Translocation Of Protein Kinase C Alpha Research Articles

Mitochondrial dysfunction increases bronchopulmonary C‐fiber excitability via PKC alpha signaling

Airway sensory nerve excitability is a key determinant of respiratory disease‐associated reflexes and sensations such as cough and dyspnea. One notable feature of peripheral terminals of sensory nerves is that they are packed with mitochondria. Mitochondrial dysfunction and subsequent oxidative stress has been reported for a variety of cell types in inflammatory diseases. We have previously shown that reactive oxygen species (ROS) produced by Antimycin A (a mitochondrial complex III Qi site inhibitor) increase excitability in bronchopulmonary nociceptive C‐fibers. This increased excitability decreases the mechanical threshold for activation and increases action potential firing in response to a P2X2/3 agonist. This Antimycin A–induced hyperexcitability is dependent on mitochondrial ROS and is blocked by intracellular antioxidants.Given that ROS are known to activate protein kinase C (PKC), we hypothesized that this increased excitability was due to PKC activation. We used dissociated vagal ganglion neurons to study activation of the PKC isoforms: alpha, beta1, beta2, delta and epsilon in response to Antimycin‐A. Cells were stained with antibodies against the specific isoforms to determine their cellular location. Of these isoforms, only PKC alpha translocated to the region of the cell membrane in response to Antimycin A treatment.We also examined the effects of Antimycin A induced ROS production on sensory nerve terminals. We recorded action potential firing from the terminals of individual bronchopulmonary C‐fibers using a mouse ex vivo lung‐vagal ganglia preparation. C‐fibers were characterized as nociceptors or non‐nociceptors based upon conduction velocity and response to transient receptor potential (TRP) channel agonists. Antimycin A–induced hyperexcitability was inhibited by the PKC inhibitor bisindolylmaleimide (BIM) I, but not by its inactive analog BIM V. Further, this BIM I inhibition occurred at a concentration that specifically blocks activity of PKC alpha over other isoforms. In conclusion, ROS evoked by mitochondrial dysfunction caused nociceptor hyperexcitability via the translocation and activation of PKC alpha.Support or Funding InformationThis work was supported by the National Heart Lung and Blood Institute in Bethesda, USA (R01‐HL119802)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Secreted PLA2 induces proliferation in astrocytoma through the EGF receptor: another inflammation-cancer link

We have investigated mechanisms that contribute to reinforce the relationship between inflammation and cancer. Secreted phospholipase A(2) group IIA (sPLA(2)-IIA) is a molecule relevant in inflammatory events and has been proposed as a marker for some of these. Previously, we reported the mitogenic properties of this sPLA(2) in the human astrocytoma cell line 1321N1. Here, we go deeper into the mechanisms that link this inflammatory protein with proliferation in one of the most aggressive types of tumors. We found that phosphorylation of the extracellular regulated kinase (ERK) was preceded by the activation of the small GTPase Ras, and both failed to be activated by inhibiting protein kinase C (PKC). Fractionation and immunofluorescence studies revealed translocation of PKC alpha, delta, and epsilon to the membrane fraction upon stimulation with sPLA(2)-IIA. Immunoprecipitation analysis showed that sPLA(2)-IIA induces phosphorylation of the epidermal growth factor receptor (EGFR) through a PKC-dependent pathway. We found that phosphorylation of this receptor contributed to Ras and ERK activation and that inhibition of ERK, PKC, and EGFR blocked the mitogenic response induced by sPLA(2)-IIA. This study showed that sPLA(2)-IIA is able to bring into play EGFR to trigger its signaling and that PKC leads the distribution of resources. Interestingly, we found that this is not a cell-specific response, because sPLA(2)-IIA was also able to transactivate EGFR in MCF7 human breast cancer cells. Therefore, this mechanism could contribute to worsen the prognosis of a tumor in an inflammatory microenvironment. We also present more links of the tumor chain possibly susceptible to targeting.

