RESUMEN
In Aplysia, persistent increases in synaptic strength are paralleled by the persistent activation of the novel protein kinase C Apl II. We raised a phosphospecific antibody against serine 725, the hydrophobic motif in protein kinase C Apl II. Phosphorylation of serine 725 increased in parallel to the persistent activation of the kinase. We expressed protein kinase C where this site was mutated to an alanine to prevent phosphorylation. The mutated protein kinase C showed decreased specific activity consistent with a model where the kinase is less stable in the absence of phosphorylation of this site. Endogenous phosphorylation of protein kinase C Apl II at serine 725 was unaffected by either activation of protein kinase C by phorbol esters, or inhibition of protein kinase C using two distinct inhibitors, suggesting the site is not autophosphorylated. Consistent with this, overexpressed kinase-dead protein kinase C Apl II still was phosphorylated at serine 725, although to a lesser extent than wild-type protein kinase C Apl II. While PDK appears to interact with the serine 725 site, it is not responsible for its phosphorylation. Finally inhibition of phosphoinositide-3 kinase or the target of rapamycin by pharmacological agents did not block basal phosphorylation of serine 725 in Aplysia ganglia. Our results suggest trans-phosphorylation of protein kinase C Apl II as Ser 725 occurs during persistent activation of the kinase, but this does not appear to be downstream of phosphoinositide-3 kinase.
Asunto(s)
Ganglios de Invertebrados/metabolismo , Isoenzimas/metabolismo , Proteína Quinasa C/metabolismo , Serina/metabolismo , Alcaloides , Secuencias de Aminoácidos/fisiología , Animales , Aplysia , Benzofenantridinas , Western Blotting/métodos , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Citosol/efectos de los fármacos , Citosol/metabolismo , Inhibidores Enzimáticos/farmacología , Lateralidad Funcional , Ganglios de Invertebrados/citología , Ganglios de Invertebrados/efectos de los fármacos , Expresión Génica/efectos de los fármacos , Mutación/fisiología , Fenantridinas/farmacología , Ésteres del Forbol/farmacología , TemperaturaRESUMEN
Activation of the extracellular signal-related kinase is important for long-term increases in synaptic strength in the Aplysia nervous system. However, there is little known about the mechanism for the activation of the kinase in this system. We examined the activation of Aplysia extracellular signal-related kinase using a phosphopeptide antibody specific to the sites required for activation of the kinase. We found that phorbol esters led to a prolonged activation of extracellular signal-related kinase in sensory cells of the Aplysia nervous system. Surprisingly, inhibitors of protein kinase C did not block this activation. Serotonin, the physiological transmitter involved in long-term synaptic facilitation, also led to prolonged activation of extracellular signal-related kinase, but inhibitors of protein kinase A or protein kinase C did not block this activation. We examined whether the protein synthesis-dependent increase in excitability stimulated by phorbol esters was dependent on phorbol ester activation of extracellular signal-related kinase, but increases in excitability were still seen in the presence of inhibitors of extracellular signal-related kinase activation. Our results suggest that prolonged phosphorylation of extracellular signal-related kinase in the Aplysia system is not mediated by either of the classic second messenger activated kinases in this system, protein kinase A or protein kinase C and that extracellular signal-related kinase is not important for phorbol ester induced long-term effects on excitability.
Asunto(s)
Aplysia , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Sistema Nervioso/efectos de los fármacos , Sistema Nervioso/metabolismo , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Proteína Quinasa C/metabolismo , Serotonina/metabolismo , Animales , Western Blotting , AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Activadores de Enzimas/farmacología , Inmunohistoquímica , Ésteres del Forbol/farmacología , Proteína Quinasa C/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacosRESUMEN
The ror receptors are a highly conserved family of receptor tyrosine kinases genetically implicated in the establishment of cellular polarity. We have cloned a ror receptor from the marine mollusk Aplysia californica. Aplysia ror (Apror) is expressed in most developing neurons and some adult neuronal populations, including the neuroendocrine bag-cell neurons. The Apror protein is present in peripheral neuronal processes and ganglionic neuropil, implicating the kinase in the regulation of growth and/or synaptic events. In cultured bag-cell neurons, the majority of the protein is stored in intracellular organelles, whereas only a small fraction is expressed on the surface. When expressed on the cell surface, the protein is clustered on neurites, suggesting that Apror is involved in the organization of functional domains within neurons. Apror immunoreactivity partially colocalizes with the P-type calcium channel BC-alpha1A at bag-cell neuron varicosities, suggesting a role for Apror in organizing neuropeptide release sites.
