested conditions, the mother centriole of the centrosome migrates to the cell surface and starts to elongate as axonemal structure composed of nine doublet microtubules enveloped within the plasma membrane, forming the mature cilium and its basal body. This is an evolutionary conserved and highly regulated process controlled by a variety of pericentriolar proteins, regulators and scaffolds, all of which contribute to the formation and maintenance of the ciliary morphology and structure (Sanchez & Dynlacht, 2016). Mutations affecting genes involved in the dynamic control of cilium biogenesis often cause developmental genetic disorders, also known as ciliopathies (Valente et al, 2014; Reiter & Leroux, 2017). The OFD1 gene encodes a component of the centrosome/basal body and pericentriolar satellites that plays a major role in cilium biogenesis (Lopes et al, 2011). Germline inactivating mutations of OFD1 cause the Oral-Facial-Digital type I (OFDI) syndrome, a developmental disorder usually characterized by typical oral-facial-digital malformations, renal cystic disease and central nervous system involvement (Macca & Franco, 2009; Bruel et al, 2017). In serum-deprived cells, removal of OFD1 from centriolar satellites through the autophagy machinery is required for the onset of ciliogenesis (Tang et al, 2013). A role for OFD1 in non-ciliary pathways has also been reported (Abramowicz et al, 2017; Iaconis et al, 2017; Alfieri et al, 2020). The ubiquitin-proteasome system (UPS) controls the levels of ciliary regulatory proteins, contributing to the dynamic assembly/disassembly of the primary cilium (Kasahara et al, 2014; Liu et al, 2014; Wheway et al, 2015; Kwon & Ciechanover, 2017; Nagai et al, 2018; Tsai et al, 2019; Wiegering et al, 2019). However, the role of signalling enzymes in OFD1-dependent functions at the ciliary compartment and the impact of the ubiquitin-proteasome system (UPS) on OFD1 stability/activity are largely unknown. Growing evidence indicates that deregulation of signalling pathways, involving Sonic Hedgehog, Wnt, Notch and cAMP cascades, generated at—or converging to—the ciliary compartment contributes to human disorders, such as developmental deficits, neurodegeneration and cancer (Anvarian et al, 2019; Jeng et al, 2020). cAMP is an ancient second messenger that controls key biological activities, including metabolism, cell growth, development, differentiation and synaptic activities. Protein Kinase A (PKA) is the main effector of cAMP action and is present in the cell as tetrameric holoenzyme composed of two regulatory (R) and two catalytic (PKAc, C) subunits. Activation of the adenylate cyclase by a GPCR ligand induces a cAMP-mediated dissociation of the PKA holoenzyme and consequent release of active PKAc subunits. Phosphorylation of cellular substrates by PKAc regulates important biological functions (Taylor et al, 2013; Newton et al, 2016; Rinaldi et al, 2019). Compartmentalization of PKA at discrete intracellular sites by A-kinase anchor proteins (AKAPs) contributes to the activation, dissemination and attenuation of cAMP signals at distal sites from signal generation (Yang & McKnight, 2015; Jones et al, 2016; Reggi & Diviani, 2017; Rinaldi et al, 2017; Torres-Quesada et al, 2017; Rinaldi et al, 2018; Bucko et al, 2019). The AKAP praja2 binds and targets PKA holoenzyme to the cell membrane, perinuclear region and cellular organelles. Co-localization of praja2•PKA complexes with PKA substrate/effector molecules ensures efficient integration and propagation of the locally generated cAMP to distinct target sites (Lignitto et al, 2011a). praja2 acts as an E3 ubiquitin ligase that controls ubiquitylation and stability of colocalized signalling enzymes, including PKA, adapter proteins and tumour suppressors (Lignitto et al, 2011b; Lignitto et al, 2013; Sepe et al, 2014; Zhang et al, 2015; Rinaldi et al, 2016; Song et al, 2019). PKA regulates different aspects of cilium biology. Thus, proteomic and functional analyses identified components of the cAMP cascade as residents and regulators of the ciliary compartment (Mukherjee et al, 2016; Siljee et al, 2018; Sherpa et al, 2020). In this context, activation of PKA within the cilium plays an inhibitory role on the Sonic Hedgehog pathway, a master regulator of embryonic development (Chen et al, 2011; Vuolo et al, 2015). The identification of orphan receptor GPCR (Gpr161) and