Shikonin reduces oedema induced by phorbol ester by interfering with IκBα degradation thus inhibiting translocation of NF‐κB to the nucleus

In the present paper we studied the effect of shikonin on ear oedema induced by 12-O-tetradecanoylphorbol-13-acetate (TPA), and determined the mechanisms through which shikonin might exert its topical anti-inflammatory action. Acute ear oedema was induced in mice by topical application of TPA. The in vitro assays used macrophages RAW 264.7 cells stimulated with lipopolysaccharide. Cyclooxygenase-2, inducible nitric oxide synthase, protein kinase Calpha, extracellular signal-regulated protein kinase (ERK), phosphorylated ERK (pERK), c-Jun N-terminal kinase (JNK), pJNK, p38, p-p38, p65, p-p65, inhibitor protein of nuclear factor-kappaB (NF-kappaB) (IkappaBalpha) and pIkappaBalpha were measured by Western blotting, activation and binding of NF-kappaB to DNA was detected by reporter gene and electrophoretic mobility shift assay, respectively, and NF-kappaB p65 localization was detected by immunocytochemistry. Shikonin reduced the oedema (inhibitory dose 50 = 1.0 mg per ear), the expression of cyclooxygenase-2 (70%) and of inducible nitric oxide synthase (100%) in vivo. It significantly decreased TPA-induced translocation of protein kinase Calpha, the phosphorylation and activation of ERK, the nuclear translocation of NF-kappaB and the TPA-induced NF-kappaB-DNA-binding activity in mouse skin. Moreover, in RAW 264.7 cells, shikonin significantly inhibited the binding of NF-kappaB to DNA in a dose-dependent manner and the nuclear translocation of p65. Shikonin exerted its topical anti-inflammatory action by interfering with the degradation of IkappaBalpha, thus inhibiting the activation of NF-kappaB.

Activation of protein kinase C-α is essential for stimulation of cell proliferation by ceramide 1-phosphate

We previously demonstrated that ceramide-1-phosphate (C1P) stimulates fibroblast and macrophage proliferation, but the mechanisms involved in this action have only been partially described. Here we demonstrate that C1P induces translocation of protein kinase C-alpha (PKC-α) from the soluble to the membrane fraction of bone marrow-derived macrophages. Translocation of this enzyme was accompanied by its phosphorylation on Ser 657 residue. Activation of PKC-α was independent of prior stimulation of phosphatidylinositol-dependent or phosphatidylcholine-dependent phospholipase C activities, but required activation of sphingomyelin synthesis. Inhibition of PKC-α activation also blocked C1P-stimulated macrophage proliferation indicating that this enzyme is essential for the mitogenic effect of C1P.

Involvement of protein kinase C isoenzymes in Trypanosoma cruzi metacyclogenesis induced by oleic acid

Previously, we showed that oleic acid (OA) induces Trypanosoma cruzi metacyclogenesis through a signaling pathway involving de novo diacylglycerol biosynthesis and simultaneous protein kinase C (PKC) activation. Herein, we demonstrated that OA also triggers a transient Ca(2+) signal in epimastigotes, necessary for parasite differentiation, that could account for PKC activation. In addition, we found that this free fatty acid (FFA) directly stimulated in vitro the activity of T. cruzi PKC in a dose-response way. We determined the presence of classical and novel PKC isoenzymes that were differentially expressed in the infective amastigotes (alpha and delta) and tripomastigotes (alpha, beta, and gamma) and in the non-infective epimastigotes (alpha, beta, gamma, and delta). We also demonstrated that OA induced in epimastigotes the translocation of PKC alpha, beta, gamma, and delta to the membrane, indicating a selective effect of this FFA. To establish a correlation between T. cruzi metacyclogenesis induced by OA and the activation of a particular PKC isoenzyme, the specific PKC inhibitors Ro 32-0432 and Rottlerin (9-30 nM and 5-35 microM, respectively) were employed. These compounds, even at the lowest concentrations assayed, abrogated both epimastigote differentiation and membrane translocation of PKC beta, gamma, and delta. These findings strongly support a key role for classical and novel PKC isoenzymes in the signaling pathways involved in T. cruzi metacyclogenesis induced by OA.

Dual regulation of the cardiac L-type calcium channel in L6 cells by protein kinase C

