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
Specific mutations in the human gene encoding the Wiskott-Aldrich syndrome protein (WASp) that compromise normal auto-inhibition of WASp result in unregulated activation of the actin-related protein 2/3 complex and increased actin polymerizing activity. These activating mutations are associated with an X-linked form of neutropenia with an intrinsic failure of myelopoiesis and an increase in the incidence of cytogenetic abnormalities. To study the underlying mechanisms, active mutant WASp(I294T) was expressed by gene transfer. This caused enhanced and delocalized actin polymerization throughout the cell, decreased proliferation, and increased apoptosis. Cells became binucleated, suggesting a failure of cytokinesis, and micronuclei were formed, indicative of genomic instability. Live cell imaging demonstrated a delay in mitosis from prometaphase to anaphase and confirmed that multinucleation was a result of aborted cytokinesis. During mitosis, filamentous actin was abnormally localized around the spindle and chromosomes throughout their alignment and separation, and it accumulated within the cleavage furrow around the spindle midzone. These findings reveal a novel mechanism for inhibition of myelopoiesis through defective mitosis and cytokinesis due to hyperactivation and mislocalization of actin polymerization.
Assuntos
Actinas/metabolismo , Citocinese , Doenças Genéticas Ligadas ao Cromossomo X/metabolismo , Mitose , Neutropenia/metabolismo , Neutropenia/patologia , Proteína da Síndrome de Wiskott-Aldrich/metabolismo , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , Aberrações Cromossômicas , Cromossomos Humanos , Citocinese/efeitos dos fármacos , DNA , Depsipeptídeos/farmacologia , Doenças Genéticas Ligadas ao Cromossomo X/patologia , Proteínas de Fluorescência Verde/metabolismo , Humanos , Camundongos , Mitose/efeitos dos fármacos , Proteínas Mutantes/metabolismo , Poliploidia , Proteínas Recombinantes de Fusão/metabolismo , TransgenesRESUMO
Podosomes are actin-based adhesions involved in migration of cells that have to cross tissue boundaries such as myeloid cells. The Wiskott Aldrich Syndrome Protein regulates de novo actin polymerization during podosome formation and it is cleaved by the protease calpain during podosome disassembly. The mechanisms that may induce the Wiskott Aldrich Syndrome Protein cleavage by calpain remain undetermined. We now report that in myeloid cells, tyrosine phosphorylation of the Wiskott Aldrich Syndrome Protein-tyrosine291 (Human)/tyrosine293 (mouse) not only enhances Wiskott Aldrich Syndrome Protein-mediated actin polymerization but also promotes its calpain-dependent degradation during podosome disassembly. We also show that activation of the Wiskott Aldrich Syndrome Protein leading to podosome formation occurs independently of tyrosine phosphorylation in spleen-derived dendritic cells. We conclude that tyrosine phosphorylation of the Wiskott Aldrich Syndrome Protein integrates dynamics of actin and cell adhesion proteins during podosome disassembly required for mobilization of myeloid cells during the immune response.
Assuntos
Citoesqueleto de Actina/fisiologia , Calpaína/metabolismo , Estruturas da Membrana Celular/metabolismo , Tirosina/metabolismo , Proteína da Síndrome de Wiskott-Aldrich/fisiologia , Animais , Adesão Celular , Movimento Celular , Células Cultivadas , Células Dendríticas/citologia , Células Dendríticas/metabolismo , Imunofluorescência , Macrófagos/citologia , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Células Mieloides/citologia , Células Mieloides/metabolismo , Fosforilação , Ligação ProteicaRESUMO
The Wiskott-Aldrich syndrome protein (WASp) is a key cytoskeletal regulator in hematopoietic cells. Covalent modification of a conserved tyrosine by phosphorylation has emerged as an important potential determinant of activity, although the physiological significance remains uncertain. In a murine knockin model, mutation resulting in inability to phosphorylate Y293 (Y293F) mimicked many features of complete WASp-deficiency. Although a phosphomimicking mutant Y293E conferred enhanced actin-polymerization, the cellular phenotype was similar due to functional dysregulation. Furthermore, steady-state levels of Y293E-WASp were markedly reduced compared to wild-type WASp and Y293F-WASp, although partially recoverable by treatment of cells with proteasome inhibitors. Consequently, tyrosine phosphorylation plays a critical role in normal activation of WASp in vivo, and is indispensible for multiple tasks including proliferation, phagocytosis, chemotaxis, and assembly of adhesion structures. Furthermore, it may target WASp for proteasome-mediated degradation, thereby providing a default mechanism for self-limiting stimulation of the Arp2/3 complex.
Assuntos
Proteína da Síndrome de Wiskott-Aldrich/metabolismo , Actinas/metabolismo , Substituição de Aminoácidos , Animais , Sítios de Ligação/genética , Células COS , Linhagem Celular , Movimento Celular , Chlorocebus aethiops , Hematopoese , Humanos , Técnicas In Vitro , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Mutagênese Sítio-Dirigida , Fagocitose , Fosforilação , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Tirosina/química , Síndrome de Wiskott-Aldrich/genética , Síndrome de Wiskott-Aldrich/metabolismo , Síndrome de Wiskott-Aldrich/patologia , Proteína da Síndrome de Wiskott-Aldrich/química , Proteína da Síndrome de Wiskott-Aldrich/genéticaRESUMO
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
The (Wiskott-Aldrich Syndrome Protein)-family verprolin homologous protein (WAVE) family of proteins occupies a pivotal position in the cell, converting extracellular signals into the formation of branched filamentous (F) actin structures. WAVE proteins contain a verprolin central acidic (VCA) domain at their C-terminus, responsible for binding to and activating the Arp2/3 complex, which in-turn nucleates the formation of new actin filaments. Here we identify five Casein Kinase 2 (CK2) phosphorylation sites within the VCA domain of WAVE2, serines 482, 484, 488, 489, and 497. Phosphorylation of these sites is required for a high affinity interaction with the Arp2/3 complex. Phosphorylation of ser 482 and 484 specifically inhibits the activation of the Arp2/3 complex by the WAVE2 VCA domain, but has no effect on the affinity for the Arp2/3 complex when the other phosphorylation sites are occupied. We demonstrate phosphorylation of all five sites on endogenous WAVE2 and show that their mutation to non-phosphorylatable alanine residues inhibits WAVE2 function in vivo, inhibiting cell ruffling and disrupting the integrity of the leading edge of migrating cells.
