RESUMO
Cellular actin assembly is tightly regulated. The study of pathogen motility has led to the identification of several cellular factors that are critical for controlling this process. Pathogens such as Listeria require Ena/VASP and Arp2/3 proteins to translate actin polymerization into movement. Recent work has extended these observations and uncovered some similarities and surprising differences in the way cells and pathogens utilize the actin cytoskeleton.
Assuntos
Actinas/metabolismo , Proteínas do Citoesqueleto , Citoesqueleto/metabolismo , Proteína 2 Relacionada a Actina , Animais , Proteínas de Bactérias/metabolismo , Proteínas de Ligação a DNA/metabolismo , Listeria monocytogenes/fisiologia , Proteínas de Membrana/metabolismo , Proteínas dos Microfilamentos/metabolismo , Ligação Proteica , Shigella flexneri/fisiologia , Fatores de Transcrição/metabolismo , Família de Proteínas da Síndrome de Wiskott-AldrichRESUMO
T cell receptor (TCR)-driven activation of helper T cells induces a rapid polarization of their cytoskeleton towards bound antigen presenting cells (APCs). We have identified the Fyn- and SLP-76-associated protein Fyb/SLAP as a new ligand for Ena/ vasodilator-stimulated phosphoprotein (VASP) homology 1 (EVH1) domains. Upon TCR engagement, Fyb/SLAP localizes at the interface between T cells and anti-CD3-coated beads, where Evl, a member of the Ena/VASP family, Wiskott-Aldrich syndrome protein (WASP) and the Arp2/3 complex are also found. In addition, Fyb/SLAP is restricted to lamellipodia of spreading platelets. In activated T cells, Fyb/SLAP associates with Ena/VASP family proteins and is present within biochemical complexes containing WASP, Nck, and SLP-76. Inhibition of binding between Fyb/SLAP and Ena/VASP proteins or WASP and the Arp2/3 complex impairs TCR-dependent actin rearrangement, suggesting that these interactions play a key role in linking T cell signaling to remodeling of the actin cytoskeleton.
Assuntos
Actinas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Proteínas de Transporte/metabolismo , Moléculas de Adesão Celular/metabolismo , Proteínas do Citoesqueleto , Citoesqueleto/metabolismo , Fosfoproteínas/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais , Proteína 2 Relacionada a Actina , Proteína 3 Relacionada a Actina , Actinas/antagonistas & inibidores , Sequência de Aminoácidos , Plaquetas/citologia , Proteínas de Transporte/antagonistas & inibidores , Proteínas de Transporte/química , Moléculas de Adesão Celular/química , Clonagem Molecular , Humanos , Ativação Linfocitária , Proteínas dos Microfilamentos , Dados de Sequência Molecular , Proteínas Oncogênicas/metabolismo , Fosfoproteínas/química , Ligação Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Proteínas/metabolismo , Pseudópodes/metabolismo , Agregação de Receptores , Linfócitos T/citologia , Linfócitos T/imunologia , Linfócitos T/metabolismo , Linfócitos T/ultraestrutura , Células Tumorais Cultivadas , Proteína da Síndrome de Wiskott-AldrichRESUMO
Intracellular propulsion of Listeria monocytogenes is the best understood form of motility dependent on actin polymerization. We have used in vitro motility assays of Listeria in platelet and brain extracts to elucidate the function of the focal adhesion proteins of the Ena (Drosophila Enabled)/VASP (vasodilator-stimulated phosphoprotein) family in actin-based motility. Immunodepletion of VASP from platelet extracts and of Evl (Ena/VASP-like protein) from brain extracts of Mena knockout (-/-) mice combined with add-back of recombinant (bacterial or eukaryotic) VASP and Evl show that VASP, Mena, and Evl play interchangeable roles and are required to transform actin polymerization into active movement and propulsive force. The EVH1 (Ena/VASP homology 1) domain of VASP is in slow association-dissociation equilibrium high-affinity binding to the zyxin-homologous, proline-rich region of ActA. VASP also interacts with F-actin via its COOH-terminal EVH2 domain. Hence VASP/ Ena/Evl link the bacterium to the actin tail, which is required for movement. The affinity of VASP for F-actin is controlled by phosphorylation of serine 157 by cAMP-dependent protein kinase. Phospho-VASP binds with high affinity (0.5 x 10(8) M-1); dephospho-VASP binds 40-fold less tightly. We propose a molecular ratchet model for insertional polymerization of actin, within which frequent attachment-detachment of VASP to F-actin allows its sliding along the growing filament.
