RESUMEN
BACKGROUND: Calcium ions play a pivotal role in cell proliferation, differentiation, and migration. Under basal conditions, the calcium level is tightly regulated; however, cellular activation by growth factors increase the ion level through calcium pumps in the plasma membrane and endoplasmic reticulum for calcium signaling. Orai1 is a major calcium channel in the cell membrane of non-excitable cells, and its activity depends on the stromal interaction molecule 1 (Stim1). Several groups reported that the store-operated calcium entry (SOCE) can be modulated through phosphorylation of Stim1 by protein kinases such as extracellular signal-regulated kinase (ERK), protein kinase A (PKA), and p21-activated kinase (PAK). PKC is a protein kinase that is activated by calcium and diacylglycerol (DAG), but it remains unclear what role activated PKC plays in controlling the intracellular calcium pool. OBJECTIVES: Here, we investigated whether PKC-ß controls intracellular calcium dynamics through Stim1. METHODS: Several biochemical methods such as immune-precipitation, site directed mutagenesis, in vitro kinase assay were employed to investigate PKC interaction with and phosphorylation of Stim1. Intracellular calcium mobilization, via Stim1 mediated SOCE channel, were studied using in the presence of PKC activator or inhibitor under a confocal microscope. RESULTS: Our data demonstrate that PKC interacts with and phosphorylates Stim1 in vitro. phosphorylation of Stim1 at its C-terminal end appears to be important in the regulation of SOCE activity in HEK293 and HeLa cells. Additionally, transient intracellular calcium mobilization assays demonstrate that the SOCE activity was inhibited by PKC activators or activated by PKC inhibitors. CONCLUSION: In sum, our data suggest a repressive role of PKC in regulating calcium entry through SOCE.
Asunto(s)
Calcio , Proteínas de Neoplasias , Calcio/metabolismo , Células HEK293 , Células HeLa , Humanos , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Fosforilación , Molécula de Interacción Estromal 1/genética , Molécula de Interacción Estromal 1/metabolismoRESUMEN
Dabie bandavirus (severe fever with thrombocytopenia syndrome virus [SFTSV]) induces an immunopathogenic disease with a high fatality rate; however, the mechanisms underlying its clinical manifestations are largely unknown. In this study, we applied targeted proteomics and single-cell transcriptomics to examine the differential immune landscape in SFTS patient blood. Serum immunoprofiling identified low-risk and high-risk clusters of SFTS patients based on inflammatory cytokine levels, which corresponded to disease severity. Single-cell transcriptomic analysis of SFTS patient peripheral blood mononuclear cells (PBMCs) at different infection stages showed pronounced expansion of B cells with alterations in B-cell subsets in fatal cases. Furthermore, plasma cells in which the interferon (IFN) pathway is downregulated were identified as the primary reservoir of SFTSV replication. This study identified not only the molecular signatures of serum inflammatory cytokines and B-cell lineage populations in SFTSV-induced fatalities but also plasma cells as the viral reservoir. Thus, this suggests that altered B-cell function is linked to lethality in SFTSV infections.IMPORTANCE SFTSV is an emerging virus discovered in China in 2009; it has since spread to other countries in East Asia. Although the fatality rates of SFTSV infection range from 5.3% to as high as 27%, the mechanisms underlying clinical manifestations are largely unknown. In this study, we demonstrated that SFTSV infection in fatal cases caused an excessive inflammatory response through high induction of proinflammatory cytokines and chemokines and the aberrant inactivation of adaptive immune responses. Furthermore, single-cell transcriptome sequencing (RNA-seq) analysis of SFTS patient PBMCs revealed that SFTSV targets the B-cell lineage population, especially plasma cells, as the potential viral reservoir in patients for whom the infection is fatal. Thus, SFTSV infection may inhibit high-affinity antibody maturation and secretion of plasma B cells, suppressing neutralizing antibody production and thereby allowing significant virus replication and subsequent fatality.
