RESUMEN
The phospholipase D (PLD) enzymatic superfamily regulates a wide range of cell biological and physiological pathways, including platelet activation, immune responses, cancer, and spermatogenesis. The three main enzymatic actions of the superfamily entail (i) hydrolyzing membrane phospholipids (phosphatidylcholine (PC) and cardiolipin) to generate choline and the second messenger signaling lipid phosphatidic acid (PA), (ii) using ethanol to transphosphatidylate PC to generate the long-lived metabolite phosphatidylethanol, and (iii) hydrolyzing RNA transcripts to generate piRNAs, the third form of endogenous RNAi. We discuss briefly previously published methods for in vitro and in vivo detection and imaging of PA, and focus on production, purification, and in vitro endonuclease activity analysis for human PLD6, a mitochondrial-tethered isoform with roles in fertility, cancer, and neuronal homeostasis.
Asunto(s)
Pruebas de Enzimas , Proteínas Mitocondriales/química , Imagen Molecular/métodos , Fosfolipasa D/química , ARN Mensajero/química , Animales , Cardiolipinas/química , Cardiolipinas/metabolismo , Diglicéridos/química , Diglicéridos/metabolismo , Activación Enzimática , Glicerofosfolípidos/química , Glicerofosfolípidos/metabolismo , Humanos , Hidrólisis , Isoenzimas/química , Isoenzimas/aislamiento & purificación , Isoenzimas/metabolismo , Proteínas Mitocondriales/aislamiento & purificación , Proteínas Mitocondriales/metabolismo , Ácidos Fosfatidicos/química , Ácidos Fosfatidicos/metabolismo , Fosfatidilcolinas/química , Fosfatidilcolinas/metabolismo , Fosfolipasa D/aislamiento & purificación , Fosfolipasa D/metabolismo , ARN Mensajero/metabolismo , ARN Interferente Pequeño/química , ARN Interferente Pequeño/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismoRESUMEN
Phospholipase D (PLD), a superfamily of signalling enzymes that most commonly generate the lipid second messenger phosphatidic acid, is found in diverse organisms from bacteria to humans and functions in multiple cellular pathways. Since the early 1980s when mammalian PLD activities were first described, most of the important insights concerning PLD function have been gained from studies on cellular models. Reports on physiological and pathophysiological roles for members of the mammalian PLD superfamily are now starting to emerge from genetic models. In this review, we summarize recent findings on PLD functions in these model systems, highlighting newly appreciated connections of the superfamily to cancer, neuronal pathophysiology, cardiovascular topics, spermatogenesis and infectious diseases.
Asunto(s)
Fosfolipasa D/fisiología , Animales , Células Germinativas/enzimología , Humanos , Metabolismo de los Lípidos/fisiología , Ratones , Mitocondrias/ultraestructura , Neoplasias/enzimología , Enfermedades Neurodegenerativas/enzimología , Ácidos Fosfatidicos/metabolismo , Fosfolipasa D/metabolismo , Transducción de Señal/fisiologíaRESUMEN
BACKGROUND AND PURPOSE: Orexin receptors potently signal to lipid messenger systems, and our previous studies have suggested that PLD would be one of these. We thus wanted to verify this by direct measurements and clarify the molecular mechanism of the coupling. EXPERIMENTAL APPROACH: Orexin receptor-mediated PLD activation was investigated in CHO cells stably expressing human OX(1) orexin receptors using [(14) C]-oleic acid-prelabelling and the transphosphatidylation assay. KEY RESULTS: Orexin stimulation strongly increased PLD activity - even more so than the phorbol ester TPA (12-O-tetradecanoyl-phorbol-13-acetate), a highly potent activator of PLD. Both orexin and TPA responses were mediated by PLD1. Orexin-A and -B showed approximately 10-fold difference in potency, and the concentration-response curves were biphasic. Using pharmacological inhibitors and activators, both orexin and TPA were shown to signal to PLD1 via the novel PKC isoform, PKCδ. In contrast, pharmacological or molecular biological inhibitors of Rho family proteins RhoA/B/C, cdc42 and Rac did not inhibit the orexin (or the TPA) response, nor did the molecular biological inhibitors of PKD. In addition, neither cAMP elevation, Gα(i/o) nor Gßγ seemed to play an important role in the orexin response. CONCLUSIONS AND IMPLICATIONS: Stimulation of OX(1) receptors potently activates PLD (probably PLD1) in CHO cells and this is mediated by PKCδ but not other PKC isoforms, PKDs or Rho family G-proteins. At present, the physiological significance of orexin-induced PLD activation is unknown, but this is not the first time we have identified PKCδ in orexin signalling, and thus some specific signalling cascade may exist between orexin receptors and PKCδ.
