RESUMEN
New coatings are obtained when graphene oxide is further oxidized at moderate anodic potentials (≤~1.3 V vs. Ag/AgCl). Based on a variety of spectroscopic and electrochemical observations, the coatings are attributed to the direct electropolymerization of graphene oxide sheets via oxidation of the phenol edge groups on graphene. Depending on the applied potential, ether or carboxylic groups are formed. The coatings obtained via further oxidation are characterized by a lower O/C ratio due to decarboxylation and a higher content of C=C bonds. These bonds extend aromatic conjugation into the combined graphene oxide sheets and are responsible for the highly conductive nature of these coatings.
RESUMEN
BACKGROUND AND PURPOSE: The HIV-envelope glycoprotein Gp120 is involved in neuronal injury and is associated with neuro-AIDS pathogenesis in the brain. Endocannabinoids are important lipid ligands in the CNS regulating neural functions, and their degeneration is controlled by hydrolysing enzymes such as the fatty acid amide hydrolase (FAAH). Here, we examined whether in vivo genetic deletion of Faah gene prevents HIV-1 Gp120-mediated effects on neurogenesis. EXPERIMENTAL APPROACH: We generated new GFAP/Gp120 transgenic (Tg) mice that have genetic deletion of Faah gene by mating glial fribillary acidic protein (GFAP)/Gp120 Tg mice with Faah-/- mice. Neurogenesis and cell death were assessed by immunocytochemical analysis. KEY RESULTS: Endocannabinoid levels in the brain of the double GFAP/Gp120//Faah-/- mice were similar to those observed in Faah-/- mice. However, unlike the impaired neurogenesis observed in GFAP/Gp120 Tg mice and Faah-/- mice, these GFAP/Gp120//Faah-/ mice showed significantly improved neurogenesis in the hippocampus, indicated by a significant increase in neuroblasts and neuronal cells, an increase in BrdU(+) cells and doublecortin positive cells (DCX(+) ), and an increase in the number of PCNA. Furthermore, a significant decrease in astrogliosis and gliogenesis was observed in GFAP/Gp120//Faah-/-mice and neurogenesis was stimulated by neural progenitor cells (NPCs) and/or the newly formed NPC niches characterized by increased COX-2 expression and elevated levels of PGE2 . CONCLUSIONS AND IMPLICATIONS: In vivo genetic ablation of Faah, resulted in enhanced neurogenesis through modulation of the newly generated NPC niches in GFAP/Gp120//Faah-/- mice. This suggests a novel approach of using FAAH inhibitors to enhance neurogenesis in HIV-1 infected brain.
Asunto(s)
Amidohidrolasas/genética , Proteína gp120 de Envoltorio del VIH/genética , Neurogénesis/fisiología , Animales , Encéfalo/metabolismo , Ciclooxigenasa 2/metabolismo , Dinoprostona/metabolismo , Proteína Doblecortina , Endocannabinoides/metabolismo , Eliminación de Gen , Proteína Ácida Fibrilar de la Glía/genética , Ratones Transgénicos , Neurogénesis/genéticaRESUMEN
BACKGROUND AND PURPOSE: Endocannabinoids such as anandamide (AEA) are important lipid ligands regulating cell proliferation, differentiation and apoptosis. Their levels are regulated by hydrolase enzymes, the fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MGL). Here, we investigated whether FAAH or AEA are involved in NF (erythroid-derived 2)-like 2 (Nrf2)/antioxidant responsive element (ARE) pathway. EXPERIMENTAL APPROACH: The aim of this study was to analyse the effects of AEA or FAAH inhibition by the URB597 inhibitor or FAAH/siRNA on the activation of Nrf2-ARE signalling pathway and heme oxygenase-1 (HO-1) induction and transcription. KEY RESULTS: Endogenous AEA was detected in the immortalized human mammary epithelial MCF-10A cells (0.034 ng per 10(6) cells) but not in MCF-7 or MDA-MB-231 breast cancer cells. Because breast tumour cells express FAAH abundantly, we examined the effects of FAAH on Nrf2/antioxidant pathway. We found that inhibition of FAAH by the URB597 inhibitor induced antioxidant HO-1 in breast cancer cells and MCF-10A cells. RNAi-mediated knockdown of FAAH or treatment with AEA-activated ARE-containing reporter induced HO-1 mRNA and protein expression, independent of the cannabinoid receptors, CB1, CB2 or TRPV1. Furthermore, URB597, AEA and siRNA-FAAH treatments induced the nuclear translocation of Nrf2, while siRNA-Nrf2 treatment and Keap1 expression blocked AEA, URB597 and si-FAAH from activation of ARE reporter and HO-1 induction. siRNA-HO-1 treatment decreased the viability of breast cancer cells and MCF-10A cells. CONCLUSIONS AND IMPLICATIONS: These data uncovered a novel mechanism by which inhibition of FAAH or exposure to AEA induced HO-1 transcripts and implicating AEA and FAAH as direct modifiers in signalling mediated activation of Nrf2-HO-1 pathway, independent of cannabinoid receptors.