Asunto(s)
Linfocitos B/inmunología , Citocinas/genética , Inflamación/genética , Phlebovirus/inmunología , Síndrome de Trombocitopenia Febril Grave/inmunología , Transcriptoma , Anciano , Anticuerpos Antivirales/sangre , Citocinas/inmunología , Reservorios de Enfermedades/virología , Femenino , Humanos , Inflamación/inmunología , Masculino , Persona de Mediana Edad , Células Plasmáticas/virología , Proteómica , Síndrome de Trombocitopenia Febril Grave/sangre , Síndrome de Trombocitopenia Febril Grave/genética , Transducción de Señal/genética , Transducción de Señal/inmunologíaRESUMEN
Genome editing is a useful tool in basic and clinical research. Among the several approaches used in genome editing, the CRISPR-Cas9 system using clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9) along with a guide RNA has been developed recently. The CRISPR/Cas9 system induces site-specific double-stranded DNA breaks, which result in DNA repair via non-homologous end joining (NHEJ) or homology-directed repair (HDR). However, HDR efficiency is lower than that of NHEJ and accordingly poses a challenge in genome modification studies. Several chemical compounds including RS-1 have been shown to enhance the HDR knock-in process by two- to six-fold in HEK 293 cells and rabbit embryos. Based on this finding, we developed an antibiotic resistance system to screen RS-1 chemical derivatives, which may promote efficient HDR. In this study, we report several chemical compounds with high knock-in efficiency at the ATG5 gene locus, using HeLa cell-based assays.
Asunto(s)
Proteína 5 Relacionada con la Autofagia/genética , Benzamidas/farmacología , Roturas del ADN de Doble Cadena/efectos de los fármacos , Reparación del ADN/efectos de los fármacos , Sulfonamidas/farmacología , Edición Génica , Células HEK293 , Células HeLa , HumanosRESUMEN
Carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) plays a crucial role in tumorigenesis of lung cancer. However, the therapeutic potential for anti CEACAM6 monoclonal antibody (mAb) has only been limitedly explored. Here, we evaluate the therapeutic potential of naked anti CEACAM6 mAb against lung adenocarcinoma. Clone 8F5, recognizing B domain of CEACAM6, is established by immunizing A549 cells and screening for clones double positive for A549 and CEACAM6-Fc recombinant protein. We found that 85.7% of 70 resected lung adenocarcinoma tissue sections were positive for CEACAM6, whereas all squamous cell carcinoma examined were negative. A549 cells with high levels of CEACAM6 demonstrated more aggressive growth nature and showed increased paclitaxel chemosensitivity upon 8F5 binding. Treatment with 8F5 to A549 decreased cellular CEACAM6 expression and reversed anoikis resistance. 8F5 also decreased cellular status of Akt phosphorylation and increased apoptosis via caspase activation. In a mouse model of lung adenocarcinoma with xenotransplanted A549 cells, 8F5 treatment alone demonstrated 40% tumor growth inhibition. When combined with paclitaxel treatment, 8F5 markedly enhanced tumor growth inhibition, up to 80%. In summary, we demonstrate that anti CEACAM6 mAb is an effective therapeutic treatment for lung adenocarcinoma whose effect is further enhanced by combined treatment with paclitaxel.
Asunto(s)
Adenocarcinoma/tratamiento farmacológico , Adenocarcinoma/patología , Anoicis , Anticuerpos Monoclonales/uso terapéutico , Antígenos CD/inmunología , Moléculas de Adhesión Celular/inmunología , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/patología , Adenocarcinoma del Pulmón , Secuencia de Aminoácidos , Animales , Anoicis/efectos de los fármacos , Anticuerpos Monoclonales/farmacología , Antígenos CD/química , Carcinogénesis/efectos de los fármacos , Carcinogénesis/patología , Caspasas/metabolismo , Moléculas de Adhesión Celular/química , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Regulación hacia Abajo/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Epítopos/química , Epítopos/inmunología , Proteínas Ligadas a GPI/química , Proteínas Ligadas a GPI/inmunología , Humanos , Masculino , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Paclitaxel/farmacología , Fosforilación/efectos de los fármacos , Unión Proteica/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/metabolismoRESUMEN
JL1, a specific epitope on CD43, is a potential biomarker for the diagnosis of acute leukemia. Although qualitative assays for detecting leukemia-specific CD43 exist, there is a need to develop quantitative assays for the same. Here, we developed two novel monoclonal antibodies (mAbs), 2C8 and 8E10, recognizing different epitopes on CD43. These clones are capable of pairing with YG5, another mAb against JL1 epitope, because they were selectively obtained using sandwich ELISA. Antigens recognized by 2C8 and 8E10 were confirmed as CD43 by western blotting using the CD43-hFC recombinant protein. When expression on various leukemic cell lines was investigated, 2C8 and 8E10 displayed a disparity in the distribution of the epitope. Enzyme assays revealed that these mAbs recognized a sialic acid-dependent epitope on CD43. Using normal thymus and lymph node paraffin-embedded tissues, we confirmed a difference in the epitopes recognized by the two mAbs that was predicted based on the maturity of the cells in the tissue. In summary, we developed and characterized two mAbs, 2C8 and 8E10, which can be used with YG5 in a sandwich ELISA for detecting leukemia-specific CD43.
