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• W2912846978 abstract "Article11 February 2019free access Source DataTransparent process Nuclear import of the DSCAM-cytoplasmic domain drives signaling capable of inhibiting synapse formation Sonja M Sachse VIB Center for Brain & Disease Research, Leuven, Belgium Department of Neurosciences, KU Leuven, Leuven, Belgium Search for more papers by this author Sam Lievens VIB Center for Medical Biotechnology, Ghent, Belgium Department of Biomolecular Medicine, Ghent University, Ghent, Belgium Search for more papers by this author Luís F Ribeiro VIB Center for Brain & Disease Research, Leuven, Belgium Department of Neurosciences, KU Leuven, Leuven, Belgium Search for more papers by this author Dan Dascenco VIB Center for Brain & Disease Research, Leuven, Belgium Department of Neurosciences, KU Leuven, Leuven, Belgium Search for more papers by this author Delphine Masschaele VIB Center for Medical Biotechnology, Ghent, Belgium Department of Biomolecular Medicine, Ghent University, Ghent, Belgium Search for more papers by this author Katrien Horré VIB Center for Brain & Disease Research, Leuven, Belgium Department of Neurosciences, KU Leuven, Leuven, Belgium Search for more papers by this author Anke Misbaer VIB Center for Brain & Disease Research, Leuven, Belgium Department of Neurosciences, KU Leuven, Leuven, Belgium Search for more papers by this author Nele Vanderroost VIB Center for Medical Biotechnology, Ghent, Belgium Department of Biomolecular Medicine, Ghent University, Ghent, Belgium Search for more papers by this author Anne Sophie De Smet VIB Center for Medical Biotechnology, Ghent, Belgium Department of Biomolecular Medicine, Ghent University, Ghent, Belgium Search for more papers by this author Evgenia Salta VIB Center for Brain & Disease Research, Leuven, Belgium Department of Neurosciences, KU Leuven, Leuven, Belgium Search for more papers by this author Maria-Luise Erfurth VIB Center for Brain & Disease Research, Leuven, Belgium Search for more papers by this author Yoshiaki Kise VIB Center for Brain & Disease Research, Leuven, Belgium Search for more papers by this author Siegfried Nebel VIB Center for Brain & Disease Research, Leuven, Belgium Department of Neurosciences, KU Leuven, Leuven, Belgium Search for more papers by this author Wouter Van Delm VIB Nucleomics Core, Leuven, Belgium Search for more papers by this author Stéphane Plaisance VIB Nucleomics Core, Leuven, Belgium Search for more papers by this author Jan Tavernier VIB Center for Medical Biotechnology, Ghent, Belgium Department of Biomolecular Medicine, Ghent University, Ghent, Belgium Search for more papers by this author Bart De Strooper orcid.org/0000-0001-5455-5819 VIB Center for Brain & Disease Research, Leuven, Belgium Department of Neurosciences, KU Leuven, Leuven, Belgium Dementia Research Institute, University College London, London, UK Search for more papers by this author Joris De Wit VIB Center for Brain & Disease Research, Leuven, Belgium Department of Neurosciences, KU Leuven, Leuven, Belgium Search for more papers by this author Dietmar Schmucker Corresponding Author [email protected] orcid.org/0000-0002-7529-6761 VIB Center for Brain & Disease Research, Leuven, Belgium Department of Neurosciences, KU Leuven, Leuven, Belgium Search for more papers by this author Sonja M Sachse VIB Center for Brain & Disease Research, Leuven, Belgium Department of Neurosciences, KU Leuven, Leuven, Belgium Search for more papers by this author Sam Lievens VIB Center for Medical Biotechnology, Ghent, Belgium Department of Biomolecular Medicine, Ghent University, Ghent, Belgium Search for more papers by this author Luís F Ribeiro VIB Center for