Asunto(s)
Aplysia/química , Ganglios de Invertebrados/metabolismo , Neuronas/metabolismo , Sistemas Neurosecretores/metabolismo , Proteínas Tirosina Quinasas Receptoras/aislamiento & purificación , Receptores de Superficie Celular/aislamiento & purificación , Factores de Edad , Secuencia de Aminoácidos/fisiología , Animales , Especificidad de Anticuerpos , Aplysia/citología , Aplysia/metabolismo , Secuencia de Bases/fisiología , Proteínas de Caenorhabditis elegans , Compartimento Celular/fisiología , Células Cultivadas/citología , Células Cultivadas/metabolismo , Clonación Molecular , Ganglios de Invertebrados/citología , Ganglios de Invertebrados/crecimiento & desarrollo , Inmunohistoquímica , Datos de Secuencia Molecular , Neuronas/citología , Sistemas Neurosecretores/citología , Sistemas Neurosecretores/crecimiento & desarrollo , ARN Mensajero/metabolismo , Proteínas Tirosina Quinasas Receptoras/química , Proteínas Tirosina Quinasas Receptoras/genética , Receptores Huérfanos Similares al Receptor Tirosina Quinasa , Receptores de Superficie Celular/química , Receptores de Superficie Celular/genéticaRESUMEN
We describe a novel mechanism for protein kinase C regulation of axonal microtubule invasion of growth cones. Activation of PKC by phorbol esters resulted in a rapid, robust advance of distal microtubules (MTs) into the F-actin rich peripheral domain of growth cones, where they are normally excluded. In contrast, inhibition of PKC activity by bisindolylmaleimide and related compounds had no perceptible effect on growth cone motility, but completely blocked phorbol ester effects. Significantly, MT advance occurred despite continued retrograde F-actin flow-a process that normally inhibits MT advance. Polymer assembly was necessary for PKC-mediated MT advance since it was highly sensitive to a range of antagonists at concentrations that specifically interfere with microtubule dynamics. Biochemical evidence is presented that PKC activation promotes formation of a highly dynamic MT pool. Direct assessment of microtubule dynamics and translocation using the fluorescent speckle microscopy microtubule marking technique indicates PKC activation results in a nearly twofold increase in the typical lifetime of a MT growth episode, accompanied by a 1.7-fold increase and twofold decrease in rescue and catastrophe frequencies, respectively. No significant effects on instantaneous microtubule growth, shortening, or sliding rates (in either anterograde or retrograde directions) were observed. MTs also spent a greater percentage of time undergoing retrograde transport after PKC activation, despite overall MT advance. These results suggest that regulation of MT assembly by PKC may be an important factor in determining neurite outgrowth and regrowth rates and may play a role in other cellular processes dependent on directed MT advance.
Asunto(s)
Conos de Crecimiento/metabolismo , Microtúbulos/metabolismo , Neuronas/citología , Neuronas/enzimología , Proteína Quinasa C/metabolismo , Actinas/metabolismo , Animales , Aplysia , Transporte Axonal , Axones/efectos de los fármacos , Axones/enzimología , Axones/metabolismo , Biopolímeros/química , Biopolímeros/metabolismo , Células Cultivadas , Citoesqueleto/efectos de los fármacos , Citoesqueleto/metabolismo , Activación Enzimática/efectos de los fármacos , Conos de Crecimiento/efectos de los fármacos , Conos de Crecimiento/enzimología , Indoles/farmacología , Cinética , Maleimidas/farmacología , Microtúbulos/efectos de los fármacos , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Nocodazol/farmacología , Octoxinol/farmacología , Forbol 12,13-Dibutirato/farmacología , Estructura Terciaria de Proteína , Solubilidad/efectos de los fármacos , Tubulina (Proteína)/metabolismoRESUMEN
The identification of tags that can specifically mark activated synapses is important for understanding how long-term synaptic changes can be restricted to specific synapses. The maintenance of synapse-specific facilitation in Aplysia sensory to motor neuron cultures can be blocked by inhibitors of translation and by the drug rapamycin, which specifically blocks a signaling pathway that regulates phosphorylation of translational regulators. One important target of rapamycin is the phosphorylation and subsequent activation of S6 kinase. To test whether S6 kinase is the target for the ability of rapamycin to block synapse-specific facilitation in Aplysia, we cloned Aplysia S6 kinase, its substrate S6, and the S6 kinase kinase phosphoinositide-dependent kinase 1 (PDK-1). Serotonin, which induces synapse-specific facilitation, increased phosphorylation of Aplysia S6 kinase at threonine 399 in a rapamycin-sensitive manner in Aplysia synaptosomes. The phosphorylation of threonine 399 by 5-HT was independent of phosphoinositide-3 kinase, dependent on PKA and PKC, and occluded by the phosphatase inhibitor calyculin-A. 5-HT also increased S6 kinase activity and led to increased phosphorylation of S6 in synaptosomes. 5-HT increased levels of S6 in synaptosomes because of a rapamycin-sensitive increase in translation-stabilization of S6. Aplysia PDK-1 bound to and phosphorylated Aplysia S6 kinase but only modulated phosphorylation of threonine 399 indirectly. These results suggest a mechanism by which the levels of translation factors can be increased specifically at activated synapses generating a long-lasting synaptic tag.