RESUMEN
FGF-2 is involved in cell survival, proliferation, apoptosis, and angiogenesis in a wide variety of cells. FRGRs, PI3K and MAP kinases are well known mediators of FGF signaling. Despite its known roles during many developmental processes, including osteogenesis, there are few known targets of FGF-2. In the present study, we identified Bcl2-A1 and Bcl-xL as two prominent targets involved in promoting cell survival. Pretreatment of ATDC5 cells with FGF-2 increased cell survival, while siRNAs specific for Bcl2-A1 and Bcl-xL compromised the anti- apoptotic effect of FGF-2, sensitized the cells to apoptosis triggered by TNF-α. Chemical inhibition of FGFR, NFkB, and PI3K activity by PD173074, pyrrolidine dithiocarbamate, and LY294002 respectively abrogated the FGF-2-mediated induction of Bcl2-A1 and Bcl-xL expression. Taken together, our data demonstrate that a subset of Bcl2 family proteins are the targets of FGF-2 signaling that promotes the survival of ATDC5 cells.
Asunto(s)
Apoptosis/efectos de los fármacos , Condrocitos/efectos de los fármacos , Factor 2 de Crecimiento de Fibroblastos/farmacología , Proteínas Proto-Oncogénicas c-bcl-2/genética , Factor de Necrosis Tumoral alfa/farmacología , Proteína bcl-X/genética , Animales , Apoptosis/genética , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Condrocitos/metabolismo , Condrocitos/fisiología , Relación Dosis-Respuesta a Droga , Regulación hacia Abajo/efectos de los fármacos , Evaluación Preclínica de Medicamentos , Factor 2 de Crecimiento de Fibroblastos/fisiología , Expresión Génica/efectos de los fármacos , Ratones , Antígenos de Histocompatibilidad Menor , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Células Madre/efectos de los fármacos , Células Madre/metabolismo , Células Madre/fisiología , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/genética , Proteína bcl-X/metabolismoRESUMEN
Anti cancer agent 5-FU (Fluoro Uracil) is a prodrug that can be metabolized and then activated to interfere with RNA and DNA homeostasis. However, the majority of administered 5-FU is known to be catabolized in vivo in the liver where Dihydropyrimidine dehydrogenase (DPD) is abundantly expressed to degrade 5-FU. The biological factors that correlate with the response to 5-FU-based chemotherapy have been proposed to include uridine phosphorylase (UPP), thymidine phosphorylase (TPP), p53 and microsatellite instability. Among these, the expression of UPP is known to be controlled by cytokines such as TNF-alpha, IL1 and IFN-gamma. Our preliminary study using a DNA microarray technique showed that basic fibroblast growth factor (bFGF) markedly induced the expression of UPP1 at the transcription level. In the present study, we investigated whether bFGF could modulate the expression of UPP1 in osteo-lineage cells and examined the sensitivity of these cells to 5-FU mediated apoptosis.
Asunto(s)
Apoptosis/efectos de los fármacos , Factor 2 de Crecimiento de Fibroblastos/farmacología , Fluorouracilo/farmacología , Regulación hacia Arriba/efectos de los fármacos , Uridina Fosforilasa/genética , Animales , Antimetabolitos Antineoplásicos/farmacología , Línea Celular , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Sinergismo Farmacológico , Citometría de Flujo , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Humanos , Mioblastos/citología , Mioblastos/efectos de los fármacos , Mioblastos/metabolismo , Osteosarcoma/genética , Osteosarcoma/patología , Interferencia de ARN , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
This study was designed to investigate the effects of polyamines on mechanical contraction and voltage-dependent calcium current (VDCC) of guinea-pig gastric smooth muscle. Mechanical contraction and calcium channel current I(Ba) were recorded by isometric tension recording and whole-cell patch clamp technique. Spermine, spermidine and putrescine inhibited spontaneous contraction of the gastric smooth muscle in a concentration-dependent manner. Spermine (2 mM) reduced high K+ (50 mM)-induced contraction to 16+/-6.4% of the control (n=9), and significantly inhibited I(Ba) in a reversible manner (p<0.05; IC50=0.8 mM). Pre- and post-treatment of tissue with spermine (2-5 mM, n=10) also inhibited acetylcholine (10 microM)-induced phasic contraction to 5+/-6.4% of the control. Inhibitory effect of spermine on I(Ba) was observed at a wide range of test potentials of current/voltage (I/V) relationship (p<0.05), and steady-state activation of I(Ba) was shifted to the right by spermine (p<0.05). Spermidine and putrescine (1 mM each) also inhibited I(Ba) to 51+/-5.7% and 81+/-5.3% of the control, respectively. And putrescine (1 mM) inhibited I(Ba) at whole tested potentials (p<0.05) without significant change of kinetics (p<0.05). Finally, 5 mM putrescine also inhibited high K+-induced contraction to 53+/-7.1% of the control (n=4). These findings suggest that polyamines inhibit contractions of guinea-pig gastric smooth muscle via inhibition of VDCC.