Brain & Disease Research, Leuven, Belgium Department of Neurosciences, KU Leuven, Leuven, Belgium Search for more papers by this author Dan Dascenco VIB Center for Brain & Disease Research, Leuven, Belgium Department of Neurosciences, KU Leuven, Leuven, Belgium Search for more papers by this author Delphine Masschaele VIB Center for Medical Biotechnology, Ghent, Belgium Department of Biomolecular Medicine, Ghent University, Ghent, Belgium Search for more papers by this author Katrien Horré VIB Center for Brain & Disease Research, Leuven, Belgium Department of Neurosciences, KU Leuven, Leuven, Belgium Search for more papers by this author Anke Misbaer VIB Center for Brain & Disease Research, Leuven, Belgium Department of Neurosciences, KU Leuven, Leuven, Belgium Search for more papers by this author Nele Vanderroost VIB Center for Medical Biotechnology, Ghent, Belgium Department of Biomolecular Medicine, Ghent University, Ghent, Belgium Search for more papers by this author Anne Sophie De Smet VIB Center for Medical Biotechnology, Ghent, Belgium Department of Biomolecular Medicine, Ghent University, Ghent, Belgium Search for more papers by this author Evgenia Salta VIB Center for Brain & Disease Research, Leuven, Belgium Department of Neurosciences, KU Leuven, Leuven, Belgium Search for more papers by this author Maria-Luise Erfurth VIB Center for Brain & Disease Research, Leuven, Belgium Search for more papers by this author Yoshiaki Kise VIB Center for Brain & Disease Research, Leuven, Belgium Search for more papers by this author Siegfried Nebel VIB Center for Brain & Disease Research, Leuven, Belgium Department of Neurosciences, KU Leuven, Leuven, Belgium Search for more papers by this author Wouter Van Delm VIB Nucleomics Core, Leuven, Belgium Search for more papers by this author Stéphane Plaisance VIB Nucleomics Core, Leuven, Belgium Search for more papers by this author Jan Tavernier VIB Center for Medical Biotechnology, Ghent, Belgium Department of Biomolecular Medicine, Ghent University, Ghent, Belgium Search for more papers by this author Bart De Strooper orcid.org/0000-0001-5455-5819 VIB Center for Brain & Disease Research, Leuven, Belgium Department of Neurosciences, KU Leuven, Leuven, Belgium Dementia Research Institute, University College London, London, UK Search for more papers by this author Joris De Wit VIB Center for Brain & Disease Research, Leuven, Belgium Department of Neurosciences, KU Leuven, Leuven, Belgium Search for more papers by this author Dietmar Schmucker Corresponding Author [email protected] orcid.org/0000-0002-7529-6761 VIB Center for Brain & Disease Research, Leuven, Belgium Department of Neurosciences, KU Leuven, Leuven, Belgium Search for more papers by this author Author Information Sonja M Sachse1,2, Sam Lievens3,4,†, Luís F Ribeiro1,2, Dan Dascenco1,2, Delphine Masschaele3,4, Katrien Horré1,2, Anke Misbaer1,2, Nele Vanderroost3,4, Anne Sophie De Smet3,4, Evgenia Salta1,2, Maria-Luise Erfurth1,†, Yoshiaki Kise1,†, Siegfried Nebel1,2,†, Wouter Van Delm5, Stéphane Plaisance5, Jan Tavernier3,4, Bart De Strooper1,2,6, Joris De Wit1,2 and Dietmar Schmucker *,1,2 1VIB Center for Brain & Disease Research, Leuven, Belgium 2Department of Neurosciences, KU Leuven, Leuven, Belgium 3VIB Center for Medical Biotechnology, Ghent, Belgium 4Department of Biomolecular Medicine, Ghent University, Ghent, Belgium 5VIB Nucleomics Core, Leuven, Belgium 6Dementia Research Institute, University College London, London, UK †Present address: Orionis Biosciences, Ghent, Belgium †Present address: Molecular Neurogenomics Group, VIB Center for Molecular Neurology, University of Antwerp, Antwerp, Belgium †Present address: Department of Biological Sciences, University