Asunto(s)
Amidohidrolasas/antagonistas & inhibidores , Antineoplásicos/farmacología , Benzamidas/farmacología , Neoplasias de la Mama/enzimología , Carbamatos/farmacología , Inhibidores Enzimáticos/farmacología , Hemo-Oxigenasa 1/metabolismo , Factor 2 Relacionado con NF-E2/metabolismo , Transducción de Señal/efectos de los fármacos , Transcripción Genética/efectos de los fármacos , Transporte Activo de Núcleo Celular , Amidohidrolasas/genética , Amidohidrolasas/metabolismo , Ácidos Araquidónicos/metabolismo , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Endocannabinoides/metabolismo , Inducción Enzimática , Femenino , Regulación Neoplásica de la Expresión Génica , Hemo-Oxigenasa 1/genética , Humanos , Células MCF-7 , Factor 2 Relacionado con NF-E2/genética , Alcamidas Poliinsaturadas/metabolismo , Interferencia de ARN , TransfecciónRESUMEN
Brain tumors are associated with genetic alterations of oncogenes and tumor suppressor genes. Accumulation of reactive oxygen species (ROS) in cells leads to oxidative stress-induced damage, resulting in tumorigenesis. Here, we showed that the nuclear matrix protein nuclear restricted protein in brain (NRP/B) was colocalized and interacted with NF-E2-related factor 2 (Nrf2). During oxidative stress response, NRP/B expression and its interaction with Nrf2 were upregulated in SH-SY5Y cells. Association of NRP/B with Nrf2 was crucial for NAD(P)H:quinone oxidoreductase 1 (NQO1) expression. NRP/B was localized predominantly in the nucleus of normal brain cells, whereas in primary brain tumors NRP/B was almost exclusively contained in the cytoplasm. In addition, unlike wild-type NRP/B, the expression of NRP/B mutants isolated from primary brain tumors was found in the cytoplasm, and these mutants failed to induce Nrf2-dependent NQO1 transcription. Thus, NRP/B mutations and their altered localization resulted in changes in NRP/B function and deregulation of Nrf2-dependent NQO1 activation in brain tumors. This study provides insights into the mechanism by which the NRP/B modulates Nrf2-dependent NQO1 induction in cellular protection against ROS in brain tumors.
Asunto(s)
Neoplasias Encefálicas/metabolismo , Proteínas de Microfilamentos/genética , Mutación/genética , NAD(P)H Deshidrogenasa (Quinona)/metabolismo , Factor 2 Relacionado con NF-E2/metabolismo , Neuropéptidos/genética , Proteínas Nucleares/genética , Animales , Neoplasias Encefálicas/patología , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Activación Enzimática , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Peróxido de Hidrógeno/farmacología , Inmunoprecipitación , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteína 1 Asociada A ECH Tipo Kelch , Ratones , Proteínas de Microfilamentos/inmunología , Proteínas de Microfilamentos/metabolismo , NAD(P)H Deshidrogenasa (Quinona)/genética , Neuropéptidos/inmunología , Neuropéptidos/metabolismo , Proteínas Nucleares/inmunología , Proteínas Nucleares/metabolismo , Oxidantes/farmacología , Estrés Oxidativo , Fosfoproteínas/metabolismo , Regiones Promotoras Genéticas , Transporte de ProteínasRESUMEN
The expression of VEGF and the relapse-free survival rate of breast cancer patients are inversely related. While VEGF induces the proliferation and migration of vascular endothelial cells, its function in breast cancer cells is not well studied. We reported previously that fibronectin increased VEGF-dependent migration in breast cancer cells. Since VEGF has an extracellular matrix (ECM)-binding domain and possesses binding affinity for heparin, we sought to determine the effects of VEGF in breast cancer cells and the role of heparin and/or fibronectin in VEGF-induced signaling. Cells grown on plastic were compared to those grown on fibronectin or to those grown on plastic in the presence of heparin, and analysed for intracellular signaling, proliferation and migration in response to VEGF(165). Both heparin and fibronectin enhanced the binding of VEGF to T47D cells. After treatment with VEGF, [(3)H]thymidine incorporation, c-fos induction, and the number of migrating cells were significantly higher ( approximately twofold) in cells grown on fibronectin or in cells grown on plastic in the presence of heparin when compared to those grown on plastic only. Likewise, tyrosine phosphorylation of VEGF receptors, MAPK activity and PI3-kinase activity were all several-fold higher in cells seeded on fibronectin or in the presence of heparin as compared to cells exposed to VEGF alone. VEGF-dependent c-fos induction was found to be regulated through a MAPK-dependent, but PI3-kinase-independent pathway. In contrast, the migration of T47D cells in response to VEGF, in the presence of ECM, was regulated through PI3-kinase. Therefore, VEGF requires ECM components to induce a mitogenic response and cell migration in T47D breast cancer cells.
Asunto(s)
Neoplasias de la Mama/patología , Factores de Crecimiento Endotelial/farmacología , Matriz Extracelular/fisiología , Fibronectinas/fisiología , Heparina/fisiología , Linfocinas/farmacología , Invasividad Neoplásica , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , División Celular , Movimiento Celular , ADN/biosíntesis , Factores de Crecimiento Endotelial/metabolismo , Femenino , Humanos , Linfocinas/metabolismo , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-fos/biosíntesis , Proteínas Proto-Oncogénicas c-fos/genética , ARN Neoplásico/biosíntesis , Transducción de Señal , Activación Transcripcional , Células Tumorales Cultivadas , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial VascularRESUMEN
In this report, we analyzed the expression and kinase activities of Csk and CHK kinases in normal breast tissues and breast tumors and their involvement in HRG-mediated signaling in breast cancer cells. Csk expression and kinase activity were abundant in normal human breast tissues, breast carcinomas, and breast cancer cell lines, whereas CHK expression was negative in normal breast tissues and low in some breast tumors and in the MCF-7 breast cancer cell line. CHK kinase activity was not detected in human breast carcinoma tissues (12 of 12) or in the MCF-7 breast cancer cell line (due to the low level of CHK protein expression), but was significantly induced upon heregulin (HRG) stimulation. We have previously shown that CHK associates with the ErbB-2/neu receptor upon HRG stimulation via its SH2 domain and that it down-regulates the ErbB-2/neu-activated Src kinases. Our new findings demonstrate that Csk has no effect on ErbB-2/neu-activated Src kinases upon HRG treatment and that its kinase activity is not modulated by HRG. CHK significantly inhibited in vitro cell growth, transformation, and invasion induced upon HRG stimulation. In addition, tumor growth of wt CHK-transfected MCF-7 cells was significantly inhibited in nude mice. Furthermore, CHK down-regulated c-Src and Lyn protein expression and kinase activity, and the entry into mitosis was delayed in the wt CHK-transfected MCF-7 cells upon HRG treatment. These results indicate that CHK, but not Csk, is involved in HRG-mediated signaling pathways, down-regulates ErbB-2/neu-activated Src kinases, and inhibits invasion and transformation of breast cancer cells upon HRG stimulation. These findings strongly suggest that CHK is a novel negative growth regulator of HRG-mediated ErbB-2/neu and Src family kinase signaling pathways in breast cancer cells.