Asunto(s)
Contracción Muscular/efectos de los fármacos , Músculo Liso/efectos de los fármacos , Poliaminas/farmacología , Antro Pilórico/efectos de los fármacos , Animales , Calcio/metabolismo , Canales de Calcio/efectos de los fármacos , Canales de Calcio/fisiología , Femenino , Cobayas , Masculino , Músculo Liso/fisiología , Potasio/farmacología , Antro Pilórico/fisiologíaRESUMEN
This study was designed to identify and characterize Na+-activated K+ current (I(K(Na))) in guinea pig gastric myocytes under whole-cell patch clamp. After whole-cell configuration was established under 110 mM intracellular Na+ concentration ([Na+]i) at holding potential of -60 mV, a large inward current was produced by external 60 mM K+([K+]degrees). This inward current was not affected by removal of external Ca2+. K+ channel blockers had little effects on the current (p>0.05). Only TEA (5 mM) inhibited steady-state current to 68+/-2.7% of the control (p<0.05). In the presence of K+ channel blocker cocktail (mixture of Ba2+, glibenclamide, 4-AP, apamin, quinidine and TEA), a large inward current was activated. However, the amplitude of the steady-state current produced under [K+]degrees (140 mM) was significantly smaller when Na+ in pipette solution was replaced with K+- and Li+ in the presence of K+ channel blocker cocktail than under 110 mM [Na+]i. In the presence of K+ channel blocker cocktail under low Cl- pipette solution, this current was still activated and seemed K+-selective, since reversal potentials (E(rev)) of various concentrations of [K+]degrees-induced current in current/voltage (I/V) relationship were nearly identical to expected values. R-56865 (10-20 microM), a blocker of I(K(Na)), completely and reversibly inhibited this current. The characteristics of the current coincide with those of I(K(Na)) of other cells. Our results indicate the presence of I(K(Na)) in guinea pig gastric myocytes.
Asunto(s)
Miocitos del Músculo Liso/fisiología , Canales de Potasio/fisiología , Sodio/farmacología , Estómago/fisiología , Animales , Calcio/metabolismo , Cloruros/farmacología , Femenino , Cobayas , Masculino , Potenciales de la Membrana , Bloqueadores de los Canales de Potasio/farmacología , Sodio/metabolismo , Compuestos de Tetraetilamonio/farmacologíaRESUMEN
To investigate whether the phosphorylation of extracellular signal-regulated kinase (ERK) is involved in autoimmune injury of the peripheral nervous system (PNS), the expression of phosphorylated ERK (p-ERK) was analyzed in experimental autoimmune neuritis (EAN) in rats. Western blot analysis showed that the level of p-ERK was increased significantly in the sciatic nerves of rats on days 14 (p<0.05) and 24 (p<0.01) post-immunization, compared with controls, and its reaction declined at day 30 post-immunization. Immunohistochemistry showed that p-ERK protein was weakly expressed in Schwann cells and vascular endothelial cells in the sciatic nerves of CFA-immunized control rats. In EAN-affected sciatic nerves, p-ERK immunoreactivity was found mainly in ED1-positive macrophages on days 14 and 24 post-immunization. Moreover, on days 24 and 30 post-immunization, p-ERK immunoreactivity increased gradually in the Schwann cells of rat sciatic nerves with EAN. Based on these results, we postulated that the phosphorylation of ERK has an important role in the differentiation and survival of cells, including inflammatory cells and Schwann cells, in the rat sciatic nerve in EAN. Specifically, the activation of ERK in the recovery phase of EAN paralysis seems to be related in the survival of Schwann cells.