of Tokyo, Tokyo, Japan †Present address: Ecole Normal Superieure, Paris, France *Corresponding author. Tel: +32 16 37 32 22; E-mail: [email protected] EMBO J (2019)38:e99669https://doi.org/10.15252/embj.201899669 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 DSCAM and DSCAML1 are immunoglobulin and cell adhesion-type receptors serving important neurodevelopmental functions including control of axon growth, branching, neurite self-avoidance, and neuronal cell death. The signal transduction mechanisms or effectors of DSCAM receptors, however, remain poorly characterized. We used a human ORFeome library to perform a high-throughput screen in mammalian cells and identified novel cytoplasmic signaling effector candidates including the Down syndrome kinase Dyrk1a, STAT3, USP21, and SH2D2A. Unexpectedly, we also found that the intracellular domains (ICDs) of DSCAM and DSCAML1 specifically and directly interact with IPO5, a nuclear import protein of the importin beta family, via a conserved nuclear localization signal. The DSCAM ICD is released by γ-secretase-dependent cleavage, and both the DSCAM and DSCAML1 ICDs efficiently translocate to the nucleus. Furthermore, RNA sequencing confirms that expression of the DSCAM as well as the DSCAML1 ICDs alone can profoundly alter the expression of genes associated with neuronal differentiation and apoptosis, as well as synapse formation and function. Gain-of-function experiments using primary cortical neurons show that increasing the levels of either the DSCAM or the DSCAML1 ICD leads to an impairment of neurite growth. Strikingly, increased expression of either full-length DSCAM or the DSCAM ICD, but not the DSCAML1 ICD, significantly decreases synapse numbers in primary hippocampal neurons. Taken together, we identified a novel membrane-to-nucleus signaling mechanism by which DSCAM receptors can alter the expression of regulators of neuronal differentiation and synapse formation and function. Considering that chromosomal duplications lead to increased DSCAM expression in trisomy 21, our findings may help uncover novel mechanisms contributing to intellectual disability in Down syndrome. Synopsis A novel membrane-to-nucleus signaling mechanism of DSCAMs affects the expression of neuronal target genes. Nuclear enrichment of the cleaved intracellular domain of DSCAM (DSCAM-ICD) in developing neurons can lead to an impairment of neurite growth and strong decrease of synapse numbers. The DYRK family kinases DYRK1A and DYRK1B, the SH2-domain adaptor SH2D2A, the Ubiquitin specific peptidase USP21, and the transcription factor STAT3 are novel cytoplasmic binding partners of DSCAM and DSCAML1. The ICDs of DSCAM and DSCAML1 interact with the importin beta IPO5 via a conserved nuclear localization signal. γ-secretase mediated intra-membrane cleavage of DSCAM results in the release of the DSCAM ICD and both the DSCAM and DSCAML1 ICDs efficiently translocate to the nucleus. In the nucleus, the DSCAM ICD alters the transcription of several genes involved in neuronal differentiation and synapse formation. Introduction The precise wiring of neuronal circuits as well as the establishment of synaptic specificity majorly relies on intracellular signaling pathways downstream of diverse families of cell surface receptors. The Down syndrome cell adhesion molecule (DSCAM) receptors (Yamakawa et al, 1998) are important examples of receptors that utilize homophilic interactions during neurite growth, but can also interact with heterologous ligands (Dascenco et al, 2015). In vertebrates, there are two paralogous DSCAM genes, DSCAM and DSCAML1 (DSCAM-Like-1; Yamakawa et al, 1998; Agarwala et al, 2001). Human DSCAM is located in the so-called