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New Partners Identified by Mass Spectrometry Assay Reveal Functions of NCAM2 in Neural Cytoskeleton Organization

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New Partners Identified by Mass Spectrometry Assay Reveal Functions of NCAM2 in Neural Cytoskeleton Organization International Journal of Molecular Sciences Article New Partners Identified by Mass Spectrometry Assay Reveal Functions of NCAM2 in Neural Cytoskeleton Organization Antoni Parcerisas 1,2,3,*,† , Alba Ortega-Gascó 1,2,† , Marc Hernaiz-Llorens 1,2 , Maria Antonia Odena 4, Fausto Ulloa 1,2, Eliandre de Oliveira 4, Miquel Bosch 3 , Lluís Pujadas 1,2 and Eduardo Soriano 1,2,* 1 Department of Cell Biology, Physiology and Immunology, University of Barcelona and Institute of Neurosciences, 08028 Barcelona, Spain; [email protected] (A.O.-G.); [email protected] (M.H.-L.); [email protected] (F.U.); [email protected] (L.P.) 2 Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), 28031 Madrid, Spain 3 Department of Basic Sciences, Universitat Internacional de Catalunya, 08195 Sant Cugat del Vallès, Spain; [email protected] 4 Plataforma de Proteòmica, Parc Científic de Barcelona (PCB), 08028 Barcelona, Spain; [email protected] (M.A.O.); [email protected] (E.d.O.) * Correspondence: [email protected] (A.P.); [email protected] (E.S.) † A.P. and A.O.-G. contributed equally. Abstract: Neuronal cell adhesion molecule 2 (NCAM2) is a membrane protein with an important role in the morphological development of neurons. In the cortex and the hippocampus, NCAM2 is essential for proper neuronal differentiation, dendritic and axonal outgrowth and synapse forma- tion. However, little is known about NCAM2 functional mechanisms and its interactive partners Citation: Parcerisas, A.; during brain development. Here we used mass spectrometry to study the molecular interactome Ortega-Gascó, A.; Hernaiz-Llorens, of NCAM2 in the second postnatal week of the mouse cerebral cortex. We found that NCAM2 M.; Odena, M.A.; Ulloa, F.; de interacts with >100 proteins involved in numerous processes, including neuronal morphogenesis Oliveira, E.; Bosch, M.; Pujadas, L.; Soriano, E. New Partners Identified and synaptogenesis. We validated the most relevant interactors, including Neurofilaments (NEFs), by Mass Spectrometry Assay Reveal Microtubule-associated protein 2 (MAP2), Calcium/calmodulin kinase II alpha (CaMKIIα), Actin Functions of NCAM2 in Neural and Nogo. An in silico analysis of the cytosolic tail of the NCAM2.1 isoform revealed specific Cytoskeleton Organization. Int. J. phosphorylation site motifs with a putative affinity for some of these interactors. Our results expand Mol. Sci. 2021, 22, 7404. https:// the knowledge of NCAM2 interactome and confirm the key role of NCAM2 in cytoskeleton organiza- doi.org/10.3390/ijms22147404 tion, neuronal morphogenesis and synaptogenesis. These findings are of interest in explaining the phenotypes observed in different pathologies with alterations in the NCAM2 gene. Academic Editor: Thomas Fath Keywords: NCAM2; mass spectrometry; cytoskeleton; neuronal morphogenesis; MAP2; CaMKIIα; Received: 15 June 2021 Neurofilaments; Nogo Accepted: 7 July 2021 Published: 9 July 2021 Publisher’s Note: MDPI stays neutral 1. Introduction with regard to jurisdictional claims in published maps and institutional affil- Neuronal differentiation, and the establishment of cell polarity and synaptic connec- iations. tions, are crucial events for the development of the brain [1,2]. These processes are tightly regulated by numerous factors, including cytoskeleton proteins, membrane receptors and elements of the extracellular matrix. Cell Adhesion Molecules (CAMs) are a family of mem- brane proteins with many functions that transduce extracellular and membrane-bound signals into cellular events, such as membrane remodeling, cytoskeletal rearrangement and Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. dynamics, vesicular transport, gene expression and cell survival [3–5]. This article is an open access article The immunoglobulin superfamily of cell adhesion molecules (IgSF CAM) includes distributed under the terms and more than 50 different members in mammals [6]. IgSF CAMs are characterized by an conditions of the Creative Commons extracellular region with one or several immunoglobulin-like (Ig) domains, followed by Attribution (CC BY) license (https:// fibronectin type III (Fn3) domains. Most IgSF CAMs present a single transmembrane creativecommons.org/licenses/by/ domain with an intracellular tail, while other members are anchored to the cell membrane 4.0/). through a glycosylphosphatidylinositol (GPI) anchor [7]. Typically, the extracellular and Int. J. Mol. Sci. 2021, 22, 7404. https://doi.org/10.3390/ijms22147404 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2021, 22, 7404 2 of 20 the intracellular domains of IgSF CAMs interact with several proteins, ligands or