Asunto(s)
Proteínas Quinasas S6 Ribosómicas/metabolismo , Serotonina/metabolismo , Sirolimus/farmacología , Sinaptosomas/enzimología , Proteínas Quinasas Dependientes de 3-Fosfoinosítido , Animales , Antibacterianos/farmacología , Aplysia , Sitios de Unión/efectos de los fármacos , Línea Celular , Inhibidores Enzimáticos/farmacología , Potenciación a Largo Plazo/efectos de los fármacos , Toxinas Marinas , Datos de Secuencia Molecular , Plasticidad Neuronal/efectos de los fármacos , Oxazoles/farmacología , Inhibidores de las Quinasa Fosfoinosítidos-3 , Fosforilación/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteína S6 Ribosómica , Proteínas Quinasas S6 Ribosómicas/efectos de los fármacos , Proteínas Quinasas S6 Ribosómicas/genética , Proteínas Ribosómicas/genética , Proteínas Ribosómicas/metabolismo , Serotonina/farmacología , Spodoptera , Sinapsis/metabolismo , Sinaptosomas/efectos de los fármacos , Treonina/metabolismoRESUMEN
Ca(2+)-independent or novel protein kinase Cs (nPKCs) contain an N-terminal C2 domain of unknown function. Removal of the C2 domain of the Aplysia nPKC Apl II allows activation of the enzyme at lower concentrations of phosphatidylserine, suggesting an inhibitory role for the C2 domain in enzyme activation. However, the mechanism for C2 domain-mediated inhibition is not known. Mapping of the autophosphorylation sites for protein kinase C (PKC) Apl II reveals four phosphopeptides in the regulatory domain of PKC Apl II, two of which are in the C2 domain at serine 2 and serine 36. Unlike most PKC autophosphorylation sites, these serines could be phosphorylated in trans. Interestingly, phosphorylation of serine 36 increased binding of the C2 domain to phosphatidylserine membranes in vitro. In cells, PKC Apl II phosphorylation at serine 36 was increased by PKC activators, and PKC phosphorylated at this position translocated more efficiently to membranes. Moreover, mutation of serine 36 to alanine significantly reduced membrane translocation of PKC Apl II. We suggest that translocation of nPKCs is regulated by phosphorylation of the C2 domain.
Asunto(s)
Proteína Quinasa C/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Membrana Celular/efectos de los fármacos , Membrana Celular/enzimología , Clonación Molecular , Cartilla de ADN , Datos de Secuencia Molecular , Mutagénesis , Forbol 12,13-Dibutirato/farmacología , Fosforilación , Proteína Quinasa C/química , Proteína Quinasa C/genética , Transporte de Proteínas , Homología de Secuencia de AminoácidoRESUMEN
We have used an antibody that specifically recognizes eukaryotic initiation factor 4E (eIF4E) when it is phosphorylated at Ser(207) to characterize eIF4E phosphorylation in the nervous system of APLYSIA: The level of phosphorylated eIF4E, but not the level of total eIF4E, was significantly correlated with the basal rate of translation measured from different animals. Serotonin (5-HT), a transmitter that regulates the rate of translation in APLYSIA: neurons, had mixed effects on eIF4E phosphorylation. 5-HT decreased eIF4E phosphorylation in sensory cell clusters through activation of protein kinase C. 5-HT increased eIF4E phosphorylation in the whole pleural ganglia. In the APLYSIA: nervous system, eIF4E phosphorylation correlated with phosphorylation of the p38 MAP kinase, but not the p42 MAP kinase (ERK). Furthermore, an inhibitor of the p38 MAP kinase significantly decreased basal eIF4E phosphorylation, but an inhibitor of the MAP or ERK kinase (MEK) did not. Despite the correlation of eIF4E phosphorylation with the basal rate of translation, inhibition of eIF4E phosphorylation by an inhibitor of the p38 MAP kinase did not significantly decrease the rate of translation.
