RESUMEN
The motility of cancer cells in 3D matrices is of two types: mesenchymal motility, in which the cells are elongated and amoeboid motility, in which the cells are round. Amoeboid motility is driven by an actomyosin-based contractile force, which is regulated by the Rho/ROCK pathway. However, the molecular mechanisms underlying the motility of elongated cells remain unknown. Here, we show that the motility of elongated cells is regulated by Rac signaling through the WAVE2/Arp2/3-dependent formation of elongated pseudopodia and cell-substrate adhesion in 3D substrates. The involvement of Rac signaling in cell motility was different in cell lines that displayed an elongated morphology in 3D substrates. In U87MG glioblastoma cells, most of which exhibit mesenchymal motility, inhibition of Rac signaling blocked the invasion of these cells in 3D substrates. In HT1080 fibrosarcoma cells, which display mixed cell motility involving both elongated and rounded cells, inhibition of Rac1 signaling not only blocked mesenchymal motility but also caused a mesenchymal-amoeboid transition. Additionally, Rac1 and RhoA signaling regulated the mesenchymal and amoeboid motility in these cells, respectively, and the inhibition of both pathways dramatically decreased cell invasion. Hence, we could conclude that Rac1 and RhoA signaling simultaneously regulate cell invasion in 3D matrices.
Asunto(s)
Movimiento Celular/fisiología , Neoplasias/patología , Proteínas de Unión al GTP rac/fisiología , Proteínas de Unión al GTP rho/fisiología , Adhesión Celular/genética , Adhesión Celular/fisiología , Técnicas de Cultivo de Célula/métodos , Movimiento Celular/genética , Forma de la Célula/genética , Forma de la Célula/fisiología , Colágeno/farmacología , Geles/farmacología , Humanos , Proteínas Mutantes/metabolismo , Proteínas Mutantes/fisiología , Invasividad Neoplásica , Neoplasias/genética , Neoplasias/metabolismo , Transducción de Señal/genética , Transducción de Señal/fisiología , Células Tumorales Cultivadas , Proteínas de Unión al GTP rac/genética , Proteínas de Unión al GTP rac/metabolismo , Proteína de Unión al GTP rac1/genética , Proteína de Unión al GTP rac1/metabolismo , Proteína de Unión al GTP rac1/fisiología , Proteínas de Unión al GTP rho/genética , Proteínas de Unión al GTP rho/metabolismo , Proteína de Unión al GTP rhoA/genética , Proteína de Unión al GTP rhoA/metabolismo , Proteína de Unión al GTP rhoA/fisiologíaRESUMEN
The retinoblastoma (Rb) gene product is a tumor suppressor that is mutated or inactivated in many types of human cancers. Although Rb is known to be an upstream negative regulator of Abl protein tyrosine kinase, we propose here that Rb also functions as a downstream effector of Abl that plays a positive role in survival of Abl-dependent human tumor cells, including Bcr/Abl-positive chronic myelogenous leukemia (CML). We show that Rb is constitutively phosphorylated at tyrosine in Abl-dependent tumor cells, and that Abl phosphorylates Rb specifically at Y805 within the C-terminal domain of the molecule. We also show that ectopic expression of Rb induces apoptosis in Abl-dependent tumor cells by inhibiting the Abl tyrosine kinase activity, and that Rb-induced apoptosis is compromised by Abl-catalysed phosphorylation of Rb at Y805. Furthermore, the silencing of endogenous Rb by RNA interference induced apoptosis in Abl-dependent tumor cells. Thus, our findings suggest that Abl-catalysed tyrosine phosphorylation of Rb is necessary for survival of Abl-dependent human tumor cells, and raises the possibility that this phosphorylated Rb can be a molecular target for cancer therapy aimed at inducing apoptosis of Abl-dependent tumor cells, such as Bcr/Abl-positive CML.
Asunto(s)
Neoplasias/patología , Proteínas Proto-Oncogénicas c-abl/fisiología , Proteína de Retinoblastoma/fisiología , Apoptosis , Catálisis , Supervivencia Celular , Células HeLa , Humanos , Neoplasias/enzimología , Fosforilación , Interferencia de ARNRESUMEN
Clathrin-mediated endocytosis starts by a recruitment of endocytic proteins to the plasma membrane to induce invagination of lipid bilayer and subsequent vesicule formation. The recruitment of these components requires PtdIns(4,5)P2, a phosphoinositide on the plasma membrane. Although it is well known that the synthesis as well as the disruption of this lipid is important, recent studies have revealed the indispensable roles of direct interaction between PtdIns(4,5)P2 and the endocytic machinery. The ENTH domain is a newly found PtdIns(4,5)P2 binding unit conserved among endocytic proteins like epsins, AP180, and the Hip1/Sla2 family. This review focuses on the essential roles of PtdIns(4,5)P2 and its specific binding partner, the ENTH domain, in clathrin-mediated endocytosis.