Assuntos
Actinas/fisiologia , Moléculas de Adesão Celular/fisiologia , Proteínas Contráteis , Proteínas do Citoesqueleto , Proteínas de Ligação a DNA/fisiologia , Listeria monocytogenes/fisiologia , Fosfoproteínas/fisiologia , Actinas/química , Actinas/ultraestrutura , Animais , Sequência de Bases , Sítios de Ligação , Plaquetas/metabolismo , Encéfalo/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/fisiologia , Moléculas de Adesão Celular/genética , Primers do DNA/genética , Proteínas de Ligação a DNA/genética , Listeria monocytogenes/genética , Camundongos , Camundongos Knockout , Proteínas dos Microfilamentos/fisiologia , Microscopia Eletrônica , Modelos Biológicos , Movimento/fisiologia , Fosfoproteínas/genética , Profilinas , Ligação Proteica , Proteínas/genética , Proteínas/fisiologiaRESUMO
The Drosophila abelson (abl) gene encodes the homolog of the mammalian c-abl cytoplasmic tyrosine kinase and is an essential gene for the development of viable adult flies. Three second-site mutations that suppress the lethality caused by the absence of abl function have been isolated, and all three map to the gene enabled (ena). The mutations are recessive embryonic lethal mutations but act as dominant mutations to compensate for the neural defects of abl mutants. Thus, mutations in a specific gene can compensate for the absence of a tyrosine kinase.
Assuntos
Drosophila/genética , Mutação , Proteínas Tirosina Quinases/genética , Proteínas Proto-Oncogênicas/genética , Supressão Genética , Animais , Drosophila/embriologia , Elementos Facilitadores Genéticos/genética , Olho/crescimento & desenvolvimento , Olho/ultraestrutura , Genes Letais , Heterozigoto , Homozigoto , Larva/crescimento & desenvolvimento , Microscopia Eletrônica , Sistema Nervoso/embriologia , Sistema Nervoso/crescimento & desenvolvimento , Fenótipo , Proteínas Proto-Oncogênicas c-ablRESUMO
Cyclin-dependent kinase 5 (Cdk5) is a small serine/threonine kinase that plays a pivotal role during development of the CNS. Cables, a novel protein, interacts with Cdk5 in brain lysates. Cables also binds to and is a substrate of the c-Abl tyrosine kinase. Active c-Abl kinase leads to Cdk5 tyrosine phosphorylation, and this phosphorylation is enhanced by Cables. Phosphorylation of Cdk5 by c-Abl occurs on tyrosine 15 (Y15), which is stimulatory for p35/Cdk5 kinase activity. Expression of antisense Cables in primary cortical neurons inhibited neurite outgrowth. Furthermore, expression of active Abl resulted in lengthening of neurites. The data provide evidence for a Cables-mediated interplay between the Cdk5 and c-Abl signaling pathways in the developing nervous system.
Assuntos
Proteínas de Transporte/fisiologia , Quinases Ciclina-Dependentes/fisiologia , Ciclinas , Neuritos/fisiologia , Fosfoproteínas/fisiologia , Fosfotransferases/metabolismo , Proteínas Proto-Oncogênicas c-abl/fisiologia , Sequência de Aminoácidos/genética , Animais , Sequência de Bases/genética , Células COS , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Células Cultivadas , Quinase 5 Dependente de Ciclina , Quinases Ciclina-Dependentes/metabolismo , Embrião de Mamíferos , Camundongos , Mitose/fisiologia , Dados de Sequência Molecular , Neurônios/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Fosforilação , Especificidade por Substrato , Tirosina/metabolismo , Regulação para CimaRESUMO
Mammalian enabled (Mena) is a member of a protein family thought to link signal transduction pathways to localized remodeling of the actin cytoskeleton. Mena binds directly to Profilin, an actin-binding protein that modulates actin polymerization. In primary neurons, Mena is concentrated at the tips of growth cone filopodia. Mena-deficient mice are viable; however, axons projecting from interhemispheric cortico-cortical neurons are misrouted in early neonates, and failed decussation of the corpus callosum as well as defects in the hippocampal commissure and the pontocerebellar pathway are evident in the adult. Mena-deficient mice that are heterozygous for a Profilin I deletion die in utero and display defects in neurulation, demonstrating an important functional role for Mena in regulation of the actin cytoskeleton.