Asunto(s)
Neoplasias de la Mama/enzimología , Proteínas Quinasas/química , Proteínas Tirosina Quinasas/química , Animales , Southern Blotting , Western Blotting , Proteína Tirosina Quinasa CSK , División Celular , Línea Celular , Transformación Celular Neoplásica , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , Regulación hacia Abajo , Humanos , Ratones , Ratones Desnudos , Mitosis , Invasividad Neoplásica , Fosforilación , Proteínas Quinasas/biosíntesis , Estructura Terciaria de Proteína , Proteínas Tirosina Quinasas/biosíntesis , Proteínas Tirosina Quinasas/metabolismo , Proteínas Recombinantes/metabolismo , Transducción de Señal , Factores de Tiempo , Transfección , Células Tumorales Cultivadas , Familia-src QuinasasRESUMEN
Platelet activation by different agonists initiates a signalling cascade involving the phosphorylation of several protein kinases, which control key regulatory events. Previously, we demonstrated that the related adhesion focal tyrosine kinase (RAFTK, Pyk2) was involved in an early phase of platelet activation, independent of integrin and glycoprotein IIb-IIIa activation. In this study, we demonstrate that RAFTK is co-immunoprecipitated with phosphoinositide 3-kinase (PI3K) upon platelet activation, and that thrombin, ADP and collagen induced the phosphorylation of both PI3K and RAFTK. A low dose of thrombin (0.015 U/ml) induced RAFTK phosphorylation and platelet aggregation in a PI3K activity-dependent manner, whereas a high dose of thrombin (0.1 U/ml) induced these events in a PI3K activity-independent manner. ADP and collagen also induced RAFTK phosphorylation and platelet aggregation in a PI3K activity-dependent manner, similar to that of the low-dose thrombin. Furthermore, protein tyrosine phosphatase activity was associated with RAFTK in response to platelet activation, and was found to be that of protein tyrosine phosphatase-2 (SHP-2). The association of SHP-2 with RAFTK was PI3K-dependent and was increased upon RAFTK phosphorylation. Taken together, our results strongly suggest that the involvement of RAFTK in platelet activation is mediated via the PI3K pathway.
Asunto(s)
Fosfatidilinositol 3-Quinasas/metabolismo , Activación Plaquetaria/fisiología , Proteínas Tirosina Quinasas/metabolismo , Transducción de Señal/fisiología , Adenosina Difosfato/farmacología , Cromonas/farmacología , Colágeno/farmacología , Inhibidores Enzimáticos/farmacología , Fibrinógeno/farmacología , Quinasa 2 de Adhesión Focal , Humanos , Péptidos y Proteínas de Señalización Intracelular , Morfolinas/farmacología , Inhibidores de las Quinasa Fosfoinosítidos-3 , Monoéster Fosfórico Hidrolasas/antagonistas & inhibidores , Fosforilación , Pruebas de Precipitina , Proteína Fosfatasa 2 , Proteína Tirosina Fosfatasa no Receptora Tipo 11 , Proteína Tirosina Fosfatasa no Receptora Tipo 6 , Proteínas Tirosina Fosfatasas/metabolismo , Trombina/farmacología , Vanadatos/farmacologíaRESUMEN
The expression of vascular endothelial growth factor (VEGF) by breast tumors has been previously correlated with a poor prognosis in the pathogenesis of breast cancer. Furthermore, VEGF secretion is a prerequisite for tumor development. Although most of the effects of VEGF have been shown to be attributable to the stimulation of endothelial cells, we present evidence here that breast tumor cells are capable of responding to VEGF. We show that VEGF stimulation of T-47D breast cancer cells leads to changes in cellular signaling and invasion. VEGF increases the cellular invasion of T-47D breast cancer cells on Matrigel/ fibronectin-coated transwell membranes by a factor of two. Northern analysis for the expression of the known VEGF receptors shows the presence of moderate levels of Flt-1 and low levels of Flk-1/KDR mRNAs in a variety of breast cancer cell lines. T-47D breast cancer cells bind 125I-labeled VEGF with a Kd of 13 x 10(-9) M. VEGF induces the activation of the extracellular regulated kinases 1,2 as well as activation of phosphatidylinositol 3'-kinase, Akt, and Forkhead receptor L1. These findings in T-47D breast cancer cells strongly suggest an autocrine role for VEGF contributing to the tumorigenic phenotype.
