RESUMO
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.
Assuntos
Células Dendríticas/patologia , Mutação de Sentido Incorreto , Proteína da Síndrome de Wiskott-Aldrich/genética , Actinas/metabolismo , Animais , Biopolímeros , Proteínas de Transporte/metabolismo , Movimento Celular , Células Cultivadas , Proteínas do Citoesqueleto , Células Dendríticas/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Fosforilação , Polimerização , Mapeamento de Interação de Proteínas , Processamento de Proteína Pós-Traducional , Estabilidade Proteica , Estrutura Terciária de Proteína , Pseudópodes/patologia , Proteínas Recombinantes de Fusão/fisiologia , Deleção de Sequência , Organismos Livres de Patógenos Específicos , Proteína da Síndrome de Wiskott-Aldrich/química , Proteína da Síndrome de Wiskott-Aldrich/deficiência , Proteína da Síndrome de Wiskott-Aldrich/metabolismo , Proteína da Síndrome de Wiskott-Aldrich/fisiologiaRESUMO
New work on the ability of IRSp53/MIM domains to induce negative membrane curvature sheds light on the mechanisms used to generate actin-rich cell protrusions.
Assuntos
Actinas/metabolismo , Membrana Celular/ultraestrutura , Endocitose/fisiologia , Proteínas do Tecido Nervoso/metabolismo , Pseudópodes/metabolismo , Animais , Membrana Celular/metabolismo , Dimerização , Humanos , Modelos Moleculares , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Pseudópodes/ultraestruturaRESUMO
Wiskott-Aldrich syndrome protein (WASP) and neural (N)-WASP regulate dynamic actin structures through the ability of their VCA domains to bind to and stimulate the actin nucleating activity of the Arp2/3 complex. Here we identify two phosphorylation sites in the VCA domain of WASP at serines 483 and 484. S483 and S484 are substrates for casein kinase 2 in vitro and in vivo. Phosphorylation of these residues increases the affinity of the VCA domain for the Arp2/3 complex 7-fold and is required for efficient in vitro actin polymerization by the full-length WASP molecule. We propose that constitutive VCA domain phosphorylation is required for optimal stimulation of the Arp2/3 complex by WASP.
Assuntos
Actinas/biossíntese , Proteínas do Citoesqueleto/metabolismo , Células Eucarióticas/metabolismo , Proteínas/metabolismo , Regulação para Cima/genética , Células 3T3 , Proteína 2 Relacionada a Actina , Proteína 3 Relacionada a Actina , Sequência de Aminoácidos/genética , Animais , Especificidade de Anticorpos/imunologia , Sítios de Ligação/genética , Células COS , Proteínas do Citoesqueleto/genética , Camundongos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Fosforilação , Polímeros/metabolismo , Estrutura Terciária de Proteína/genética , Proteínas/genética , Serina/metabolismo , Proteína da Síndrome de Wiskott-Aldrich , Proteína Neuronal da Síndrome de Wiskott-AldrichRESUMO
Wiskott-Aldrich Syndrome protein (WASp) is a key regulator of the Arp2/3 complex and the actin cytoskeleton in hematopoietic cells. WASp is capable of forming an auto-inhibited conformation, which can be disrupted by binding of Cdc42 and phosphatidylinositol 4,5-bisphosphate, leading to its activation. Stimulation of the collagen receptor on platelets and crosslinking the B-cell receptor induce tyrosine phosphorylation of WASp. Here we show that the Src family kinase Hck induces phosphorylation of WASp-Tyr(291) independently of Cdc42 and that this causes a shift in the mobility of WASp upon SDS-PAGE. A phospho-mimicking mutant, WASp-Y291E, exhibited an enhanced ability to stimulate actin polymerization in a cell-free system and when microinjected into primary macrophages induced extensive filopodium formation with greater efficiency than wild-type WASp or a Y291F mutant. We propose that phosphorylation of Tyr(291) directly regulates WASp function.