Down syndrome critical region (DSCR) on chromosome 21 and belongs thereby to a group of genes present in three copies in Down syndrome (DS) individuals (Yamakawa et al, 1998). Notably, DSCAM levels are increased in brains of DS patients and it has been speculated that this may contribute to the cognitive disabilities observed in DS (Saito et al, 2000; Bahn et al, 2002). DSCAM is also overexpressed in brain of DS mouse models (Amano et al, 2004; Alves-Sampaio et al, 2010), and in mice that overexpress Amyloid precursor protein (Jia et al, 2011), a causative gene in Alzheimer's disease. Studies using segmental trisomy mouse models of DS showed that triplication of genes of the DSCR including Dscam has a dose-dependent effect on the segregation of retinal ganglion cell (RGC) axons in the lateral geniculate nucleus (LGN) in the developing retina (Blank et al, 2011). Moreover, dendrite arborization and soma spacing of subtypes of mouse RGCs have been found to be highly sensitive to both increased and decreased Dscam gene dosage (Blank et al, 2011). Overexpression of DSCAM in mice disrupts dendrite targeting and leads to increased neuronal cell death of retinal neurons (Li et al, 2015). Loss of DSCAM or DSCAML1 on the other hand results in increased neuron numbers, self-avoidance defects, and disorganized layers in the retina (Fuerst et al, 2008, 2009), demonstrating that deregulated DSCAM levels result in altered neuronal wiring. In Drosophila, expression of three Dscam1 copies in vivo alters synaptic function at the neuromuscular junction (Lowe et al, 2018), causes synaptic targeting defects of sensory axons (Cvetkovska et al, 2013), and increased presynaptic arbor enlargement (Kim et al, 2013; Sterne et al, 2015). Thus, deregulated DSCAM levels strongly alter dendritic, axonal, and synaptic development, which may explain how DSCAM gene-dosage imbalance could potentially contribute to the pathogenesis of neurological disorders. Drosophila Dscam1 has been studied most extensively because of its extraordinary molecular diversity generated by alternative splicing (Schmucker et al, 2000; Hattori et al, 2008; Kise & Schmucker, 2013; Sun et al, 2013). Drosophila Dscam1 is essential for key aspects of neuronal wiring including axonal growth, guidance, targeting, and branching (Schmucker et al, 2000; Hummel et al, 2003; Chen et al, 2006; He et al, 2014; Dascenco et al, 2015), dendritic field organization (Hughes et al, 2007; Matthews et al, 2007; Soba et al, 2007), and synaptic connectivity (Millard et al, 2010). Although vertebrate DSCAMs lack extensive alternative splicing, many functions important for neuronal wiring are highly conserved from flies to mammals. Homophilic self-recognition of DSCAMs is required in flies and mice for neurite repulsion and self-avoidance of sister-neurites in vivo (Hughes et al, 2007; Fuerst et al, 2008, 2010; Hattori et al, 2008; Yamagata & Sanes, 2008; Simmons et al, 2017). Despite the conserved roles of DSCAMs in shaping neuronal circuitry, no coherent downstream signaling pathway has been identified. Vertebrate DSCAM has been implicated in the regulation of actin cytoskeleton dynamics (Ly et al, 2008; Liu et al, 2009; Purohit et al, 2012), yet in vivo evidence remains sparse. In Drosophila Dscam1 is thought to affect actin cytoskeleton dynamics through the SH2/SH3 adaptor protein Dock/Nck acting upstream of P21 activated kinase (Pak1) and Rho GTPases (Manser et al, 1994; Hall, 1998; Hing et al, 1999; Schmucker et al, 2000). However, loss of Dock or Pak1 does not lead to phenotypic defects such as dendrite self-crossing (Hughes et al, 2007). Dscam1 also physically and genetically interacts with tubulin folding co-factor D (TBCD), which is required