Asunto(s)
Proteínas Portadoras/metabolismo , Endocitosis/fisiología , Fosfatidilinositol 4,5-Difosfato/metabolismo , Animales , Proteínas Portadoras/química , Humanos , Lípidos de la Membrana/metabolismo , Modelos Biológicos , Modelos Moleculares , Estructura Terciaria de ProteínaRESUMEN
Down syndrome (DS) is the most frequent genetic disorder with mental retardation and caused by trisomy 21. Although the gene dosage effect hypothesis has been proposed to explain the impact of extra chromosome 21 on the pathology of DS, a series of evidence that challenge this hypothesis has been reported. The availability of the complete sequences of genes on chromosome 21 serves now as starting point to find functional information of the gene products, but information on gene products is limited so far. We therefore evaluated expression levels of six proteins whose genes are encoded on chromosome 21 (synaptojanin-1, chromosome 21 open reading frame 2, oligomycin sensitivity confering protein, peptide 19, cystatin B and adenosine deaminase RNA-specific 2) in fetal cerebral cortex from DS and controls at 18-19 weeks of gestational age using Western blot analysis. Synaptojanin-1 and C21orf2 were increased in DS, but others were comparable between DS and controls, suggesting that the DS phenotype cannot be simply explained by gene dosage effects. We are systematically quantifying all proteins whose genes are encoded on chromosome 21 in order to provide a better understanding of the pathobiochemistry of DS at the protein level. These studies are of significance as they show for the first time protein levels that are carrying out specific function in human fetal brain with DS.
Asunto(s)
Encéfalo/metabolismo , Cromosomas Humanos Par 21 , Síndrome de Down/genética , Dosificación de Gen , Western Blotting , Encéfalo/embriología , Estudios de Casos y Controles , Femenino , HumanosRESUMEN
Phosphoinositide plays a critical role not only in generating second messengers, such as inositol 1,4,5-trisphosphate and diacylglycerol, but also in modulating a variety of cellular functions including cytoskeletal organization and membrane trafficking. Many inositol lipid kinases and phosphatases appear to regulate the concentration of a variety of phosphoinositides in a specific area, thereby inducing spatial and temporal changes in their availability. For example, local concentration changes in phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) in response to extracellular stimuli cause the reorganization of actin filaments and a change in cell shape. PI(4,5)P(2) uncaps the barbed end of actin filaments and increases actin nucleation by modulating a variety of actin regulatory proteins, leading to de novo actin polymerization. PI(4,5)P(2) also plays a key role in membrane trafficking processes. In endocytosis, PI(4,5)P(2) targets clathrin-associated proteins to endocytic vesicles, leading to clathrin-coated pit formation. On the contrary, PI(4,5)P(2) must be dephosphorylated when they shed clathrin coats to fuse endosome. Thus, through regulating actin cytoskeleton organization and membrane trafficking, phosphoinositides play crucial roles in a variety of cell functions such as growth, polarity, movement, and pattern formation.
Asunto(s)
Actinas/metabolismo , Membrana Celular/metabolismo , Citoesqueleto/metabolismo , Fosfatidilinositoles/fisiología , Citoesqueleto de Actina/metabolismo , Actinas/química , Animales , Calcio/metabolismo , Endocitosis , Exocitosis , Humanos , Modelos Químicos , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Transducción de SeñalRESUMEN
Mutations of Wiskott-Aldrich syndrome protein (WASP) underlie the severe thrombocytopenia and immunodeficiency of the Wiskott-Aldrich syndrome. WASP, a specific blood cell protein, and its close homologue, the broadly distributed N-WASP, function in dynamic actin polymerization processes. Here it is demonstrated that N-WASP is expressed along with WASP, albeit at low levels, in human blood cells. The presence of approximately 160 nmol/L rapidly acting N-WASP molecules may explain the normal capacity of WASP-negative patient platelets for early agonist-induced aggregation and filopodia formation. Ex vivo experiments revealed a significant difference between WASP and N-WASP in sensitivity to calpain, the Ca++-dependent protease activated in agonist-stimulated platelets. Through the use of a series of calpain-containing broken cell systems, it is shown that WASP is cleaved in a Ca++-dependent reaction inhibitable by calpeptin and E64d and that N-WASP is not cleaved, suggesting that the cleavage of WASP by calpain functions in normal platelets as part of a Ca++-dependent switch mechanism that terminates the surface projection phase of blood cell activation processes.