Asunto(s)
Neoplasias de la Mama/metabolismo , Comunicación Celular/fisiología , Factores de Crecimiento Endotelial/farmacología , Linfocinas/farmacología , Proteínas de la Membrana , Invasividad Neoplásica/fisiopatología , Neovascularización Patológica/inducido químicamente , Transducción de Señal/fisiología , Células Tumorales Cultivadas/efectos de los fármacos , Neoplasias de la Mama/patología , Neoplasias de la Mama/fisiopatología , Comunicación Celular/efectos de los fármacos , Colágeno/farmacología , Combinación de Medicamentos , Factores de Crecimiento Endotelial/metabolismo , Femenino , Fibronectinas/metabolismo , Fibronectinas/farmacología , Humanos , Inmunohistoquímica , Proteína 1 Similar al Receptor de Interleucina-1 , Laminina/farmacología , Linfocinas/metabolismo , Proteínas Quinasas Activadas por Mitógenos/efectos de los fármacos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Invasividad Neoplásica/patología , Neovascularización Patológica/patología , Neovascularización Patológica/fisiopatología , Neurregulina-1/metabolismo , Neurregulina-1/farmacología , Fosfatidilinositol 3-Quinasas/efectos de los fármacos , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación/efectos de los fármacos , Proteínas/efectos de los fármacos , Proteínas/genética , Proteínas/metabolismo , Proteoglicanos/farmacología , ARN Mensajero/efectos de los fármacos , ARN Mensajero/metabolismo , Ensayo de Unión Radioligante , Proteínas Tirosina Quinasas Receptoras/efectos de los fármacos , Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptores de Superficie Celular , Receptores de Factores de Crecimiento/efectos de los fármacos , Receptores de Factores de Crecimiento/metabolismo , Receptores de Factores de Crecimiento Endotelial Vascular , Transducción de Señal/efectos de los fármacos , Células Tumorales Cultivadas/metabolismo , Células Tumorales Cultivadas/patología , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial VascularRESUMEN
PTPs and PTKs control the level of tyrosine phosphorylation of cellular proteins. Although many substrates for PTKs have been identified, the specific targets of individual PTP family members, along with the consequences of protein dephosphorylation for cellular physiology, remain largely unknown. Fine regulation of tyrosine phosphorylation events is required for the proper progression of hematopoiesis. In this review, we have summarized the characterization of tyrosine phosphatases in hematopoietic cells and delineated their potential role in the process of hematopoiesis and the development of hematopoietic disorders.
Asunto(s)
Células Madre Hematopoyéticas/enzimología , Proteínas Tirosina Quinasas Receptoras/fisiología , Hematopoyesis , Células Madre Hematopoyéticas/fisiología , Humanos , Leucemia/enzimología , Leucemia/fisiopatologíaRESUMEN
Endothelium of the cerebral blood microvessels, which constitutes the major component of the blood-brain barrier, controls leukocyte and metastatic cancer cell adhesion and trafficking into the brain parenchyma. In this study, using rat primary brain microvascular endothelial cells (BMEC), we demonstrate that the vascular endothelial growth factor (VEGF), a potent promoter of angiogenesis, up-regulates the expression of the intracellular adhesion molecule-1 (ICAM-1) through a novel pathway that includes phosphatidylinositol 3 OH-kinase (PI3K), AKT, and nitric oxide (NO), resulting in the migration of BMEC. Upon VEGF treatment, AKT is phosphorylated in a PI3K-dependent manner. AKT activation leads to NO production and release and activation-deficient AKT attenuates NO production stimulated by VEGF. Transfection of the constitutive myr-AKT construct significantly increased basal NO release in BMEC. In these cells, VEGF and the endothelium-derived NO synergistically up-regulated the expression of ICAM-1, which was mediated by the PI3K pathway. This activity was blocked by the PI3K-specific inhibitor, wortmannin. Furthermore, VEGF and NO significantly increased BMEC migration, which was mediated by the up-regulation of ICAM-1 expression and was dependent on the integrity of the PI3K/AKT/NO pathway. This effect was abolished by wortmannin, by the specific ICAM-1 antibody, by the specific inhibitor of NO synthase, N(G)-l-monomethyl-arginine (l-NMMA) or by a combination of wortmannin, ICAM-1 antibody, and l-NMMA. These findings demonstrate that the angiogenic factor VEGF up-regulates ICAM-1 expression and signals to ICAM-1 as an effector molecule through the PI3K/AKT/NO pathway, which leads to brain microvessel endothelial cell migration. These observations may contribute to a better understanding of BMEC angiogenesis and the physiological as well as pathophysiological function of the blood-brain barrier, whose integrity is crucial for normal brain function.