for the formation of alpha- and beta-tubulin heterodimers in flies. This interaction is required in vivo for glomerular targeting of olfactory neurons (Okumura et al, 2015). Here we identify several novel cytoplasmic signaling effectors of DSCAM and DSCAML1 (DSCAM/L1). We report that Importin 5 (IPO5)-mediated membrane-to-nucleus translocation of DSCAM/L1 may provide a novel signaling mode of this important receptor class. We show that the ICD of mammalian DSCAM is liberated by γ-secretase-mediated cleavage and that both the DSCAM and DSCAML1 ICD efficiently translocate to the nucleus. Considering that increased DSCAM levels have been proposed to contribute to intellectual disability in Down syndrome patients, we tested in gain-of-function experiments how increased DSCAM levels, and in particular nuclear import, might affect neurite growth and synapse formation. We show that nuclear enrichment of the DSCAM and DSCAML1 ICD in cell lines profoundly alters the transcription of genes associated with neuronal differentiation and function. Increased nuclear levels of either the DSCAM or DSCAML1 ICD strongly impair neurite outgrowth in primary mouse cortical cultures. Interestingly, only increased expression of either full-length DSCAM or the DSCAM ICD, but not of the DSCAML1 ICD leads to a strong decrease in synapse numbers in primary mouse hippocampal neurons. Thus, these studies uncover how increased DSCAM membrane-to-nucleus signaling is capable of altering synaptic connectivity and suggest a novel scenario how gain of DSCAM function may contribute to neurological pathologies. Results Identifying human signaling effectors of DSCAM and DSCAML1 In order to identify novel cytoplasmic binding partners of vertebrate DSCAMs, we employed the mammalian protein-protein interaction trap (MAPPIT) to monitor protein–protein interactions in mammalian cells (Fig 1A; Eyckerman et al, 2001; Lievens et al, 2009). We generated MAPPIT receptors with the intracellular domains (ICDs) of mouse DSCAM or DSCAML1 serving as baits (Fig 1A) and performed high-throughput MAPPIT screens in which the DSCAM/L1 baits were screened against a library of about 10,000 preys (Lievens et al, 2009, 2011). From these screens, we further selected and validated IPO5, DYRK1A, DYRK1B, SH2D2A, STAT3, and USP21 as binding partners of both DSCAM and DSCAML1 in binary MAPPIT experiments including control baits and preys (Table 1; Fig 1B and G; Appendix Fig S1A and B). We also tested the interaction of the DSCAM/L1 bait receptors with preys of known binding partners including PAK1 (DSCAM) and MAGI-1 (DSCAM and DSCAML1; Yamagata & Sanes, 2010; Purohit et al, 2012). The DSCAM/L1 baits gave rise to MAPPIT signals with MAGI-1 and the DSCAM bait also interacted with the PAK1 prey, but these interactions caused weaker luciferase induction compared to newly identified candidates (Appendix Fig S1C and D). Recruitment of kinases and SH2-domain adaptor proteins were expected based on previous studies on Drosophila Dscam1 (Schmucker et al, 2000); however, neither DYRK1A (also referred to as “Down syndrome kinase”) nor SH2D2A have been previously implicated in DSCAM signaling. Figure 1. DSCAM and DSCAML1 interact with DYRK1A, DYRK1B, and IPO5 A. Schematic of the MAPPIT technique. Bait receptors consist of the mouse DSCAM or DSCAML1 ICDs fused to the C-terminus of a signaling-deficient leptin receptor (LR) fragment, in which the tyrosines of STAT3 recruitment motifs were mutated to phenylalanine. The preys are tethered to a gp130 cytokine receptor fragment containing functional STAT3 recruitment sites. Upon leptin stimulation, association of bait and prey restores a functional leptin receptor complex resulting in STAT3 