A double point mutation in PCL-gamma1 (Y509A/F510A) enhances Y783 phosphorylation and inositol phospholipid-hydrolyzing activity upon EGF stimulation

Growth factor stimulation induces Y783 phosphorylation of phosphoinositide-specific PLC-gamma1, and the subsequent activation of this enzyme in a cellular signaling cascade. Previously, we showed that a double point mutation, Y509A/F510A, of PLC-gamma1, abolished interactions with translational elongation factor 1-alpha. Here, we report that the Y509A/F510A mutant PLC-gamma1 displayed extremely high levels of Y783 phosphorylation and enhanced catalytic activity, compared to wild-type PLC-gamma1, upon treatment of COS7 cells with EGF. In quiescent COS7 cells, the Y509A/F510A mutant PLC-gamma1 exhibited a constitutive hydrolytic activity, whereas the wild-type counterpart displayed a basal level of activity. Upon treatment of COS7 cells with EGF, the Y783F mutation in Y509A/F510A PLC-gamma1 (Y509A/F510A/Y783F triple mutant) cells also led to an enhanced catalytic activity, whereas Y783F mutation alone displayed a basal level of activity. Our results collectively suggest that the Y509A/F510A mutant is more susceptible to receptor tyrosine kinase-induced Y783 phosphorylation than is wild-type PLC-gamma1, but no longer requires Y783 phosphorylation step for the Y509A/F510A mutant PLC-gamma1 activation in vivo.

Mass spectrometry based cellular phosphoinositides profiling and phospholipid analysis: A brief review

Phospholipids are key components of cellular membrane and signaling. Among cellular phospholipids, phosphoinositides, phosphorylated derivatives of phosphatidylinositol are important as a participant in essential metabolic processes in animals. However, due to its low abundance in cells and tissues, it is difficult to identify the composition of phosphoinositides. Recent advances in mass spectrometric techniques, combined with established separation methods, have allowed the rapid and sensitive detection and quantification of a variety of lipid species including phosphoinositides. In this mini review, we briefly introduce progress in profiling of cellular phosphoinositides using mass spectrometry. We also summarize current progress of matrices development for the analysis of cellular phospholipids using matrix-assisted laser desorption/ionization mass spectrometry. The phosphoinositides profiling and phospholipids imaging will help us to understand how they function in a biological system and will provide a powerful tool for elucidating the mechanism of diseases such as diabetes, cancer and neurodegenerative diseases. The investigation of cellular phospholipids including phosphoinositides using electrospray ionization mass spectrometry and matrix-assisted laser desorption/ionization mass spectrometry will suggest new insights on human diseases, and on clinical application through drug development of lipid related diseases.

Type II phosphatidylinositol 4-kinase(s) in cell signaling cascades.

Phosphorylated derivatives of phosphatidylinositol (PtdIns) are key components of many signaling cascades. Many isoforms of PtdIns kinases, PtdIns phosphate kinases and phosphatases use these lipids in amazing networks of signaling cascades that are yet to be understood fully. PtdIns 4-kinase(s) phosphorylates PtdIns at the 4th -OH position of inositol head group and are classified in to type II and III PtdIns 4-kinases. While type III PtdIns 4-kinases are implicated in vesicular trafficking, type II PtdIns 4-kinases are suggested to play a role in cell signaling, cytoskeletal rearrangements, cell motility and in microbial pathogenicity. This paper reviews the role of type II PtdIns 4-kinases in cell signaling cascades in health and disease.

Effect of aflatoxin B1 on phosphoinositide signal transduction pathway during regeneration of liver cells following partial hepatectomy.

Aflatoxin B1 (AFB1) when administered to partially hepatectomised rats 4 hr prior to sacrifice, activated the signalling pathway in regenerating rat liver. The activity of phosphatidylinositol (PI) kinase was found decreased at 30 min but increased at 24 hr and returned to normal at 48 hr. At 30 min, inositol-1,4,5-triphosphate (IP3) level increased significantly whereas diacylglycerol (DAG) level dropped. However, at 24 hr and 48 hr, DAG and IP3 showed the same trend i.e. an increase in their levels. Phosphatidylinositol-4-phosphate levels were found to increase at 24 hr. Protein kinase C (PKC), activity from the particulate fraction was significantly inhibited at 30 min, followed by increase in activity at 24 hr and return to normal at 48 hr. Cytosolic PKC showed a decrease at 24 hr and a significant increase at 48 hr. At the peak of DNA synthesis (24 hr) following partial hepatectomy, all these signalling steps had earlier been found to be inhibited, but the present study shows that aflatoxin B1 administration 4 hr prior to sacrifice reverses the action. Activation of PKC by aflatoxin B1, during regeneration of liver cells when PKC in normally inhibited, may possibly create conditions conducive to carcinogenesis.