Asunto(s)
Factores de Crecimiento Endotelial/farmacología , Molécula 1 de Adhesión Intercelular/genética , Linfocinas/farmacología , Androstadienos/farmacología , Animales , Encéfalo/metabolismo , Movimiento Celular/efectos de los fármacos , Células Cultivadas , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Molécula 1 de Adhesión Intercelular/metabolismo , Músculo Liso Vascular/metabolismo , Óxido Nítrico/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Ratas , Transducción de Señal , Transfección , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial Vascular , Wortmanina , omega-N-Metilarginina/farmacologíaRESUMEN
The related adhesion focal tyrosine kinase (RAFTK), a member of the focal adhesion kinase (FAK) family and highly expressed in brain, is a key mediator of various extracellular signals that elevate intracellular Ca(2+) concentration. We investigated RAFTK and FAK signaling upon nerve growth factor (NGF) stimulation of PC12 cells. NGF induced the tyrosine phosphorylation of RAFTK in a time- and dose-dependent manner, whereas no change in the tyrosine phosphorylation of FAK was observed. Chemical inhibition showed that RAFTK phosphorylation was inhibited by blocking phospholipase Cgamma activity or intracellular Ca(2+). Blocking of extracellular Ca(2+) or phosphatidylinositol 3-kinase activity partially reduced the phosphorylation of RAFTK. In addition, disruption of actin polymerization abolished RAFTK phosphorylation, indicating that an intact actin-based cytoskeletal organization is required for RAFTK phosphorylation. The focal adhesion molecule paxillin was co-immunoprecipitated with RAFTK, and its tyrosine phosphorylation was increased in a Ca(2+)-dependent manner upon NGF stimulation. Confocal microscopic analysis demonstrated that RAFTK translocated from the cytoplasm to potential neurite initiation sites at the cell periphery, where RAFTK co-localized with paxillin and bundled actin in the early phase (within 5 min) of NGF stimulation, whereas FAK co-localized with paxillin at "point contacts," which are the primary cell adhesion sites in neuronal cells. Significant distribution of RAFTK was observed in the neurites and growth cones of differentiated PC12 cells. Furthermore, potassium depolarization induced the tyrosine phosphorylation of both RAFTK and paxillin in an intracellular Ca(2+)-dependent manner in the differentiated PC12 cells. Taken together, these results demonstrate that RAFTK is involved in NGF-induced cytoskeletal organization and may play a role in neurite and growth cone function(s).
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Factor de Crecimiento Nervioso/metabolismo , Neuronas/metabolismo , Proteínas Tirosina Quinasas/química , Proteínas Tirosina Quinasas/metabolismo , Receptor trkA , Actinas/metabolismo , Animales , Calcio/metabolismo , Proteínas Portadoras/metabolismo , Adhesión Celular , Diferenciación Celular/fisiología , Membrana Celular/metabolismo , Medio de Cultivo Libre de Suero/metabolismo , Citoplasma/metabolismo , Proteínas del Citoesqueleto/metabolismo , Citoesqueleto/metabolismo , ADN Complementario/metabolismo , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Quinasa 1 de Adhesión Focal , Quinasa 2 de Adhesión Focal , Proteína-Tirosina Quinasas de Adhesión Focal , Isoenzimas/metabolismo , Proteínas de la Membrana/metabolismo , Modelos Biológicos , Células PC12 , Paxillin , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfolipasa C gamma , Fosfoproteínas/metabolismo , Fosforilación/efectos de los fármacos , Proteínas Tirosina Quinasas/fisiología , Ratas , Factores de Tiempo , Transfección , Fosfolipasas de Tipo C/metabolismo , Tirosina/metabolismoRESUMEN
Intracellular signal transduction following extracellular ligation by a wide variety of surface molecules involves the activation and tyrosine phosphorylation of protein tyrosine kinases (PTKs). Tyrosine phosphorylation, controlled by the coordinated actions of protein tyrosine phosphatases (PTPs) and tyrosine kinases, is a critical regulatory mechanism for various physiological processes, including cell growth, differentiation, metabolism, cell cycle regulation and cytoskeleton function. The focal adhesion PTK family consists of the focal adhesion kinase (FAK) and the RAFTK/Pyk2 kinase (also known as CAK-beta and CADTK). RAFTK/Pyk2 can be activated by a variety of extracellular signals that elevate intracellular calcium concentration, and by stress signals. RAFTK/Pyk2 is expressed mainly in the central nervous system and in cells derived from hematopoietic lineages, while FAK is widely expressed in various tissues and links transmembrane integrin receptors to intracellular pathways. This review describes the role of RAFTK/Pyk2 in various signalling cascades and details the differential signalling by FAK and RAFTK/Pyk2.
Asunto(s)
Proteínas Tirosina Quinasas/fisiología , Transducción de Señal/fisiología , Animales , Moléculas de Adhesión Celular/fisiología , Quinasa 1 de Adhesión Focal , Quinasa 2 de Adhesión Focal , Proteína-Tirosina Quinasas de Adhesión Focal , Humanos , FosforilaciónRESUMEN
Focal adhesions and actin cytoskeleton are involved in cell growth, shape and movement and in tumor invasion. Mitogen-induced changes in actin cytoskeleton are accompanied by changes in the tyrosine phosphorylation of several focal adhesion proteins. In this study, we have investigated the role of RAFTK, a cytoplasmic tyrosine kinase related to focal adhesion kinase (FAK), in heregulin-mediated signal transduction in breast cancer cells. Stimulation of T47D cells with heregulin (HRG) induced the tyrosine phosphorylation of RAFTK and the formation of a multiprotein complex. Analyses of the members of the HRG-stimulated complex revealed that RAFTK is associated with p190 RhoGAP (p190), RasGAP and ErbB-2, and plays an essential role in mediating the tyrosine phosphorylation of p190 by Src. Mutation of the Src binding site within RAFTK (402) abolished the phosphorylation of p190. In addition, upon HRG stimulation of T47D cells, association of ErbB-2 with RAFTK was observed and found to be indirect and mediated by Src. Expression of wild-type RAFTK (WT) significantly increased MDA-MB-435 and MCF-7 breast cancer cell invasion, while expression of the kinase-mutated RAFTK-R457 (KM) or the Src binding site mutant RAFTK (402) did not affect this cell invasion. Furthermore, HRG leads to the activation of MAP kinase which is mediated by RAFTK. These findings indicate that RAFTK serves as a mediator and an integration point between the GAP proteins and HRG-mediated signaling in breast cancer cells, and implicate RAFTK involvement in the MAP kinase pathway and in breast cancer cell invasion.