activation, which can be monitored by a STAT3-responsive luciferase reporter. P, phospho-tyrosine. JAK, janus kinase. B. DSCAM interacts with DYRK1A and DYRK1B. LR-DSCAM and LR-eDHFR (negative control bait) baits were introduced in HEK cells together with a DYRK1A prey, DYRK1B prey, empty prey (negative control only consisting of the gp130 fragment), or an EFHA1 prey (positive control) and binding was tested in MAPPIT. Results for DSCAML1, see Appendix Fig S1B. C. Schematic of full-length (FL) DYRK1A, DYRK1B, and truncated DYRK1A/B preys. DYRK1A contains a nuclear localization signal, a protein kinase domain, a leucine zipper motif, and a highly conserved poly-histidine repeat; DYRK1B lacks the poly-histidine repeat. D, E. The interactions between truncated DYRK1B (aa 558–629) (D) or DYRK1A (aa 1–482) (E) preys and the DSCAM (LR-DSCAMY1746F) bait (i.e., suppressing STAT3-mediated background) were assayed using MAPPIT. Results for DSCAML1, see Appendix Fig S3A and B. The C-terminus of DYRK1B is sufficient for the interaction with DSCAM, and the kinase domain is not required (D). The DYRK1A kinase domain is not sufficient to bind to DSCAM (E). F. Mutation of the potential DYRK kinase substrate site does not inhibit the interaction between DSCAM and DYRK1A or DYRK1B. A potential substrate site for DYRKs was mutated in the DSCAM bait (RPGT to AAGA). The interactions of mutant or wild-type DSCAM baits with DYRK1A, DYRK1B, and IPO5 (positive control prey) were tested in a binary MAPPIT experiments. Results for DSCAML1, see Appendix Fig S3C and D. G. DSCAM and DSCAML1 robustly interact with IPO5. LR-DSCAM, LR-DSCAML1, and LR-eDHFR (negative control bait) baits were introduced in HEK cells together with an IPO5 prey, an empty prey (negative control), or an EFHA1 prey (positive control) and binding was tested in MAPPIT. H. DSCAMs exhibit a conserved NLS. Green, putative monopartite NLS motif. I. Loss of the NLSs of DSCAM/L1 inhibits binding of IPO5. Binding of IPO5 to wt or mutant DSCAM/L1 baits lacking the NLS (LR-DSCAMΔNLS, LR-DSCAML1ΔNLS) was tested in MAPPIT. Negative control, empty prey. Positive control, STAT3 prey. J. The interaction between DSCAM/L1 and IPO5 is specific. The interaction between 9 importin preys and the DSCAMY1746F or the DSCAML1Y1744/1937F bait (i.e., suppressing STAT3-mediated background signal) was evaluated in a binary MAPPIT experiment, and expression of the importin preys was confirmed by Western blot (lower panel). Data information: In (B, D–G, and I, J), bar graphs show the mean ± SD of fold induction from samples assayed in triplicates. Fold induction is the ratio between the average luciferase activities of the ligand-treated and the ligand-untreated samples. Results shown are representative for three independent experiments. Source data are available online for this figure. Source Data for Figure 1J [embj201899669-sup-0003-SDataFig1.pdf] Download figure Download PowerPoint Table 1. Binding partners of DSCAM/L1 identified with MAPPIT. Further validation of the candidates can be found in Fig EV1 and Appendix Figs S1–S3 Symbol Name Function Interaction with DSCAM Interaction with DSCAML1 IPO5 Importin 5 Nuclear protein import. Binds to cargo NLS Binds to NLS of DSCAM: RRRRREQR (Fig 1I) Binds to NLS of DSCAML1: RKKRKEKR (Fig 1I) STAT3 Signal transducer and activator of transcription 3 Transcriptional activator in response to cytokines and growth factors Binds to YASQ motif of DSCAM (Appendix Fig S2B) Binds to YSSQ/YHTQ motifs of DSCAML1 (Appendix Fig S2E) SH2D2A SH2 domain containing 2A SH2 domain adaptor protein for VEGF receptor KDR. Functions in T-cell signal transduction N/A (Appendix Fig S2J) Binds to YCNL motif of DSCAML1 (Appendix Fig S2I) DYRK1A