Assuntos
Encéfalo/embriologia , Proteínas de Transporte/fisiologia , Proteínas Contráteis , Proteínas do Citoesqueleto , Sistema Nervoso/embriologia , Animais , Animais Recém-Nascidos/fisiologia , Axônios/fisiologia , Proteínas de Transporte/genética , Embrião de Mamíferos/fisiologia , Desenvolvimento Embrionário e Fetal/fisiologia , Deleção de Genes , Cones de Crescimento/fisiologia , Camundongos/embriologia , Proteínas dos Microfilamentos/genética , Mutação/fisiologia , Profilinas , Distribuição TecidualRESUMO
A class of proteins dubbed pipmodulins bind to and sequester the phospholipid PIP2 in the plasma membrane. Local release of PIP2 controls actin dynamics in specific subcellular regions and plays a critical role in regulating actin-based cell motility and morphogenesis.
Assuntos
Actinas/metabolismo , Membrana Celular/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas de Membrana , Fosfatidilinositol 4,5-Difosfato/metabolismo , Proteína Quinase C/metabolismo , Transdução de Sinais , Animais , Proteína GAP-43/metabolismo , Microdomínios da Membrana , Substrato Quinase C Rico em Alanina Miristoilada , Proteínas/metabolismoRESUMO
We report our molecular characterization of the Drosophila melanogaster Abelson gene (abl), a gene in which recessive loss-of-function mutations result in lethality at the pupal stage of development. This essential gene consists of 10 exons extending over 26 kilobase pairs of genomic DNA. The DNA sequence encodes a protein of 1,520 amino acids with strong sequence similarity to the human c-abl proto-oncogene beginning in the type lb 5' exon and extending through the region essential for tyrosine kinase activity. When the tyrosine kinase homologous region was expressed in Escherichia coli, phosphorylation of proteins on tyrosine residues was observed with an antiphosphotyrosine antibody. These results show that the abl gene is highly conserved through evolution and encodes a functional tyrosine protein kinase required for Drosophila development.
Assuntos
Drosophila melanogaster/genética , Genes , Proteínas Tirosina Quinases/genética , Proto-Oncogenes , Sequência de Aminoácidos , Animais , Sequência de Bases , Enzimas de Restrição do DNA , Drosophila melanogaster/enzimologia , Genes Letais , Dados de Sequência Molecular , Mutação , Proto-Oncogene Mas , PupaRESUMO
The enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase in Drosophila melanogaster synthesizes mevalonate for the production of nonsterol isoprenoids, which are essential for growth and differentiation. To understand the regulation and developmental role of HMG CoA reductase, we cloned the D. melanogaster HMG CoA reductase gene. The nucleotide sequence of the Drosophila HMG CoA reductase was determined from genomic and cDNA clones. A 2,748-base-pair open reading frame encoded a polypeptide of 916 amino acids (Mr, 98,165) that was similar to the hamster HMG CoA reductase. The C-terminal region had 56% identical residues and the N-terminal region had 7 potential transmembrane domains with 32 to 60% identical residues. In hamster HMG CoA reductase, the membrane regions were essential for posttranslational regulation. Since the Drosophila enzyme is not regulated by sterols, the strong N-terminal similarity was surprising. Two HMG CoA reductase mRNA transcripts, approximately 3.2 and 4 kilobases, were differentially expressed throughout Drosophila development. Mevalonate-fed Schneider cells showed a parallel reduction of both enzyme activity and abundance of the 4-kilobase mRNA transcript.