Asunto(s)
Fosfolipasa D/metabolismo , Proteína Quinasa C-delta/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Neuropéptido/metabolismo , Acetofenonas/farmacología , Animales , Benzopiranos/farmacología , Benzoxazoles/farmacología , Células CHO , Cricetinae , Cricetulus , Humanos , Péptidos y Proteínas de Señalización Intracelular/farmacología , Naftiridinas , Neuropéptidos/farmacología , Receptores de Orexina , Orexinas , Proteína Quinasa C-delta/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Receptores Acoplados a Proteínas G/antagonistas & inhibidores , Receptores de Neuropéptido/antagonistas & inhibidores , Acetato de Tetradecanoilforbol/farmacología , Urea/análogos & derivados , Urea/farmacologíaRESUMEN
Cardiolipin (CL) is a mitochondria-specific phospholipid synthesized by CL synthase (CLS). We describe here a human gene for CLS and its analysis via RNAi knockdown on apoptotic progression. Although mitochondrial membrane potential is unchanged in cells containing only 25% of the normal amount of CL, free cytochrome c (cyt. c) is detected in the intermembrane space and the mitochondria exhibit signs of reorganized cristae. However, the release of cyt. c from the mitochondria still requires apoptotic stimulation. Increased sensitivity to apoptotic signals and accelerated rates of apoptosis are observed in CL-deficient cells, followed by elevated levels of secondary necrosis. Apoptosis is thought to progress via binding of truncated Bid (tBid) to mitochondrial CL, followed by CL oxidation which results in cyt. c release. The exaggerated and accelerated apoptosis observed in CL-deficient cells is matched by an accelerated reduction in membrane potential and increased cyt. c release, but not by decreased tBid binding. This study suggests that the CL/cyt. c relationship is important in apoptotic progression and that regulating CL oxidation or/and deacylation could represent a possible therapeutic target.
Asunto(s)
Cardiolipinas/metabolismo , Citocromos c/metabolismo , Proteínas de la Membrana/metabolismo , Mitocondrias/metabolismo , Membranas Mitocondriales/metabolismo , Transferasas (Grupos de Otros Fosfatos Sustitutos)/metabolismo , Animales , Apoptosis , Cardiolipinas/fisiología , Células HeLa , Humanos , Potencial de la Membrana Mitocondrial , Proteínas de la Membrana/genética , Ratones , Células 3T3 NIH , Interferencia de ARN , Transferasas (Grupos de Otros Fosfatos Sustitutos)/genéticaRESUMEN
Phospholipase D (PLD) hydrolyzes the phosphodiester bond of the glycerolipid phosphatidylcholine, resulting in the production of phosphatidic acid and free choline. Phosphatidic acid is widely considered to be the intracellular lipid mediator of many of the biological functions attributed to PLD. However, phosphatidic acid is a tightly regulated lipid in cells and can be converted to other potentially bioactive lipids, including diacylglycerol and lysophosphatidic acid. PLD activities have been described in multiple organisms, including plants, mammals, bacteria and yeast. In mammalian systems, PLD activity regulates the actin cytoskeleton, vesicle trafficking for secretion and endocytosis, and receptor signaling. PLD is in turn regulated by phosphatidylinositol-4,5-bisphosphate, protein kinase C and ADP Ribosylation Factor and Rho family GTPases. This review focuses on the lipid precursors and products of mammalian PLD metabolism, especially phosphatidic acid and the roles this lipid performs in the mediation of the functions of PLD.