adenylate cyclases within the cilium suggested that locally generated cAMP microdomains directly controls the activation of ciliary PKA and the signal dissemination to co-targeted ciliary effector proteins (Mukhopadhyay et al, 2013). cAMP signalling also contributes to cilium biogenesis and dynamics (Pal & Mukhopadhyay, 2015; Bachmann et al, 2016; Tschaikner et al, 2020). A link between PKA signalling and the ubiquitin-proteasome system at ciliary compartment has been recently discovered (Porpora et al, 2018). Thus, PKA phosphorylation of NIMA-related kinase NEK10 promotes its ubiquitylation by the E3 ligase CHIP/Stub1. Ubiquitylated NEK10 undergoes proteasomal degradation, leading to primary cilium resorption (Porpora et al, 2018). However, the role of the PKA-ubiquitin system in cilium biogenesis and its relevant targets are still unknown. Here, we report the identification of a novel multifaceted signalling complex assembled at the centrosome by TBC1D31 that finely controls the PKA-mediated phosphorylation of OFD1 and its ubiquitin-dependent proteolysis through the proteasome. Interfering with this control mechanism affects cilium biogenesis and Medaka fish development. Results TBC1D31 anchors praja2 to centrosome and centriolar satellites A yeast two-hybrid screening using the C-terminus of praja2 as bait and a human brain cDNA library identified a clone encoding for the C-terminus (residues 940–970) of TBC1D31, a protein with unknown functions that localizes to the centrosome and centriolar satellites(Gupta et al, 2015). First, we asked whether praja2 and TBC1D31 interact in cell lysates. Co-immunoprecipitation (CoIp) experiments confirmed the interaction between praja2 and TBC1D31 (Fig 1A). By deletion mutagenesis and CoIp assays, we identified residues 530–630 as the praja2 segment that binds to TBC1D31 (Fig 1B and C). GST pull-down experiments confirmed that residues 940–970 of TBC1D31 interact with praja2 (Fig 1D). praja2 is known to anchor PKA to specific intracellular sites (Lignitto et al, 2011a). Accordingly, we tested whether PKA was present in the praja2/TBC1D31 complex. As suspected, the PKAc subunit, along with praja2, was recovered in the TBC1D31 immunoprecipitates using antibodies raised against residues 239–358 of human TBC1D31 (Fig 1E, Appendix Fig S1A and B). In situ immunostaining analysis confirmed that TBC1D31 is localized at the centrosome and centriolar satellites (Fig 1F, upper panels, Appendix Fig S1B). Moreover, a fraction of the praja2 signal colocalizes with GFP-TBC1D31, supporting the presence of a praja2/TBC1D31 complex within the same intracellular compartment (Fig 1F, lower panels). A similar partial immunostaining pattern of praja2 at centrosome was also observed (Fig EV1A). TBC1D31 acts as an anchor for praja2. Thus, the genetic silencing of TBC1D31 dramatically reduced the localization of praja2 at the centrosome and centriolar satellites (Figs 1G and EV1B–D). In contrast, praja2 silencing had no significant impact on the intracellular localization of TBC1D31 (Fig EV1E and F), supporting the model by which TBC1D31 acts as an anchor for praja2 at the centrosome and centriolar satellites (Fig 1H). Figure 1. TBC1D31 binds and targets praja2 to the centrosome A. Co-immunoprecipitation of flag-praja2 and GFP-TBC1D31 from lysates of HEK293 cells. The immunoprecipitation (Ip) was performed using an anti-flag antibody or control IgG. B, C. Same as in (A), with the exception that cells expressing flag-praja2rm or praja2 deletion mutants (praja21–530, praja21–630 and praja2Δ530–630) were included in the analysis. D. Lysates expressing flag-praja2 were subjected to pull down assay with GST and GST-TBC1D31940–970 polypeptides. E. Co-immunoprecipitation of endogenous TBC1D31/praja2/PKAc complex from cell lysates. F. Staining of HEK293 cells with anti-TBC1D31, anti-γ-tubulin and anti-praja2 antibodies. Nuclei were stained with DRAQ5 (blue). Where indicated, cells were transfected with GFP-TBC1D31. Arrows indicate the pool of praja2 colocalizing with TBC1D31 staining at the centrosome. G. Cells transfected with control siRNA (siCNT) or siRNA targeting TBC1D31 (siTBC1D31) were stained for praja2, anti-γ-tubulin and DRAQ5. H. Schematic picture of TBC1D31/praja2/PKA complex. Source data are available online for this figure. Source Data for Figure 1 [embj2020106503-sup-0003-SDataFig1.pdf] Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Localization