Assuntos
Drosophila melanogaster/genética , Regulação da Expressão Gênica , Hidroximetilglutaril-CoA Redutases/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Drosophila melanogaster/enzimologia , Indução Enzimática , Ácido Mevalônico/farmacologia , Dados de Sequência Molecular , Homologia de Sequência do Ácido Nucleico , Transcrição Gênica/efeitos dos fármacosRESUMO
Disabled gene products are important for nervous system development in drosophila and mammals. In mice, the Dab1 protein is thought to function downstream of the extracellular protein Reln during neuronal positioning. The structures of Dab proteins suggest that they mediate protein-protein or protein-membrane docking functions. Here we show that the amino-terminal phosphotyrosine-binding (PTB) domain of Dab1 binds to the transmembrane glycoproteins of the amyloid precursor protein (APP) and low-density lipoprotein receptor families and the cytoplasmic signaling protein Ship. Dab1 associates with the APP cytoplasmic domain in transfected cells and is coexpressed with APP in hippocampal neurons. Screening of a set of altered peptide sequences showed that the sequence GYXNPXY present in APP family members is an optimal binding sequence, with approximately 0.5 microM affinity. Unlike other PTB domains, the Dab1 PTB does not bind to tyrosine-phosphorylated peptide ligands. The PTB domain also binds specifically to phospholipid bilayers containing phosphatidylinositol 4P (PtdIns4P) or PtdIns4,5P2 in a manner that does not interfere with protein binding. We propose that the PTB domain permits Dab1 to bind specifically to transmembrane proteins containing an NPXY internalization signal.
Assuntos
Precursor de Proteína beta-Amiloide/metabolismo , Glicoproteínas/metabolismo , Glicoproteínas de Membrana/metabolismo , Lipídeos de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Fosfolipídeos/metabolismo , Sequência de Aminoácidos , Precursor de Proteína beta-Amiloide/química , Animais , Sítios de Ligação , Clonagem Molecular , Citoplasma/metabolismo , Células HeLa , Humanos , Ligantes , Camundongos , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/genética , Peptídeos/metabolismo , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases , Fosfatidilinositóis/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Fosforilação , Fosfotirosina/metabolismo , Receptores de LDL/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteína Reelina , Saccharomyces cerevisiae , Frações Subcelulares , Células Tumorais CultivadasRESUMO
The Abl tyrosine kinase plays an important role in axonogenesis. Recent reports indicate that this role involves interaction with several different protein families, including LAR phosphatases, catenin/cadherin cell adhesion complexes, Trio family GEFs, and Ena/VASP family actin regulatory proteins. These findings suggest that Abl and its associated proteins may regulate cell adhesion and actin polymerization, thereby regulating growth cone motility during axonogenesis.
Assuntos
Actinas/metabolismo , Cones de Crescimento/enzimologia , Cones de Crescimento/fisiologia , Proteínas Proto-Oncogênicas c-abl/metabolismo , Animais , Adesão Celular , Moléculas de Adesão Celular/metabolismo , Movimento Celular , Citoesqueleto/metabolismo , Proteínas de Drosophila , Fosforilação , Ligação Proteica , Proteínas Tirosina Fosfatases/metabolismo , Proteínas Tirosina Fosfatases Semelhantes a Receptores , Proteínas rho de Ligação ao GTP/metabolismoRESUMO
Cancer invasion is a hallmark of metastasis. The mesenchymal mode of cancer cell invasion is mediated by elongated membrane protrusions driven by the assembly of branched F-actin networks. How deregulation of actin regulators promotes cancer cell invasion is still enigmatic. We report that increased expression and membrane localization of the actin regulator Lamellipodin correlate with reduced metastasis-free survival and poor prognosis in breast cancer patients. In agreement, we find that Lamellipodin depletion reduced lung metastasis in an orthotopic mouse breast cancer model. Invasive 3D cancer cell migration as well as invadopodia formation and matrix degradation was impaired upon Lamellipodin depletion. Mechanistically, we show that Lamellipodin promotes invasive 3D cancer cell migration via both actin-elongating Ena/VASP proteins and the Scar/WAVE complex, which stimulates actin branching. In contrast, Lamellipodin interaction with Scar/WAVE but not with Ena/VASP is required for random 2D cell migration. We identified a phosphorylation-dependent mechanism that regulates selective recruitment of these effectors to Lamellipodin: Abl-mediated Lamellipodin phosphorylation promotes its association with both Scar/WAVE and Ena/VASP, whereas Src-dependent phosphorylation enhances binding to Scar/WAVE but not to Ena/VASP. Through these selective, regulated interactions Lamellipodin mediates directional sensing of epidermal growth factor (EGF) gradients and invasive 3D migration of breast cancer cells. Our findings imply that increased Lamellipodin levels enhance Ena/VASP and Scar/WAVE activities at the plasma membrane to promote 3D invasion and metastasis.