Asunto(s)
Ácidos Fosfatidicos/metabolismo , Fosfolipasa D/metabolismo , Animales , Metabolismo de los Lípidos , Fosfolipasa D/análisis , Fosfolipasa D/químicaRESUMEN
Phosphatidic acid produced by phospholipase D (PLD) as a result of signaling activity is thought to play a role in membrane vesicle trafficking, either as an intracellular messenger or as a cone-shaped lipid that promotes membrane fusion. We recently described that, in neuroendocrine cells, plasma membrane-associated PLD1 operates at a stage of Ca(2+)-dependent exocytosis subsequent to cytoskeletal-mediated recruitment of secretory granules to exocytotic sites. We show here that PLD1 also plays a crucial role in neurotransmitter release. Using purified rat brain synaptosomes subjected to hypotonic lysis and centrifugation, we found that PLD1 is associated with the particulate fraction containing the plasma membrane. Immunostaining of rat cerebellar granule cells confirmed localization of PLD1 at the neuronal plasma membrane in zones specialized for neurotransmitter release (axonal neurites, varicosities, and growth cone-like structures). To determine the potential involvement of PLD1 in neurotransmitter release, we microinjected catalytically inactive PLD1(K898R) into Aplysia neurons and analyzed its effects on evoked acetylcholine (ACh) release. PLD1(K898R) produced a fast and potent dose-dependent inhibition of ACh release. By analyzing paired-pulse facilitation and postsynaptic responses evoked by high-frequency stimulations, we found that the exocytotic inhibition caused by PLD1(K898R) was not the result of an alteration in stimulus-secretion coupling or in vesicular trafficking. Analysis of the fluctuations in amplitude of the postsynaptic responses revealed that the PLD1(K898R) blocked ACh release by reducing the number of active presynaptic-releasing sites. Our results provide evidence that PLD1 plays a major role in neurotransmission, most likely by controlling the fusogenic status of presynaptic release sites.
Asunto(s)
Neurotransmisores/metabolismo , Fosfolipasa D/metabolismo , Acetilcolina/metabolismo , Animales , Aplysia , Catálisis , Células Cultivadas , Técnica del Anticuerpo Fluorescente , Microscopía Confocal , Neuronas/enzimología , Neuronas/metabolismo , Ratas , Ratas Wistar , Fracciones Subcelulares/enzimología , Sinapsis/metabolismoRESUMEN
BACKGROUND: In cholinergic neurons, the hydrolysis of phosphatidylcholine (PC) by a phospholipase D (PLD)-type enzyme generates some of the precursor choline used for the synthesis of the neurotransmitter acetylcholine (ACh). We sought to determine the molecular identity of the relevant PLD using murine basal forebrain cholinergic SN56 cells in which the expression and activity of the two PLD isoforms, PLD1 and PLD2, were experimentally modified. ACh levels were examined in cells incubated in a choline-free medium, to ensure that their ACh was synthesized entirely from intracellular choline. RESULTS: PLD2, but not PLD1, mRNA and protein were detected in these cells and endogenous PLD activity and ACh synthesis were stimulated by phorbol 12-myristate 13-acetate (PMA). Introduction of a PLD2 antisense oligonucleotide into the cells reduced PLD2 mRNA and protein expression by approximately 30%. The PLD2 antisense oligomer similarly reduced basal- and PMA-stimulated PLD activity and ACh levels. Overexpression of mouse PLD2 by transient transfection increased basal- (by 74%) and PMA-stimulated (by 3.2-fold) PLD activity. Moreover, PLD2 transfection increased ACh levels by 26% in the absence of PMA and by 2.1-fold in the presence of PMA. Overexpression of human PLD1 by transient transfection increased PLD activity by 4.6-fold and ACh synthesis by 2.3-fold in the presence of PMA as compared to controls. CONCLUSIONS: These data identify PLD2 as the endogenous enzyme that hydrolyzes PC to generate choline for ACh synthesis in cholinergic cells, and indicate that in a model system choline generated by PLD1 may also be used for this purpose.