Asunto(s)
Ganglios de Invertebrados/fisiología , Neuronas/fisiología , Factores de Iniciación de Péptidos/metabolismo , Biosíntesis de Proteínas , Alcaloides , Animales , Aplysia , Benzofenantridinas , Colforsina/farmacología , AMP Cíclico/análogos & derivados , AMP Cíclico/farmacología , Inhibidores Enzimáticos/farmacología , Factor 4E Eucariótico de Iniciación , Técnicas In Vitro , Cinética , Metionina/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Neuronas/efectos de los fármacos , Fenantridinas/farmacología , Forbol 12,13-Dibutirato/farmacología , Fosforilación , Serotonina/farmacología , Tionucleótidos/farmacología , Proteínas Quinasas p38 Activadas por MitógenosRESUMEN
Phosphorylation of calcium-activated protein kinase Cs (PKCs) at threonine 634 and/or threonine 641 increases during long term potentiation or associative learning in rodents. In the marine mollusk Aplysia, persistent activation of the calcium-activated PKC Apl I occurs during long term facilitation. We have raised an antibody to a peptide from PKC Apl I phosphorylated at threonines 613 and 620 (sites homologous to threonines 634 and 641). This antibody recognizes PKC Apl I only when it is phosphorylated at threonine 613. Both phorbol esters and serotonin increase the percentage of kinase phosphorylated at threonine 613 in Aplysia neurons. Furthermore, the pool of PKC that is phosphorylated at threonine 613 in neurons is resistant to both membrane translocation and down-regulation. Replacement of threonine 613 with alanine increased the affinity of PKC Apl I for calcium, suggesting that phosphorylation of this site may reduce the ability of PKC Apl I to translocate to membranes in the presence of calcium. We propose that phosphorylation of this site is important for removal of PKC from the membrane and may be a mechanism for negative feedback of PKC activation.
Asunto(s)
Fosfotreonina/metabolismo , Proteína Quinasa C/química , Treonina/química , Secuencia de Aminoácidos , Animales , Anticuerpos/inmunología , Aplysia , Calcio/metabolismo , Datos de Secuencia Molecular , Mutación , Neuronas/metabolismo , Forbol 12,13-Dibutirato/farmacología , Fosfopéptidos/inmunología , Fosforilación , Proteína Quinasa C/inmunología , Serotonina/farmacologíaRESUMEN
Nerve growth factor (NGF) is released through the constitutive secretory pathway from cells in peripheral tissues and nerves where it can act as a target-derived survival factor. In contrast, brain-derived neurotrophic factor (BDNF) appears to be processed in the regulated secretory pathway of brain neurons and secreted in an activity-dependent manner to play a role in synaptic plasticity. To determine whether sorting differences are intrinsic to the neurotrophins or reflect differences between cell types, we compared NGF and BDNF processing in cultured hippocampal neurons using a Vaccinia virus expression system. Three independent criteria (retention or release from cells after pulse-chase labeling, depolarization-dependent release, and immunocytochemical localization) suggest that the bulk of newly synthesized NGF is sorted into the constitutive pathway, whereas BDNF is primarily sorted into the regulated secretory pathway. Similar results occurred with AtT 20 cells, including those transfected with cDNAs encoding neurotrophin precursor-green fluorescent protein fusions. The NGF precursor, but not the BDNF precursor, is efficiently cleaved by the endoprotease furin in the trans-Golgi network (TGN). Blocking furin activity in AtT 20 cells with alpha1-PDX as well as increasing the expression of NGF precursor partially directed NGF into the regulated secretory pathway. Therefore, neurotrophins can be sorted into either the constitutive or regulated secretory pathways, and sorting may be regulated by the efficiency of furin cleavage in the TGN. This mechanism may explain how neuron-generated neurotrophins can act both as survival factors and as neuropeptides.