Assuntos
Proteínas de Transporte/genética , Proteínas de Ligação a DNA/genética , Neoplasias Mamárias Animais/genética , Proteínas de Membrana/genética , Família de Proteínas da Síndrome de Wiskott-Aldrich/genética , Citoesqueleto de Actina/genética , Animais , Moléculas de Adesão Celular/genética , Movimento Celular/genética , Fator de Crescimento Epidérmico/genética , Humanos , Neoplasias Mamárias Animais/patologia , Camundongos , Invasividade Neoplásica/genética , Fosforilação , Mapas de Interação de Proteínas/genéticaRESUMO
Here, we identify a mouse homolog of the Drosophila Disabled (Dab) protein, mDab1, and show it is an adaptor molecule functioning in neural development. We find that mDab1 is expressed in certain neuronal and hematopoietic cell lines, and is localized to the growing nerves of embryonic mice. During mouse embryogenesis, mDab1 is tyrosine phosphorylated when the nervous system is undergoing dramatic expansion. However, when nerve tracts are established, mDab1 lacks detectable phosphotyrosine. Tyrosine-phosphorylated mDab1 associates with the SH2 domains of Src, Fyn and Abl. An interaction between mDab1 and Src is observed when P19 embryonal carcinoma (EC) cells undergo differentiation into neuronal cell types. mDab1 can also form complexes with cellular phosphotyrosyl proteins through a domain that is related to the phosphotyrosine binding (PTB) domains of the Shc family of adaptor proteins. The mDab1 PTB domain binds to phosphotyrosine-containing proteins of 200, 120 and 40 kDa from extracts of embryonic mouse heads. The properties of mDab1 and genetic analysis of Dab in Drosophila suggest that these molecules function in key signal transduction pathways involved in the formation of neural networks.
Assuntos
Proteínas do Tecido Nervoso/metabolismo , Sistema Nervoso/embriologia , Domínios de Homologia de src , Sequência de Aminoácidos , Animais , Linhagem Celular , Mapeamento Cromossômico , Clonagem Molecular , Desenvolvimento Embrionário e Fetal , Expressão Gênica , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Rede Nervosa/metabolismo , Proteínas do Tecido Nervoso/genética , Sistema Nervoso/metabolismo , Fosforilação , Ligação Proteica , Proteínas Tirosina Quinases/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae/genética , Homologia de Sequência de Aminoácidos , Tirosina/metabolismoRESUMO
In the absence of the Drosophila abl protein-tyrosine kinase (PTK), loss-of-function mutations in either disabled or prospero have dominant phenotypic effects on embryonic development. Molecular and genetic characterizations indicate that the products of these genes interact with the abl PTK by different mechanisms. The interaction between abl and prospero, which encodes a nuclear protein required for correct axonal outgrowth, is likely to be indirect. In contrast, the product of disabled may be a substrate for the abl PTK. The disabled protein is colocalized with abl in axons, its predicted amino acid sequence contains 10 motifs similar to the major autophosphorylation site of abl, and the protein is recognized by antibodies to phosphotyrosine.
Assuntos
Axônios/fisiologia , Proteínas de Drosophila , Drosophila melanogaster/genética , Genes Reguladores , Genes abl , Proteínas do Tecido Nervoso/genética , Proteínas Oncogênicas v-abl/genética , Proteínas Tirosina Quinases/genética , Sequência de Aminoácidos , Animais , Mapeamento Cromossômico , Cruzamentos Genéticos , DNA/genética , Drosophila melanogaster/enzimologia , Drosophila melanogaster/crescimento & desenvolvimento , Feminino , Biblioteca Gênica , Genótipo , Técnicas In Vitro , Dados de Sequência Molecular , Mutagênese Insercional , Proteínas do Tecido Nervoso/metabolismo , Sistema Nervoso/crescimento & desenvolvimento , Proteínas Nucleares/metabolismo , Proteínas Oncogênicas v-abl/metabolismo , Fases de Leitura Aberta , Fosfotirosina , Proteínas Tirosina Quinases/metabolismo , Deleção de Sequência , Especificidade por Substrato , Tirosina/análogos & derivados , Tirosina/análiseRESUMO
The Abelson gene in Drosophila (abl) consists of ten exons extending over 26 kb of genomic DNA. The DNA sequence encodes a protein of 1520 amino acids with sequence homology to the human c-abl proto-oncogene product, beginning at the amino terminus and extending 656 amino acids through the region essential for tyrosine kinase activity. Mutant lesions in the abl gene were identified first by their failure to complement chromosomal deletions that overlap the abl DNA sequence and then by rescue of the mutant phenotypes with an abl minigene in transgenic flies. Elimination of abl zygotic function by mutations produces some recessive lethality at the pharate adult pupal stage, and mutant adults with reduced longevity, reduced fecundity, and an irregular pattern of retinal cells.