Asunto(s)
Plaquetas/metabolismo , Calpaína/metabolismo , Proteínas del Tejido Nervioso/fisiología , Proteínas/fisiología , Síndrome de Wiskott-Aldrich/sangre , Plaquetas/ultraestructura , Calcio/metabolismo , Sistema Libre de Células , Dipéptidos/metabolismo , Femenino , Células HeLa/metabolismo , Humanos , Masculino , Proteínas del Tejido Nervioso/sangre , Activación Plaquetaria , Proteínas/análisis , Seudópodos/ultraestructura , Sensibilidad y Especificidad , Especificidad por Sustrato , Proteína del Síndrome de Wiskott-Aldrich , Proteína Neuronal del Síndrome de Wiskott-AldrichRESUMEN
Reversibility of protein denaturation is a prerequisite for all applications that depend on reliable enzyme catalysis, particularly, for using steam to sterilize enzyme reactors or enzyme sensor tips, and for developing protein-based devices that perform on-off switching of the protein function such as enzymatic activity, ligand binding and so on. In this study, we have successfully constructed an immobilized protein that retains full enzymatic activity even after thermal treatments as high as 120 degrees C. The key for the complete reversibility was the development of a new reaction that allowed a protein to be covalently attached to a surface through its C-terminus and the protein engineering approach that was used to make the protein compatible with the new attachment chemistry.
Asunto(s)
Enzimas Inmovilizadas/química , Ingeniería de Proteínas , Tetrahidrofolato Deshidrogenasa/química , Sustitución de Aminoácidos , Estabilidad de Medicamentos , Enzimas Inmovilizadas/genética , Calor , Humanos , Mutagénesis Sitio-Dirigida , Conformación Proteica , Desnaturalización Proteica , Renaturación de Proteína , Tetrahidrofolato Deshidrogenasa/genética , Tetrahidrofolato Deshidrogenasa/metabolismoRESUMEN
Synaptojanin is a highly abundant polyphosphoinositide phosphatase in nerve terminals, and has been thought to play roles in clathrin-mediated synaptic vesicle endocytosis and signaling. In order to determine the broader role of synaptojanin in the central nervous system, we examined synaptojanin expression in the cerebrum and cerebellum from the fetal to the adult period by means of immunohistochemical and Western blot analyses. Immunohistochemistry consistently revealed the localization of synaptojanin in Cajal--Retzius cells, cortical plate neurons, subplate neurons, intermediate neurons, germinal matrix cells and the ventricular neuroepithelium of the fetal cerebrum. In the fetal cerebellum, synaptojanin immunoreactivity was localized in the external granular cell layer, Purkinje cell layer neuropil, cytoplasm of Purkinje cells and internal granular cells. The immunoreactivity in these structures was decreased around birth. After birth, the synaptojanin immunoreactivity of cortical neurons in the cerebrum, Purkinje cell layer neuropil, and internal granular cells and Purkinje cells in the cerebellum increased and reached a plateau after 11 years of age. These results were consistent with the intensity observed on Western blot analysis. These developmental changes of synaptojanin suggest a broader role in not only synaptic vesicle recycling, but also the regulation of neuronal migration and synaptogenesis in the fetal cerebrum and cerebellum.
