RESUMEN
Melatonin induces nuclear exclusion of the androgen receptor (AR) via activation of protein kinase C (PKC). The specific members of the PKC superfamily involved in AR nuclear exclusion were investigated in prostate cancer PC3 cells stably transfected with the wild-type androgen receptor (PC3-AR). PKCalpha was essentially cytoplasmic whereas PKCbeta and PKCepsilon were essentially membranal, suggesting their constitutive activity in the PC3-AR cells. Melatonin treatment induced membrane association of PKCalpha in a time and dose dependent manner. The PKCalpha and PKCbeta1 specific inhibitor GO6976 and the PKCbeta isoform-specific inhibitor hispidin had no effects on AR localization under basal conditions. However, GO6976 but not hispidin negated the melatonin-mediated nuclear exclusion of the AR. These data indicate that PKCalpha activation is a critical step in AR nuclear exclusion by melatonin. They also imply that PKCalpha-activation is a potentially effective way to control of the AR activity in prostate cancer cells.
Asunto(s)
Melatonina/fisiología , Transducción de Señal/fisiología , Línea Celular Tumoral , Membrana Celular/enzimología , Citosol/enzimología , Humanos , Cinética , Masculino , Neoplasias de la Próstata , Proteína Quinasa C-alfa/fisiologíaRESUMEN
Insulin stimulates glucose uptake into skeletal muscle tissue mainly through the translocation of glucose transporter 4 (GLUT4) to the plasma membrane. The precise mechanism involved in this process is presently unknown. In the cascade of events leading to insulin-induced glucose transport, insulin activates specific protein kinase C (PKC) isoforms. In this study we investigated the roles of PKC zeta in insulin-stimulated glucose uptake and GLUT4 translocation in primary cultures of rat skeletal muscle. We found that insulin initially caused PKC zeta to associate specifically with the GLUT4 compartments and that PKC zeta together with the GLUT4 compartments were then translocated to the plasma membrane as a complex. PKC zeta and GLUT4 recycled independently of one another. To further establish the importance of PKC zeta in glucose transport, we used adenovirus constructs containing wild-type or kinase-inactive, dominant-negative PKC zeta (DNPKC zeta) cDNA to overexpress this isoform in skeletal muscle myotube cultures. We found that overexpression of PKC zeta was associated with a marked increase in the activity of this isoform. The overexpressed, active PKC zeta coprecipitated with the GLUT4 compartments. Moreover, overexpression of PKC zeta caused GLUT4 translocation to the plasma membrane and increased glucose uptake in the absence of insulin. Finally, either insulin or overexpression of PKC zeta induced serine phosphorylation of the GLUT4-compartment-associated vesicle-associated membrane protein 2. Furthermore, DNPKC zeta disrupted the GLUT4 compartment integrity and abrogated insulin-induced GLUT4 translocation and glucose uptake. These results demonstrate that PKC zeta regulates insulin-stimulated GLUT4 translocation and glucose transport through the unique colocalization of this isoform with the GLUT4 compartments.
Asunto(s)
Glucosa/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Transporte de Monosacáridos/metabolismo , Proteínas Musculares , Músculo Esquelético/metabolismo , Proteína Quinasa C/metabolismo , Serina/metabolismo , Animales , Transporte Biológico , Fraccionamiento Celular , Células Cultivadas , Activación Enzimática , Expresión Génica , Transportador de Glucosa de Tipo 4 , Membranas Intracelulares/metabolismo , Músculo Esquelético/citología , Fosforilación , Proteína Quinasa C/genética , Proteínas R-SNARE , RatasRESUMEN
Insulin and insulin-like growth factor-1 (IGF-1) are members of the family of the insulin family of growth factors, which activate similar cellular downstream pathways. In this study, we analyzed the effects of insulin and IGF-1 on the proliferation of murine skin keratinocytes in an attempt to determine whether these hormones trigger the same signaling pathways. Increasing doses of insulin and IGF-1 promote keratinocyte proliferation in an additive manner. We identified downstream pathways specifically involved in insulin signaling that are known to play a role in skin physiology; these include activation of the Na+/K+ pump and protein kinase C (PKC). Insulin, but not IGF-1, stimulated Na+/K+ pump activity. Furthermore, ouabain, a specific Na+/K+ pump inhibitor, abolished the proliferative effect of insulin but not that of IGF-1. Insulin and IGF-1 also differentially regulated PKC activation. Insulin, but not IGF-1, specifically activated and translocated the PKCB isoform to the membrane fraction. There was no effect on PKC isoforms alpha, eta, epsilon, and zeta, which are expressed in skin. PKC8 overexpression increased keratinocyte proliferation and Na+/K+ pump activity to a degree similar to that induced by insulin but had no affect on IGF-1-induced proliferation. Furthermore, a dominant negative form of PKCdelta abolished the effects of insulin on both proliferation and Na+/K+ pump activity but did not abrogate induction of keratinocyte proliferation induced by other growth factors. These data indicate that though insulin or IGF-1 stimulation induce keratinocyte proliferation, only insulin action is specifically mediated via PKC8 and involves activation of the Na+/K+ pump.
