RESUMEN
Decades of research have indicated that gap junction channels contribute to the propagation of apoptosis between neighboring cells. Inositol 1,4,5-trisphosphate (IP3) has been proposed as the responsible molecule conveying the apoptotic message, although conclusive results are still missing. We investigated the role of IP3 in a model of gap junction-mediated spreading of cytochrome C-induced apoptosis. We used targeted loading of high-molecular-weight agents interfering with the IP3 signaling cascade in the apoptosis trigger zone and cell death communication zone of C6-glioma cells heterologously expressing connexin (Cx)43 or Cx26. Blocking IP3 receptors or stimulating IP3 degradation both diminished the propagation of apoptosis. Apoptosis spread was also reduced in cells expressing mutant Cx26, which forms gap junctions with an impaired IP3 permeability. However, IP3 by itself was not able to induce cell death, but only potentiated cell death propagation when the apoptosis trigger was applied. We conclude that IP3 is a key necessary messenger for communicating apoptotic cell death via gap junctions, but needs to team up with other factors to become a fully pro-apoptotic messenger.
Asunto(s)
Apoptosis , Uniones Comunicantes/metabolismo , Inositol 1,4,5-Trifosfato/metabolismo , Animales , Comunicación Celular , Permeabilidad de la Membrana Celular , Conexina 26 , Conexina 43/metabolismo , Conexinas/genética , Conexinas/metabolismo , Citocromos c/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Ratas , Transducción de SeñalRESUMEN
SHIP-1 (SH2 (Src homology 2)-containing inositol 5'-phosphatase-1) functions as a negative regulator of immune responses by hydrolyzing phosphatidylinositol-3,4,5-triphosphate generated by phosphoinositide-3 (PI 3)-kinase activity. As a result, SHIP-1 deficiency in mice results in myeloproliferation and B-cell lymphoma. On the other hand, SHIP-1-deficient mice have a reduced T-cell population, but the underlying mechanisms are unknown. In this work, we hypothesized that SHIP-1 plays anti-apoptotic functions in T cells upon stimulation of the death receptor CD95/APO-1/Fas. Using primary T cells from SHIP-1(-/-) mice and T leukemic cell lines, we report that SHIP-1 is a potent inhibitor of CD95-induced death. We observed that a small fraction of the SHIP-1 pool is localized to the endoplasmic reticulum (ER), in which it promotes CD95 glycosylation. This post-translational modification requires an intact SH2 domain of SHIP-1, but is independent of its phosphatase activity. The glycosylated CD95 fails to oligomerize upon stimulation, resulting in impaired death-inducing signaling complex (DISC) formation and downstream apoptotic cascade. These results uncover an unanticipated inhibitory function for SHIP-1 and emphasize the role of glycosylation in the regulation of CD95 signaling in T cells. This work may also provide a new basis for therapeutic strategies using compounds inducing apoptosis through the CD95 pathway on SHIP-1-negative leukemic T cells.
Asunto(s)
Apoptosis , Linfoma de Células T/patología , Monoéster Fosfórico Hidrolasas/fisiología , Linfocitos T/patología , Receptor fas/antagonistas & inhibidores , Animales , Western Blotting , Células Cultivadas , Proteínas Adaptadoras de Señalización del Receptor del Dominio de Muerte/metabolismo , Retículo Endoplásmico , Citometría de Flujo , Glicosilación , Humanos , Inositol Polifosfato 5-Fosfatasas , Linfoma de Células T/metabolismo , Ratones , Ratones Noqueados , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatasas , Fosforilación , Procesamiento Proteico-Postraduccional , ARN Interferente Pequeño/farmacología , Transducción de Señal , Linfocitos T/metabolismo , Receptor fas/metabolismoRESUMEN
SHIP1 [SH2 (Src homology 2)-containing inositol phosphatase-1], an inositol 5-phosphatase expressed in haemopoietic cells, acts by hydrolysing the 5-phosphates from PtdIns(3,4,5)P(3) and Ins(1,3,4,5)P(4), thereby negatively regulating the PI3K (phosphoinositide 3-kinase) pathway. SHIP1 plays a major role in inhibiting proliferation of myeloid cells. As a result, SHIP1(-/-) mice have an increased number of neutrophils and monocytes/macrophages due to enhanced survival and proliferation of their progenitors. Although SHIP1 contributes to PtdIns(3,4,5)P(3) metabolism in T-lymphocytes, its exact role in this cell type is much less explored. Jurkat cells have recently emerged as an interesting tool to study SHIP1 function in T-cells because they do not express SHIP1 at the protein level, thereby allowing reintroduction experiments in a relatively easy-to-use system. Data obtained from SHIP1 reintroduction have revealed that SHIP1 not only acts as a negative player in T-cell lines proliferation, but also regulates critical pathways, such as NF-kappaB (nuclear factor kappaB) activation, and also appears to remarkably inhibit T-cell apoptosis. On the other hand, experiments using primary T-cells from SHIP1(-/-) mice have highlighted a new role for SHIP1 in regulatory T-cell development, but also emphasize that this protein is not required for T-cell proliferation. In support of these results, SHIP1(-/-) mice are lymphopenic, suggesting that SHIP1 function in T-cells differs from its role in the myeloid lineage.