Asunto(s)
Cerebelo/crecimiento & desarrollo , Cerebelo/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Monoéster Fosfórico Hidrolasas/metabolismo , Telencéfalo/crecimiento & desarrollo , Telencéfalo/metabolismo , Adulto , Anciano , Western Blotting , Cerebelo/citología , Niño , Preescolar , Feto/química , Feto/metabolismo , Humanos , Inmunohistoquímica , Lactante , Recién Nacido , Proteínas del Tejido Nervioso/análisis , Neuronas/química , Neuronas/metabolismo , Monoéster Fosfórico Hidrolasas/análisis , Terminales Presinápticos/química , Terminales Presinápticos/metabolismo , Telencéfalo/citologíaRESUMEN
Neural Wiskott-Aldrich syndrome protein (N-WASP) is an essential regulator of actin cytoskeleton formation via its association with the actin-related protein (Arp) 2/3 complex. It is believed that the C-terminal Arp2/3 complex-activating domain (verprolin homology, cofilin homology, and acidic (VCA) or C-terminal region of WASP family proteins domain) of N-WASP is usually kept masked (autoinhibition) but is opened upon cooperative binding of upstream regulators such as Cdc42 and phosphatidylinositol 4,5-bisphosphate (PIP2). However, the mechanisms of autoinhibition and association with Arp2/3 complex are still unclear. We focused on the acidic region of N-WASP because it is thought to interact with Arp2/3 complex and may be involved in autoinhibition. Partial deletion of acidic residues from the VCA portion alone greatly reduced actin polymerization activity, demonstrating that the acidic region contributes to Arp2/3 complex-mediated actin polymerization. Surprisingly, the same partial deletion of the acidic region in full-length N-WASP led to constitutive activity comparable with the activity seen with the VCA portion. Therefore, the acidic region in full-length N-WASP plays an indispensable role in the formation of the autoinhibited structure. This mutant contains WASP-homology (WH) 1 domain with weak affinity to the Arp2/3 complex, leading to activity in the absence of part of the acidic region. Furthermore, the actin comet formed by the DeltaWH1 mutant of N-WASP was much smaller than that of wild-type N-WASP. Partial deletion of acidic residues did not affect actin comet size, indicating the importance of the WH1 domain in actin structure formation. Collectively, the acidic region of N-WASP plays an essential role in Arp2/3 complex activation as well as in the formation of the autoinhibited structure, whereas the WH1 domain complements the activation of the Arp2/3 complex achieved through the VCA portion.
Asunto(s)
Actinas/química , Actinas/metabolismo , Proteínas del Citoesqueleto , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/metabolismo , Proteína 2 Relacionada con la Actina , Proteína 3 Relacionada con la Actina , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Sitios de Unión , Humanos , Cinética , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Proteínas del Tejido Nervioso/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Spodoptera , Transfección , Síndrome de Wiskott-Aldrich/genética , Proteína Neuronal del Síndrome de Wiskott-AldrichRESUMEN
WASP family proteins activate nucleation by the Arp2/3 complex, inducing rapid actin polymerization in vitro. Although the C-terminal portion of WASP family proteins (VCA) activates nucleation by the Arp2/3 complex in pure systems, we find that this fragment lacks activity in cell extracts. Thus, polystyrene beads coated with VCA did not move in brain cytosol, while beads coated with N-WASP or WAVE2 did move. The basic clusters between the WH1 domain and the CRIB domain of N-WASP were critical for movement since beads coated with N-WASP or WAVE2 constructs missing the basic clusters (Delta basic) also did not move. Furthermore, VCA and N-WASP/WAVE2 Delta basic constructs were much less able than wild-type N-WASP and WAVE2 to induce actin polymerization in cytosol. All of the proteins, with or without the basic domain, were potent activators of nucleation by purified Arp2/3 complex.
Asunto(s)
Actinas/fisiología , Proteínas de Microfilamentos/química , Proteínas de Microfilamentos/fisiología , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/fisiología , Animales , Encéfalo/fisiología , Bovinos , Citoesqueleto/química , Citoesqueleto/fisiología , Técnicas In Vitro , Proteínas de Microfilamentos/genética , Microesferas , Modelos Biológicos , Movimiento/fisiología , Mutación , Proteínas del Tejido Nervioso/genética , Estructura Terciaria de Proteína , Eliminación de Secuencia , Proteína Neuronal del Síndrome de Wiskott-AldrichRESUMEN
Several phospholipase C (PLC) isoforms have been found in male and female mammalian gametes, and splicing isoforms of PLCdelta4 are predominantly expressed in testis. Here we report that male mice in which the PLCdelta4 gene had been disrupted either produced few small litters or were sterile. In vitro fertilization studies showed that insemination with PLCdelta4-/- sperm resulted in significantly fewer eggs becoming activated and that the calcium transients associated with fertilization were absent or delayed. PLCdelta4-/- sperm were unable to initiate the acrosome reaction, an exocytotic event required for fertilization and induced by interaction with the egg coat, the zona pellucida. These data demonstrate that PLCdelta4 functions in the acrosome reaction that is induced by the zona pellucida during mammalian fertilization.