Dual-Specificity-Tyrosine-Phosphorylation Regulated Kinase 1A S/T/Y-kinase. Plays important roles in neuronal development. Down Syndrome candidate gene Interaction through C-terminus of DYRK1A (Fig 1E) Interaction through C-terminus of DYRK1A (Appendix Fig S3B) DYRK1B Dual-Specificity-Tyrosine-Phosphorylation Regulated Kinase 1B S/T/Y-kinase. Regulates transcription in the nucleus Interaction through C-terminus of DYRK1B (Fig 1D) Interaction through C-terminus of DYRK1B (Appendix Fig S3A) USP21 Ubiquitin specific peptidase 21 Cysteine protease with dual specificity to cleave Ubiquitin and Nedd8 Binds to YASQ motif of DSCAM (Appendix Fig S2C) Binds to YSSQ/YHTQ motifs of DSCAML1 (Appendix Fig S2F) We further validated the MAPPIT screen candidates by co-immunoprecipitation from cell lines co-transfected with HA-tagged DSCAM/L1 and their novel interaction partners (Fig EV1). Additionally, we identified potential binding motifs for STAT3, SH2D2A, and USP21 (Table 1; Appendix Fig S2A–J). Mutation of those motifs in the bait receptors confirmed that specific tyrosine residues within the DSCAM and DSCAML1 ICDs function as binding sites for STAT3, USP21, and SH2D2A (Table 1; Appendix Fig S2A–J). Furthermore, we analyzed the interactions between DSCAM/L1 and DYRK1A/B in more detail. We found that a truncated DYRK1B prey, which consists of the most C-terminal 72 amino acids and lacks the kinase domain (Fig 1C), was sufficient to interact with DSCAM and DSCAML1 baits in MAPPIT (Fig 1D; Appendix Fig S3A). Moreover, a truncated DYRK1A prey, consisting of its N-terminal and its kinase domains but lacking its C-terminal domains (Fig 1C), failed to bind to the DSCAM or DSCAML1 baits in contrast to full-length (FL) DYRK1A (Fig 1E; Appendix Fig S3B). Taken together, this suggests that not the kinase domains, but the C-terminal domains of DYRK1A and B mediate the interaction with DSCAM/L1. Both DSCAM and DSCAML1 ICDs exhibit a potential DYRK substrate motif (RPGTNP). A triple alanine substitution of the RPx(T/S/x)P motif previously shown to abolish the interaction of DYRK1A with a substrate peptide (Soundararajan et al, 2013) was generated (RPGTNP to AAGANP) for both DSCAM and DSCAML1. However, the AAGANP mutant baits could still interact with DYRKs (Fig 1F, Appendix Fig S3C and D), further suggesting that binding of DYRKs to DSCAM/L1 might be independent of their kinase domains, as previously suggested for other DYRK1A interactors (Aranda et al, 2008). In conclusion, the MAPPIT approach leads to the identification of IPO5, STAT3, DYRK1A/B, SH2D2A, and USP21 as novel potential downstream effectors of DSCAM/L1. Click here to expand this figure. Figure EV1. Validation of MAPPIT Interactions by co-immunoprecipitation (related to Fig 1) A, B. HEK293T cells or SH-SY5Y cells were co-transfected with either DSCAM-HA or DSCAML1-HA together with gp130-Flag-tagged SVT (SV40 large T antigen, unrelated negative control), DYRK1A, DYRK1B, STAT3, USP21, SH2D2A, or Flag-tagged IPO5. HA-tagged DSCAM/L1 proteins were precipitated using anti-HA magnetic beads and co-precipitated protein complexes were analyzed by Western blot using HA- and Flag-tag specific antibodies. (A) DSCAM-HA co-precipitates with gp130-Flag-tagged DYRK1A, DYRK1B, STAT3, SH2D2A and USP21 in HEK293T cells or Flag-tagged IPO5 in SH-SY5Y cells. (B) DSCAML1-HA co-precipitates with gp130-Flag-tagged DYRK1A, DYRK1B, STAT3, USP21, SH2D2A, or Flag-tagged IPO5 in HEK293T cells. Source data are available online for this figure. Download figure Download PowerPoint DSCAM and DSCAML1 interact with IPO5 via a nuclear localization signal Since binary MAPPIT assays confirmed the interactions of both DSCAM and DSCAML1 with Importin 5 (IPO5; Fig 1G), we