Asunto(s)
Neoplasias de la Mama/patología , Factores de Intercambio de Guanina Nucleótido , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Proteínas Activadoras de ras GTPasa/metabolismo , Neoplasias de la Mama/metabolismo , Femenino , Quinasa 2 de Adhesión Focal , Proteínas Activadoras de GTPasa , Humanos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Invasividad Neoplásica , Neurregulina-1/farmacología , Fosforilación , Unión Proteica , Proteínas Proto-Oncogénicas pp60(c-src)/metabolismo , Receptor ErbB-2/metabolismo , Proteínas Represoras , Tirosina , ras-GRF1RESUMEN
The breast cancer susceptibility gene BRCA1 encodes a nuclear phosphoprotein that acts as a tumor suppressor. Phosphorylation of BRCA1 has been implicated in altering its function, however, the pathway(s) that leads to the phosphorylation of BRCA1 has not been described. Here, a signaling pathway by which heregulin induces cell cycle-independent phosphorylation of BRCA1 was delineated. We showed that heregulin stimulation induced the phosphorylation of BRCA1 and concomitant activation of the serine/threonine kinase AKT in T47D human breast cancer cells. Heregulin-induced phosphorylation of BRCA1 was abrogated by phosphatidylinositol 3-kinase (PI3K) inhibitors and by a dominant-negative AKT. In the absence of heregulin, the ectopic expression of the constitutively active p110 subunit of PI3K was sufficient to induce BRCA1 phosphorylation. Furthermore, the purified glutathione S-transferase/AKT kinase phosphorylated BRCA1 in vitro. We have also shown that the phosphorylation of BRCA1 by AKT occurs on the residue Thr-509, which is located in the nuclear localization signal. These results reveal a novel signaling pathway that links extracellular signals to the phosphorylation of BRCA1 in breast cancer cells.
Asunto(s)
Proteína BRCA1/metabolismo , Neoplasias de la Mama/metabolismo , Neurregulina-1/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Femenino , Humanos , Señales de Localización Nuclear , Fosforilación , Treonina/metabolismo , Células Tumorales CultivadasRESUMEN
We have isolated the cDNAs encoding human and mouse homologues of a yeast protein, termed peroxisomal membrane protein 20 (PMP20). Comparison of the amino acid sequences of human (HsPMP20) and mouse (MmPMP20) PMP20 proteins revealed a high degree of identity (93%), whereas resemblance to the yeast Candida boidinii PMP20A and PMP20B (CbPMP20A and CbPMP20B) was less (30% identity). Both HsPMP20 and MmPMP20 lack transmembrane regions, as do CbPMP20A and CbPMP20B. HsPMP20 mRNA expression was low in human fetal tissues, especially in the brain. In adult tissues, HsPMP20 mRNA was expressed in the majority of tissues tested. HsPMP20 and MmPMP20 contained the C-terminal tripeptide sequence Ser-Gln-Leu (SQL), which is similar to the peroxisomal targeting signal 1 utilized for protein import into peroxisomes. HsPMP20 bound directly to the human peroxisomal targeting signal 1 receptor, HsPEX5. Mutagenesis analysis showed that the C-terminal tripeptide sequence, SQL, of HsPMP20 is necessary for its binding to HsPEX5. Subcellular fractionation of HeLa cells, expressing epitope-tagged PMP20, revealed that HsPMP20 is localized in the cytoplasm and in a particulate fraction containing peroxisomes. Double-staining immunofluorescence studies showed colocalization of HsPMP20 and thiolase, a bona fide peroxisomal protein. The amino acid sequence alignment of HsPMP20, MmPMP20, CbPMP20A, and CbPMP20B displayed high similarity to thiol-specific antioxidant proteins. HsPMP20 exerted an inhibitory effect on the inactivation of glutamine synthetase in the thiol metal-catalyzed oxidation system but not in the nonthiol metal-catalyzed oxidation system, suggesting that HsPMP20 possesses thiol-specific antioxidant activity. In addition, HsPMP20 removed hydrogen peroxide by its thiol-peroxidase activity. These results indicate that HsPMP20 is imported into the peroxisomal matrix via PEX5p and may work to protect peroxisomal proteins against oxidative stress. Because some portion of PMP20 might also be present in the cytosol, HsPMP20 may also have a protective effect in the cytoplasm.