modifiers, which determine their important roles during development [5,8,9]. The Neural Cell Adhesion Molecule (NCAM) family has two members, NCAM1 and NCAM2 [10,11]. Both proteins present a similar extracellular structure with five Ig domains and two Fn3 domains. NCAM1 has three different isoforms (180, 140 and 120 KDa), whereas NCAM2 has two isoforms: NCAM2.1 (with a transmembrane domain and a cytoplasmic tail) and NCAM2.2 (a shorter isoform with no transmembrane domain but a GPI-anchor motif) [12,13]. A large number of studies have investigated the functions of NCAM1, which play a fundamental role in both neural development and plasticity [8,14,15]. NCAM1 inter- acts with many extracellular ligands and adaptors, such as the Fibroblast Growth Factor Receptor (FGFR), Prion Protein (PRNP), Homer1, Tyrosine-protein kinase Fyn or Focal adhesion kinase 1 (FAK) [16–20]. By contrast, NCAM2 is less studied. NCAM2 is widely expressed in the Central Nervous System (CNS) during brain development. In the olfactory system NCAM2 is necessary for the formation and maintenance of dendritic and axonal compartments [21–26], and in synapse formation and maintenance [26–28]. Genetic studies suggest that the NCAM2 gene is implicated in the intellectual disability phenotype in Down syndrome and Autism Spectrum Disorders, as well as in other neurodevelopmental dis- eases [24,29–32]. In addition, NCAM2 has been involved in synaptic deficits in Alzheimer’s disease [28]. Although NCAM2 functions have begun to be clarified [25,26,28], the ex- tracellular ligands and intracellular adaptors that interact with NCAM2 remain largely unknown. It has been recently shown that NCAM2 regulates neurite outgrowth through the kinase Src in the cerebral cortex [26,27], and that it mediates dendritic morphogenesis via the formation of molecular complexes with Microtubule-associated protein 2 (MAP2) and 14-3-3 family proteins [25]. To gain insight into NCAM2 functions, here we investigate the interactome of the NCAM2 protein during postnatal cortical development using proteomic and molecular approaches. We found more than 100 proteins that interact with NCAM2 using mass spectrometry. We further validated the more relevant interactions for cytoskeleton organi- zation (CaMKIIα, NEFs, Actin and MAP2) by means of immunoprecipitation. To better characterize the NCAM2 interactome, our proteomic data were analyzed by bioinformatic tools; we detected significant enrichments in gene ontology terms and in cellular pathways linked to the cytoskeleton, as well as to other important neural functions. In addition, we identify putative phosphorylation sites of the NCAM2.1 cytosolic tail using in silico analysis. These data increase our knowledge about the interactome of the NCAM2 protein and open new perspectives for the study of NCAM2 functions. 2. Results 2.1. In Silico Analysis of the Cytoplasmic Domains of NCAM2.1 Previous studies showed the importance of the cytoplasmic domains of CAMs, such as NCAM1 (NCAM140 or NCAM180 isoforms). We thus performed a structural analysis of the amino acid sequence of the NCAM2.1 cytosolic domain (Figure1A). The residues S765, T780 and S786 have already been described as phosphorylation sites [33]. We used different bioinformatic tools (Scansite 4.0 and Disphos 1.3 analysis, Koch Institute and MIT, Cambridge, MA, USA) to identify additional phosphorylatable residues. High scores for S746 and T818, which match the consensus sequences of 14-3-3ζ and CaMKIIγ, respectively, were detected with Scansite 4.0. Disphos analysis showed that approximately 70% (12/17) of the phosphorylatable residues have significant scores to be phosphorylated (serines 7/9, threonines 4/7 and tyrosine 1/1), as shown in Figure1B–D. We also detected different motifs that match the consensus sequences of the kinases Cyclin dependent kinase 5 (CDK5), Protein kinase C alpha (PRKCA), Casein kinase II (CSNK2) and Glycogen synthase 3 (GSK3), using Scansite. These are kinases known to phosphorylate other CAMs, such as Cadherins, Epithelial cell adhesion molecule (EpCAM), Integrins or Neuronglia cell adhesion molecule (NgCAM) [34–39]. Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 3 of 21 We also detected different motifs that match the consensus sequences of the kinases Cyclin dependent kinase 5 (CDK5), Protein kinase C alpha (PRKCA), Casein kinase II Int. J. Mol. Sci. 2021, 22, 7404 (CSNK2) and Glycogen synthase 3 (GSK3), using Scansite. These are kinases known to 3 of 20 phosphorylate other CAMs, such as Cadherins, Epithelial cell adhesion molecule (EpCAM), Integrins or Neuronglia cell adhesion molecule (NgCAM) [34–39]. FigureFigure 1. In silico1. In silico analysis analysis of NCAM2.1of NCAM2.1 cytoplasmatic cytoplasmatic domain. domain. (A ()A Schematic) Schematic representation representation of phosphorylation of phosphorylation sites in sites in NCAM2.1NCAM2.1 cytoplasmatic
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