Asunto(s)
Factor I del Crecimiento Similar a la Insulina/fisiología , Insulina/fisiología , Isoenzimas/metabolismo , Queratinocitos/citología , Proteína Quinasa C/metabolismo , Transducción de Señal/fisiología , Animales , Transporte Biológico/efectos de los fármacos , División Celular/efectos de los fármacos , División Celular/fisiología , Células Cultivadas , Activación Enzimática , Genes Dominantes , Insulina/farmacología , Factor I del Crecimiento Similar a la Insulina/farmacología , Isoenzimas/genética , Ratones , Ratones Endogámicos BALB C , Proteína Quinasa C/genética , Proteína Quinasa C-delta , Rubidio/farmacocinética , ATPasa Intercambiadora de Sodio-Potasio/efectos de los fármacos , ATPasa Intercambiadora de Sodio-Potasio/metabolismoRESUMEN
Certain protein kinase C (PKC) isoforms, in particular PKCs beta II, delta, and zeta, are activated by insulin stimulation. In primary cultures of skeletal muscle, PKCs beta II and zeta, but not PKC delta, are activated via a phosphatidylinositol 3-kinase (PI3K)-dependent pathway. The purpose of this study was to investigate the possibility that PKC delta may be activated upstream of PI3K by direct interaction with insulin receptor (IR). Experiments were done on primary cultures of newborn rat skeletal muscle, age 5--6 days in vitro. The time course of insulin-induced activation of PKC delta closely paralleled that of IR. Insulin stimulation caused a selective coprecipitation of PKC delta with IR, and these IR immunoprecipitates from insulin-stimulated cells displayed a striking induction of PKC activity due specifically to PKC delta. To examine the involvement of PKC delta in the IR signaling cascade, we used recombinant adenovirus constructs of wild-type (W.T.) or dominant negative (D.N.) PKC delta. Overexpression of W.T.PKC delta induced PKC delta activity and coassociation of PKC delta and IR without addition of insulin. Overexpression of D.N.PKC delta abrogated insulin- induced coassociation of PKC delta and IR. Insulin-induced tyrosine phosphorylation of IR was greatly attenuated in cells overexpressing W.T.PKC delta, whereas in myotubes overexpressing D.N.PKC delta, tyrosine phosphorylation occurred without addition of insulin and was sustained longer than that in control myotubes. In control myotubes IR displayed a low level of serine phosphorylation, which was increased by insulin stimulation. In cells overexpressing W.T.PKC delta, serine phosphorylation was strikingly high under basal conditions and did not increase after insulin stimulation. In contrast, in cells overexpressing D.N.PKC delta, the level of serine phosphorylation was lower than that in nonoverexpressing cells and did not change notably after addition of insulin. Overexpression of W.T.PKC delta caused IR to localize mainly in the internal membrane fractions, and blockade of PKC delta abrogated insulin-induced IR internalization. We conclude that PKC delta is involved in regulation of IR activity and routing, and this regulation may be important in subsequent steps in the IR signaling cascade.