Asunto(s)
Acrosoma/enzimología , Fertilización , Isoenzimas/metabolismo , Fosfolipasas de Tipo C/metabolismo , Zona Pelúcida/fisiología , Reacción Acrosómica , Animales , Calcio/metabolismo , Señalización del Calcio , Epidídimo/enzimología , Femenino , Marcación de Gen , Fosfatos de Inositol/metabolismo , Isoenzimas/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Óvulo/fisiología , Fosfolipasa C delta , Capacitación Espermática , Inyecciones de Esperma Intracitoplasmáticas , Espermatozoides/enzimología , Espermatozoides/metabolismo , Testículo/enzimología , Fosfolipasas de Tipo C/genéticaRESUMEN
Induction of filopodia is dependent on activation of the small GTPase Cdc42 and on neural Wiskott-Aldrich-syndrome protein (N-WASP). Here we show that WASP-interacting protein (WIP) interacts directly with N-WASP and actin. WIP retards N-WASP/Cdc42-activated actin polymerization mediated by the Arp2/3 complex, and stabilizes actin filaments. Microinjection of WIP into NIH 3T3 fibroblasts induces filopodia; this is inhibited by microinjection of anti-N-WASP antibody. Microinjection of anti-WIP antibody inhibits induction of filopodia by bradykinin, by an active Cdc42 mutant (Cdc42(V12)) and by N-WASP. Our results indicate that WIP and N-WASP may act as a functional unit in filopodium formation, which is consistent with their role in actin-tail formation in cells infected with vaccinia virus or Shigella.
Asunto(s)
Actinas/metabolismo , Proteínas Portadoras/metabolismo , Proteínas del Citoesqueleto , Proteínas del Tejido Nervioso/metabolismo , Seudópodos/metabolismo , Células 3T3 , Proteína 2 Relacionada con la Actina , Proteína 3 Relacionada con la Actina , Animales , Western Blotting , Bradiquinina/farmacología , Línea Celular , Relación Dosis-Respuesta a Droga , Electroforesis en Gel de Poliacrilamida , Activación Enzimática , Glutatión Transferasa/metabolismo , Ratones , Microscopía Fluorescente , Mutación , Unión Proteica , Estructura Terciaria de Proteína , Conejos , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes/metabolismo , Shigella/metabolismo , Transducción de Señal , Factores de Tiempo , Técnicas del Sistema de Dos Híbridos , Proteína Neuronal del Síndrome de Wiskott-Aldrich , Proteína de Unión al GTP cdc42/metabolismoRESUMEN
Reorganization of cortical actin filaments plays critical roles in cell movement and pattern formation. Recently, the WASP and WAVE family proteins WASP and N-WASP, and WAVE1, WAVE2 and WAVE3 have been shown to regulate cortical actin filament reorganization in response to extracellular stimuli. These proteins each have a verprolin-homology (V) domain, cofilin-homology (C) domain and an acidic (A) region at the C-terminus, through which they activate the Arp2/3 complex, leading to rapid actin polymerization. N-WASP is usually present as an inactive form in which the VCA region is masked. Cooperative binding of Cdc42 and phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)) exposes the VCA region, activating N-WASP. In addition to this activation mechanism, WISH also activates N-WASP independently of Cdc42 and PtdIns(4,5)P(2), by binding to the proline-rich region of N-WASP. N-WASP activation induces formation of filopodia in vivo. In contrast, the ubiquitously expressed form of WAVE2 is activated downstream of Rac, leading to formation of lamellipodia. In this case, IRSp53 transmits a signal from Rac to WAVE2 through formation of a ternary Rac-IRSp53-WAVE2 complex. Thus, N-WASP, which is activated downstream of Cdc42 or independently by WISH, induces formation of filopodia and WAVE2, which is activated via IRSp53 downstream of Rac, induces formation of lamellipodia.