Asunto(s)
Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Neuritis Autoinmune Experimental/enzimología , Nervio Ciático/enzimología , Animales , Activación Enzimática/fisiología , Quinasas MAP Reguladas por Señal Extracelular/inmunología , Femenino , Neuritis Autoinmune Experimental/inmunología , Ratas , Ratas Endogámicas Lew , Nervio Ciático/inmunologíaRESUMEN
Amphiphysin1, which can simultaneously bind to dynamin1 and the clathrin adaptor AP-2, is essential for dynamin1 recruitment during receptor-mediated endocytosis, but little is known about its regulatory mechanism. Here, we purified a 120-kDa mitogen-activated protein kinase (MAPK) substrate protein from porcine brains and identified the protein as amphiphysin1. Serine phosphorylation of amphiphysin1 was rapidly induced by nerve growth factor (NGF) in PC12 cells, and the induction was blocked by a MAPK inhibitor. Furthermore, when phosphorylated by MAPK in vitro or by NGF treatment in vivo, amphiphysin1 failed to bind to AP-2, but its association with dynamin1 was unaffected. Consistent with this, mutation of consensus MAPK phosphorylation sites increased amphiphysin1 binding to AP-2 and their intracellular colocalization. Thus, we propose that MAPK phosphorylation of amphiphysin1 controls NGF receptor/TrkA-mediated endocytosis by terminating the amphiphysin1-AP-2 interaction. This perhaps helps to regulate the availability of amphiphysin1-dynamin1 complexes for binding to the endocytic vesicle.
Asunto(s)
Endocitosis , Regulación de la Expresión Génica , Sistema de Señalización de MAP Quinasas/fisiología , Factor de Crecimiento Nervioso/metabolismo , Proteínas del Tejido Nervioso/biosíntesis , Complejo 2 de Proteína Adaptadora/metabolismo , Animales , Electroforesis en Gel de Poliacrilamida , Inhibidores Enzimáticos/farmacología , Flavonoides/farmacología , Glutatión Transferasa/metabolismo , Immunoblotting , Inmunohistoquímica , Microscopía Fluorescente , Modelos Biológicos , Mutación , Proteínas del Tejido Nervioso/metabolismo , Células PC12 , Ácidos Fosfoaminos/metabolismo , Fosforilación , Pruebas de Precipitina , Unión Proteica , Ratas , Fracciones Subcelulares , Porcinos , TransfecciónRESUMEN
p21-activated kinase (PAK)-interacting exchange factor (PIX) is known to be involved in regulation of Cdc42/Rac GTPases and PAK activity. PIX binds to the proline-rich region of PAK, and regulates biological events through activation of Cdc42/Rac GTPase. To further investigate the role of PIX we produced monoclonal antibodies (Mab) against bPIX. Three clones; N-C6 against N-terminal half and C-A3 and C-B7 against C- terminal half of bPIX were generated and characterized. N-C6 Mab detected bPIX as a major band in most cell lines. C-A3 Mab recognizes GIT-binding domain (GBD), but it does not interfere with GIT binding to bPIX. Using C-A3 Mab possible bPIX interaction with actin in PC12 cells was examined. bPIX Mab (C-A3) specifically precipitated actin of the PC12 cell lysates whereas actin Mab failed to immunoprecipitate bPIX. Co-sedimentation of PC12 cell lysates with the polymerized F-actin resulted in the recovery of most of bPIX in the cell lysates. These results suggest that bPIX may not interact with soluble actin but with polymerized F-actin and revealed that bPIX constitutes a functional complex with actin. These data indicate real usefulness of the bPIX Mab in the study of bPIX role(s) in regulation of actin cyoskeleton.
