RESUMEN
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.
Asunto(s)
Proteínas Portadoras/genética , Proteínas de Unión al ADN/genética , Neoplasias Mamarias Animales/genética , Proteínas de la Membrana/genética , Familia de Proteínas del Síndrome de Wiskott-Aldrich/genética , Citoesqueleto de Actina/genética , Animales , Moléculas de Adhesión Celular/genética , Movimiento Celular/genética , Factor de Crecimiento Epidérmico/genética , Humanos , Neoplasias Mamarias Animales/patología , Ratones , Invasividad Neoplásica/genética , Fosforilación , Mapas de Interacción de Proteínas/genéticaRESUMEN
SCF ubiquitin ligases target phosphorylated substrates for ubiquitin-dependent proteolysis by means of adapter subunits called F-box proteins. The F-box protein Cdc4 captures phosphorylated forms of the cyclin-dependent kinase inhibitor Sic1 for ubiquitination in late G1 phase, an event necessary for the onset of DNA replication. The WD40 repeat domain of Cdc4 binds with high affinity to a consensus phosphopeptide motif (the Cdc4 phospho-degron, CPD), yet Sic1 itself has many sub-optimal CPD motifs that act in concert to mediate Cdc4 binding. The weak CPD sites in Sic1 establish a phosphorylation threshold that delays degradation in vivo, and thereby establishes a minimal G1 phase period needed to ensure proper DNA replication. Multisite phosphorylation may be a more general mechanism to set thresholds in regulated protein-protein interactions.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Replicación del ADN/fisiología , Proteínas F-Box , Proteínas Fúngicas/fisiología , Proteínas de Saccharomyces cerevisiae , Ubiquitina-Proteína Ligasas , Sitios de Unión , Ciclo Celular , Proteínas de Ciclo Celular/antagonistas & inhibidores , Secuencia de Consenso , Proteínas Inhibidoras de las Quinasas Dependientes de la Ciclina , ADN de Hongos/biosíntesis , Inhibidores Enzimáticos , Proteínas Fúngicas/antagonistas & inhibidores , Proteínas Fúngicas/metabolismo , Fosforilación , Estructura Terciaria de Proteína , Especificidad por Sustrato , Ubiquitina/metabolismoRESUMEN
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.
Asunto(s)
Actinas/metabolismo , Proteínas del Citoesqueleto , Citoesqueleto/metabolismo , Proteína 2 Relacionada con la Actina , Animales , Proteínas Bacterianas/metabolismo , Proteínas de Unión al ADN/metabolismo , Listeria monocytogenes/fisiología , Proteínas de la Membrana/metabolismo , Proteínas de Microfilamentos/metabolismo , Unión Proteica , Shigella flexneri/fisiología , Factores de Transcripción/metabolismo , Familia de Proteínas del Síndrome de Wiskott-AldrichRESUMEN
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.
Asunto(s)
Actinas/metabolismo , Membrana Celular/fisiología , Péptidos y Proteínas de Señalización Intracelular , Proteínas de la Membrana , Fosfatidilinositol 4,5-Difosfato/metabolismo , Proteína Quinasa C/metabolismo , Transducción de Señal , Animales , Proteína GAP-43/metabolismo , Microdominios de Membrana , Sustrato de la Proteína Quinasa C Rico en Alanina Miristoilada , Proteínas/metabolismoRESUMEN
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.
Asunto(s)
Actinas/metabolismo , Proteínas Portadoras/química , Moléculas de Adhesión Celular/química , Proteínas Contráctiles , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Proteínas del Citoesqueleto , Fosfoproteínas/química , Proteínas/metabolismo , Dominios Homologos src , Secuencia de Aminoácidos , Animales , Unión Competitiva , Biopolímeros/metabolismo , Linfocitos T CD4-Positivos/citología , Linfocitos T CD4-Positivos/enzimología , Linfocitos T CD4-Positivos/metabolismo , Células Cultivadas , Fibroblastos/metabolismo , Técnica del Anticuerpo Fluorescente , Activación de Linfocitos , Ratones , Proteínas de Microfilamentos/metabolismo , Datos de Secuencia Molecular , Fosforilación , Profilinas , Prolina/metabolismo , Unión Proteica , Proteínas/química , Proteínas/genética , Ratas , TransfecciónRESUMEN
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.