Participation of the phosphoinositide metabolism in the hypersensitive response of Citrus limon against Alternaria alternata

Lemon seedlings inoculated with Alternaria alternata develop a hypersensitive response (HR) that includes the induction of Phenylalanine ammonia-lyase (PAL, E. C. 4.3.1.5) and the synthesis of scoparone. The signal transduction pathway involved in the development of this response is unknown. We used several inhibitors of the Phosphoinositide (PI) animal system to study a possible role of Inositol-1,4,5-triphosphate (IP3) in the transduction of the fungal conidia signal in Citrus limon. The HR was only partially inhibited by EGTA, suggesting that not only external but internal calcium as well are necessary for a complete development of the HR. In this plant system, Alternaria alternata induced an early accumulation of the second messenger IP3. When lemon seedlings were watered long term with LiCl, an inhibitor of the phosphoinositide cycle, the IP3 production was reduced, and the LiCl-watered plants could neither induce PAL nor synthesize scoparone in response to fungal conidia. Furthermore, neomycin, a Phospholipase C (PLC, E. C. 3.1.4.3) inhibitor, also inhibited PAL induction and scoparone synthesis in response to A. alternata. These results suggest that IP3 could be involved in the signal transduction pathway for the development of the HR of Citrus limon against A. alternata.

Espectroscopia de prótons (1H) por ressonância magnética, neuroquímica cerebral e diagnóstico de doenças neurológicas / Proton (1H) magnetic resonance spectroscopy, brain neurochemistry, and diagnosis of neurological diseases

A técnica de espectroscopia de prótons (1H) por ressonância magnética do cérebro permite identificar, in vivo e de modo näo-invasivo, neurometabolitos pertencentes a diversas vias do metabolismo intermediário. A análise desses achados é o objetivo da presente revisäo. As bases do método e os principais metabolitos que constituem o espectro säo considerados, assim como as vias neuroquímicas relacionadas com importantes funçöes metabólicas e os neurometabolitos representativos das mesmas, possíveis de serem observados no espectro em condiçöes normais e patológicas. O conhecimento dessas relaçöes aponta para aspectos neuroquímicos da amostra de tecido nervoso examinada e permite hipóteses fisiológicas e fisiopatológicas relativas às variaçöes dos principais metabolitos. Säo descritas variaçöes regionais e em relaçäo ao envelhecimento normal, importantes na seleçäo e comparaçäo de amostras adequadamente pareadas, sobretudo em situaçäo de pesquisa. A aplicaçäo da técnica no diagnóstico em neurologia também é considerada, com ênfase em doenças degenerativas. Conclui-se ser uma técnica de grande utilidade clínica e que contribui de modo significativo no aprofundamento disgnóstico, assim como na monitorizaçäo terapêutica e no acompanhamento evolutivo de doenças neurológicas

Perturbations in phosphoinositide metabolism and protein kinase C activity in mouse liver following whole body irradiation.

The involvement of the signal transduction pathway in mouse liver following whole body irradition was investigated. Mice were exposed to 60Co gamma rays (3 Gy) and sacrificed after different time intervals. Various elements of phosphatidyl inositol signal transduction pathway were investigated. Alterations could be seen as early as 15 min of irradiation. These changes are reflected in elevation in DAG levels and increased activation of PKC, an enzyme which is involved in tumorigenesis. The chronological appearance of various transducers following whole body irradiation is of significance since these early effects may set the stage for radiation-induced tumorigenesis and hence may be used to manipulate tumor response to radiotherapy.

Alterations of phosphatidylinositol signal transduction pathway in hepatic mitochondria following aflatoxin B1 administration.

A single dose of aflatoxin B1 (7 mg/kg body wt) to male rats significantly stimulated the turnover of mitochondrial phosphoinositides 1-7 hr following its administration. The elevation of phosphatidylinositol 3,4,5-trisphosphate was most pronounced whose level continued to be moderately high even at 17 hr period. The level of diacylglycerol showed a marked increase from 4 hr till 7 hr after carcinogen treatment, whereas that of inositol 1,4,5-trisphosphate recorded an increase with a maximum at 7 hr followed by a gradual decrease to near normal level at 24 hr period. The activation of phosphatidylinositol cycle together with an activation of PI 3-kinase, whose product PIP3 is known to be involved in apoptosis might contribute to the early step in the manifestation of toxicity and/or carcinogenicity.

