title: Disease-associated missense mutations in the EVH1 domain disrupt intrinsic WASp function causing dysregulated actin dynamics and impaired dendritic cell migration.
abstract: Wiskott Aldrich syndrome (WAS), an X-linked immunodeficiency, results from loss-of-function mutations in the human hematopoietic cytoskeletal regulator gene WAS. Many missense mutations in the Ena Vasp homology1 (EVH1) domain preserve low-level WAS protein (WASp) expression and confer a milder clinical phenotype. Although disrupted binding to WASp-interacting protein (WIP) leads to enhanced WASp degradation in vivo, the intrinsic function of EVH1-mutated WASp is poorly understood. In the present study, we show that, despite mediating enhanced actin polymerization compared with wild-type WASp in vitro, EVH1 missense mutated proteins did not support full biologic function in cells, even when levels were restored by forced overexpression. Podosome assembly was aberrant and associated with dysregulated lamellipodia formation and impaired persistence of migration. At sites of residual podosome-associated actin polymerization, localization of EVH1-mutated proteins was preserved even after deletion of the entire domain, implying that WIP-WASp complex formation is not absolutely required for WASp localization. However, retention of mutant proteins in podosomes was significantly impaired and associated with reduced levels of WASp tyrosine phosphorylation. Our results indicate that the EVH1 domain is important not only for WASp stability, but also for intrinsic biologic activity in vivo.
authors.affiliation: Molecular Immunology Unit, Institute of Child Health, University College London, London, United Kingdom. a.worth@ucl.ac.uk
authors.forename: Austen J J
authors.initials: AJ
authors.lastname: Worth
authors.forename: Joao
authors.initials: J
authors.lastname: Metelo
authors.forename: Gerben
authors.initials: G
authors.lastname: Bouma
authors.forename: Dale
authors.initials: D
authors.lastname: Moulding
authors.forename: Marco
authors.initials: M
authors.lastname: Fritzsche
authors.forename: Bertrand
authors.initials: B
authors.lastname: Vernay
authors.forename: Guillaume
authors.initials: G
authors.lastname: Charras
authors.forename: Giles O C
authors.initials: GO
authors.lastname: Cory
authors.forename: Adrian J
authors.initials: AJ
authors.lastname: Thrasher
authors.forename: Siobhan O
authors.initials: SO
authors.lastname: Burns
mesh_terms: D000199:Actins / Q000378:metabolism; D000818:Animals; D001704:Biopolymers; D002352:Carrier Proteins / Q000378:metabolism; D002465:Cell Movement; D002478:Cells, Cultured; D003598:Cytoskeletal Proteins; D003713:Dendritic Cells / Q000378:metabolism / Q000473:pathology*; D006801:Humans; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D008822:Mice, Transgenic; D020125:Mutation, Missense; D010766:Phosphorylation; D058105:Polymerization; D025941:Protein Interaction Mapping; D011499:Protein Processing, Post-Translational; D055550:Protein Stability; D017434:Protein Structure, Tertiary; D011554:Pseudopodia / Q000473:pathology; D011993:Recombinant Fusion Proteins / Q000502:physiology; D017384:Sequence Deletion; D013047:Specific Pathogen-Free Organisms; D051316:Wiskott-Aldrich Syndrome Protein / Q000737:chemistry / Q000172:deficiency / Q000235:genetics* / Q000378:metabolism / Q000502:physiology
chemical_list: D000199:Actins; D001704:Biopolymers; D002352:Carrier Proteins; D003598:Cytoskeletal Proteins; D011993:Recombinant Fusion Proteins; C495767:WAS protein, human; C495768:Was protein, mouse; C491671:Waspip protein, mouse; D051316:Wiskott-Aldrich Syndrome Protein