Asunto(s)
Citoesqueleto de Actina/metabolismo , Movimiento Celular/fisiología , Células Eucariotas/fisiología , Proteínas de Microfilamentos/metabolismo , Proteínas/metabolismo , Animales , Células Eucariotas/citología , Humanos , Transducción de Señal/fisiología , Proteína del Síndrome de Wiskott-Aldrich , Familia de Proteínas del Síndrome de Wiskott-AldrichRESUMEN
WASP- and Ena/VASP-family proteins have been reported to regulate the cortical actin cytoskeleton as downstream effectors of the Rho-family small G-proteins Rac and Cdc42, but their functions are little understood. We observed the localization of the WASP family proteins, N-WASP and WAVE, and the Ena/VASP family protein, Mena, in protruding lamellipodia. Rat fibroblast cell line 3Y1 protruded lamellipodia on poly-L-lysine-coated substrate without any trophic factor. N-WASP and Cdc42 were concentrated along the actin filament bundles of microspikes but not at the tips. By immunofluorescence and immunoelectron microscopy, both WAVE and Mena were observed to localize at the lamellipodium edge. Interestingly, Mena tended to concentrate at the microspike tips but WAVE did not. At the edge of the lamellipodium, the correlation between the fluorescence from Mena and actin filaments stained with the specific antibody and rhodamine-phalloidin, respectively, was much higher than that between WAVE and actin filament. The Ena/VASP homology 2 (EVH2) domain of avian Ena, an avian homolog of Mena, was localized to the lamellipodium edge and concentrated at the tip of microspikes. The SCAR homology domain (SHD) of human WAVE was distributed along the lamellipodium edge. These results indicate that N-WASP, WAVE and Mena have different roles in the regulation of the cortical actin cytoskeleton in the protruding lamellipodium. WAVE and Mena should be recruited to the lamellipodium edge through SHD and the EVH2 domain, respectively, to regulate the actin polymerization near the cell membrane. N-WASP should regulate the formation of the actin filament bundle in addition to activating Arp2/3 complex in lamellipodium under the control of Cdc42.
Asunto(s)
Proteínas Portadoras/análisis , Proteínas del Citoesqueleto , Citoesqueleto/química , Proteínas de Microfilamentos/análisis , Proteínas del Tejido Nervioso/análisis , Seudópodos/ultraestructura , Actinas/química , Animales , Proteínas Portadoras/fisiología , Moléculas de Adhesión Celular/análisis , Moléculas de Adhesión Celular/fisiología , Línea Celular , Citoesqueleto/metabolismo , Citoesqueleto/ultraestructura , Fibroblastos/citología , Fibroblastos/metabolismo , Proteínas de Microfilamentos/química , Proteínas de Microfilamentos/metabolismo , Proteínas de Microfilamentos/fisiología , Proteínas del Tejido Nervioso/fisiología , Fosfoproteínas/análisis , Fosfoproteínas/fisiología , Proteínas/análisis , Proteínas/metabolismo , Proteínas Proto-Oncogénicas/análisis , Seudópodos/química , Seudópodos/metabolismo , Ratas , Familia de Proteínas del Síndrome de Wiskott-Aldrich , Proteína Neuronal del Síndrome de Wiskott-Aldrich , Proteína de Unión al GTP cdc42/metabolismoRESUMEN
Injection of a porcine cytosolic sperm factor (SF) or of a porcine testicular extract into mammalian eggs triggers oscillations of intracellular free calcium ([Ca(2+)](i)) similar to those initiated by fertilization. To elucidate whether SF activates the phosphoinositide (PI) pathway, mouse eggs or SF were incubated with U73122, an inhibitor of events leading to phospholipase C (PLC) activation and/or of PLC itself. In both cases, U73122 blocked the ability of SF to induce [Ca(2+)](i) oscillations, although it did not inhibit Ca(2+) release caused by injection of inositol 1,4,5-triphosphate (IP(3)). The inactive analogue, U73343, had no effect on SF-induced Ca(2+) responses. To determine at the single cell level whether SF triggers IP(3) production concomitantly with a [Ca(2+)](i) rise, SF was injected into Xenopus oocytes and IP(3) concentration was determined using a biological detector cell combined with capillary electrophoresis. Injection of SF induced a significant increase in [Ca(2+)](i) and IP(3) production in these oocytes. Using ammonium sulfate precipitation, chromatographic fractionation, and Western blotting, we determined whether PLCgamma1, PLCgamma2, or PLCdelta4 and/or its splice variants, which are present in sperm and testis, are responsible for the Ca(2+) activity in the extracts. Our results revealed that active fractions do not contain PLCgamma1, PLCgamma2, or PLCdelta4 and/or its splice variants, which were present in inactive fractions. We also tested whether IP(3) could be the sensitizing stimulus of the Ca(2+)-induced Ca(2+) release mechanism, which is an important feature of fertilized and SF-injected eggs. Eggs injected with adenophostin A, an IP(3) receptor agonist, showed enhanced Ca(2+) responses to CaCl(2) injections. Thus, SF, and probably sperm, induces [Ca(2+)](i) rises by persistently stimulating IP(3) production, which in turn results in long-lasting sensitization of Ca(2+)-induced Ca(2+) release. Whether SF is itself a PLC or whether it acts upstream of the egg's PLCs remains to be elucidated.