Asunto(s)
Acetilcolina/biosíntesis , Colina/biosíntesis , Fosfolipasa D/metabolismo , Acetato de Tetradecanoilforbol/análogos & derivados , Animales , Línea Celular , Activación Enzimática/efectos de los fármacos , Expresión Génica/efectos de los fármacos , Líquido Intracelular/metabolismo , Isoenzimas/genética , Isoenzimas/metabolismo , Ratones , Datos de Secuencia Molecular , Neuronas/citología , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Fosfolipasa D/genética , ARN Mensajero/metabolismo , Acetato de Tetradecanoilforbol/farmacología , TransfecciónRESUMEN
Phospholipase D (PLD) plays a central role in the control of vesicle budding and protein transit. We previously showed that in resting epithelial HT29-cl19A cells, PLD is implicated in the control of constitutive protein transit, from the trans-Golgi network to the plasma membrane, and that phorbol ester stimulation of protein transit is correlated with PLD activation (Auger, R., Robin, P., Camier, B., Vial, G., Rossignol, B., Tenu, J.-P., and Raymond, M.-N. (1999) J. Biol. Chem. 274, 28652-28659). In this paper we demonstrate that: 1) PLD is not implicated in the earliest phases of protein transit; 2) PLD controls apical but not basolateral protein transit; 3) HT29-cl19A cells express PLD1b and PLD2a mRNAs and proteins; 4) the expression of a catalytically inactive mutant of PLD2 (mPLD2-K758R) significantly inhibited apical constitutive protein transit whereas expression of a catalytically inactive mutant of PLD1 (hPLD1b-K898R) prevented increases in the rate of apical transit as triggered by phorbol esters; 5) PLD2 appears to be located in a perinuclear region containing the Golgi whereas PLD1, which is scattered in the cytoplasm in resting cells, is translocated to the plasma membrane after phorbol ester stimulation. Taken together, these data lead to the conclusion that in HT29-cl19A cells, both PLDs regulate protein transit between the trans-Golgi network and the apical plasma membrane, but that they do so at different steps in the pathway.
Asunto(s)
Células Epiteliales/metabolismo , Fosfolipasa D/metabolismo , Transporte de Proteínas/fisiología , alfa 1-Antitripsina/metabolismo , Secuencia de Aminoácidos , Membrana Celular/metabolismo , Polaridad Celular , Retículo Endoplásmico/metabolismo , Células Epiteliales/efectos de los fármacos , Etanol/farmacología , Genes Reporteros , Aparato de Golgi/metabolismo , Células HT29 , Humanos , Inmunohistoquímica , Datos de Secuencia Molecular , Fosfolipasa D/genética , Isoformas de Proteínas , Radioisótopos/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Transfección , Inhibidores de Tripsina/metabolismoRESUMEN
Phospholipase D (PLD) has been proposed to mediate cytoskeletal remodeling and vesicular trafficking along the secretory pathway. We recently described the activation of an ADP ribosylation factor-regulated PLD at the plasma membrane of chromaffin cells undergoing secretagogue-stimulated exocytosis. We show here that the isoform involved is PLD1b, and, using a real-time assay for individual cells, that PLD activation and exocytosis are closely correlated. Moreover, overexpressed PLD1, but not PLD2, increases stimulated exocytosis in a phosphatidylinositol 4,5-bisphosphate-dependent manner, whereas catalytically inactive PLD1 inhibits it. These results provide the first direct evidence that PLD1 is an important component of the exocytotic machinery in neuroendocrine cells.
Asunto(s)
Células Cromafines/enzimología , Exocitosis/fisiología , Fosfolipasa D/metabolismo , Actinas/metabolismo , Animales , Catálisis , Bovinos , Células Cultivadas , Células Cromafines/citología , Células Cromafines/fisiología , Inhibidores Enzimáticos/farmacología , Líquido Intracelular/enzimología , Isoenzimas/antagonistas & inhibidores , Isoenzimas/metabolismo , Sistemas Neurosecretores/citología , Células PC12 , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfolipasa D/antagonistas & inhibidores , Proteína Quinasa C/metabolismo , Ratas , Esfingosina/análogos & derivados , Esfingosina/farmacologíaRESUMEN
G protein-coupled and tyrosine kinase receptor activation of phospholipase D1 (PLD1) play key roles in agonist-stimulated cellular responses such as regulated exocytosis, actin stress fiber formation, and alterations in cell morphology and motility. Protein Kinase C, ADP-ribosylation factor (ARF), and Rho family members activate PLD1 in vitro; however, the actions of the stimulators on PLD1 in vivo have been proposed to take place through indirect pathways. We have used the yeast split-hybrid system to generate PLD1 alleles that fail to bind to or to be activated by RhoA but that retain wild-type responses to ARF and PKC. These alleles then were employed in combination with alleles unresponsive to PKC or to both stimulators to examine the activation of PLD1 by G protein-coupled receptors. Our results demonstrate that direct stimulation of PLD1 in vivo by RhoA (and by PKC) is critical for significant PLD1 activation but that PLD1 subcellular localization and regulated phosphorylation occur independently of these stimulatory pathways.