Asunto(s)
Monoéster Fosfórico Hidrolasas/fisiología , Linfocitos T/citología , Animales , Apoptosis , Muerte Celular , Línea Celular , Supervivencia Celular , Humanos , Inositol Polifosfato 5-Fosfatasas , Células Jurkat , Linfopenia/genética , Ratones , Ratones Noqueados , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatasas , Monoéster Fosfórico Hidrolasas/deficiencia , Monoéster Fosfórico Hidrolasas/genética , Dominios Homologos srcRESUMEN
Nuclear factor-kappa B (NF-kappaB) is an important prosurvival transcription factor activated in response to a large array of external stimuli, including reactive oxygen species (ROS). Previous works have shown that NF-kappaB activation by ROS involved tyrosine phosphorylation of the inhibitor IkappaBalpha through an IkappaB kinase (IKK)-independent mechanism. In the present work, we investigated with more details NF-kappaB redox regulation in human leukemic cells. By using different cell lines (CEM, Jurkat and the subclone Jurkat JR), we clearly showed that NF-kappaB activation by hydrogen peroxide (H2O2) is cell-type dependent: it activates NF-kappaB through tyrosine phosphorylation of IkappaBalpha in Jurkat cells, whereas it induces an IKK-mediated IkappaBalpha phosphorylation on S32 and 36 in CEM and Jurkat JR cells. We showed that this H2O2-induced IKK activation in CEM and Jurkat JR cells is mediated by SH2-containing inositol 5'-phosphatase 1 (SHIP-1), a lipid phosphatase that is absent in Jurkat cells. Indeed, the complementation of SHIP-1 in Jurkat cells made them shift to an IKK-dependent mechanism upon oxidative stress stimulation. We also showed that Jurkat cells expressing SHIP-1 are more resistant to H2O2-induced apoptosis than the parental cells, suggesting that SHIP-1 has an important role in leukemic cell responses to ROS in terms of signal transduction pathways and apoptosis resistance, which can be of interest in improving ROS-mediated chemotherapies.
Asunto(s)
Supervivencia Celular/efectos de los fármacos , Peróxido de Hidrógeno/farmacología , FN-kappa B/metabolismo , Estrés Oxidativo , Monoéster Fosfórico Hidrolasas/metabolismo , Apoptosis/efectos de los fármacos , Western Blotting , Citometría de Flujo , Prueba de Complementación Genética , Humanos , Quinasa I-kappa B/metabolismo , Proteínas I-kappa B/metabolismo , Inositol Polifosfato 5-Fosfatasas , Células Jurkat , Inhibidor NF-kappaB alfa , Oxidación-Reducción , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatasas , Monoéster Fosfórico Hidrolasas/genética , Fosforilación , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal , Factor de Necrosis Tumoral alfa/farmacología , Vanadatos/farmacologíaRESUMEN
Oncogenic mutations of the receptor tyrosine kinase KIT are encountered in myeloid leukemia and various solid tumors, including gastrointestinal stromal tumors. We previously identified the human oncogenic germ line mutant KIT(K642E), a substitution in the tyrosine kinase 1 domain (TK1D) in a familial form of gastrointestinal stromal tumors. The effects of oncogenic KIT mutants on cell signaling and regulation are complex. Cellular models are valuable basic tools to tailor novel strategies on specific cellular and molecular bases for tumors expressing KIT oncogenic mutants. Murine KIT(WT) and the murine homologues of human KIT oncogenic mutants, further referred to as KIT(K641E) and KIT(del559), a point deletion in the juxtamembrane domain (JMD), were stably expressed in IL-3-dependent Ba/F3 cells. Major differences in the constitutively activation of Akt/PKB, MAP kinases and STATs pathways were observed between KIT(K641E) and KIT(del559), whereas KIT ligand elicited responses in both mutants. Noteworthy, the protein level of the phosphoinositide phosphatase SHIP1, but not SHIP2 and PTEN, was reduced in KIT(K641E) only while inhibition of KIT phosphorylation reversibly raised SHIP1 level in both JMD and TK1D oncogenic mutants, unraveling the control of SHIP protein level by KIT phosphorylation.