reasoned that DSCAMs might have nuclear functions. Such membrane-to-nucleus signaling mechanism would be similar to Neogenin and DCC, two neuronal CAMs that are closely related to vertebrate DSCAMs and cleaved by proteases resulting in the release of C-terminal ICD fragments, which then translocate into the nucleus where they regulate gene transcription (Taniguchi et al, 2003; Goldschneider et al, 2008). IPO5 is an importin beta mediating nuclear protein import (Yaseen & Blobel, 1997) and can directly interact with the nuclear localization signal (NLS) of its cargo (Chao et al, 2012). We therefore considered that the ICDs of DSCAMs might contain an NLS serving as docking site for IPO5. Indeed, upon sequence analysis we identified a potential monopartite NLS within the membrane-proximal regions enriched in conserved Arginine and Lysine residues (Fig 1H). Interestingly, Drosophila Dscam1 also exhibits a predicted NLS within the cytoplasmic portion that is common to all its isoforms, indicating that a membrane-proximal NLS is highly conserved from insects to vertebrates (Fig 1H). To test its functional relevance, we generated NLS-deficient MAPPIT bait versions (i.e., LR-DSCAMΔNLS and LR-DSCAML1ΔNLS), which failed to interact with the IPO5 prey but could still interact with the STAT3 control prey (Fig 1I), demonstrating that the NLSs are required for the interaction between IPO5 and DSCAM/L1. To determine specificity, we tested all full-length importin alpha and beta preys present in the human ORFeome collection 5.1 and 8.1. Out of the 9 importins tested, only IPO5 interacted with DSCAM and DSCAML1 (Fig 1J). Together, these results show that IPO5 can bind to the membrane-proximal NLS motifs of DSCAM and DSCAML1 with high specificity. DSCAM is cleaved by γ-secretase Several neuronal transmembrane proteins including APP, DCC, Neogenin, and Notch undergo ectodomain cleavage directly followed by γ-secretase-mediated intra-membrane cleavage leading to the release of their ICDs (De Strooper et al, 1999; Cupers et al, 2001; Taniguchi et al, 2003; Goldschneider et al, 2008). Shedding of the DSCAM ectodomain has been reported in flies and mice (Watson et al, 2005; Schramm et al, 2012); however, it is currently unknown whether the ICD of DSCAMs could be released by proteolytic cleavage. To investigate this, we fused HA-epitopes to the very C-terminus of the DSCAM/L1 ICDs as well as to FL DSCAM/L1 (Fig 2A) and expressed these fusion proteins in HEK293T cells. Immunoprecipitation with an HA-specific antibody and Western blot (WB) analysis showed FL DSCAM migrating at approximately 250 kDA as well as a C-terminal fragment at 55 kDa, which co-migrates with the DSCAM ICD construct at approximately the same molecular weight (Fig 2B). Likewise, FL DSCAML1 migrated at nearly 250 kDa and we detected two additional C-terminal fragments migrating at 65 kDa and at 60 kDa, co-migrating with two fragments originating from the DSCAML1 ICD HA-fusion (Fig 2B). This suggests that DSCAM and DSCAML1 are substrates for a protease and cleavage generates C-terminal ICD fragments. Figure 2. DSCAM family IgCAMs are cleaved releasing their ICDs Schematic of C-terminally HA-tagged DSCAM and DSCAML1 constructs. ECD, extracellular domain. TM, transmembrane domain. ICD, intracellular domain. HA, human influenza hemagglutinin epitope. Immunoblot showing C-termina" @default.
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• W2912846978 date "2019-02-11" @default.
• W2912846978 modified "2023-10-15" @default.
• W2912846978 title "Nuclear import of the <scp>DSCAM</scp> ‐cytoplasmic domain drives signaling capable of inhibiting synapse formation" @default.
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