Asunto(s)
Insulina/metabolismo , Isoenzimas/metabolismo , Músculo Esquelético/metabolismo , Proteína Quinasa C/metabolismo , Receptor de Insulina/metabolismo , Acetofenonas/farmacología , Animales , Benzopiranos/farmacología , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Células Cultivadas , Inhibidores Enzimáticos/farmacología , Insulina/farmacología , Factor I del Crecimiento Similar a la Insulina/farmacología , Isoenzimas/efectos de los fármacos , Isoenzimas/genética , Músculo Esquelético/citología , Músculo Esquelético/efectos de los fármacos , Fosforilación , Pruebas de Precipitina , Proteína Quinasa C/efectos de los fármacos , Proteína Quinasa C/genética , Proteína Quinasa C-delta , Ratas , Receptor de Insulina/efectos de los fármacos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Serina/metabolismo , Tirosina/metabolismoRESUMEN
Insulin activates certain protein kinase C (PKC) isoforms that are involved in insulin-induced glucose transport. In this study, we investigated the possibility that activation of PKCdelta by insulin participates in the mediation of insulin effects on glucose transport in skeletal muscle. Studies were performed on primary cultures of rat skeletal myotubes. The role of PKCdelta in insulin-induced glucose uptake was evaluated both by selective pharmacological blockade and by over-expression of wild-type and point-mutated inactive PKCdelta isoforms in skeletal myotubes. We found that insulin induces tyrosine phosphorylation and translocation of PKCdelta to the plasma membrane and increases the activity of this isoform. Insulin-induced effects on translocation and phosphorylation of PKCdelta were blocked by a low concentration of rottlerin, whereas the effects of insulin on other PKC isoforms were not. This selective blockade of PKCdelta by rottlerin also inhibited insulin-induced translocation of glucose transporter 4 (GLUT4), but not glucose transporter 3 (GLUT3), and significantly reduced the stimulation of glucose uptake by insulin. When overexpressed in skeletal muscle, PKCdelta and PKCdelta were both active. Overexpression of PKCdelta induced the translocation of GLUT4 to the plasma membrane and increased basal glucose uptake to levels attained by insulin. Moreover, insulin did not increase glucose uptake further in cells overexpressing PKCdelta. Overexpression of PKCdelta did not affect basal glucose uptake or GLUT4 location. Stimulation of glucose uptake by insulin in cells overexpressing PKCdelta was similar to that in untransfected cells. Transfection of skeletal myotubes with dominant negative mutant PKCdelta did not alter basal glucose uptake but blocked insulin-induced GLUT4 translocation and glucose transport. These results demonstrate that insulin activates PKCdelta and that activated PKCdelta is a major signaling molecule in insulin-induced glucose transport.
Asunto(s)
Glucosa/metabolismo , Insulina/farmacología , Isoenzimas/metabolismo , Proteínas Musculares , Músculo Esquelético/enzimología , Proteínas del Tejido Nervioso , Proteína Quinasa C/metabolismo , Acetofenonas/farmacología , Animales , Benzopiranos/farmacología , Transporte Biológico/efectos de los fármacos , Membrana Celular/enzimología , Células Cultivadas , Activación Enzimática , Inhibidores Enzimáticos/farmacología , Expresión Génica , Transportador de Glucosa de Tipo 3 , Transportador de Glucosa de Tipo 4 , Isoenzimas/antagonistas & inhibidores , Isoenzimas/genética , Proteínas de Transporte de Monosacáridos/metabolismo , Músculo Esquelético/efectos de los fármacos , Fosforilación , Mutación Puntual , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C/genética , Proteína Quinasa C-delta , Ratas , TransfecciónRESUMEN
Several reports indicate that protein kinase C (PKC) plays a role in insulin-induced glucose transport in certain cells. The precise effects of insulin on specific PKC isoforms are as yet unknown. Utilizing primary cultures of rat skeletal muscle, we investigated the possibility that insulin may influence the activation state of PKC isoenzymes by inducing their translocation and tyrosine phosphorylation. This, in turn, may mediate insulin effects on glucose transport. We identified and determined the glucose transporters and PKC isoforms affected by insulin and 12-O-tetradecanoylphorbol-13-acetate (TPA). Insulin and TPA each caused an increase in glucose uptake. Insulin translocated GLUT3 and GLUT4 without affecting GLUT1. In contrast, TPA translocated GLUT1 and GLUT3 without affecting GLUT4. Insulin translocated and tyrosine phosphorylated and activated PKC-beta2 and -zeta; these effects were blocked by phosphatidylinositol 3-kinase (PI3K) inhibitors. TPA translocated and activated PKC-alpha, -beta2, and -delta; these effects were not noticeably affected by PI3K inhibitors. Furthermore, wortmannin significantly inhibited both insulin and TPA effects on GLUT translocation and glucose uptake. Finally, insulin-induced glucose transport was blocked by the specific PKC-beta2 inhibitor LY379196. These results indicate that specific PKC isoenzymes, when tyrosine-phosphorylated, are implicated in insulin-induced glucose transport in primary cultures of skeletal muscle.