Asunto(s)
Proteínas Portadoras/fisiología , Quinasas Ciclina-Dependientes/fisiología , Ciclinas , Neuritas/fisiología , Fosfoproteínas/fisiología , Fosfotransferasas/metabolismo , Proteínas Proto-Oncogénicas c-abl/fisiología , Secuencia de Aminoácidos/genética , Animales , Secuencia de Bases/genética , Células COS , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Células Cultivadas , Quinasa 5 Dependiente de la Ciclina , Quinasas Ciclina-Dependientes/metabolismo , Embrión de Mamíferos , Ratones , Mitosis/fisiología , Datos de Secuencia Molecular , Neuronas/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Fosforilación , Especificidad por Sustrato , Tirosina/metabolismo , Regulación hacia ArribaRESUMEN
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.
Asunto(s)
Moléculas de Adhesión Celular/fisiología , Movimiento Celular/fisiología , Proteínas de Unión al ADN/fisiología , Fibroblastos/citología , Fibroblastos/fisiología , Fosfoproteínas/fisiología , Animales , Adhesión Celular/fisiología , Expresión Génica , Regulación de la Expresión Génica/fisiología , Listeria monocytogenes , Proteínas de Microfilamentos/fisiologíaRESUMEN
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.
Asunto(s)
Actinas/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Proteínas Portadoras/metabolismo , Moléculas de Adhesión Celular/metabolismo , Proteínas del Citoesqueleto , Citoesqueleto/metabolismo , Fosfoproteínas/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Transducción de Señal , Proteína 2 Relacionada con la Actina , Proteína 3 Relacionada con la Actina , Actinas/antagonistas & inhibidores , Secuencia de Aminoácidos , Plaquetas/citología , Proteínas Portadoras/antagonistas & inhibidores , Proteínas Portadoras/química , Moléculas de Adhesión Celular/química , Clonación Molecular , Humanos , Activación de Linfocitos , Proteínas de Microfilamentos , Datos de Secuencia Molecular , Proteínas Oncogénicas/metabolismo , Fosfoproteínas/química , Unión Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Proteínas/metabolismo , Seudópodos/metabolismo , Agregación de Receptores , Linfocitos T/citología , Linfocitos T/inmunología , Linfocitos T/metabolismo , Linfocitos T/ultraestructura , Células Tumorales Cultivadas , Proteína del Síndrome de Wiskott-AldrichRESUMEN
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.
Asunto(s)
Actinas/metabolismo , Conos de Crecimiento/enzimología , Conos de Crecimiento/fisiología , Proteínas Proto-Oncogénicas c-abl/metabolismo , Animales , Adhesión Celular , Moléculas de Adhesión Celular/metabolismo , Movimiento Celular , Citoesqueleto/metabolismo , Proteínas de Drosophila , Fosforilación , Unión Proteica , Proteínas Tirosina Fosfatasas/metabolismo , Proteínas Tirosina Fosfatasas Similares a Receptores , Proteínas de Unión al GTP rho/metabolismoRESUMEN
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.
Asunto(s)
Precursor de Proteína beta-Amiloide/metabolismo , Glicoproteínas/metabolismo , Glicoproteínas de Membrana/metabolismo , Lípidos de la Membrana/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Fosfolípidos/metabolismo , Secuencia de Aminoácidos , Precursor de Proteína beta-Amiloide/química , Animales , Sitios de Unión , Clonación Molecular , Citoplasma/metabolismo , Células HeLa , Humanos , Ligandos , Ratones , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/genética , Péptidos/metabolismo , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatasas , Fosfatidilinositoles/metabolismo , Monoéster Fosfórico Hidrolasas/metabolismo , Fosforilación , Fosfotirosina/metabolismo , Receptores de LDL/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteína Reelina , Saccharomyces cerevisiae , Fracciones Subcelulares , Células Tumorales CultivadasRESUMEN
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.