Asunto(s)
Factor Neurotrófico Derivado del Encéfalo/metabolismo , Hipocampo/metabolismo , Factores de Crecimiento Nervioso/metabolismo , Neuronas/metabolismo , Animales , Electrofisiología , Furina , Hipocampo/citología , Hipocampo/fisiología , Inmunohistoquímica , Ratones/embriología , Neuronas/fisiología , Precursores de Proteínas/metabolismo , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Subtilisinas/farmacología , Distribución TisularRESUMEN
We have cloned eIF4E from the marine mollusk, Aplysia californica. The sequence of eIF4E from Aplysia is more similar to vertebrate eIF4Es than to other invertebrate sequences. Aplysia eIF4E is encoded by two tissue-specific RNAs. Antibodies raised to the carboxyl terminus of eIF4E recognize a 29-kDa protein that can bind to 7-methyl-GTP caps. The phosphorylation site identified in mammalian eIF4E is conserved in the Aplysia homologue, and an Aplysia eIF4E fusion protein is phosphorylated well by both Aplysia protein kinase C isoforms. However, protein kinase C phosphorylates both Ser-207 and Thr-208 in vitro, while only Ser-207 is phosphorylated in vivo. We have confirmed that Ser-207 is phosphorylated in vivo by raising a phosphopeptide antibody to this site. This antibody will be useful in determining the signal transduction pathways leading to eIF4E phosphorylation in Aplysia.
Asunto(s)
Secuencia Conservada , Factores de Iniciación de Péptidos/metabolismo , Serina/metabolismo , Animales , Aplysia , Secuencia de Bases , Cartilla de ADN , Factor 4E Eucariótico de Iniciación , Datos de Secuencia Molecular , Factores de Iniciación de Péptidos/química , Fosforilación , Proteína Quinasa C/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
Recently, two of the 10 vertebrate protein kinase C (PKC) isoforms, PKC betaII and PKC epsilon, have been shown to bind specifically to actin filaments, suggesting that these kinases may regulate cytoskeletal dynamics. Here, we present evidence that two PKCs from the marine mollusk Aplysia californica, PKC Apl I, a Ca2+-activated PKC, and PKC Apl II, a Ca2+-independent PKC most similar to PKC epsilon and eta, also bind F-actin. First, they both cosedimented with purified actin filaments in a phorbol ester-dependent manner. Second, they both translocated to the Triton-insoluble fraction of the nervous system after phorbol ester treatment. PKC Apl II could also partially translocate to actin filaments and associate with the Triton-insoluble fraction in the absence of phorbol esters. Translocation to purified actin filaments was increased in the presence of a PKC inhibitor, suggesting that PKC phosphorylation reduces PKC bound to actin. Although both kinases bound F-actin, actin was not sufficient to activate the kinases. In support of a physiological role for actin-PKC interactions, immunochemical localization of PKC Apl II in neuronal growth cones revealed a striking colocalization with F-actin. Our results are consistent with a role for actin-PKC interactions in regulating cytoskeletal dynamics in Aplysia.
Asunto(s)
Actinas/metabolismo , Aplysia/metabolismo , Isoenzimas/metabolismo , Proteína Quinasa C/metabolismo , Actinas/fisiología , Animales , Transporte Biológico/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Inmunohistoquímica , Neuronas/enzimología , Forbol 12,13-Dibutirato/farmacología , Distribución TisularRESUMEN
In the nervous system of the marine mollusk Aplysia there are two protein kinase C (PKC) isoforms, the Ca2+-activated PKC Apl I and the Ca2+-independent PKC Apl II. PKC Apl I, but not PKC Apl II is activated by a short-term application of the neurotransmitter serotonin. This may be explained by the fact that purified PKC Apl II requires a higher mole percentage of phosphatidylserine to stimulate enzyme activity than does PKC Apl I. In order to understand the molecular basis for this difference, we have compared the ability of lipids to interact with the purified kinases and with regulatory domain fusion proteins derived from the kinases using a variety of assays including kinase activity, phorbol dibutyrate binding, and liposome binding. We found that a C2 domain fusion protein derived from PKC Apl I binds to lipids constitutively, while a C2 domain fusion protein derived from PKC Apl II does not. In contrast, fusion proteins containing the C1 domains of PKC Apl I and PKC Apl II showed only small differences in lipid interactions. Thus, while the presence of a C2 domain assists lipid-mediated activation of PKC Apl I, it inhibits activation of PKC Apl II.
