title: Nuclear Localization of the Autism Candidate Gene Neurobeachin and Functional Interaction with the NOTCH1 Intracellular Domain Indicate a Role in Regulating Transcription.
abstract: BACKGROUND
  Neurobeachin (NBEA) is an autism spectrum disorders (ASD) candidate gene. NBEA deficiency affects regulated secretion, receptor trafficking, synaptic architecture and protein kinase A (PKA)-mediated phosphorylation. NBEA is a large multidomain scaffolding protein. From N- to C-terminus, NBEA has a concanavalin A-like lectin domain flanked by armadillo repeats (ACA), an A-kinase anchoring protein domain that can bind to PKA, a domain of unknown function (DUF1088) and a BEACH domain, preceded by a pleckstrin homology-like domain and followed by WD40 repeats (PBW). Although most of these domains mediate protein-protein interactions, no interaction screen has yet been performed.
  
  
  METHODS
  Yeast two-hybrid screens with the ACA and PBW domain modules of NBEA gave a list of interaction partners, which were analyzed for Gene Ontology (GO) enrichment. Neuro-2a cells were used for confocal microscopy and nuclear extraction analysis. NOTCH-mediated transcription was studied with luciferase reporter assays and qRT-PCR, combined with NBEA knockdown or overexpression.
  
  
  RESULTS
  Both domain modules showed a GO enrichment for the nucleus. PBW almost exclusively interacted with transcription regulators, while ACA interacted with a number of PKA substrates. NBEA was partially localized in the nucleus of Neuro-2a cells, albeit much less than in the cytoplasm. A nuclear localization signal was found in the DUF1088 domain, which was shown to contribute to the nuclear localization of an EGFP-DPBW fusion protein. Yeast two-hybrid identified the Notch1 intracellular domain as a physical interactor of the PBW domain and a role for NBEA as a negative regulator in Notch-mediated transcription was demonstrated.
  
  
  CONCLUSIONS
  Defining novel interaction partners of conserved NBEA domain modules identified a role for NBEA as transcriptional regulator in the nucleus. The physical interaction of NBEA with NOTCH1 is most relevant for ASD pathogenesis because NOTCH signaling is essential for neural development.
authors.affiliation: Department of Human Genetics, KU Leuven, Leuven, Belgium.|Leuven Autism Research consortium (LAuRes), KU Leuven, Leuven, Belgium.
authors.forename: Krizia
authors.initials: K
authors.lastname: Tuand
authors.affiliation: Department of Human Genetics, KU Leuven, Leuven, Belgium.
authors.forename: Pieter
authors.initials: P
authors.lastname: Stijnen
authors.affiliation: Department of Human Genetics, KU Leuven, Leuven, Belgium.|Leuven Autism Research consortium (LAuRes), KU Leuven, Leuven, Belgium.
authors.forename: Karolien
authors.initials: K
authors.lastname: Volders
authors.affiliation: Department of Human Genetics, KU Leuven, Leuven, Belgium.
authors.forename: Jeroen
authors.initials: J
authors.lastname: Declercq
authors.affiliation: Department of Human Genetics, KU Leuven, Leuven, Belgium.|Leuven Autism Research consortium (LAuRes), KU Leuven, Leuven, Belgium.
authors.forename: Kim
authors.initials: K
authors.lastname: Nuytens
authors.affiliation: Department of Human Genetics, KU Leuven, Leuven, Belgium.
authors.forename: Sandra
authors.initials: S
authors.lastname: Meulemans
authors.affiliation: Department of Human Genetics, KU Leuven, Leuven, Belgium.
authors.forename: John
authors.initials: J
authors.lastname: Creemers
mesh_terms: D000818:Animals; D001321:Autistic Disorder / Q000235:genetics*; D002352:Carrier Proteins / Q000378:metabolism*; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism*; D055785:Gene Knockdown Techniques; D056726:Genetic Association Studies; D006801:Humans; D008565:Membrane Proteins; D051379:Mice; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D019913:Nuclear Localization Signals / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D051881:Receptor, Notch1 / Q000737:chemistry* / Q000378:metabolism*; D014158:Transcription, Genetic; D020798:Two-Hybrid System Techniques
chemical_list: D002352:Carrier Proteins; D008565:Membrane Proteins; C403265:Nbea protein, mouse; D009419:Nerve Tissue Proteins; C497762:Notch1 protein, mouse; D019913:Nuclear Localization Signals; D051881:Receptor, Notch1