Asunto(s)
Adenosina/análogos & derivados , Factores Biológicos/farmacología , Calcio/metabolismo , Fosfatidilinositoles/metabolismo , Adenosina/farmacología , Empalme Alternativo , Animales , Western Blotting , Cloruro de Calcio/farmacología , Citosol/química , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Estrenos/farmacología , Femenino , Inositol 1,4,5-Trifosfato/biosíntesis , Inositol 1,4,5-Trifosfato/farmacología , Isoenzimas/metabolismo , Masculino , Microinyecciones , Oocitos/metabolismo , Pirrolidinonas/farmacología , Semen/química , Porcinos , Testículo/química , Extractos de Tejidos/farmacología , Fosfolipasas de Tipo C/antagonistas & inhibidores , Fosfolipasas de Tipo C/genética , Fosfolipasas de Tipo C/metabolismo , XenopusRESUMEN
We have characterized a novel Sac domain-containing inositol phosphatase, hSac2. It was ubiquitously expressed but especially abundant in the brain, heart, skeletal muscle, and kidney. Unlike other Sac domain-containing proteins, hSac2 protein exhibited 5-phosphatase activity specific for phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate. This is the first time that the Sac domain has been reported to possess 5-phosphatase activity. Its 5-phosphatase activity for phosphatidylinositol 4,5-bisphosphate (K(m) = 14.3 microm) was comparable with those of Type II 5-phosphatases. These results imply that hSac2 functions as an inositol polyphosphate 5-phosphatase.
Asunto(s)
Fosfatidilinositoles/metabolismo , Fosfoproteínas Fosfatasas/metabolismo , Monoéster Fosfórico Hidrolasas/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , ADN Complementario , Humanos , Inositol Polifosfato 5-Fosfatasas , Datos de Secuencia Molecular , Monoéster Fosfórico Hidrolasas/química , Monoéster Fosfórico Hidrolasas/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
Endocytic proteins such as epsin, AP180, and Hip1R (Sla2p) share a conserved modular region termed the epsin NH2-terminal homology (ENTH) domain, which plays a crucial role in clathrin-mediated endocytosis through an unknown target. Here, we demonstrate a strong affinity of the ENTH domain for phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P2]. With nuclear magnetic resonance analysis of the epsin ENTH domain, we determined that a cleft formed with positively charged residues contributed to phosphoinositide binding. Overexpression of a mutant, epsin Lys76 --> Ala76, with an ENTH domain defective in phosphoinositide binding, blocked epidermal growth factor internalization in COS-7 cells. Thus, interaction between the ENTH domain and PtdIns(4,5)P2 is essential for endocytosis mediated by clathrin-coated pits.
Asunto(s)
Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Endocitosis , Neuropéptidos/química , Neuropéptidos/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Proteínas de Transporte Vesicular , Proteínas Adaptadoras del Transporte Vesicular , Secuencias de Aminoácidos , Sustitución de Aminoácidos , Animales , Células COS , Chlorocebus aethiops , Clatrina/metabolismo , Invaginaciones Cubiertas de la Membrana Celular/metabolismo , Proteínas de Unión al ADN/metabolismo , Factor de Crecimiento Epidérmico/metabolismo , Fosfatos de Inositol/metabolismo , Liposomas/metabolismo , Modelos Moleculares , Resonancia Magnética Nuclear Biomolecular , Conformación Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Factores de Transcripción/metabolismo , Dedos de ZincRESUMEN
We identified a novel adaptor protein that contains a Src homology (SH)3 domain, SH3 binding proline-rich sequences, and a leucine zipper-like motif and termed this protein WASP interacting SH3 protein (WISH). WISH is expressed predominantly in neural tissues and testis. It bound Ash/Grb2 through its proline-rich regions and neural Wiskott-Aldrich syndrome protein (N-WASP) through its SH3 domain. WISH strongly enhanced N-WASP-induced Arp2/3 complex activation independent of Cdc42 in vitro, resulting in rapid actin polymerization. Furthermore, coexpression of WISH and N-WASP induced marked formation of microspikes in Cos7 cells, even in the absence of stimuli. An N-WASP mutant (H208D) that cannot bind Cdc42 still induced microspike formation when coexpressed with WISH. We also examined the contribution of WISH to a rapid actin polymerization induced by brain extract in vitro. Arp2/3 complex was essential for brain extract-induced rapid actin polymerization. Addition of WISH to extracts increased actin polymerization as Cdc42 did. However, WISH unexpectedly could activate actin polymerization even in N-WASP-depleted extracts. These findings suggest that WISH activates Arp2/3 complex through N-WASP-dependent and -independent pathways without Cdc42, resulting in the rapid actin polymerization required for microspike formation.