Asunto(s)
Monoéster Fosfórico Hidrolasas/metabolismo , Proteínas Proto-Oncogénicas c-kit/metabolismo , Transducción de Señal/fisiología , Animales , Línea Celular , Humanos , Inositol Polifosfato 5-Fosfatasas , Ratones , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatasas , Monoéster Fosfórico Hidrolasas/genética , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Proto-Oncogénicas c-kit/genéticaRESUMEN
Receptor-mediated production of inositol 1,4,5-trisphosphate (InsP(3)) initiates Ca(2+) release and is responsible for cytosolic Ca(2+) oscillations. InsP(3) oscillations have also been observed in some cells. One of the enzymes controlling InsP(3) catabolism, the InsP(3) 3-kinase, is stimulated by Ca(2+); this regulation is presumably part of the reason for InsP(3) oscillations that have been observed in some cells. Here, we investigate the possible role of Ca(2+)-activated InsP(3) catabolism on the characteristics of the InsP(3)-induced Ca(2+) oscillations. Numerical simulations show that if it is assumed that the Ca(2+)-independent InsP(3) catabolism is predominant, Ca(2+) oscillations remain qualitatively unchanged although the relative amplitude of the oscillations in InsP(3) concentrations becomes minimal. We tested this prediction in hepatocytes by masking the Ca(2+)-dependent InsP(3) catabolism by 3-kinase through the injection of massive amounts of InsP(3) 5-phosphatase, which is not stimulated by Ca(2+). We find that in such injected hepatocytes, Ca(2+) oscillations generated by modest agonist levels are suppressed, presumably because of the decreased dose in InsP(3), but that at higher doses of agonist, oscillations reappear, with characteristics similar to those of untreated cells at low agonist doses. Altogether, these results suggest that oscillations in InsP(3) concentration due to Ca(2+)-stimulated InsP(3) catabolism do not play a major role for the oscillations in Ca(2+) concentration.
Asunto(s)
Hepatocitos/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Animales , Calcio/metabolismo , Señalización del Calcio , Células Cultivadas , Femenino , Hepatocitos/efectos de los fármacos , Inositol 1,4,5-Trifosfato/metabolismo , Inositol Polifosfato 5-Fosfatasas , Modelos Biológicos , Norepinefrina/farmacología , Monoéster Fosfórico Hidrolasas/farmacología , Ratas , Ratas WistarRESUMEN
The germinative ventricular zone of embryonic brain contains neural lineage progenitor cells that give rise to neurons, astrocytes and oligodendrocytes. The ability to generate neurons persists at adulthood in restricted brain areas. During development, many growth factors exert their effects by interacting with tyrosine kinase receptors and activate the phosphatidylinositol 3-kinase and the Ras/MAP kinase pathways. By its ability to modulate these pathways, the recently identified Src homology 2 domain-containing inositol polyphosphate 5-phosphatase 2, SHIP2, has the potential to regulate neuronal development. Using in situ hybridization technique with multiple synthetic oligonucleotides, we demonstrated that SHIP2 mRNA was highly expressed in the ventricular zone at early embryonic stages and subventricular zones at latter stages of brain and spinal cord and in the sympathetic chain. No significant expression was seen in differentiated fields. This restricted expression was maintained from embryonic day 11.5 to birth. In the periphery, large expression was detected in muscle and kidney and moderate expression in thyroid, pituitary gland, digestive system and bone. In the adult brain, SHIP2 was mainly restricted in structures containing neural stem cells such as the anterior subventricular zone, the rostral migratory stream and the olfactory tubercle. SHIP2 was also detected in the choroid plexuses and the granular layer of the cerebellum. The specificity of SHIP2 expression in neural stem cells was further demonstrated by (i) the dramatic increase in SHIP2 mRNA signal in neural cell adhesion molecule (N-CAM)-deficient mice, which present an accumulation of progenitor cells in the anterior subventricular zone and the rostral migratory stream, (ii) the abundant expression of 160-kDa SHIP2 by western blotting in proliferating neurospheres in culture and its downregulation in non-proliferating differentiated neurospheres. In conclusion, the close correlation between the pattern of SHIP2 expression in the brain and the proliferative and early differentiative events suggests that the phosphatase SHIP2 may have important roles in neural development.