Assuntos
Drosophila melanogaster/genética , Genes , Proto-Oncogenes , Alelos , Animais , Animais Geneticamente Modificados/genética , Mapeamento Cromossômico , Drosophila melanogaster/crescimento & desenvolvimento , Mutação , Fenótipo , Proto-Oncogene Mas , Homologia de Sequência do Ácido NucleicoRESUMO
During Drosophila embryogenesis, the Abelson tyrosine kinase (abl) is localized in the axons of the central nervous system (CNS). Mutations in abl have no detectable effect on the morphology of the embryonic CNS, and the mutant animals survive to the pupal and adult stages. In the absence of abl function, however, heterozygous mutations or deletions of disabled (dab) exert dominant effects, disrupting axonal organization and shifting the lethal phase of the animals to embryonic and early larval stages. Embryos that are homozygous mutant for both abl and dab fail to develop any axon bundles in the CNS, although the peripheral nervous system and the larval cuticle appear normal. The genetic interaction between these two genes begins to define a process in which both the abl tyrosine kinase and the dab gene product participate in establishing axonal connections in the embryonic CNS of Drosophila.
Assuntos
Drosophila melanogaster/embriologia , Neurônios/enzimologia , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Animais , Axônios/enzimologia , Sistema Nervoso Central/embriologia , Sistema Nervoso Central/enzimologia , Mapeamento Cromossômico , Drosophila melanogaster/enzimologia , Imuno-Histoquímica , Morfogênese , Mutação , Proteínas Proto-Oncogênicas c-ablRESUMO
Drosophila Enabled is required for proper formation of axonal structures and is genetically implicated in signaling pathways mediated by Drosophila AbI. We have identified two murine proteins, Mena and Evl, that are highly related to Enabled as well as VASP (Vasodilator-Stimulated Phosphoprotein). A conserved domain targets Mena to localized proteins containing a specific proline-rich motif. The association of Mena with the surface of the intracellular pathogen Listeria monocytogenes and the G-actin binding protein profilin suggests that this molecule may participate in bacterial movement by facilitating actin polymerization. Expression of neural-enriched isoforms of Mena in fibroblasts induces the formation of abnormal F-actin-rich outgrowths, supporting a role for this protein in microfilament assembly and cell motility.