Activación plaquetaria por trombina / Platelet activation by thrombin

Cuando un agonista se une a su receptor específico sobre la membrana plaquetaria se inician una serie de cambios morfológicosy metabólicos que llevan al cambio de forma, agregación y secreción de contenidos granulares. La trombina, serinoproteasa multifuncional y fuerte agonosta plaquetario, tiene dos tipos de receptores sobre la membrana plaquetaria: de alta y de moderada afinidad. Este último pertenece a la familia de receptores ß2 adrenérgicos que presentan siete dominios de intramembrana, e inician la activación a través de G proteínas específicas. De esta manera se desencadenan diversos pasos metabólicos a través de varias enzimas claves. La actividad de la fosfolipasa Cß (PLCß) origina dos segundos mensajeros: Inositol 3 fosfato (IP3) que promueve la movilización de calcio del sistema tubular denso al citosol y el diacilglicerol (DG) que activa proteína quinasa C (PKC). Si bien la plaqueta no prolifera se han detectado enzimas relacionadas a oncogenes. De esta manera se han estudiado y comprendido nuevos caminos de activación. La familia de la tirosina quinasas, relacionas a la proliferación celular y oncogenes, fosforilan residuos tirosinas; en su mayoría son quinasas del tipo no receptor que se encuentran en el citosol como ser: Scr, Syk y FAK. La fosfolipasa Cy necesita la presencia de RasGAP, Rap 1b para hidrolizar fosfoinosítidos de membrana. La formación de este complejo trimérico se induce por trombina. La fosfoinositol-3-quinasa fosforila la posición 3 del anillo del inositol generando nuevos compuestos. La regulación completa de estos mecanismos de activación llevan a la respuesta hemostática plaquetaria. Su conocimiento hace posible el desarrollo de moléculas inhibitorias como terapéutica en los procesos trombóticos y tromboembólicos

Inhibition of carbachol-induced inositol phosphate accumulation in the embryonic retina promoted by kainate and veratridine

In the present study, we report that low concentrations of the glutamate ionotropic agonist kainate decreased the turnover of [3H]-phosphoinositides ([3H]-InsPs) induced by muscarinic receptors in the chick embryonic retina. When 100 muM carbachol was used, the estimated IC50 value for kainate was 0.2 muM and the maximal inhibition of ~50 percent was obtained with 1 muM or higher concentrations of the glutamatergic agonist. Our data also show that veratridine, a neurotoxin that increases the permeability of voltage-sensitive sodium channels, had no effect on [3H]-InsPs levels of the embryonic retina. However, 50 muM veratridine, but not 50 mM KCl, inhibited ~65 percent of the retinal response to carbachol. While carbachol increased [3H]-InsPs levels from 241.2 + 38.0 to 2044.5 + 299.9 cpm/mg protein, retinal response decreased to 861.6 + 113.9 cpm/mg protein when tissues were incubated with carbachol plus veratridine. These results suggest that the accumulation of phosphoinositides induced by activation of muscarinic receptors can be inhibited by the influx of Na+ ions triggered by activation of kainate receptors or opening of voltage-sensitive sodium channels in the chick embryonic retina.

Importancia de alteracoes do ciclo intracelular do fosfoinositol na fisiopatologia do disturbio bipolar: estudos preliminares em modelos plaquetarios / Importance of intracellular cycle alterations of phosphatidylinositols in bipolar disorder physiopathology: blood platelet models: preliminary studies

O ciclo intracelular do fosfoinositol (PI) e uma via de segundos mensageiros que pode estar desregulada em pacientes com disturbio bipolar. Essa hipotese tem sido investigada em alguns laboratorios com o uso de modelos plaquetarios, celulas sanguineas ou tecidos cerebrais de autopsia. Recentemente novos dados tem sido publicados sugerindo um aumento dos niveis ou da atividade de alguns intermediarios desta via em pacientes com disturbio bipolar; relatos de aumento da atividade da proteina quinase C (PKC) em pacientes nao medicados na fase maniaca e aumento da liberacao de Ca++ intracelular, em conjunto com achados de aumento do conteudo de fosfoinositois na membrana, sao condizentes com a hipotese de uma hiperfuncao do ciclo intracelular do PI nesse disturbio...