Asunto(s)
Envejecimiento/metabolismo , Encéfalo/embriología , Encéfalo/metabolismo , Monoéster Fosfórico Hidrolasas/metabolismo , Animales , Células Cultivadas , Técnicas Citológicas , Femenino , Feto/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Sondas Moleculares , Moléculas de Adhesión de Célula Nerviosa/deficiencia , Neuronas/metabolismo , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatasas , Monoéster Fosfórico Hidrolasas/genética , ARN Mensajero/metabolismo , Distribución TisularRESUMEN
Glycogenolytic agonists induce coordinated Ca(2+) oscillations in multicellular rat hepatocyte systems as well as in the intact liver. The coordination of intercellular Ca(2+) signals requires functional gap-junction coupling. The mechanisms ensuring this coordination are not precisely known. We investigated possible roles of Ca(2+) or inositol 1,4,5-trisphosphate (InsP(3)) as a coordinating messengers for Ca(2+) spiking among connected hepatocytes. Application of ionomycin or of supra-maximal concentrations of agonists show that Ca(2+) does not significantly diffuse between connected hepatocytes, although gap junctions ensure the passage of small signaling molecules, as demonstrated by FRAP experiments. By contrast, coordination of Ca(2+) spiking among connected hepatocytes can be favored by a rise in the level of InsP(3), via the increase of agonist concentrations, or by a shift in the affinity of InsP(3) receptor for InsP(3). In the same line, coordination cannot be achieved if the InsP(3) is rapidly metabolized by InsP(3)-phosphatase in one cell of the multiplet. These results demonstrate that even if small amounts of Ca(2+) diffuse across gap junctions, they most probably do not play a significant role in inducing a coordinated Ca(2+) signal among connected hepatocytes. By contrast, coordination of Ca(2+) oscillations is fully dependent on the diffusion of InsP(3) between neighboring cells.
Asunto(s)
Señalización del Calcio , Calcio/metabolismo , Hepatocitos/metabolismo , Inositol 1,4,5-Trifosfato/metabolismo , Animales , Canales de Calcio/metabolismo , Señalización del Calcio/efectos de los fármacos , Permeabilidad de la Membrana Celular/efectos de los fármacos , Difusión/efectos de los fármacos , Conductividad Eléctrica , Fluorescencia , Fura-2/metabolismo , Uniones Comunicantes/efectos de los fármacos , Uniones Comunicantes/metabolismo , Hepatocitos/citología , Hepatocitos/efectos de los fármacos , Hepatocitos/enzimología , Receptores de Inositol 1,4,5-Trifosfato , Inositol Polifosfato 5-Fosfatasas , Ionomicina/farmacología , Hígado/citología , Microscopía Confocal , Monoéster Fosfórico Hidrolasas/metabolismo , Ratas , Receptores Citoplasmáticos y Nucleares/metabolismoRESUMEN
D-myo-inositol 1,4,5-trisphosphate (Ins(1,4,5)P(3)) and D-myo-inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P(4)) are both substrates of the 43-kDa type I inositol polyphosphate 5-phosphatase. Transient and okadaic acid-sensitive inhibition by 70-85% of Ins(1,4,5)P(3) and Ins(1,3,4,5)P(4) 5-phosphatase activities was observed in homogenates from rat cortical astrocytes, human astrocytoma 1321N1 cells, and rat basophilic leukemia RBL-2H3 cells after incubation with carbachol. The effect was reproduced in response to UTP in rat astrocytic cells and Chinese hamster ovary cells overexpressing human type I 5-phosphatase. Immunodetection as well as mass spectrometric peptide mass fingerprinting and post-source decay (PSD) sequence data analysis after immunoprecipitation permitted unambiguous identification of the major native 5-phosphatase isoform hydrolyzing Ins(1,4,5)P(3) and Ins(1,3,4,5)P(4) as type I inositol polyphosphate 5-phosphatase. In ortho-(32)P-preincubated cells, the phosphorylated 43 kDa-enzyme could be identified after receptor activation by immunoprecipitation followed by electrophoretic separation. Phosphorylation of type I 5-phosphatase was blocked after cell preincubation in the presence of Ca(2+)/calmodulin kinase II inhibitors (i.e. KN-93 and KN-62). In vitro phosphorylation of recombinant type I enzyme by Ca(2+)/calmodulin kinase II resulted in an inhibition (i.e. 60-80%) of 5-phosphatase activity. In this study, we demonstrated for the first time a novel regulation mechanism of type I 5-phosphatase by phosphorylation in intact cells.