Asunto(s)
Antioxidantes/metabolismo , Proteínas de la Membrana/genética , Peroxidasas , Peroxisomas/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , ADN Complementario , Humanos , Proteínas de la Membrana/metabolismo , Ratones , Datos de Secuencia Molecular , Peroxirredoxinas , Receptor de la Señal 1 de Direccionamiento al Peroxisoma , Unión Proteica , Receptores Citoplasmáticos y Nucleares/metabolismo , Saccharomyces cerevisiae/genética , Homología de Secuencia de Aminoácido , Fracciones Subcelulares/metabolismo , Compuestos de Sulfhidrilo/metabolismoRESUMEN
The Flt-3 receptor is expressed in primitive haematopoietic cells and its ligand exerts proliferative effects on these cells in vitro in synergy with other cytokines. To increase our knowledge of the functional properties of the human Flt-3 ligand (FL) as relating to in vitro expansion of haematopoietic stem cells, the effects on murine haematopoiesis of FL alone or in combination with other growth factors were studied. Analysis of Flk-2/Flt-3 mRNA expression indicated that Flk-2/Flt-3 was preferentially expressed in primitive haematopoietic cell populations. To examine the expression of the Flk-2/Flt-3 receptor on megakaryocyte progenitors (CFU-Meg), Flk-2/Flt-3 positive and negative CD34(+)populations were separated from human bone marrow and cultured in a plasma clot culture system. CFU-Meg colonies were found in the Flk-2/Flt-3 negative fraction. Myeloid (CFU-GM) derived colonies appeared in the presence of FL alone. Neither FL+IL-3 nor FL+IL-3+IL-6 had any effect on the generation of megakaryocyte colonies (CFU-MK), due to the lack of FL receptor expression on megakaryocyte progenitors. Bone marrow cells remaining after 5-fluorouracil (5-FU) treatment of mice represent a very primitive population of progenitors enriched for reconstituting stem cells. This cell population expressed FL receptors, as revealed by RT-PCR analysis. Addition of FL alone did not enhance the replication of such cells in liquid cultures as compared to controls. However, a significantly greater generation of myeloid progenitors (CFU-GM) in clonogenic assays was observed in the presence of FL+IL-3, FL+GM-CSF or FL+CSF-1. In addition, the effects of FL on in vitro expansion of murine haematopoietic stem cells were studied using lineage-negative (lin(-)) Sca-1 positive (Sca-1(+)) c-kit positive (c-kit(+)) marrow cells from 5-FU treated mice. FL enhanced the survival of primitive murine lin(-)Sca-1(+)c-kit(+)cells. FL and IL-6 were able to significantly expand murine progenitor stem cells in vitro and promote their survival. These studies strongly suggest that FL significantly and selectively enhanced the generation of myeloid progenitors in vitro and increased myeloid progenitor responsiveness to later acting growth factors. In addition, FL synergized with IL-6 to support in vitro expansion of haematopoietic progenitors and promoted the survival of lin(-)Sca-1(+)c-kit(+)cells.
Asunto(s)
Células Madre Hematopoyéticas/efectos de los fármacos , Proteínas de la Membrana/farmacología , Proteínas Proto-Oncogénicas/efectos de los fármacos , Proteínas Tirosina Quinasas Receptoras/efectos de los fármacos , Animales , Enfermedades de la Médula Ósea/inducido químicamente , Enfermedades de la Médula Ósea/patología , Linaje de la Célula , Células Cultivadas , Ensayo de Unidades Formadoras de Colonias , Inducción Enzimática/efectos de los fármacos , Fluorouracilo/toxicidad , Factores de Crecimiento de Célula Hematopoyética/farmacología , Células Madre Hematopoyéticas/citología , Humanos , Interleucina-3/farmacología , Interleucina-6/farmacología , Megacariocitos/citología , Ratones , Ratones Endogámicos C57BL , ARN Mensajero/biosíntesis , Proteínas Recombinantes/farmacología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Tirosina Quinasa 3 Similar a fmsRESUMEN
We have recently isolated a cDNA encoding a novel human receptor-type tyrosine phosphatase, termed PTP-RO (for a protein tyrosine phosphatase receptor omicron), from 5-fluorouracil-treated murine bone marrow cells. PTP-RO is a human homologue of murine PTPlambda and is related to the homotypically adhering kappa and mu receptor-type tyrosine phosphatases. PTP-RO is expressed in human megakaryocytic cell lines, primary bone marrow megakaryocytes, and stem cells. PTP-RO mRNA and protein expression are upregulated upon phorbol 12-myristate 13-acetate (PMA) treatment of the megakaryocytic cell lines CMS, CMK, and Dami. To elucidate the function of PTP-RO in megakaryocytic cells and its potential involvement in the stem cell factor (SCF)/c-Kit receptor pathway, COS-7 and 293 cells were cotransfected with the cDNAs of both the c-Kit tyrosine kinase receptor and PTP-RO. PTP-RO was found to be associated with the c-Kit receptor in these transfected cells and the SCF/Kit ligand induced a rapid tyrosine phosphorylation of PTP-RO. Interestingly, these transfected cells demonstrated a decrease in their proliferative response to the SCF/Kit ligand. In addition, we assessed the association of PTP-RO with c-Kit in vivo. The results demonstrated that PTP-RO associates with c-Kit but not with the tyrosine kinase receptor FGF-R and that PTP-RO is tyrosine-phosphorylated after SCF stimulation of Mo7e and CMK cells. Antisense oligonucleotides directed against PTP-RO mRNA sequences significantly inhibited megakaryocyte progenitor proliferation. Therefore, these data show that the novel tyrosine kinase phosphatase PTP-RO is involved in megakaryocytopoiesis and that its function is mediated by the SCF/c-Kit pathway.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Megacariocitos/citología , Proteínas Tirosina Fosfatasas/biosíntesis , Proteínas Proto-Oncogénicas c-kit/metabolismo , Transactivadores , Adolescente , Animales , Células COS , Cadherinas/metabolismo , Diferenciación Celular/efectos de los fármacos , Línea Celular , Chlorocebus aethiops , Proteínas del Citoesqueleto/metabolismo , Inducción Enzimática/efectos de los fármacos , Femenino , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Hematopoyesis , Humanos , Leucemia Mieloide Aguda/patología , Megacariocitos/enzimología , Ratones , Oligonucleótidos Antisentido/farmacología , Fosforilación , Procesamiento Proteico-Postraduccional , Proteínas Tirosina Fosfatasas/química , Proteínas Tirosina Fosfatasas/genética , Proteínas Proto-Oncogénicas c-kit/genética , Proteínas Tirosina Fosfatasas Clase 2 Similares a Receptores , Proteínas Tirosina Fosfatasas Clase 3 Similares a Receptores , Proteínas Recombinantes de Fusión/metabolismo , Factor de Células Madre/farmacología , Acetato de Tetradecanoilforbol/farmacología , Transfección , Células Tumorales Cultivadas , beta CateninaRESUMEN