Asunto(s)
Aplysia/enzimología , Calcio/metabolismo , Isoenzimas/metabolismo , Proteína Quinasa C/metabolismo , Animales , Sitios de Unión , Activación Enzimática , Metabolismo de los Lípidos , Forbol 12,13-Dibutirato/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Serotonina/metabolismoRESUMEN
Protein kinases A (PKA) and C (PKC) play a central role as intracellular transducers during simple forms of learning in Aplysia. These two proteins seem to cooperate in mediating the different forms of plasticity underlying behavioral modifications of defensive reflexes in a state- and time-dependent manner. Although short- and long-term changes in the synaptic efficacy of the connections between mechanosensory neurons and motoneurons of the reflex have been well characterized, there is also a distinct intermediate phase of plasticity that is not as well understood. Biochemical and physiological experiments have suggested a role for PKC in the induction and expression of this form of facilitation. In this report, we demonstrate that PKC activation can induce both intermediate- and long-term changes in the excitability of sensory neurons (SNs). Short application of 4beta-phorbol ester 12,13-dibutyrate (PDBU), a potent activator of PKC, produced a long-lasting increase in the number of spikes fired by SNs in response to depolarizing current pulses. This effect was observed in isolated cell culture and in the intact ganglion; it was blocked by a selective PKC inhibitor (chelerythrine). Interestingly, the increase in excitability measured at an intermediate-term time point (3 h) after treatment was independent of protein synthesis, while it was disrupted at the long-term (24 h) time point by the general protein synthesis inhibitor, anisomycin. In addition to suggesting that PKC as well as PKA are involved in long-lasting excitability changes, these findings support the idea that memory formation involves multiple stages that are mechanistically distinct at the biochemical level.
Asunto(s)
Potenciales de Acción/fisiología , Potenciales Evocados/fisiología , Ganglios de Invertebrados/fisiología , Neuronas Aferentes/fisiología , Forbol 12,13-Dibutirato/farmacología , Proteína Quinasa C/metabolismo , Potenciales de Acción/efectos de los fármacos , Animales , Aplysia , Células Cultivadas , Activación Enzimática , Potenciales Evocados/efectos de los fármacos , Potenciales Postsinápticos Excitadores , Modelos Neurológicos , Neuronas Motoras/efectos de los fármacos , Neuronas Motoras/fisiología , Estereoisomerismo , Transmisión Sináptica/efectos de los fármacos , Transmisión Sináptica/fisiologíaRESUMEN
There are two protein kinase Cs (PKCs) in the Aplysia nervous system, PKC Apl I, which is homologous to the Ca(2+)-activated PKC family, and PKC Apl II, which is homologous to the Ca(2+)-independent PKCs epsilon and eta. Purified PKC Apl I requires much less phosphatidylserine for activation than does purified PKC Apl II, and this may explain why the neurotransmitter serotonin activates PKC Apl I but not PKC Apl II in the intact nervous system [Sossin, W. S., Fan, X., and Baseri, F. (1996) J. Neurosci. 16, 10-18]. PKC Apl II's requirement for high levels of phosphatidylserine may be mediated by its C2 domain, since removal of this domain allows PKC Apl II to be activated at lower concentrations of phosphatidylserine. To begin to understand how this inhibition is mediated, we generated fusion proteins containing the C1 and C2 domains from PKC Apl II and determined their lipid dependence for phorbol ester binding. Our results indicate that the presence of the C2 domain lowers the affinity of protein kinase C activators for the C1 domains and this inhibition can be removed by phosphatidylserine. Phosphatidic acid, however, is much more potent than phosphatidylserine in reducing C2 domain-mediated inhibition, suggesting that phosphatidic acid may be a required cofactor for the activation of PKC Apl II.
Asunto(s)
Aplysia/enzimología , Forbol 12,13-Dibutirato/metabolismo , Ácidos Fosfatidicos/farmacología , Proteína Quinasa C/farmacología , Estructura Terciaria de Proteína , Animales , Unión Competitiva/efectos de los fármacos , Calcio/metabolismo , Fosfatidilserinas/metabolismo , Proteína Quinasa C/genética , Proteínas Recombinantes de Fusión/químicaRESUMEN
In the marine mollusk Aplysia californica, serotonin initiates three phases of translational regulation: an initial decrease in translation, followed by a transient increase in protein synthesis, both of which are independent of transcription, followed by a later increase in protein synthesis that is dependent on transcription. These increases in protein synthesis may underlie translation-dependent changes in synaptic plasticity. We have characterized the second messenger pathways that underlie these changes in the pleural ganglia of Aplysia. Activation of protein kinase C was both necessary and sufficient for the initial decrease in translation. Protein kinase C, cyclic AMP-dependent protein kinase, and a tyrosine kinase were all required for the second phase, a transient increase in protein synthesis. The late increase in protein synthesis required both protein kinase A and spaced applications of serotonin. Rapamycin, a specific inhibitor of a downstream translational regulator, blocked the transient increase in protein synthesis (second phase), suggesting that this drug may be useful in determining the specific physiological consequences of this translational regulation. Indeed, we used rapamycin to demonstrate that one type of intermediate form of synaptic plasticity induced by serotonin did not require the rapamycin-sensitive increase in translation.