Asunto(s)
Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Monoéster Fosfórico Hidrolasas/metabolismo , Animales , Células CHO , Calcio/agonistas , Células Cultivadas , Corteza Cerebral/metabolismo , Cricetinae , ADN Complementario/metabolismo , Relación Dosis-Respuesta a Droga , Electroforesis en Gel de Poliacrilamida , Humanos , Inositol Polifosfato 5-Fosfatasas , Espectrometría de Masas , Ácido Ocadaico/farmacología , Péptidos/química , Fosforilación , Pruebas de Precipitina , Unión Proteica , Ratas , Proteínas Recombinantes/metabolismo , Transducción de Señal , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Factores de Tiempo , Células Tumorales Cultivadas , Uridina Trifosfato/farmacología , Dominios Homologos srcRESUMEN
The consequences of the rapid 3-phosphorylation of inositol 1,4,5-trisphosphate (IP(3)) to produce inositol 1,3,4,5-tetrakisphosphate (IP(4)) via the action of IP(3) 3-kinases involve the control of calcium signals. Using green fluorescent protein constructs of full-length and truncated IP(3) 3-kinase isoform A expressed in HeLa cells, COS-7 cells, and primary neuronal cultures, we have defined a novel N-terminal 66-amino acid F-actin-binding region that localizes the kinase to dendritic spines. The region is necessary and sufficient for binding F-actin and consists of a proline-rich stretch followed by a predicted alpha-helix. We also localized endogenous IP(3) 3-kinase A to the dendritic spines of pyramidal neurons in primary hippocampal cultures, where it is co-localized postsynaptically with calcium/calmodulin-dependent protein kinase II. Our experiments suggest a link between inositol phosphate metabolism, calcium signaling, and the actin cytoskeleton in dendritic spines. The phosphorylation of IP(3) in dendritic spines to produce IP(4) is likely to be important for modulating the compartmentalization of calcium at synapses.
Asunto(s)
Actinas/metabolismo , Neuronas/enzimología , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Animales , Células COS , Señalización del Calcio/fisiología , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Extractos Celulares , Citoesqueleto/metabolismo , Detergentes/química , Proteínas Fluorescentes Verdes , Células HeLa , Hipocampo/enzimología , Hipocampo/metabolismo , Humanos , Proteínas Luminiscentes/metabolismo , Neuronas/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/química , Pruebas de Precipitina , Isoformas de Proteínas/metabolismo , Estructura Terciaria de Proteína , Ratas , Sinapsis/metabolismo , TransfecciónRESUMEN
The lipid phosphatase SHIP2 (SH2 domain containing inositol 5-phosphatase 2) has recently been shown to be a potent negative regulator of insulin signaling and insulin sensitivity in vivo. We show here that SHIP2 is expressed in Chinese hamster ovary cells overexpressing the insulin receptor (CHO-IR cells) and tyrosine phosphorylated upon insulin stimulation. We show that SHIP2, which is recruited in anti-phosphotyrosine immunoprecipitates in insulin-stimulated cells, accounts for the insulin sensitivity or apparent increase in activity reported by Guilherme et al. (J. Biol. Chem. 271, 29533-29536, 1996). Overexpression of SHIP2 led to a decrease of the insulin-dependent PIP3 production as well as Akt/PKB activation and MAPK stimulation.
Asunto(s)
Insulina/farmacología , Fosfatos de Fosfatidilinositol/metabolismo , Monoéster Fosfórico Hidrolasas/química , Monoéster Fosfórico Hidrolasas/metabolismo , Proteínas Serina-Treonina Quinasas , Receptor de Insulina/metabolismo , Animales , Células CHO , Cricetinae , Expresión Génica , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatasas , Monoéster Fosfórico Hidrolasas/genética , Fosforilación , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-akt , Receptor de Insulina/genética , Transfección , Tirosina/metabolismo , Dominios Homologos srcRESUMEN
Insulin is the primary hormone involved in glucose homeostasis, and impairment of insulin action and/or secretion has a critical role in the pathogenesis of diabetes mellitus. Type-II SH2-domain-containing inositol 5-phosphatase, or 'SHIP2', is a member of the inositol polyphosphate 5-phosphatase family. In vitro studies have shown that SHIP2, in response to stimulation by numerous growth factors and insulin, is closely linked to signalling events mediated by both phosphoinositide-3-OH kinase and Ras/mitogen-activated protein kinase. Here we report the generation of mice lacking the SHIP2 gene. Loss of SHIP2 leads to increased sensitivity to insulin, which is characterized by severe neonatal hypoglycaemia, deregulated expression of the genes involved in gluconeogenesis, and perinatal death. Adult mice that are heterozygous for the SHIP2 mutation have increased glucose tolerance and insulin sensitivity associated with an increased recruitment of the GLUT4 glucose transporter and increased glycogen synthesis in skeletal muscles. Our results show that SHIP2 is a potent negative regulator of insulin signalling and insulin sensitivity in vivo.