The cytoskeleton plays an important role in neuronal morphogenesis. We have identified and characterized a novel actin-binding protein, termed Mayven, predominantly expressed in brain. Mayven contains a BTB (broad complex, tramtrack, bric-a-brac)/POZ (poxvirus, zinc finger) domain-like structure in the predicted N terminus and "kelch repeats" in the predicted C-terminal domain. Mayven shares 63% identity (77% similarity) with the Drosophila ring canal ("kelch") protein. Somatic cell-hybrid analysis indicated that the human Mayven gene is located on chromosome 4q21.2, whereas the murine homolog gene is located on chromosome 8. The BTB/POZ domain of Mayven can self-dimerize in vitro, which might be important for its interaction with other BTB/POZ-containing proteins. Confocal microscopic studies of endogenous Mayven protein revealed a highly dynamic localization pattern of the protein. In U373-MG astrocytoma/glioblastoma cells, Mayven colocalized with actin filaments in stress fibers and in patchy cortical actin-rich regions of the cell margins. In primary rat hippocampal neurons, Mayven is highly expressed in the cell body and in neurite processes. Binding assays and far Western blotting analysis demonstrated association of Mayven with actin. This association is mediated through the "kelch repeats" within the C terminus of Mayven. Depolarization of primary hippocampal neurons with KCl enhanced the association of Mayven with actin. This increased association resulted in dynamic changes in Mayven distribution from uniform to punctate localization along neuronal processes. These results suggest that Mayven functions as an actin-binding protein that may be translocated along axonal processes and might be involved in the dynamic organization of the actin cytoskeleton in brain cells.
Asunto(s)
Encéfalo/metabolismo , Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/metabolismo , Proteínas del Tejido Nervioso , Secuencia de Aminoácidos , Animales , Astrocitos/metabolismo , Astrocitoma/genética , Astrocitoma/metabolismo , Axones/metabolismo , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Polaridad Celular/efectos de los fármacos , Células Cultivadas , Mapeo Cromosómico , Cromosomas Humanos Par 4 , Clonación Molecular , Cricetinae , Citoesqueleto/metabolismo , Dimerización , Glioblastoma/genética , Glioblastoma/metabolismo , Proteínas Fluorescentes Verdes , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Humanos , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos , Datos de Secuencia Molecular , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Especificidad de Órganos , Cloruro de Potasio/farmacología , Pruebas de Precipitina , Ratas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Células Tumorales CultivadasRESUMEN
The Csk Homologous Kinase (CHK) has been shown to have an enzymatic activity similar to the tyrosine kinase Csk in that it down-regulates Src family kinase activity by causing phosphorylation of the Src C-terminal tyrosine residue. In megakaryocytic Mo7e cells, CHK associates with a specific phosphotyrosine juxtamembrane sequence of the SCF/KL-activated c-Kit receptor. Here, we show that in Mo7e cells, the major Src family kinase activity is p53/56(Lyn). Studies using immobilized c-Kit phosphopeptides show that Lyn is able to specifically associate with the tyrosine-phosphorylated juxtamembrane 568Y*VY*IDPT sequence of c-Kit which has previously been shown to associate with CHK. In cells over-expressing CHK by means of a recombinant vaccinia virus, we observed an elimination of the SCF/KL-stimulated Lyn kinase peak of activity observed at 2-5 minutes in cells infected with the helper T7-expressing vaccinia virus by itself. Examination of total tyrosine phosphorylation by Western blotting showed that over-expression of CHK resulted in a reduction in the levels of tyrosine phosphorylations in the range of 50-60 kDa, but had no apparent effect on c-Kit autophosphorylation. Taken together, these findings show that CHK is able to down-regulate SCF/KL-stimulated Lyn activity in megakaryocytes.
Asunto(s)
Regulación hacia Abajo , Megacariocitos/metabolismo , Proteínas Tirosina Quinasas/fisiología , Proteínas Proto-Oncogénicas pp60(c-src) , Familia-src Quinasas/metabolismo , Línea Celular , Factores de Tiempo , Virus Vaccinia/metabolismoRESUMEN
Csk homologous kinase (CHK), a member of the Csk regulatory tyrosine kinase family, is expressed primarily in brain and hematopoietic cells. The role of CHK in the nervous system is as yet unknown. Using PC12 cells as a model system of neuronal cells, we show that CHK participates in signaling mediated by TrkA receptors. CHK was found to be associated with tyrosine-phosphorylated TrkA receptors in PC12 cells upon stimulation with NGF. Binding assays and far Western blotting analysis, using glutathione S-transferase fusion proteins containing the Src homology 2 (SH2) and SH3 domains of CHK, demonstrate that the SH2 domain of CHK binds directly to the tyrosine-phosphorylated TrkA receptors. Site-directed mutagenesis of TrkA cDNA, as well as phosphopeptide inhibition of the in vitro interaction of the CHK-SH2 domain or native CHK with TrkA receptors, indicated that the residue Tyr-785 on TrkA is required for its binding to the CHK-SH2 domain upon NGF stimulation. In addition, overexpression of CHK resulted in enhanced activation of the mitogen-activated protein kinase pathway upon NGF stimulation, and microinjection of anti-CHK antibodies, but not anti-Csk antibodies, inhibited neurite outgrowth of PC12 cells in response to NGF. Thus, CHK is a novel signaling molecule that participates in TrkA signaling, associates directly with TrkA receptors upon NGF stimulation, and is involved in neurite outgrowth of PC12 cells in response to NGF.