Assuntos
Família de Proteínas da Síndrome de Wiskott-Aldrich/química , Família de Proteínas da Síndrome de Wiskott-Aldrich/metabolismo , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Sequência de Aminoácidos , Animais , Células COS , Caseína Quinase II/metabolismo , Movimento Celular/efeitos dos fármacos , Chlorocebus aethiops , Humanos , Camundongos , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Dados de Sequência Molecular , Células NIH 3T3 , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Estrutura Terciária de Proteína , Pseudópodes/efeitos dos fármacos , Pseudópodes/enzimologia , RatosRESUMO
The WAVE family of proteins has long been implicated in the stimulus-dependent generation of lamellipodia at the leading edge of migrating cells, with WAVE2 in particular implicated in the formation of peripheral ruffles and chemotactic migration. However, the lack of direct visualisation of cell migration in WAVE2 mutants or knockdowns has made defining the mechanisms of WAVE2 regulation during cell migration difficult. We have characterised three MAP kinase phosphorylation sites within WAVE2 and analysed fibroblast behaviour in a scratch-wound model following introduction of transgenes encoding phospho-defective WAVE2. The cells exhibited an increase in migration speed, a decrease in the persistence of migration, and disruption of polarisation of the Golgi apparatus. All these effects could be mimicked by acute knockdown of endogenous WAVE2 expression with RNAi, indicating that phosphorylation of WAVE2 by MAP kinases regulates cell polarity during migration.
Assuntos
Movimento Celular , Polaridade Celular , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Família de Proteínas da Síndrome de Wiskott-Aldrich/metabolismo , Animais , Células COS , Chlorocebus aethiops , Fibroblastos/citologia , Fibroblastos/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Camundongos , Células NIH 3T3 , Fosforilação , Testes de Precipitina , Estrutura Terciária de Proteína , Interferência de RNA , RNA Interferente Pequeno/farmacologia , Transgenes , Família de Proteínas da Síndrome de Wiskott-Aldrich/químicaRESUMO
Severe congenital neutropenia (SCN) is characterized by neutropenia, recurrent bacterial infections, and maturation arrest in the bone marrow. Although many cases have mutations in the ELA2 gene encoding neutrophil elastase, a significant proportion remain undefined at a molecular level. A mutation (Leu270Pro) in the gene encoding the Wiskott-Aldrich syndrome protein (WASp) resulting in an X-linked SCN kindred has been reported. We therefore screened the WAS gene in 14 young SCN males with wild-type ELA2 and identified 2 with novel mutations, one who presented with myelodysplasia (Ile294Thr) and the other with classic SCN (Ser270Pro). Both patients had defects of immunologic function including a generalized reduction of lymphoid and natural killer cell numbers, reduced lymphocyte proliferation, and abrogated phagocyte activity. In vitro culture of bone marrow progenitors demonstrated a profound reduction in neutrophil production and increased levels of apoptosis, consistent with an intrinsic disturbance of normal myeloid differentiation as the cause of the neutropenia. Both mutations resulted in increased WASp activity and produced marked abnormalities of cytoskeletal structure and dynamics. Furthermore, these results also suggest a novel cause of myelodysplasia and that male children with myelodysplasia and disturbance of immunologic function should be screened for such mutations.
Assuntos
Mutação , Neutropenia/congênito , Neutropenia/genética , Proteína da Síndrome de Wiskott-Aldrich/genética , Animais , Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Células COS , Proliferação de Células , Criança , Pré-Escolar , Chlorocebus aethiops , Humanos , Elastase de Leucócito/metabolismo , Linfócitos/citologia , Masculino , Neutropenia/metabolismo , Células U937 , Proteína da Síndrome de Wiskott-Aldrich/metabolismoRESUMO
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.
Assuntos
Actinas/metabolismo , Proteínas/metabolismo , Pseudópodes/metabolismo , Tirosina/metabolismo , Animais , Linhagem Celular , Tamanho Celular , Humanos , Macrófagos/citologia , Macrófagos/metabolismo , Camundongos , Microinjeções , Mutação , Fosforilação , Polímeros/metabolismo , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , Proteínas/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-hck , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteína da Síndrome de Wiskott-Aldrich , Proteína cdc42 de Ligação ao GTP/genética , Proteína cdc42 de Ligação ao GTP/metabolismoRESUMO
The Wiskott-Aldrich syndrome protein (WASp) is a key regulator of actin polymerization in hematopoietic cells. The dynamic nature of cytoskeletal changes during a variety of cellular processes demands complex mechanisms for coordinated integration of input signals, precise localization within the cell, and regulated activation of the Arp2/3 complex. Mutations in the Wiskott-Aldrich syndrome gene either inhibit or dysregulate normal WASp function, resulting in clinical diseases with complex and disparate phenotypes. This review highlights recent advances that have enhanced our understanding of the mechanisms by which these molecular defects cause hematologic and immunologic disease.