Asunto(s)
Insulina/fisiología , Monoéster Fosfórico Hidrolasas/fisiología , Animales , Animales Recién Nacidos , Glucemia/metabolismo , Femenino , Eliminación de Gen , Marcación de Gen , Hipoglucemia/etiología , Hipoglucemia/genética , Insulina/sangre , Hígado/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Músculo Esquelético/metabolismo , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatasas , Fosfoenolpiruvato Carboxiquinasa (GTP)/metabolismo , Monoéster Fosfórico Hidrolasas/genética , Transducción de Señal , Células MadreRESUMEN
The lipid phosphatase SHIP2 (Src homology 2 domain containing inositol 5-phosphatase 2) has been shown to be expressed in nonhemopoietic and hemopoietic cells. It has been implicated in signaling events initiated by several extracellular signals, such as epidermal growth factor (EGF) and insulin. In COS-7 cells, SHIP2 was tyrosine-phosphorylated at least at two separated tyrosine phosphorylation sites in response to EGF. SHIP2 was coimmunoprecipitated with the EGF receptor (EGFR) and also with the adaptor protein Shc. A C-terminal truncated form of SHIP2 that lacks the 366 last amino acids, referred to as tSHIP2, was also precipitated with the EGFR when transfected in COS-7 cells. The Src homology 2 domain of SHIP2 was unable to precipitate the EGFR in EGF-stimulated cells. Moreover, when transfected in COS-7 cells, it could not be detected in immunoprecipitates of the EGFR. When the His-tagged full-length enzyme was expressed in COS-7 cells and stained with anti-His6 monoclonal antibody, a signal was observed at plasma membranes in EGF-stimulated cells that colocalize with the EGFR by double staining. Upon stimulation by EGF, phosphatidylinositol 3,4,5-trisphosphate and protein kinase B activity were decreased in SHIP2-transfected COS-7 cells as compared with the vector alone. SHIP2 appears therefore in a tyrosine-phosphorylated complex with at least two other proteins, the EGFR and Shc.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Proteínas Adaptadoras del Transporte Vesicular , Factor de Crecimiento Epidérmico/metabolismo , Receptores ErbB/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Monoéster Fosfórico Hidrolasas/biosíntesis , Proteínas Serina-Treonina Quinasas , Animales , Western Blotting , Células COS , Cromatografía de Afinidad , Clonación Molecular , Relación Dosis-Respuesta a Droga , Eliminación de Gen , Vectores Genéticos , Histidina/química , Microscopía Confocal , Microscopía Fluorescente , Mutagénesis Sitio-Dirigida , Mutación , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatasas , Fosforilación , Pruebas de Precipitina , Unión Proteica , Estructura Terciaria de Proteína , Proteínas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-akt , Proteínas Adaptadoras de la Señalización Shc , Transfección , Tirosina/metabolismoRESUMEN
Arg82, a nuclear regulator of diverse cellular processes in yeast, is an inositol polyphosphate kinase. Some defects such as the regulation of arginine metabolism observed in an arg82Delta, result from a lack of Mcm1 and Arg80 stability. We show here that neither the kinase activity of Arg82 nor inositol phosphates are required for the control of arginine metabolism. Arg82 mutations keeping kinase active affect the expression of arginine genes, whereas mutations in the kinase domain do not impair this metabolic control.
Asunto(s)
Arginina/metabolismo , Proteínas Fúngicas/metabolismo , Fosfatos de Inositol/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Sitios de Unión/genética , División Celular/genética , Proteínas Fúngicas/genética , Eliminación de Gen , Mutagénesis Sitio-Dirigida , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Mutación Puntual , Fosfolipasas de Tipo C/deficiencia , Fosfolipasas de Tipo C/genética , Fosfolipasas de Tipo C/metabolismoRESUMEN
Inositol 1,4,5-trisphosphate [Ins(1,4,5)P(3)] 3-kinase catalyses the phosphorylation of Ins(1,4,5)P(3) to Ins(1,3,4,5)P(4). cDNAs encoding two isoenzymes of Ins(1,4,5)P(3) 3-kinase (3-kinases A and B) have been described previously. In the present study, we report the cloning of a full-length 2052 bp cDNA encoding a third human isoenzyme of the Ins(1,4,5)P(3) 3-kinase family, referred to as isoform C. This novel enzyme has a calculated molecular mass of 75. 207 kDa and a K(m) for Ins(1,4,5)P(3) of 6 microM. Northern-blot analysis showed the presence of a transcript of approx. 3.9 kb in various human tissues. Inositol trisphosphate 3-kinase C demonstrates enzymic activity when expressed in DH5alphaF' bacteria or COS-7 cells. Calcium alone decreases the Ins(1,4,5)P(3) 3-kinase activity of the 3-kinase C isoenzyme in transfected COS-7 cells. This inhibitory effect is reversed in the presence of calmodulin. The recombinant bacterial 3-kinase C can be adsorbed on calmodulin-Sepharose in the presence of calcium. The present data show that Ins(1,4,5)P(3) 3-kinase C: (i) shares a conserved catalytic domain of about 275 amino acids with the two other mammalian isoforms, (ii) could be purified on a calmodulin-Sepharose column and (iii) could be distinguished from the A and B isoenzymes by the effects of calcium and of calmodulin.