Asunto(s)
Citoesqueleto de Actina/fisiología , Actinas/metabolismo , Proteínas de Ciclo Celular/fisiología , Factores de Intercambio de Guanina Nucleótido/fisiología , Animales , Anticuerpos Monoclonales/inmunología , Proteínas de Ciclo Celular/inmunología , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Proteínas del Citoesqueleto/metabolismo , Mapeo Epitopo , Factores de Intercambio de Guanina Nucleótido/inmunología , Factores de Intercambio de Guanina Nucleótido/metabolismo , Inmunoprecipitación , Ratones , Estructura Terciaria de Proteína , Ratas , Factores de Intercambio de Guanina Nucleótido RhoRESUMEN
Guanine nucleotide exchange factors (GEFs) have been implicated in growth factor-induced neuronal differentiation through the activation of small GTPases. Although phosphorylation of these GEFs is considered an activation mechanism, little is known about the upstream of PAK-interacting exchange factor (PIX), a member of the Dbl family of GEFs. We report here that phosphorylation of p85 betaPIX/Cool/p85SPR is mediated via the Ras/ERK/PAK2 pathway. To understand the role of p85 betaPIX in basic fibroblast growth factor (bFGF)-induced neurite outgrowth, we established PC12 cell lines that overexpress the fibroblast growth factor receptor-1 in a tetracycline-inducible manner. Treatment with bFGF induces the phosphorylation of p85 betaPIX, as determined by metabolic labeling and mobility shift upon gel electrophoresis. Interestingly, phosphorylation of p85 betaPIX is inhibited by PD98059, a specific MEK inhibitor, suggesting the involvement of the ERK cascade. PAK2, a major PAK isoform in PC12 cells as well as a binding partner of p85 betaPIX, also functions upstream of p85 betaPIX phosphorylation. Surprisingly, PAK2 directly binds to ERK, and its activation is dependent on ERK. p85 betaPIX specifically localizes to the lamellipodia at neuronal growth cones in response to bFGF. A mutant form of p85 betaPIX (S525A/T526A), in which the major phosphorylation sites are replaced by alanine, shows significant defect in targeting. Moreover, expression of the mutant p85 betaPIX efficiently blocks PC12 cell neurite outgrowth. Our study defines a novel signaling pathway for bFGF-induced neurite outgrowth that involves activation of the PAK2-p85 betaPIX complex via the ERK cascade and subsequent translocation of this complex.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Neuronas/fisiología , Transducción de Señal , Animales , Diferenciación Celular , División Celular , Células Cultivadas , Activación Enzimática , Immunoblotting , Microscopía Fluorescente , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Modelos Biológicos , Neuronas/citología , Células PC12 , Fosforilación , Plásmidos/metabolismo , Pruebas de Precipitina , Unión Proteica , Isoformas de Proteínas , Transporte de Proteínas , Ratas , Factores de Intercambio de Guanina Nucleótido Rho , Serina/metabolismo , Tetraciclina/farmacología , Treonina/metabolismo , Factores de Tiempo , TransfecciónRESUMEN
Amphiphysin II (Amph2) is known to undergo rapid dephosphorylation and phosphorylation at nerve terminals. After in vivo electroconvulsive shock (ECS) in the rat cerebellum, we found an electrophoretic mobility retardation of Amph2, which suggested an increased degree of phosphorylation above the non-stimulated level. This shifted signal was observed from 1 min, reached the maximum level at 5 min and extended beyond 2 h after ECS. The shifted band was markedly decreased by the phosphatase treatment. Pretreatment with cyclosporin A augmented the mobility retardation of Amph2 after ECS. Our results indicate that ECS induces the phosphorylation of Amph2 in the rat cerebellum.
Asunto(s)
Cerebelo/metabolismo , Electrochoque , Proteínas del Tejido Nervioso/metabolismo , Animales , Western Blotting , Ciclosporina/farmacología , Ensayo de Cambio de Movilidad Electroforética/métodos , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Masculino , Monoéster Fosfórico Hidrolasas/efectos de los fármacos , Monoéster Fosfórico Hidrolasas/metabolismo , Fosforilación , Ratas , Ratas Sprague-DawleyRESUMEN
p21-activated kinase (PAK) targeting to the plasma membrane is essential for PC12 cell neurite outgrowth. Phospholipase C-gamma1 (PLC-gamma1) can mediate the PAK translocation in response to growth factors, since PLC-gamma1 binds to both tyrosine-phosphorylated receptor tyrosine kinases and PAK through its SH2 and SH3 domain, respectively. In the present study, we examined a potential role for PLC-gamma1 in the basic fibroblast growth factor (bFGF)-induced PAK translocation using stable PC12 cell lines that overexpress in a tetracycline-inducible manner either the wild-type FGFR-1 or the Y766F FGFR-1 mutant. Phosphatidylinositol hydrolysis was increased 6.5-fold in response to bFGF in the wild type cells but negligible in the mutant cells. The recombinant GST-PLC-gamma1 SH3 was able to bind to PAK1 but not GST alone. However, examination of PLC-gamma1 as an adaptor for translocation of PAK1 in cells showed that both cells transfected with pEGFP-PAK1 was able to differentiate for 24 h, as visualized by laser confocal microscopy. Translocation of PAK1 to growth cones occurs at similar levels in both wild and mutant cells. These results suggest that a protein(s) other than PLC-gamma1 is functionally relevant for PAK targeting.