The role of protein kinase C (PKC) in the regulation of cardiac L-type Ca(2+) channel activity (LCC) was investigated in L6 rat neonatal myoblasts. Depolarization of fura-2 loaded cells with 140mM KCl activated a Ba(2+) influx pathway that was blocked by nifedipine and stimulated by (-) Bay K 8644. At least two splice variants of the alpha(1C) subunit of the cardiac LCC were identified by PCR; the alpha(1S) subunit of the skeletal muscle LCC was not detected. Peptides that specifically inhibit translocation of the novel, Ca(2+)-independent delta and epsilon PKC isozymes reduced Ba(2+) influx by 27% and 19%, respectively, whereas a corresponding peptide directed against translocation of classical PKC alpha had no effect. Ingenol 3,20-dibenzoate, an agent reported to selectively activate novel PKCs, increased Ba(2+) uptake by 31% while ethanol, a PKC epsilon agonist, enhanced uptake by 38%. In contrast, selective activation of classical PKCs with thymeleatoxin or an agonist peptide reduced Ba(2+) influx by 23-33%. Ba(2+) influx was reduced by 30-40% when cells were treated with either a PKC inhibitor (Gö 6983, bisindolylmaleimide) or the PKC activator phorbol-12-myristate-13-acetate. We propose that novel, Ca(2+)-insensitive PKC(s) enhance cardiac Ca(2+) channel activity in L6 cells under basal conditions while activation of the classical, Ca(2+)-sensitive PKC(s) inhibits channel activity. These findings provide the first evidence that different PKC isozymes exert class-specific opposing effects on cardiac L-type Ca(2+) channel activity in L6 myoblasts.

ERK1/2 regulates two sequential steps promoting monocyte survival to peroxynitrite

Previous studies from our laboratory indicate that cytosolic phospholipase A(2) (cPLA(2))-released arachidonic acid promotes monocyte/macrophage survival in the presence of peroxynitrite. In particular, the lipid messenger is metabolised by 5-lipoxygenase (5-LO) to 5-hydroxyeicosatetraenoic acid and causes the mitochondrial translocation of protein kinase Calpha (PKCalpha), an event associated with the cytosolic accumulation of Bad and Bax. Here we show that phosphorylation reactions driven by extracellular regulated kinase 1/2 (ERK1/2) critically regulate the activation/nuclear translocation of 5-LO. Inhibition of ERK1/2 was invariably associated with the cytosolic localisation of PKCalpha, the mitochondrial accumulation of Bad and Bax and with a rapid mitochondrial permeability transition-dependent necrosis. All these events were prevented by nanomolar concentrations of 5-hydroxyeicosatetraenoic acid. Hence, in addition to the previously characterised effects on cPLA(2), ERK1/2 critically regulates 5-LO activity in the absence of additional downstream targets in the survival signalling preventing peroxynitrite toxicity.

Apolipoprotein A‐I increases association of cytosolic cholesterol and caveolin‐1 with microtubule cytoskeletons in rat astrocytes

Apolipoprotein (apo) A-I induces rapid translocation of protein kinase Calpha and phospholipase Cgamma, and slow translocation of caveolin-1 and newly synthesized cholesterol to the cytosolic lipid-protein particle (CLPP) fraction in rat astrocytes. In order to understand the function of CLPP, we investigated the interaction with cytoskeletons of CLPP-related proteins such as caveolin-1 and protein kinase Calpha and of CLPP-related lipids in rat astrocytes. Under the conditions that microtubules were depolymerized, association of cytosolic caveolin-1 with protein kinase Calpha and alpha-tubulin was enhanced when the cells were treated with apoA-I for 5 min. This association was suppressed by a scaffolding domain-peptide of caveolin-1. Association with the microtubule-like filaments of cytosolic lipids, caveolin-1 and protein kinase Calpha was also increased by the apoA-I treatment and inhibited by the scaffolding domain peptide. Paclitaxel (taxol), a compound to stabilize microtubules, suppressed the apoA-I-mediated intracellular translocation and release from the cells of the de novo synthesized cholesterol and phospholipid. The findings suggested that the association of CLPP with microtubules is mediated by a scaffolding domain of caveolin-1, induced by apoA-I and involved in regulation of intracellular cholesterol trafficking for assembly of cellular lipids to apoA-I-high-density lipoprotein (HDL).

Sevoflurane Stimulates MAP Kinase Signal transduction through the Activation of PKC .ALPHA. and .BETA.II in Fetal Rat Cerebral Cortex Cultured Neuron