Asunto(s)
Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Células COS , Calcio/farmacología , Cromatografía Líquida de Alta Presión , Clonación Molecular , Cartilla de ADN , ADN Complementario , Humanos , Datos de Secuencia Molecular , Fosfotransferasas (Aceptor de Grupo Alcohol)/antagonistas & inhibidores , Fosfotransferasas (Aceptor de Grupo Alcohol)/química , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
p130 was originally identified as an Ins(1,4,5)P(3)-binding protein similar to phospholipase C-delta but lacking any phospholipase activity. In the present study we have further analysed the interactions of p130 with inositol compounds in vitro. To determine which of the potential ligands interacts with p130 in cells, we performed an analysis of the cellular localization of this protein, the isolation of a protein-ligand complex from cell lysates and studied the effects of p130 on Ins(1,4,5)P(3)-mediated Ca(2+) signalling by using permeabilized and transiently or stably transfected COS-1 cells (COS-1(p130)). In vitro, p130 bound Ins(1,4,5)P(3) with a higher affinity than that for phosphoinositides. When the protein was isolated from COS-1(p130) cells by immunoprecipitation, it was found to be associated with Ins(1,4,5)P(3). Localization studies demonstrated the presence of the full-length p130 in the cytoplasm of living cells, not at the plasma membrane. In cell-based assays, p130 had an inhibitory effect on Ca(2+) signalling. When fura-2-loaded COS-1(p130) cells were stimulated with bradykinin, epidermal growth factor or ATP, it was found that the agonist-induced increase in free Ca(2+) concentration, observed in control cells, was inhibited in COS-1(p130). This inhibition was not accompanied by the decreased production of Ins(1,4,5)P(3); the intact p130 pleckstrin homology domain, known to be the ligand-binding site in vitro, was required for this effect in cells. These results suggest that Ins(1,4,5)P(3) could be the main p130 ligand in cells and that this binding has the potential to inhibit Ins(1,4,5)P(3)-mediated Ca(2+) signalling.
Asunto(s)
Calcio/metabolismo , Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Fosfolipasas de Tipo C/metabolismo , Células 3T3 , Adenosina Trifosfato/farmacología , Animales , Proteínas Sanguíneas/química , Bradiquinina/farmacología , Células COS , Canales de Calcio/química , Catálisis , Línea Celular , Membrana Celular/metabolismo , Citoplasma/metabolismo , Perros , Relación Dosis-Respuesta a Droga , Factor de Crecimiento Epidérmico/farmacología , Proteínas Fluorescentes Verdes , Inositol 1,4,5-Trifosfato/metabolismo , Receptores de Inositol 1,4,5-Trifosfato , Ligandos , Liposomas/metabolismo , Proteínas Luminiscentes/metabolismo , Ratones , Microscopía Fluorescente , Fosfoproteínas/química , Plásmidos/metabolismo , Pruebas de Precipitina , Unión Proteica , Estructura Terciaria de Proteína , Receptores Citoplasmáticos y Nucleares/química , Proteínas Recombinantes de Fusión/metabolismo , Transducción de Señal , Factores de Tiempo , TransfecciónRESUMEN
Fc gammaRIIB are single-chain low-affinity receptors for IgG that bear an immunoreceptor tyrosine-based inhibition motif (ITIM) in their intracytoplasmic domain and that negatively regulate immunoreceptor tyrosine-based activation motif (ITAM)-dependent cell activation. In B cells, coaggregation of the B cell receptor (BCR) and Fc gammaRIIB leads to an inhibition of B cell activation. Inhibitory properties of Fc gammaRIIB have been related to the recruitment of SHIP, an SH2 domain-containing inositol 5-phosphatase (referred to as SHIP1), via ITIM phosphorylated Fc gammaRIIB. Here, we demonstrate that the second SH2 domain-containing inositol 5-phosphatase SHIP2 could also bind to the Fc gammaRIIB ITIM. As a model, a Fc gammaRIIB deficient B cell line (IIA1.6), transfected with a cDNA encoding either w.t. Fc gammaRIIB1' or Fc gammaRIIB1' whose ITIM tyrosine was mutated has been used. SHIP2 tyrosine phosphorylation and association to the adaptator protein Shc were only found in transfectants expressing w.t. Fc gammaRIIB1'. SHIP2 was also found to bind to a phosphopeptide corresponding to the ITIM sequence of Fc gammaRIIB. There was no binding to the nonphosphorylated peptide. Finally, both SHIP2 and SHIP1 were coprecipitated with Fc gammaRIIB1' upon coaggregation with BCR in IIA1.6 transfectants.