Asunto(s)
Proteínas de Unión al GTP Heterotriméricas/metabolismo , Fosfolipasa D/metabolismo , Proteína Quinasa C/fisiología , Transducción de Señal , Proteína de Unión al GTP rhoA/fisiología , Factor 1 de Ribosilacion-ADP/fisiología , Alelos , Animales , Células COS , Línea Celular , Membranas Intracelulares/enzimología , Mutación , Fosfolipasa D/genética , Fosforilación , Receptores de Superficie Celular/metabolismo , Técnicas del Sistema de Dos HíbridosRESUMEN
Phospholipase D (PLD)1 is quiescent in vitro and in vivo until stimulated by classical protein kinase C (PKC) isoforms, ADP-ribosylation factor or Rho family members. By contrast, PLD2 has high basal activity, and the mechanisms involved in agonist-induced activation of PLD2 are poorly understood. Using transiently transfected human embryonic kidney (HEK)-293 cells as a model system, we report in the present study that PLD2 overexpressed in HEK-293 cells exhibits regulatory properties similar to PLD1 when stimulated in response to insulin and phorbol ester. Co-expression of PLD1 or PLD2 with PKC alpha results in constitutive activation of both PLD isoforms, which cannot be further stimulated by insulin. Co-expression of PLD1 with phospholipase C (PLC)gamma has the same effect, while co-expression of PLD2 with PLC gamma allows PLD2 activity to be stimulated in an insulin-dependent manner. The PKC-specific inhibitors bisindolylmaleimide and Gö 6976 abolish insulin-induced PLD2 activation in HEK-293 cells co-expressing the insulin receptor, PLC gamma and PLD2, confirming that not only PLD1, but PLD2 as well, is regulated in a PKC-dependent manner. Finally, we provide evidence that PKC alpha is constitutively associated with PLD2. In summary, we demonstrate that insulin treatment results in activation of both PLD1 and PLD2 in appropriate cell types when the appropriate upstream intermediate signalling components, i.e. PKC alpha and PLC gamma, are expressed at sufficient levels.
Asunto(s)
Insulina/farmacología , Isoenzimas/metabolismo , Riñón/enzimología , Fosfolipasa D/biosíntesis , Proteína Quinasa C/metabolismo , Transducción de Señal , Fosfolipasas de Tipo C/metabolismo , Línea Celular , Humanos , Riñón/citología , Fosfolipasa C gamma , Fosfolipasa D/metabolismo , Unión Proteica , Proteína Quinasa C-alfaAsunto(s)
Fosfolipasa D/metabolismo , Proteína de Unión al GTP rhoA/metabolismo , Animales , Baculoviridae/genética , Activación Enzimática , Fosfatidilinositol 4,5-Difosfato/aislamiento & purificación , Fosfatos de Fosfatidilinositol/aislamiento & purificación , Fosfolipasa D/genética , Unión Proteica , Ratas , Proteínas Recombinantes/metabolismo , Schizosaccharomyces/genética , Spodoptera , Técnicas del Sistema de Dos HíbridosRESUMEN
Phospholipase D (PLD), a signal-transducing membrane-associated enzyme, is implicated in diverse processes including apoptosis, ERK activation, and glucose transport. Prior studies have identified specific PLD activators and repressors that directly regulate its enzymatic activity. Using two-hybrid screens, we have identified PEA-15 as a PLD interactor that unexpectedly functions to alter its level of expression. PEA-15 is a widely expressed death effector domain-containing phosphoprotein involved in signal transduction, apoptosis, ERK activation, and glucose transport. The PLD1-interacting site on PEA-15 consists of part of the death effector domain domain plus additional C-terminal flanking sequences, whereas the PEA-15-interacting site on PLD1 overlaps the previously identified RhoA-interacting site. PEA-15 did not affect basal or stimulated in vitro PLD1 enzymatic activation. However, co-expression of PEA-15 increased levels of PLD1 activity. This increased activation correlated with higher PLD1 protein expression levels, as marked by faster accumulation and longer persistence of PLD1 when PEA-15 was present. PEA-15 similarly increased protein expressions level of PLD2 and co-immunoprecipitated with it. These results suggest that PEA-15 may stabilize PLD or act as a PLD chaperone. The common involvement of PEA-15 and PLD in apoptosis, ERK activation, and glucose transport additionally suggests functional significance.