Phosphoinositide signal transduction pathway during regeneration following partial hepatectomy.

Phosphorylation of endogenous phosphatidylinositol was transiently increased following partial hepatectomy but was suppressed during peak DNA synthesis. Formation of inositol trisphosphate was decreased while generation of diacylglycerol and its breakdown to phosphatidic acid was increased. In response to partial hepatectomy protein kinase C was activated due to translocation from cytosol to particulate fraction, but the membrane bound activity was decreased during regeneration. Alteration of certain parameters in the signal transduction pathway apparently facilitates cell proliferation.

Intracellular signalling: phosphatidylinositol lipid metabolism in cancer cells.

Evidence for heightened capacity for signal transduction in rat hepatoma as well as in human breast and ovarian carcinoma cells as reflected by coordinate increases in PI kinase and PIP kinase in the PI phosphorylation sequence leading to the production of second messengers IP3 and DAG is shown. The linkage of signal transduction enzymes with malignant growth is also seen as MDA-MB- 435 human breast carcinoma or ovarian OVCAR-5 cells express their proliferative capacity in tissue culture in the log phase. In both cases, quercetin inhibit cell proliferation with a decline in PI kinase activity and IP3 levels preceding the growth inhibition seen with quercetin. The elevated steady state activities of PI and PIP kinase indicate a metabolic up-regulation in signal transduction capacity of cancer cells which is down-regulated by quercetin. Since the gain in function manifested in the over-expressed capacity for signal transduction confers selective growth advantage to cancer cells, increased activities of PI and PIP kinases may be considered as sensitive targets for cancer chemotherapy. The potential of quercetin as an interceptor of intracellular signal transduction mechanisms needs to be explored.

Rol de las plaquetas en la hemostasia / The role of the platelets in hemostasis

Luego de la injuria del vaso sanguíneo, las plaquetas son activadas, cambian su forma discoide a esférica y se adhieren al endotelio expuesto por un proceso denominado adhesión. Este proceso involucra la interacción de un componente plasmático, el FvW, y una glicoproteína específica de membrana, la GPIb, sobre la superficie plaquetaria. La adhesión es seguida por el reclutamiento de plaquetas adicionales que se adhieren entre sí (proceso denominado agregación). Este proceso comprende, entre otros elementos, la unión del fibrinógeno a receptores plaquetarios específicos GP IIb y IIIa. La adhesión y agregación comprometen la interacción con las proteínas (FG y FvW) que están presentes en el plasma y en los gránulos alfa. Las plaquetas activadas liberan el contenido de sus gránulos, por un proceso llamado secreción. Esto libera sustancias como el ADP, que pueden causar activación adicional de plaquetas. La interacción de las plaquetas con sus agonistas produce una serie de fenómenos que preceden a respuestas como la agregación o secreción. Una de las respuestas plaquetarias más tempranas es la activación de la fosfolipasa C, llevando a la hidrólisis del fosfatidilinositol y a la generación de mensajeros moleculares como el IP3y el DG. El IP3 media el aumento de la concentración del calcio ionizado en la plaqueta, lo cual se considera un factor regulador en varias respuestas plaquetarias como la movilización mediada por fosfolipasa A2 de AA libre, desde los fosfolípidos unidos a la membrana y la fosforilación de la cadena liviana de miosina, que está involucrada en la secreción plaquetaria. El DG activa a la proteinquinasa C, la cual produce la fosforilación de una proteína de 47 kD. Esta se sabe que tiene un rol sinérgico con la movilización de calcio intracelular. Otra respuesta a la estimulación plaquetaria es la liberación de AA de los fosfolípidos de la membrana y su oxigenación a tromboxano A2 por las enzimas cicloxigenasa y tromboxano sintetasa. De este modo la activación plaquetaria termina en la formación y liberación de sustancias activantes (ejemplos: ADP y TXA2), los cuales producen un mecanismo de feed-back positivo, que amplifica el proceso de activación. El rol más importante de las plaquetas en la hemostasia es su contribución a la activación de la cascada de coagulación y los fenómenos que conducen a la generación de trombina. Varias reacciones enzimáticas de la coagulación ocurren sobre la superficie plaquetaria...

Is aging related to cell signalling alterations in amphibian oocytes?