Assuntos
Citoesqueleto de Actina/fisiologia , Proteínas de Transporte/fisiologia , Moléculas de Adesão Celular/fisiologia , Proteínas Contráteis , Proteínas do Citoesqueleto , Proteínas de Ligação a DNA/fisiologia , Fosfoproteínas/fisiologia , Proteínas/fisiologia , Citoesqueleto de Actina/ultraestrutura , Actinas/fisiologia , Sequência de Aminoácidos , Animais , Técnica Indireta de Fluorescência para Anticorpo , Ligantes , Listeria monocytogenes/ultraestrutura , Camundongos , Proteínas dos Microfilamentos/metabolismo , Dados de Sequência Molecular , Fosforilação , Profilinas , Ratos , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Domínios de Homologia de srcRESUMO
Proteins of the Ena/VASP family are implicated in processes that require dynamic actin remodeling such as axon guidance and platelet activation. In this work, we explored some of the pathways that likely regulate actin dynamics in part via EVL (Ena/VASP-like protein). Two isoforms, EVL and EVL-I, were highly expressed in hematopoietic cells of thymus and spleen. In CD3-activated T-cells, EVL was found in F-actin-rich patches and at the distal tips of the microspikes that formed on the activated side of the T-cells. Like the other family members, EVL localized to focal adhesions and the leading edge of lamellipodia when expressed in fibroblasts. EVL was a substrate for the cAMP-dependent protein kinase, and this phosphorylation regulated several of the interactions between EVL and its ligands. Unlike VASP, EVL nucleated actin polymerization under physiological conditions, whereas phosphorylation of both EVL and VASP decreased their nucleating activity. EVL bound directly to the Abl, Lyn, and nSrc SH3 domains; the FE65 WW domain; and profilin, likely via its proline-rich core. Binding of Abl and nSrc SH3 domains, but not profilin or other SH3 domains, was abolished by cAMP-dependent protein kinase phosphorylation of EVL. We show strong cooperative binding of two profilin dimers on the polyproline sequence of EVL. Additionally, profilin competed with the SH3 domains for binding to partially overlapping binding sites. These data suggest that the function of EVL could be modulated in a complex manner by its interactions with multiple ligands and through phosphorylation by cyclic nucleotide dependent kinases.
Assuntos
Actinas/metabolismo , Proteínas de Transporte/química , Moléculas de Adesão Celular/química , Proteínas Contráteis , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Proteínas do Citoesqueleto , Fosfoproteínas/química , Proteínas/metabolismo , Domínios de Homologia de src , Sequência de Aminoácidos , Animais , Ligação Competitiva , Biopolímeros/metabolismo , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/enzimologia , Linfócitos T CD4-Positivos/metabolismo , Células Cultivadas , Fibroblastos/metabolismo , Imunofluorescência , Ativação Linfocitária , Camundongos , Proteínas dos Microfilamentos/metabolismo , Dados de Sequência Molecular , Fosforilação , Profilinas , Prolina/metabolismo , Ligação Proteica , Proteínas/química , Proteínas/genética , Ratos , TransfecçãoRESUMO
Ena/VASP proteins have been implicated in cell motility through regulation of the actin cytoskeleton and are found at focal adhesions and the leading edge. Using overexpression, loss-of-function, and inhibitory approaches, we find that Ena/VASP proteins negatively regulate fibroblast motility. A dose-dependent decrease in movement is observed when Ena/VASP proteins are overexpressed in fibroblasts. Neutralization or deletion of all Ena/VASP proteins results in increased cell movement. Selective depletion of Ena/VASP proteins from focal adhesions, but not the leading edge, has no effect on motility. Constitutive membrane targeting of Ena/VASP proteins inhibits motility. These results are in marked contrast to current models for Ena/VASP function derived mainly from their role in the actin-driven movement of Listeria monocytogenes.
Assuntos
Moléculas de Adesão Celular/fisiologia , Movimento Celular/fisiologia , Proteínas de Ligação a DNA/fisiologia , Fibroblastos/citologia , Fibroblastos/fisiologia , Fosfoproteínas/fisiologia , Animais , Adesão Celular/fisiologia , Expressão Gênica , Regulação da Expressão Gênica/fisiologia , Listeria monocytogenes , Proteínas dos Microfilamentos/fisiologiaRESUMO
The ActA protein of the intracellular pathogen Listeria monocytogenes induces a dramatic reorganization of the actin-based cytoskeleton. Two profilin binding proteins, VASP and Mena, are the only cellular proteins known so far to bind directly to ActA. This interaction is mediated by a conserved module, the EVH1 domain. We identify E/DFPPPPXD/E, a motif repeated 4-fold within the primary sequence of ActA, as the core of the consensus ligand for EVH1 domains. This motif is also present and functional in at least two cellular proteins, zyxin and vinculin, which are in this respect major eukaryotic analogs of ActA. The functional importance of the novel protein-protein interaction was examined in the Listeria system. Removal of EVH1 binding sites on ActA reduces bacterial motility and strongly attenuates Listeria virulence. Taken together we demonstrate that ActA-EVH1 binding is a paradigm for a novel class of eukaryotic protein-protein interactions involving a proline-rich ligand that is clearly different from those described for SH3 and WW/WWP domains. This class of interactions appears to be of general importance for processes dependent on rapid actin remodeling.