Asunto(s)
Regulación Enzimológica de la Expresión Génica , Fosfolipasa D/metabolismo , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Alelos , Secuencia de Aminoácidos , Animales , Apoptosis , Proteínas Reguladoras de la Apoptosis , Sitios de Unión , Transporte Biológico , Western Blotting , Células COS , Línea Celular , Activación Enzimática , Glucosa/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular , Microscopía Fluorescente , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Datos de Secuencia Molecular , Fosfolipasa D/genética , Fosfoproteínas/genética , Plásmidos/metabolismo , Pruebas de Precipitina , Unión Proteica , Estructura Terciaria de Proteína , Homología de Secuencia de Aminoácido , Transducción de Señal , Factores de Tiempo , Técnicas del Sistema de Dos HíbridosRESUMEN
Myocardial phospholipase D (PLD) has been implicated in the regulation of Ca(2+) mobilization and contractile performance in the heart. However, the molecular identity of this myocardial PLD and the mechanisms that regulate it are not well understood. Using subcellular fractionation and Western blot analysis, we found that PLD2 is the major myocardial PLD and that it localizes primarily to sarcolemmal membranes. A 100-kDa PLD2-interacting cardiac protein was detected using a protein overlay assay employing purified PLD2 and then identified as alpha-actinin using peptide-mass fingerprinting with matrix-assisted laser desorption/ionization mass spectroscopy. The direct association between PLD2 and alpha-actinin was confirmed using an in vitro binding assay and localized to PLD2's N-terminal 185 amino acids. Purified alpha-actinin potently inhibits PLD2 activity (IC(50) = 80 nm) in an interaction-dependent and ADP-ribosylation factor-reversible manner. Finally, alpha-actinin co-localizes with actin and with PLD2 in the detergent-insoluble fraction from sarcolemmal membranes. These results suggest that PLD2 is reciprocally regulated in sarcolemmal membranes by alpha-actinin and ARF1 and accordingly that a major role for PLD2 in cardiac function may involve reorganization of the actin cytoskeleton.
Asunto(s)
Actinina/metabolismo , Miocardio/enzimología , Fosfolipasa D/metabolismo , Sarcolema/enzimología , Actinina/química , Adenosina Difosfato Ribosa/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Células COS , Fraccionamiento Celular/métodos , Línea Celular , Membrana Celular/enzimología , Ventrículos Cardíacos , Humanos , Cinética , Datos de Secuencia Molecular , Miocardio/ultraestructura , Fosfolipasa D/análisis , Fosfolipasa D/antagonistas & inhibidores , Ratas , Proteínas Recombinantes/análisis , Proteínas Recombinantes/metabolismo , Sarcolema/ultraestructura , Spodoptera , TransfecciónRESUMEN
Two distinct proteins inhibiting phospholipase D (PLD) activity in rat brain cytosol were previously purified and identified as synaptojanin and AP180, which are specific to nerve terminals and associate with the clathrin coat. Two additional PLD-inhibitory proteins have now been purified and identified as the amphiphysins I and II, which forms a heterodimer that also associates with the clathrin coat. Bacterially expressed recombinant amphiphysins inhibited both PLD1 and PLD2 isozymes in vitro with a potency similar to that of brain amphiphysin (median inhibitory concentration of approximately 15 nm). Expressions of either amphiphysin in COS-7 cells reduced activity of endogenous PLD as well as exogenously expressed PLD1 and PLD2. Coprecipitation experiments suggested that the inhibitory effect of amphiphysins results from their direct interaction with PLDs. The NH(2) terminus of amphiphysin I was critical for both inhibition of and binding to PLD. Phosphatidic acid formed by signal-induced PLD is thought to be required for the assembly of clathrin-coated vesicles during endocytosis. Thus, the inhibition of PLD by amphiphysins, synaptojanin, and AP180 might play an important role in synaptic vesicle trafficking.