Protein kinase C (PKC) is a key enzyme that participates in various neuronal functions. PKC has also been identified as a target molecule for general anesthetic actions. Raf, mitogen-activated protein kinase (MEK) and extracellular signal-regulated kinase (ERK1/2) have been thought to be target effectors of PKC. In the present study, we attempted to evaluate the effect of sevoflurane on PKC/MAPK cascade signaling in cultured fetal rat cerebral ­cortex neurons, prepared from embryonic day 18 fetuses. The effects of sevoflurane on the translocation of 7 PKC isoforms (α, βI, βII, γ, δ, ɛ and ζ) were observed by immunoblotting using isoform-selective antibodies to PKCs. The treatment of neurons with sevoflurane induced the translocation of PKC α and PKC βII species from the cytosol to the membrane fraction, which indicated the activation of these PKC isoforms. In contrast, there was no clear change in the distribution of other PKC isoforms. We next examined whether the specific activation of PKC α and βII by sevoflurane could stimulate the MAP kinase signaling pathway in cultured neurons. Raf phosphorylation was increased by the administration of 0.25 mM sevoflurane. The phosphorylation of Raf proteins reached a maximum at 5–10 min. Subsequently, the phosphorylation of MEK proteins was increased at 10–15 min after sevoflurane treatments. That of ERK proteins was induced at 15–60 min. Moreover, the phosphorylation of ERK induced by sevoflurane was significantly decreased by the treatment of PKC inhibitor (staurosporine) and MEK inhibitor (PD98059). On the other hand, the contents of total Raf, MEK and ERK proteins were relatively constant at all times examined. To examine the ­localization of phosphorylated-ERK protein, immunohistochemical staining of sevoflurane-treated cultured neurons was performed. The phosphorylated-ERK proteins were markedly accumulated in both the cytosol of the cell body and the neurites in the neuronal cells with time after 0.25 mM sevoflurane-treatment. These results demonstrated that sevoflurane induced the phosphorylation of the MAP kinase cascade through the activation of the PKC α and PKC βII species.

The Role of the Protein Kinase C (PKC) Family in Glucocorticoid-Induced Apoptosis of Human Leukemic Cells.

Glucocorticoids (GC's) are among the most important clinical agents for the treatment of human leukemias. Cells exist, however, that have a functional glucocorticoid (GC) receptor, but are resistance to GC-induced apoptosis—suggesting that major signal transduction pathways (ie PKC family) may influence cellular response to GC. In order to determine whether glucocorticoid-induced apoptosis is mediated by members of the PKC family, we first screened the CEM acute T-cell lymphoblastic leukemic cell line for the presence or absence of all known PKC isoforms. We used two clonal cell lines that were derived from the same parent, CEM-CCRF, but which subsequently developed a pronounced difference in their sensitivity to glucocorticoids. One of these cell lines, CEM C7-14 is sensitive to dexamethasone-induced apoptosis, while the second cell line, CEM C1-15, is resistant. Of the 12 known PKC isoforms, 5 were detected at the basal level, and furthermore that specific members (PKC iota) was up-regulated during resistance, while PKC theta was up-regulated during apoptosis. Selectively inhibiting the entire PKC family caused increased cellular susceptibility to GC-induced apoptosis. Selectively targeting specific isoforms (PKC iota) caused resistant cells to become sensitive to apoptosis. Using immunoflouresence imaging, we showed the selective translocation of PKC's iota, theta, and alpha after treatment with glucocorticoids. In addition, we showed specific phosphorylation of the isoforms iota, theta, and alpha in cells undergoing DEX-induced apoptosis. Two-dimensional proteomic analysis indicated a large number of proteins in CEM cells that show significant change after DEX treatment. We successfully identified several proteins which were regulated both after treatment with DEX alone, and after treatment with DEX + the PKC specific inhibitor Safingol. These proteins may represent the potential cross talk between the glucocorticoid and PKC apoptosis pathways. Taken together, these studies show that the PKC family is directly involved in glucocorticoid-induced apoptosis.

Prostaglandin E2Enhances Neurotrophin-4 Production via EP3 Receptor in Human Keratinocytes