O armazenamento de oócitos de sapo Bufo arenarum diminuiu sua habilidade para serem fertilizados "in vitro". A estimulaçäo com carbachol em "oócitos jovens" mostrou uma hidrólisis persistente de fosfatidilinositol 4,5 bifosfato (PIP2) näo foram hidrolizados até um nível significativo. Estes resultados e os menores níveis de 32P-fosfoinositideos achados nos "oócitos envelhecidos" num tempo zero de estimulaçäo, condordam com uma diminuiçäo nas atividades das quinases de fosfoinositideos e da fosfolipase C, como consequência de uma hidrólisis näo específica de fosfoinositidos que ocorreria durante o armazenamento

Signal transduction in host cells mediated by glycosylphosphatidylinositols of the parasitic protozoa, or why do the parasitic protozoa have so many GPI molecules?

Considerable circumstantial evidence indicates that glycosylphosphatidylinositol (GPI) molecules of mammalian origin are able to mediate signal transduction in lymphoid cells. For example, pertubation of GPI-anchored surface proteins, but not transmembrane forms of these molecules, can lead to the activation of T lymphocytes. GPIs appear also to be precursors of pharmacologically active phosphoinositol-glycans which mediate responses to hormones such as insulin, nerve growth factor and IL-2. Nonetheless, the biochemical mechanisms of signal transduction by GPIs remain obscure. We have shown that structurally defined GPIs of protozoal parasite origin are able to mediate signal transduction in host macrophages and lymphocytes, by substituting for the putative endogenous GPI-based signalling mechanisms of the host. Signalling by parasite GPIs appears to involve the activation of protein tyrosine kinase and protein C. Evidence from other sources indicates that structurally variant GPIs may provide anergic signals to down-regulate host cell function. These phenomena may represent mechanisms by which eukaryotic parasites regulate host cell function, and can explain a variety of pathological and immunological features of protozoal infections. Furthermore, protozoal GPIs may prove to be an informative model system for the analysis of GPI-mediated signal transduction in lymphocytes and macrophages

Early molecular signals induced by antibodies to GPI-anchored proteins

We have examined intracellular biochemical and metabolic changes induced by antibodies specific for glycosylphosphatidylinositol (GPI)-anchored cell surface molecules. In lymphoid cells the earliest detectable responses are phosphorylation of intracellular substrates. The GPI-linked target antigens are also rapidly redistributed into patches and caps on the cell surface and then internalised. Between two and five hours later, cytokine receptors are expressed. Later, cells become metabolically active and begin to proliferate and express endogenous cytokines, thus promoting autocrine growth. Very early events, such as kinase activity, are induced by antibody binding alone and are characteristic of the cell surface molecule recognised by antibodies. Thus, the initial events in the activation cascade are critical in selecting the metabolic route. Progression down the activation cascade requires further signals such as cross-linking antibodies, exogenous cytokines, phorbol esters, or accessory cells. Once in cycle, cells no longer display evidence of their original route of activation. Activated T lymphocytes acquire resitance to cleavage by GPI-specific phospholipase C, suggesting a possible feedback mechanism to limit cell proliferation

Characterisation of phosphoprotein complexes associated with rat thymocyte Thy-1

We have previously demonstrated the non-covalent association of the protein tyrosine kinases p56lck and p60fyn together with a number of substrates for phosphorylation with rat thymocyte Thy-1. Here we present evidence that one of these associated phosphoproteins, p85, is associated by disulphide bridging with another polypeptide, demonstrating that it is an integral membrane protein with an extracellular domain. We also show that phosphatidylinositol 3 kinase activity may be coprecipitated with Thy-1 in Brij 96 thymocyte lysates

A role of Ly-6A.2 in T lymphocyte activation and development

Ly-6A.2 is a glycosylphosphatidylinositol (GPI)-anchored protein expressed on the plasma membrane of T lymphocytes. The precise function of this molecule is not known. A role of Ly-6A.2 in T cell signaling is discussed

The fate of GPI-anchored molecules

Glycosylphosphatidylinositol (GPI)-anchored proteins comprise a diverse class of membrane molecules. They protect cells from complement-mediated lysis, control cell to cell adhesion, activate T cells, and play a role in the etiology of slow viral diseases. Despite their functional diversity, GPI-anchored proteins are all attached to the plasma membrane by a common glycolipid anchor. We will examine one aspect of GPI-anchor metabolism, namely, the processing of the molecule after it arrives at the plasma membrane. After biosynthesis and transport to the plasma membrane, the GPI-anchored protein can be endocytosed and degraded or cleaved and released. The enzymatic machinery controlling the catabolism of GPI-anchored molecules at the plasma membrane is likely to play a central role in regulating the cell surface expression of these molecules