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Isoenzimas/antagonistas & inhibidores , Proteínas del Tejido Nervioso/farmacología , Fosfolipasa D/antagonistas & inhibidores , Secuencia de Aminoácidos , Animales , Células COS , Línea Celular , Cromatografía DEAE-Celulosa , Cromatografía Líquida de Alta Presión , Clatrina/metabolismo , Cricetinae , Endocitosis , Glutatión Transferasa/química , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/aislamiento & purificación , Ratas , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/farmacologíaRESUMEN
Short seizure episodes are associated with remodeling of neuronal connections. One region where such reorganization occurs is the hippocampus, and in particular, the mossy fiber pathway. Using genetic and pharmacological approaches, we show here a critical role in vivo for tissue plasminogen activator (tPA), an extracellular protease that converts plasminogen to plasmin, to induce mossy fiber sprouting. We identify DSD-1-PG/phosphacan, an extracellular matrix component associated with neurite reorganization, as a physiological target of plasmin. Mice lacking tPA displayed decreased mossy fiber outgrowth and an aberrant band at the border of the supragranular region of the dentate gyrus that coincides with the deposition of unprocessed DSD-1-PG/phosphacan and excessive Timm-positive, mossy fiber termini. Plasminogen-deficient mice also exhibit the laminar band and DSD- 1-PG/phosphacan deposition, but mossy fiber outgrowth through the supragranular region is normal. These results demonstrate that tPA functions acutely, both through and independently of plasmin, to mediate mossy fiber reorganization.
Asunto(s)
Proteoglicanos Tipo Condroitín Sulfato/fisiología , Fibrinolisina/metabolismo , Hipocampo/fisiología , Fibras Nerviosas/fisiología , Neuritas/fisiología , Plasminógeno/metabolismo , Convulsiones/fisiopatología , Activador de Tejido Plasminógeno/metabolismo , Amígdala del Cerebelo/efectos de los fármacos , Amígdala del Cerebelo/fisiología , Amígdala del Cerebelo/fisiopatología , Animales , Hipocampo/fisiopatología , Hipocampo/ultraestructura , Ácido Kaínico/farmacología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fibras Nerviosas/efectos de los fármacos , Fibras Nerviosas/ultraestructura , Neuritas/efectos de los fármacos , Neuritas/ultraestructura , Plasminógeno/deficiencia , Plasminógeno/genética , Proteínas Tirosina Fosfatasas Clase 5 Similares a Receptores , Convulsiones/inducido químicamente , Activador de Tejido Plasminógeno/deficiencia , Activador de Tejido Plasminógeno/genéticaRESUMEN
Synthesis of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2], a signaling phospholipid, is primarily carried out by phosphatidylinositol 4-phosphate 5-kinase [PI(4)P5K], which has been reported to be regulated by RhoA and Rac1. Unexpectedly, we find that the GTPgammaS-dependent activator of PI(4)P5Kalpha is the small G protein ADP-ribosylation factor (ARF) and that the activation strictly requires phosphatidic acid, the product of phospholipase D (PLD). In vivo, ARF6, but not ARF1 or ARF5, spatially coincides with PI(4)P5Kalpha. This colocalization occurs in ruffling membranes formed upon AIF4 and EGF stimulation and is blocked by dominant-negative ARF6. PLD2 similarly translocates to the ruffles, as does the PH domain of phospholipase Cdelta1, indicating locally elevated PI(4,5)P2. Thus, PI(4)P5Kalpha is a downstream effector of ARF6 and when ARF6 is activated by agonist stimulation, it triggers recruitment of a diverse but interactive set of signaling molecules into sites of active cytoskeletal and membrane rearrangement.