Asunto(s)
Encéfalo/embriología , Proteínas Portadoras/fisiología , Proteínas Contráctiles , Proteínas del Citoesqueleto , Sistema Nervioso/embriología , Animales , Animales Recién Nacidos/fisiología , Axones/fisiología , Proteínas Portadoras/genética , Embrión de Mamíferos/fisiología , Desarrollo Embrionario y Fetal/fisiología , Eliminación de Gen , Conos de Crecimiento/fisiología , Ratones/embriología , Proteínas de Microfilamentos/genética , Mutación/fisiología , Profilinas , Distribución TisularRESUMEN
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.
Asunto(s)
Actinas/fisiología , Moléculas de Adhesión Celular/fisiología , Proteínas Contráctiles , Proteínas del Citoesqueleto , Proteínas de Unión al ADN/fisiología , Listeria monocytogenes/fisiología , Fosfoproteínas/fisiología , Actinas/química , Actinas/ultraestructura , Animales , Secuencia de Bases , Sitios de Unión , Plaquetas/metabolismo , Encéfalo/metabolismo , Proteínas Portadoras/genética , Proteínas Portadoras/fisiología , Moléculas de Adhesión Celular/genética , Cartilla de ADN/genética , Proteínas de Unión al ADN/genética , Listeria monocytogenes/genética , Ratones , Ratones Noqueados , Proteínas de Microfilamentos/fisiología , Microscopía Electrónica , Modelos Biológicos , Movimiento/fisiología , Fosfoproteínas/genética , Profilinas , Unión Proteica , Proteínas/genética , Proteínas/fisiologíaRESUMEN
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.
Asunto(s)
Proteínas Bacterianas/metabolismo , Proteínas del Citoesqueleto/metabolismo , Listeria monocytogenes/patogenicidad , Proteínas de la Membrana/metabolismo , Prolina , Secuencia de Aminoácidos , Animales , Sitios de Unión , Proteínas Portadoras/metabolismo , Moléculas de Adhesión Celular/metabolismo , Glicoproteínas , Células HeLa , Humanos , Metaloproteínas/metabolismo , Ratones , Ratones Endogámicos , Proteínas de Microfilamentos , Imitación Molecular , Datos de Secuencia Molecular , Oligopéptidos/metabolismo , Fosfoproteínas/metabolismo , Unión Proteica , Eliminación de Secuencia , Vinculina/metabolismo , ZixinaRESUMEN
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.
Asunto(s)
Proteínas del Tejido Nervioso/metabolismo , Sistema Nervioso/embriología , Dominios Homologos src , Secuencia de Aminoácidos , Animales , Línea Celular , Mapeo Cromosómico , Clonación Molecular , Desarrollo Embrionario y Fetal , Expresión Génica , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Red Nerviosa/metabolismo , Proteínas del Tejido Nervioso/genética , Sistema Nervioso/metabolismo , Fosforilación , Unión Proteica , Proteínas Tirosina Quinasas/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Saccharomyces cerevisiae/genética , Homología de Secuencia de Aminoácido , Tirosina/metabolismoRESUMEN
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.