Asunto(s)
Ganglios de Invertebrados/fisiología , Regulación de la Expresión Génica/efectos de los fármacos , Neuronas Aferentes/metabolismo , Biosíntesis de Proteínas/efectos de los fármacos , Serotonina/farmacología , 8-Bromo Monofosfato de Adenosina Cíclica/farmacología , Animales , Aplysia , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Inhibidores Enzimáticos/farmacología , Ganglios de Invertebrados/efectos de los fármacos , Técnicas In Vitro , Cinética , Metionina/metabolismo , Plasticidad Neuronal , Neuronas Aferentes/efectos de los fármacos , Forbol 12,13-Dibutirato/farmacología , Polienos/farmacología , Proteína Quinasa C/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Sirolimus , Sinapsis/efectos de los fármacos , Sinapsis/fisiología , Factores de TiempoRESUMEN
Synaptojanin is a nerve-terminal enriched inositol 5-phosphatase thought to function in synaptic vesicle endocytosis, in part through interactions with the Src homology 3 domain of amphiphysin. We have used synaptojanin purified from Sf9 cells after baculovirus mediated expression in overlay assays to identify two major synaptojanin-binding proteins in rat brain. The first, at 125 kDa, is amphiphysin. The second, at 40 kDa, is the major synaptojanin-binding protein detected, is highly enriched in brain, is concentrated in a soluble synaptic fraction, and co-immunoprecipitates with synaptojanin. The 40-kDa protein does not bind to a synaptojanin construct lacking the proline-rich C terminus, suggesting that its interaction with synaptojanin is mediated through an Src homology 3 domain. The 40-kDa synaptojanin-binding protein was partially purified from rat brain cytosol through a three-step procedure involving ammonium sulfate precipitation, sucrose density gradient centrifugation, and DEAE ion-exchange chromatography. Peptide sequence analysis identified the 40-kDa protein as SH3P4, a member of a novel family of Src homology 3 domain-containing proteins. These data suggest an important role for SH3P4 in synaptic vesicle endocytosis.
Asunto(s)
Química Encefálica , Proteínas Portadoras/aislamiento & purificación , Proteínas del Tejido Nervioso/metabolismo , Monoéster Fosfórico Hidrolasas/metabolismo , Secuencia de Aminoácidos , Animales , Proteínas Portadoras/química , Células Cultivadas , Datos de Secuencia Molecular , Peso Molecular , Proteínas del Tejido Nervioso/química , Ratas , Spodoptera , Dominios Homologos srcRESUMEN
Prolonged treatment with serotonin leads to long-term facilitation of sensory-to-motor neuron synapses in Aplysia. We have shown previously that there is a protein synthesis-dependent increase in an autonomous kinase activity that phosphorylates a protein kinase C substrate during an intermediate phase of this facilitation. Here, I report that the increase in autonomous activity was independent of RNA synthesis, suggesting it may play a role in the maintenance phase of synaptic facilitation. Immunoprecipitation experiments using an antibody specific to the Ca(2+)-independent protein kinase C, Apl II, demonstrated that the autonomous kinase activity increased by serotonin emanated from Apl II. Chelerythrine, an inhibitor targeted to the substrate binding site of protein kinase C, also blocked the autonomous kinase activity increased by serotonin. Using immunoblotting experiments and calphostin-C, an inhibitor targeted to the regulatory domain of protein kinase C, the autonomous activity is shown not to be a catalytic fragment of Apl II. Furthermore, a higher concentration of calphostin-C was required to inhibit autonomous kinase activity than regulated kinase activity, suggesting that calphostin-C's binding site in the regulatory domain of Apl II is modified in the autonomous kinase. These data suggest that an autonomous kinase derived from Apl II may play a role in synaptic facilitation in Aplysia.