Asunto(s)
Actinas/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Proteínas Portadoras/metabolismo , Proteínas del Citoesqueleto , Proteínas Musculares , Proteínas del Tejido Nervioso/metabolismo , Proteína de Unión al GTP cdc42/metabolismo , Proteína 2 Relacionada con la Actina , Proteína 3 Relacionada con la Actina , Secuencia de Aminoácidos , Animales , Northern Blotting , Western Blotting , Química Encefálica , Proteínas Portadoras/química , Proteínas Portadoras/genética , Bovinos , Línea Celular , Extensiones de la Superficie Celular/metabolismo , Medio de Cultivo Libre de Suero , Proteína Adaptadora GRB2 , Genes Reporteros , Humanos , Leucina Zippers , Datos de Secuencia Molecular , Polímeros/metabolismo , Unión Proteica , Proteínas/metabolismo , Ratas , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Síndrome de Wiskott-Aldrich , Proteína Neuronal del Síndrome de Wiskott-Aldrich , Dominios Homologos srcRESUMEN
Phosphatidylinositol 4-phosphate 5-kinase (PIPK) catalyzes a final step in the synthesis of phosphatidylinositol 4,5-bisphosphate (PIP(2)), a lipid signaling molecule. Strict regulation of PIPK activity is thought to be essential in intact cells. Here we show that type I enzymes of PIPK (PIPKI) are phosphorylated by cyclic AMP-dependent protein kinase (PKA), and phosphorylation of PIPKI suppresses its activity. Serine 214 was found to be a major phosphorylation site of PIPK type Ialpha (PIPKIalpha) that is catalyzed by PKA. In contrast, lysophosphatidic acid-induced protein kinase C activation increased PIPKIalpha activity. Activation of PIPKIalpha was induced by dephosphorylation, which was catalyzed by an okadaic acid-sensitive phosphatase, protein phosphatase 1 (PP1). In vitro dephosphorylation of PIPKIalpha with PP1 increased PIPK activity, indicating that PP1 plays a role in lysophosphatidic acid-induced dephosphorylation of PIPKIalpha. These results strongly suggest that activity of PIPKIalpha in NIH 3T3 cells is regulated by the reversible balance between PKA-dependent phosphorylation and PP1-dependent dephosphorylation.
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Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Animales , Línea Celular , Proteínas Quinasas Dependientes de AMP Cíclico/fisiología , Activación Enzimática , Lisofosfolípidos/farmacología , Ratones , Modelos Biológicos , Fosfoproteínas Fosfatasas/metabolismo , Fosfoproteínas/metabolismo , Fosforilación , Fosfoserina/metabolismo , Proteína Quinasa C/metabolismo , Proteína Fosfatasa 1RESUMEN
The actin-related protein (Arp) 2/3 complex is an essential regulator of de novo actin filament formation. Arp2/3 nucleates the polymerization of actin and creates branched actin filaments when activated by Arp2/3-complex activating domain (VCA) of Wiskott-Aldrich syndrome proteins (WASP family proteins). We found that the branching of actin filaments on pre-existing ADP filaments mediated by the Arp2/3 complex is twice as efficient when Arp2/3 was activated by wild-type neural WASP (N-WASP) or WASP-family verprolin-homologous protein (WAVE) 2 than when activated by the VCA domain alone. By contrast, there was no difference between wild-type N-WASP or WAVE2 and VCA in the branching efficiency on de novo filaments, which are thought to consist mainly of ADP-phosphate filaments. This increased branching efficiency on ADP filaments is due to the basic region located in the center of N-WASP and WAVE2, which was found to associate with ADP actin filaments. Actin filaments and phosphatidylinositol bisphosphate (PIP2) associate with N-WASP at different sites. This association of N-WASP and WAVE2 with actin filaments enhanced recruitment of Arp2/3 to the pre-existing filaments, presumably leading to efficient nucleation and branch formation on pre-existing filaments. These data together suggest that the actin filament binding activity of N-WASP and WAVE2 in the basic region increases the number of barbed ends created on pre-existing filaments. Efficient branching on ADP filaments may be important for initiation of actin-based motility.