Atopic dermatitis is characterized by increased skin innervation. The expression of neurotrophin-4 is enhanced in the epidermal keratinocytes of lesions with atopic dermatitis and may be related to hyperinnervation in these lesions. Prostaglandin E(2) (PGE(2)) levels are increased in lesions with atopic dermatitis; thus, PGE(2) may be involved in the development of this disease. We examined the in vitro effects of PGE(2) on neurotrophin-4 production in human keratinocytes. PGE(2) and EP1/EP3 agonist sulprostone increased neurotrophin-4 secretion and mRNA levels without altering its mRNA stability. Antisense Sp1 oligodeoxynucleotide and Sp1 inhibitor mithramycin A suppressed PGE(2) and sulprostone-induced neurotrophin-4 expression, indicating the requirement for Sp1 for expression. PGE(2) or sulprostone markedly enhanced the phosphorylation, DNA binding, and transcriptional activity of Sp1 and modestly increased Sp1 mRNA and protein levels. PGE(2) or sulprostone induced the membrane translocation of protein kinase Calpha and the phosphorylation of extracellular signal-regulated kinase (ERK). PGE(2)-induced increases in neurotrophin-4 expression, Sp1 transcriptional and DNA-binding activity, Sp1 mRNA and protein levels, and ERK phosphorylation were suppressed by antisense EP3 oligodeoxynucleotide, inhibitors of phosphatidylinositol-specific phospholipase C, conventional protein kinase C, and mitogen-activated protein kinase/ERK kinase 1 (MEK1). These results suggest that PGE(2) enhances neurotrophin-4 production by activating Sp1 via the EP3/phosphatidylinositol-specific phospholipase C/protein kinase Calpha/MEK1/ERK pathway. PGE(2) may promote innervation in skin lesions with atopic dermatitis via the induction of neurotrophin-4.

Chronic exposure to ammonia induces isoform‐selective alterations in the intracellular distribution and NMDA receptor‐mediated translocation of protein kinase C in cerebellar neurons in culture

Hyperammonemia is responsible for most neurological alterations in patients with hepatic encephalopathy by mechanisms that remain unclear. Hyperammonemia alters phosphorylation of neuronal protein kinase C (PKC) substrates and impairs NMDA receptor-associated signal transduction. The aim of this work was to analyse the effects of hyperammonemia on the amount and intracellular distribution of PKC isoforms and on translocation of each isoform induced by NMDA receptor activation in cerebellar neurons. Chronic hyperammonemia alters differentially the intracellular distribution of PKC isoforms. The amount of all isoforms (except PKC zeta) was reduced (17-50%) in the particulate fraction. The contents of alpha, beta1, and epsilon isoforms decreased similarly in cytosol (65-78%) and membranes (66-83%), whereas gamma, delta, and theta; isoforms increased in cytosol but decreased in membranes, and zeta isoform increased in membranes and decreased in cytosol. Chronic hyperammonemia also affects differentially NMDA-induced translocation of PKC isoforms. NMDA-induced translocation of PKC alpha and beta is prevented by ammonia, whereas PKC gamma, delta, epsilon, or theta; translocation is not affected. Inhibition of phospholipase C did not affect PKC alpha translocation but reduced significantly PKC gamma translocation, indicating that NMDA-induced translocation of PKC alpha is mediated by Ca2+, whereas PKC gamma translocation is mediated by diacylglycerol. Chronic hyperammonemia reduces Ca+2-mediated but not diacylglycerol-mediated translocation of PKC isoforms induced by NMDA.

Apolipoprotein A-I induces translocation of protein kinase Cα to a cytosolic lipid-protein particle in astrocytes

Apolipoprotein A-I (apoA-I) induces the translocation of newly synthesized cholesterol as well as caveolin-1 to the cytosolic lipid-protein particle (CLPP) fraction in astrocytes before its appearance in high density lipoprotein generated in the medium (Ito, J., Y. Nagayasu, K. Kato, R. Sato, and S. Yokoyama. 2002. Apolipoprotein A-I induces translocation of cholesterol, phospholipid, and caveolin-1 to cytosol in rat astrocytes. J. Biol. Chem. 277: 7929-7935). We here report the association of signal-related molecules with CLPP. ApoA-I induces rapid translocation of protein kinase Calpha to the CLPP fraction and its phosphorylation in astrocytes. ApoA-I also induces the translocation of phospholipase Cgamma to CLPP. Diacylglyceride (DG) production is increased by apoA-I in the cells, with a maximum at 5 min after the stimulation, and the increase takes place also in the CLPP fraction. An inhibitor of receptor-coupled phospholipase C, U73122, inhibited all the apoA-I-induced events, such as DG production, cholesterol translocation to the cytosol, release of cholesterol, and translocation of protein kinase Calpha into the CLPP fraction. CLPP may thus be involved in the apoA-I-initiated signal transduction in astrocytes that is related to intracellular cholesterol trafficking for the generation of high density lipoprotein in the brain.