Asunto(s)
Antígenos CD/metabolismo , Linfocitos B/inmunología , Monoéster Fosfórico Hidrolasas/metabolismo , Receptores de IgG/metabolismo , Transducción de Señal , Dominios Homologos src , Secuencias de Aminoácidos , Animales , Antígenos CD/química , Antígenos CD/genética , Linfocitos B/metabolismo , Activación de Linfocitos , Ratones , Mutación , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatasas , Fosforilación , Fosfotirosina/metabolismo , Pruebas de Precipitina , Receptores de Antígenos de Linfocitos B/química , Receptores de Antígenos de Linfocitos B/metabolismo , Receptores de IgG/química , Receptores de IgG/genética , Transfección , Células Tumorales Cultivadas , Dominios Homologos src/inmunologíaRESUMEN
SH2-containing inositol-5-phosphatase 1 (SHIP1) was originally identified as a 145 kDa protein that became tyrosine-phosphorylated in response to multiple cytokines. It is now well established that SHIP1 is specifically expressed in haemopoietic cells and is important as a negative regulator of signalling. We found recently that SHIP1 was present in human blood platelets as an Ins(1,3,4, 5)P(4)-phosphatase and a PtdIns(3,4,5)P(3)-5-phosphatase that became tyrosine-phosphorylated and was relocated to the cytoskeleton in an integrin-dependent manner. Here we report biochemical and pharmacological evidence that the tyrosine kinase pp60(c-src) is constitutively associated with SHIP1 and is involved in its tyrosine phosphorylation downstream of integrin engagement in thrombin-activated human platelets. The use of cytochalasin D allowed us to demonstrate that the actin cytoskeleton reorganization induced on thrombin stimulation was not required for its integrin-mediated phosphorylation. Moreover, the integrin-dependent relocation of SHIP1 to the cytoskeleton did not require its tyrosine phosphorylation. These results suggest that SHIP1 is first recruited to the integrin-linked signalling complexes and then becomes tyrosine-phosphorylated through a Src-kinase-dependent mechanism but independently of the actin cytoskeleton reorganization.
Asunto(s)
Plaquetas/metabolismo , Monoéster Fosfórico Hidrolasas/metabolismo , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/metabolismo , Proteínas Proto-Oncogénicas pp60(c-src)/metabolismo , Dominios Homologos src/fisiología , Actinas/metabolismo , Transporte Biológico , Citoesqueleto , Humanos , Técnicas In Vitro , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatasas , Fosforilación/efectos de los fármacos , Proteínas Proto-Oncogénicas pp60(c-src)/antagonistas & inhibidores , Trombina/metabolismo , Tirosina/metabolismoRESUMEN
The activation of many hematopoietic cells via cytokine receptors, as well as B and T cell receptors, leads to the tyrosine phosphorylation of Shc and its association with both Grb2-Sos1 complexes and with a 145 kDa protein referred to as the SH2 containing inositol 5-phosphatase (SHIP1). In a search of putative 5-phosphatase isoenzymes, we have isolated a second SH2 domain containing inositol 5-phosphatase, referred to as (SHIP2). Both SHIP1 and SHIP2 are coexpressed in human T lymphocytes. This was shown at the protein level by Western blot analysis in transformed T cell lines and in peripheral blood T lymphocytes either unstimulated or after in vitro activation through TCR-CD3 complex. SHIP1 protein level was not modulated after activation of T lymphocytes, in contrast to SHIP2, which was increased after long-term stimulation. SHIP1 was tyrosine phosphorylated in resting naive T cells. This was not observed in the transformed T cell lines. T lymphocyte is therefore a model of coexpression of the two SH2-containing inositol 5-phosphatases SHIP1 and SHIP2.