Asunto(s)
Factores de Ribosilacion-ADP/metabolismo , Membrana Celular/fisiología , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Factor 1 de Ribosilacion-ADP/aislamiento & purificación , Factor 6 de Ribosilación del ADP , Factores de Ribosilacion-ADP/aislamiento & purificación , Secuencia de Aminoácidos , Animales , Transporte Biológico , Bovinos , Compartimento Celular , Activación Enzimática , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Isoenzimas/metabolismo , Datos de Secuencia Molecular , Ácidos Fosfatidicos/metabolismo , Fosfolipasa D/metabolismo , Homología de Secuencia de Aminoácido , Transducción de SeñalRESUMEN
Phosphoinositides are both substrates for second messenger-generating enzymes and spatially localized membrane signals that mediate vital steps in signal transduction, cytoskeletal regulation and membrane trafficking. Phosphatidylcholine-specific phospholipase D (PLD) activity is stimulated by phosphoinositides, but the mechanism and physiological requirement for such stimulation to promote PLD-dependent cellular processes is not known. To address these issues, we have identified a site at which phosphoinositides interact with PLD and have assessed the role of this region in PLD function. This interacting motif contains critical basic amino acid residues that are required for stimulation of PLD activity by phosphoinositides. Although PLD alleles mutated at this site fail to bind to phosphoinositides in vitro, they are membrane-associated and properly localized within the cell but are inactive against cellular lipid substrates. Analogous mutations of this site in yeast PLD, Spo14p, result in enzymes that localize normally, but with catalytic activity that has dramatically reduced responsiveness to phosphoinositides. The level of responsiveness to phosphoinositides in vitro correlated with the ability of PLD to function in vivo. Taken together, these results provide the first evidence that phosphoinositide regulation of PLD activity observed in vitro is physiologically important in cellular processes in vivo including membrane trafficking and secretion.
Asunto(s)
Fosfatidilinositoles/farmacología , Fosfolipasa D/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Línea Celular , Reactivos de Enlaces Cruzados , Activación Enzimática/efectos de los fármacos , Técnica del Anticuerpo Fluorescente , Proteínas Fúngicas/química , Isoenzimas/metabolismo , Liposomas/metabolismo , Datos de Secuencia Molecular , Fragmentos de Péptidos/metabolismo , Fosfatidilinositol 4,5-Difosfato/farmacología , Fosfolipasa D/genética , Unión Proteica , Proteínas Recombinantes , Saccharomyces cerevisiae , TransfecciónRESUMEN
Activation of phosphatidylcholine-specific phospholipase D (PLD) constitutes an important part of the cellular response to agonist signaling. PLD1 is stimulated in vitro in a direct and synergistic manner by protein kinase C (PKC), ADP-ribosylation factor (ARF) and Rho family members. However, the direct and specific role of each of these effectors in agonist-stimulated PLD activation is poorly understood. We have used transposon mutagenesis to generate a library of PLD1 alleles containing random pentapeptide insertions. Forty-five alleles were characterized to identify functionally important regions. Use of an allele unresponsive to PKC, but otherwise seemingly normal, to examine coupling of PLD1 to a subset of G-protein-coupled receptors demonstrates for the first time direct stimulation of PLD1 in vivo by PKC and reveals that this direct stimulation is unexpectedly critical for PLD1 activation.
Asunto(s)
Fosfolipasa D/metabolismo , Proteína Quinasa C/metabolismo , Receptores de Superficie Celular/fisiología , Alelos , Secuencia de Aminoácidos , Animales , Células COS , Línea Celular , Activación Enzimática , Proteínas de Unión al GTP/metabolismo , Biblioteca de Genes , Humanos , Datos de Secuencia Molecular , Mutagénesis Insercional , Fosfolipasa D/química , Fosfolipasa D/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Transducción de Señal , TransfecciónRESUMEN
The recent identification of cDNA clones for phospholipase D1 and 2 has opened the door to new studies on its structure and regulation. PLD activity is encoded by at least two different genes that contain catalytic domains that relate their mechanism of action to phosphodiesterases. In vivo roles for PLD suggest that it may be important for multiple specialized steps in receptor dependent and constitutive processes of secretion, endocytosis, and membrane biogenesis.