Asunto(s)
Glucosa/metabolismo , Insulina/farmacología , Isoenzimas/metabolismo , Músculo Esquelético/metabolismo , Proteína Quinasa C/metabolismo , Tirosina/metabolismo , Animales , Transporte Biológico Activo/efectos de los fármacos , Células Cultivadas , Inhibidores Enzimáticos/farmacología , Músculo Esquelético/efectos de los fármacos , Inhibidores de las Quinasa Fosfoinosítidos-3 , Fosforilación , Ratas , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
Studies from this laboratory have shown that the physiological expression of the Na+/K+ pump in primary cultures of rat skeletal muscle increases with development. The molecular mechanisms underlying these changes are not known. Therefore, we have examined the expression of alpha and beta subunits of the Na+/K+ pump at both the protein and mRNA levels during myogenesis of primary skeletal muscle cell cultures obtained from newborn rats. Protein isoforms were identified by Western blotting techniques with specific monoclonal and polyclonal antibodies and subunit mRNA was studied with specific cDNA probes. Freshly isolated skeletal muscle from newborn rats expressed both alpha1 and alpha2 protein subunits. From day 1 after plating, primary cultures expressed only the alpha1 protein isoform. In contrast, both beta1 and beta2 isoforms were expressed in freshly isolated muscle and in primary cultures, with beta1 expression being stronger in both preparations. Studies on RNA expression showed that mRNA for alpha1, alpha2, beta1, and beta2 isoforms was identified both in freshly isolated muscle and after plating of cells in culture. These findings indicate that the lack of alpha2 protein expression in primary muscle cell cultures reflects a form of posttranscriptional regulation. There did not appear to be a quantitative difference in isoform expression as a function of age or of fusion in spite of developmental increases in Na+/K+ pump activity and its dependence on cell fusion. The lack of expression of the alpha2 subunit isoform suggests that the developmental changes in physiological expression of the Na+/K+ pump in primary cultures of skeletal muscle may be attributable either to the changes in activity of the alpha1 subunit or to differential activities of alphabeta complexes involving either of the beta subunits.
Asunto(s)
Isoenzimas/genética , Músculo Esquelético/citología , Músculo Esquelético/enzimología , ATPasa Intercambiadora de Sodio-Potasio/genética , Animales , Animales Recién Nacidos , Anticuerpos Monoclonales , Northern Blotting , Western Blotting , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/fisiología , Células Cultivadas , Quelantes/farmacología , Ácido Egtácico/farmacología , Regulación Enzimológica de la Expresión Génica , Isoenzimas/análisis , Fibras Musculares Esqueléticas/citología , Fibras Musculares Esqueléticas/efectos de los fármacos , Fibras Musculares Esqueléticas/enzimología , ARN Mensajero/análisis , Ratas , Proteínas Recombinantes de Fusión/análisis , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , ATPasa Intercambiadora de Sodio-Potasio/análisis , ATPasa Intercambiadora de Sodio-Potasio/inmunologíaRESUMEN
We investigated the effects of nerve growth factor (NGF) on expression of K+ channels in cultured skeletal muscle. The channels studied were (1) charybdotoxin (ChTx)-sensitive channels by using a polyclonal antibody raised in rabbits against ChTx, (2) Kv1.5 voltage-sensitive channels, and (3) apamin-sensitive (afterhyperpolarization) channels. Crude homogenates were prepared from cultures made from limb muscles of 1-2-day-old rat pups for identification of ChTx-sensitive and Kv1.5 channels by Western blotting techniques. Apamin-sensitive K+ channels were studied by measurement of specific [125I]-apamin binding by whole cell preparations. ChTx-sensitive channels display a fusion-related increase in expression, and NGF downregulates these channels in both myoblasts and myotubes. Voltage-dependent Kv1.5 channel expression is low in myoblasts and increases dramatically with fusion; NGF induces early expression of these channels and causes expression after fusion to increase even further. NGF downregulates apamin-sensitive channels. NGF increases the rate of fall of the action potential recorded intracellularly from single myotubes with intracellular microelectrodes. The results confirm and extend those of previous studies in showing a functional role for NGF in the regulation of membrane properties of skeletal muscle. Moreover, the findings demonstrate that the different K+ channels in this preparation are regulated in a discoordinate manner. The divergent effects of NGF on expression of different K+ channels, however, do not appear sufficient to explain the NGF-induced increase in the rate of fall of the action potential. The changes during the falling phase may rather be due to increases in channel properties or may result from an increased driving force on the membrane potential secondary to the NGF-induced hyperpolarization.