Asunto(s)
Citoesqueleto de Actina/fisiología , Proteínas Portadoras/fisiología , Moléculas de Adhesión Celular/fisiología , Proteínas Contráctiles , Proteínas del Citoesqueleto , Proteínas de Unión al ADN/fisiología , Fosfoproteínas/fisiología , Proteínas/fisiología , Citoesqueleto de Actina/ultraestructura , Actinas/fisiología , Secuencia de Aminoácidos , Animales , Técnica del Anticuerpo Fluorescente Indirecta , Ligandos , Listeria monocytogenes/ultraestructura , Ratones , Proteínas de Microfilamentos/metabolismo , Datos de Secuencia Molecular , Fosforilación , Profilinas , Ratas , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Dominios Homologos srcRESUMEN
Genetic screens for dominant second-site mutations that suppress the lethality of Abl mutations in Drosophila identified alleles of only one gene, enabled (ena). We report that the ena protein contains proline-rich motifs and binds to Abl and Src SH3 domains, ena is also a substrate for the Abl kinase; tyrosine phosphorylation of ena is increased when it is coexpressed in cells with human or Drosophila Abl and endogenous ena tyrosine phosphorylation is reduced in Abl mutant animals. Like Abl, ena is expressed at highest levels in the axons of the embryonic nervous system and ena mutant embryos have defects in axonal architecture. We conclude that a critical function of Drosophila Abl is to phosphorylate and negatively regulate ena protein during neural development.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Drosophila/enzimología , Genes Supresores/genética , Genes abl/genética , Proteínas Tirosina Quinasas/metabolismo , Secuencia de Aminoácidos , Animales , Línea Celular , Clonación Molecular , Proteínas de Unión al ADN/análisis , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Drosophila/embriología , Drosophila/genética , Femenino , Regulación Enzimológica de la Expresión Génica , Genes de Insecto/genética , Humanos , Masculino , Datos de Secuencia Molecular , Sistema Nervioso/química , Sistema Nervioso/embriología , Fosforilación , Fosfotirosina , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/metabolismo , Análisis de Secuencia de ADN , Especificidad por Sustrato , Tirosina/análogos & derivados , Tirosina/análisisRESUMEN
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.
Asunto(s)
Axones/fisiología , Proteínas de Drosophila , Drosophila melanogaster/genética , Genes Reguladores , Genes abl , Proteínas del Tejido Nervioso/genética , Proteínas Oncogénicas v-abl/genética , Proteínas Tirosina Quinasas/genética , Secuencia de Aminoácidos , Animales , Mapeo Cromosómico , Cruzamientos Genéticos , ADN/genética , Drosophila melanogaster/enzimología , Drosophila melanogaster/crecimiento & desarrollo , Femenino , Biblioteca de Genes , Genotipo , Técnicas In Vitro , Datos de Secuencia Molecular , Mutagénesis Insercional , Proteínas del Tejido Nervioso/metabolismo , Sistema Nervioso/crecimiento & desarrollo , Proteínas Nucleares/metabolismo , Proteínas Oncogénicas v-abl/metabolismo , Sistemas de Lectura Abierta , Fosfotirosina , Proteínas Tirosina Quinasas/metabolismo , Eliminación de Secuencia , Especificidad por Sustrato , Tirosina/análogos & derivados , Tirosina/análisisAsunto(s)
Drosophila/genética , Genes abl , Proteínas Oncogénicas v-abl/genética , Proteínas Tirosina Quinasas/genética , Proteínas Proto-Oncogénicas c-abl/genética , Animales , Secuencia de Bases , Western Blotting , Elementos Transponibles de ADN , Drosophila/embriología , Drosophila/enzimología , Embrión no Mamífero/fisiología , Ojo/embriología , Ojo/ultraestructura , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Sondas de Oligonucleótidos , Fenotipo , Proteínas Tirosina Quinasas/metabolismo , Fracciones Subcelulares/enzimologíaRESUMEN
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.
Asunto(s)
Drosophila/genética , Mutación , Proteínas Tirosina Quinasas/genética , Proteínas Proto-Oncogénicas/genética , Supresión Genética , Animales , Drosophila/embriología , Elementos de Facilitación Genéticos/genética , Ojo/crecimiento & desarrollo , Ojo/ultraestructura , Genes Letales , Heterocigoto , Homocigoto , Larva/crecimiento & desarrollo , Microscopía Electrónica , Sistema Nervioso/embriología , Sistema Nervioso/crecimiento & desarrollo , Fenotipo , Proteínas Proto-Oncogénicas c-ablRESUMEN
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.