Asunto(s)
Aplysia/fisiología , Calcio/fisiología , Isoenzimas/química , Potenciación a Largo Plazo/fisiología , Fosfotransferasas/química , Fosfotransferasas/metabolismo , Proteína Quinasa C/química , Animales , Activación Enzimática/fisiología , Inhibidores Enzimáticos/farmacología , Isoenzimas/inmunología , Naftalenos/farmacología , Pruebas de Precipitina , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C/inmunología , Proteína Quinasa C/metabolismo , ARN/biosíntesisRESUMEN
We report here the sequences of two new proteins from Aplysia, aplycalcin and Aplysia neurocalcin. These proteins belong to a family of calcium-binding proteins, found primarily in vertebrate brain and retina, that have been proposed to play a role in calcium-dependent regulation of enzymes in signal transduction pathways. Like other members of this family, the Aplysia proteins have consensus sequences for myristoylation, bind calcium, and translocate from cytosol to membrane when the calcium level is raised above the resting intracellular concentration. Both proteins are relatively enriched in Aplysia nervous system, but are also found to a significant degree in other tissues. The expression of mRNA for these proteins in Aplysia nervous tissue is regulated during development, roughly paralleling the reported emergence of several forms of synaptic plasticity. The messages are present at low levels in stage 11, show a large increase by late stage 12, and decline to a plateau of approximately 30% of the peak value afterward. On the basis of the properties of these proteins and by analogy with proposed functions of some of the retinal homologues, we suggest that these proteins may play a role in mediating calcium-dependent processes in neuronal function. The presence of both proteins in other tissues may suggest analogous roles for the proteins in other cell types.
Asunto(s)
Proteínas de Unión al Calcio/química , Animales , Aplysia , Secuencia de Bases , Western Blotting , Calcio/metabolismo , Proteínas de Unión al Calcio/genética , Calmodulina/química , Calmodulina/genética , Clonación Molecular , ADN Complementario/genética , Regulación del Desarrollo de la Expresión Génica/fisiología , Biblioteca de Genes , Inmunohistoquímica , Proteínas de la Membrana/metabolismo , Datos de Secuencia Molecular , Sistema Nervioso/citología , Sistema Nervioso/embriología , Fenómenos Fisiológicos del Sistema Nervioso , Neuronas/química , Neuronas/fisiología , ARN Mensajero/metabolismo , Conejos , Análisis de Secuencia de ADN , Homología de Secuencia de AminoácidoRESUMEN
Activation of tyrosine kinase-linked receptors has been shown to stimulate Ca2+-independent protein kinase C isoforms in nonneuronal cells. We have examined this signaling pathway in the nervous system. Incubating bag cell neurons from the marine mollusk Aplysia californica with concentrations of insulin known to stimulate a tyrosine kinase-linked receptor in these cells persistently activated and down-regulated the Ca2+-independent protein kinase C (Apl II), whereas insulin only transiently activated and did not down-regulate the Ca2+-activated protein kinase C (Apl I). The effects of insulin may be mediated by activation of phosphoinositide 3-kinase because (a) diC16phosphatidylinositol 3,4,5-trisphosphate, a synthetic phosphoinositide 3-kinase product, stimulated autophosphorylation of baculovirus-expressed Apl II, but not of Apl I, and (b) wortmannin, an inhibitor of phosphoinositide 3-kinase, blocked the activation and down-regulation of Apl II by insulin but not the transient activation of Apl I. These results suggest that activators of tyrosine kinase-linked receptors may mediate some of their effects in neurons through activation of Ca2+-independent protein kinase C isoforms.
Asunto(s)
Proteína Quinasa C/metabolismo , Receptor de Insulina/fisiología , Transducción de Señal/fisiología , Androstadienos/farmacología , Animales , Aplysia , Calcio/fisiología , Regulación hacia Abajo/fisiología , Inhibidores Enzimáticos/farmacología , Neuronas/química , Neuronas/enzimología , Proteínas Tirosina Quinasas/metabolismo , Sensibilidad y Especificidad , WortmaninaRESUMEN
Since long-term memory (LTM) depends on transcription, signals required for LTM must emanate from the cell body. In some cases, signals from the cell body are not only required, but are also sufficient for LTM. However, It is difficult to reconcile this finding with the need to modify synapses independently. To retain synapse specificity during LTM, a form of memory is required that is independent of transcription, and that produces a synaptic 'mark' which interacts with molecules synthesized in the cell body. To reconcile the sufficiency of transcriptional signals for LTM with the need for synapse specificity, I propose that not all LTM is synapse-specific; activation of transcription presynaptically results in cell-wide LTM, whereas postsynaptic transcriptional activation leads to synapse-specific LTM.