of praja2 and TBC1D31 at centrosome A. HEK293 cells were fixed and stained with anti-praja2 and anti-γ-tubulin antibodies. Nuclei were stained with DRAQ5. B. HEK293 cells transiently transfected with control siRNA or siRNA targeting endogenous praja2 were stained with anti-praja2 antibody and DRAQ5. C. Immunoblot analysis of TBC1D31 and Hsp90 in siRNA-silenced cells. D. Cells were transiently transfected with control siRNA or siRNA targeting endogenous TBC1D31. Total RNA was extracted and analysed by quantitative RT–PCR. E. Cells transiently transfected with control siRNA or siRNA targeting endogenous praja2 were stained for TBC1D31, γ-tubulin and DRAQ5. F. Immunoblot analysis of praja2 and Hsp90 in siRNA-silenced cells. Download figure Download PowerPoint Click here to expand this figure. Figure EV2. Centrosomal localization of praja2, TBC1D31, OFD1 and PKAc A. HEK293 cells transfected with GFP-TBC1D31 were fixed and immunostained with anti-OFD1 and anti-γ-tubulin antibodies. B. HEK293 cells transfected with GFP-TBC1D31 were fixed and immunostained with anti-praja2 and anti-γ-tubulin antibodies. C. HEK293 cells transfected with GFP-TBC1D31 were fixed and immunostained with anti-OFD1 and anti-PKAc antibodies. Download figure Download PowerPoint Click here to expand this figure. Figure EV3. Phosphorylation of endogenous OFD1 A. HEK293 cells were starved for 24 h and then treated with FSK (40 μM) for 1 h. Lysates were immunoprecipitated with anti-OFD1 antibody or with control IgG. Lysates and precipitates were immunoblotted with anti-phospho-(K/R)(K/R)X(S*/T*) and anti-OFD1 antibodies. B. Western blot analysis of affinity-isolated endogenous OFD1. Hela cells transiently expressing PKAc-YFP were serum-deprived for 48 h and lysed. Total lysates were immunoprecipitated with anti-HA (control IP) or with anti-OFD1 antibody. An aliquot of whole cell lysate (WCL) and the precipitates were immunoblotted for OFD1. Gel-isolated OFD1-containing fragments were subjected to mass spectrometric analysis. C. Fragment spectrum of m/z 426.5708 [M + 3H]3+ with the identified y- and b-fragment ions of the phosphorylated OFD1 peptide LpSSTPLPKAKR. b2*: b2-fragment ion containing dehydrolalanine, which represents the formerly phosphorylated serine residue. Dehydroalanine is formed from phosphoserine during collision-induced dissociation in HCD (Higher-energy Collisional Dissociation) due to the neutral loss of H3PO4. The phosphorylated peptide was identified by a Sequest data base search using Percolator with a q-value of 7e-4. D. Extracted ion chromatograms (EICs) of the b- and y-fragment ions showing that all fragment ions co-elute. Download figure Download PowerPoint Click here to expand this figure. Figure EV4. cAMP stimulation regulates OFD1 stability A. HEK293 cells were serum-deprived for 24 h, treated with cycloheximide (100 μM) with or without FSK (40 μM) and harvested at the indicated time points. Lysates were immunoblotted with anti-OFD1 and anti-α-tubulin antibodies. B. Quantitative analysis of the experiments shown in (A). A mean value of two independent experiments that gave similar results is shown. C. Same as in (A), with the exception that PGE2 (1 μM) was used instead of FSK. Lysates were immunoblotted with anti-OFD1 and anti-Hsp70 antibodies. D. Quantitative analysis of the experiments shown in (C). A mean value of two independent experiments that gave similar results is shown. E. HEK293 cells were serum-deprived for 24 h, treated with cycloheximide (100 μM) and treated with FSK (40 μM) for the indicated times. Where indicated, MG132 (20 μM) was added to the medium. Lysates were immunoblotted with anti-OFD1 and anti-Hsp90 antibodies. F. Quantitative analysis of the experiments shown in (E). A mean value ± SD of three independent experiments is shown. Student's t test *P < 0.05, **P < 0.01. Download figure Download PowerPoint Click here to expand this figure. Figure EV5. Role of TBC1D31, OFD1 and FSK in ciliogenesis and cilium morphology A. Immunostaining analysis of serum-deprived HEK293 cells for TBC1D31, acetylated α-tubulin and DRAQ5. B. Human U-87MG glioblastoma cells transfected with control siRNA (siCNT) or siRNA targeting TBC1D31 (siTBC1D31) were serum-deprived for 36 h, fixed and stained for acetylated-tubulin, TBC1D31 and DRAQ5. C. Quantitative analysis of the experiments shown in (B). A mean value ± SD of three independent experiments is shown. Student's t test **P < 0.01. D. HEK293 cells were transiently transfected with flag-OFD1 or flag-S735A, serum-deprived for 36 h, fixed and immunostained for flag, acetylated-tubulin and DRAQ5. E. NIH3T3 cells transiently expressing flag-OFD1 or flag-S735A were serum-deprived for 36 h, treated with FSK (6 h), fixed and immunostained for flag, acetylated-tubulin and DRAQ5. F. Statistical analysis of the experiments shown in (E). A mean value ± SD of three independent experiments is shown. Student's t test, ***P < 0.001, **P < 0.01. Download figure Download PowerPoint Binding modules of TBC1D31/praja2 complex Next, we tested whether praja2 and TBC1D31 interact in vitro. A fusion protein carrying residues 531–631 of praja2 fused to the C-terminus of glutathione S-transferase polypeptide (GST) coprecipitated GFP-TBC1D31 from cell lysates (Fig 2A). To identify the minimal core domain on praja2 that binds TBC1D31, we performed in vitro microscale thermophoresis binding experiments using partially overlapping synthetic peptides spanning the praja2530–630 domain. As shown in Fig 2B, praja2530–570 and praja2550–610 peptides bind the C-terminus domain of TBC1D31 with micromolar affinity (KD 37 µM and KD 80 µM, respectively), whereas no binding was observed with the praja2590–630 peptide. This finding suggested that praja2550–570 segment contributes to the interaction with TBC1D31. As predicted, deletion of residues 550–570 of praja2 (praja2Δ550–570) dramatically reduced the binding to GFP-TBC1D31 (Fig 2C). Figure 2. Modelling TBC1D31/praja2 binding in vitro A. Lysates from HEK293 cells expressing GFP-TBC1D31 were subjected to pull down assay with GST and GST-praja2531–631 polypeptides. B. MST signal (normalized fluorescence) of P1 (red curve), P2 (green curve) and P3 (cyan curve) plotted against TBC1D31, at increasing concentrations of peptides. The threading modelled structure of the overlapping binding segment of praja2 (praja2550–570) is shown. C. Co-immunoprecipitation of GFP-TBC1D31 and flag-praja2 ring mutant (flag-praja2rm) or praja2Δ550–570. D. Threading modelled structure of TBC1D31, with a zoom of its C-terminus. Mutated residues are highlighted in stick coloured by atom type. E. MD derived binding mode of praja2530–570 (green cartoon) to the C-terminal region of TBC1D31 (red cartoon). F. MST signal of P1 plotted against increasing concentrations of TBC1D31 peptides: wild-type (red curve), R948A-R951A (violet curve) and R957-R959D-H960A (orange curve). Source data are available online for this figure. Source Data for Figure 2 [embj2020106503-sup-0004-SDataFig2.pdf] Download figure Download PowerPoint The molecular basis of praja2 and TBC1D31 interaction was investigated by docking and molecular dynamics (MD) studies. praja2550–570 (Fig 2B) and TBC1D31941–970 (Fig 2D) 3D structures were generated using the threading approach implemented in I-TASSER website. praja2550–570 3D structure resulted mostly coiled, while the TBC1D31941–970 domain was modelled as α-helix with a kink at the level of Q941. Accordingly, CD spectra showed that TBC1D31941–970 domain assumed a partial helical structure (Fig 2E, Appendix Fig S2A and B). A two-step docking procedure followed by 2 µs classical MD simulations reported a binding mode represented by three main clusters (Appendix Fig S2C and D, Movie EV1). The binding is mainly driven by the arginine-rich stretch R957-R961 (RARHR) of TBC1D31 that establishes cation-π and ionic interactions with the praja2 stretch F553-D558 and with E564. Moreover, only discontinuous interactions of TBC1D31 R948 and R951 residues, mainly with the praja2 D570 residue, were observed (Movie EV1). To validate the proposed binding mode, we designed two different mutants of the C-terminal TBC1D31 peptide: 1. TBC1D31ADA triple mutant (R957A, R959D and H960A) peptide; 2. TBC1D31AA double-mutant (R948A and R951A) peptide. Microscale thermophoresis experiments showed that the interaction between praja2530–570 and TBC1D31AA mutant was preserved, whereas it was almost abolished with TBC1D31ADA (Fig 2F, Appendix Fig S3A). In addition, CD spectra showed a partial helical structure for both mutant peptides, without any appreciable difference with wild-type (Appendix Fig S3B), supporting the MD-derived hypothesis of a specific role of residues
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