Histamine Enhances the Production of Granulocyte-Macrophage Colony-Stimulating Factor via Protein Kinase Cα and Extracellular Signal-Regulated Kinase in Human Keratinocytes

The production of granulocyte-macrophage colony-stimulating factor (GM-CSF) in keratinocytes is related to the chronicity of atopic dermatitis. Mast cell-derived histamine contributes to the cross-talk between mast cells and keratinocytes. We examined the effects of histamine on GM-CSF production in human keratinocytes. Histamine increased GM-CSF secretion, mRNA stability and promoter activity. Activator protein-1 (AP-1) and nuclear factor-kappaB (NF-kappaB) elements on the promoter were responsible for the activation by histamine. Histamine enhanced transcriptional activity and DNA binding of AP-1 and NF-kappaB. Histamine shifted AP-1 composition form c-Jun homodimers to c-Fos/c-Jun heterodimers, and transiently expressed c-Fos protein. Histamine rapidly induced the phosphorylation and degradation of inhibitory kappaB. Histamine induced membrane translocation of protein kinase Calpha. Histamine-induced GM-CSF production was completely abolished by H1 antagonist pyrilamine and conventional protein kinase C inhibitor Gö6976, and partially suppressed by PD98059 which inhibits the activation of extracellular signal-regulated kinase. Gö6976 and PD98059 suppressed histamine-induced c-Fos expression and AP-1 activation. Gö6976 and PD98059 suppressed histamine-induced enhancement of NF-kappaB transcriptional activity. Histamine-induced phosphorylation and degradation of inhibitory kappaB was suppressed by Gö6976, but not by PD98059. These results suggest that histamine may enhance GM-CSF production at transcriptional and posttranscriptional levels via H1 receptor, protein kinase Calpha and extracellular signal-regulated kinase.

Magnesium sulfate induces translocation of protein kinase C isoenzymes alpha and delta in myometrial cells from pregnant women

ObjectivesThe purpose of this study was to determine the effect of magnesium sulfate on protein kinase C (PKC) translocation in myometrial cells from pregnant women. Study designMyometrium was obtained at the time of cesarean delivery from women at term before labor. Cultured myometrial cells were treated with magnesium sulfate (3, 5, and 10 mmol/L), oxytocin (0.1 μmol/L), or 12-O-tetradecanoylphorbol-13-acetate (TPA, 0.1 μmol/L). The translocation of PKC isozymes alpha (calcium dependent) and delta (calcium independent) from cytosol to membrane fractions was assessed with use of Western blot analysis. ResultsIn unexposed control cells, the majority of PKC alpha and delta was located in the cytosol fraction. Exposure to magnesium sulfate for 60 minutes induced translocation of both PKC alpha and delta at concentrations as low as 5 and 3 mmol/L, respectively. The magnitude of magnesium sulfate induced translocation for PKC delta is similar to that seen after oxytocin or TPA exposure but less for PKC alpha. Exposure to oxytocin for 30 minutes and 60 minutes induced translocation of PKC alpha and delta, respectively. Exposure to TPA for 5 and 30 minutes induced translocation of PKC alpha and PKC delta, respectively. In calcium-free media, only TPA induced translocation of these two isoenzymes. ConclusionMagnesium sulfate stimulates PKC translocation in cultured myometrial cells from pregnant women. Magnesium sulfate and oxytocin require extracellular calcium to induce translocation of both PKC alpha and delta.

Thyrotropin-releasing hormone increases phospholipase D activity through stimulation of protein kinase C in GH3 cells.

Activation of phospholipase D was investigated after treatment of GH3 cells with thyrotropin-releasing hormone. Thyrotropin-releasing hormone treatment resulted in both time- and dose-dependent increases of phospholipase D activity, translocation of protein kinase C-alpha and -beta I isozymes from cytosol to membrane within 30 min, and approx 43-fold increase of phosphatidylinositol-specific phospholipase C activity. Intracellular calcium concentration was rapidly increased and diacyglycerol level remained high up to 3 h after the treatment. Pretreatment of the cells with U73122, a potent inhibitor of phosphatidylinositol-specific phospholipase C, inhibited thyrotropin-releasing hormone-induced phospholipase D activation. Protein kinase C activity was down-regulated by pretreatment of the GH3 cells with either protein kinase C inhibitors (RO320432, GF109203X) or preincubation of the cells with phorbol myristrate acetate (500 nM) for 24 h. This treatment largely abolished the thyrotropin-releasing hormone-induced activation of phospholipase D, thus further confirming the involvement of protein kinase C in the activation. These results suggest that thyrotropin-releasing hormone-induced phospholipase D activation may be due to phosphatidylinositol-specific phospholipase C, and activation of protein kinase C isozymes is responsible for this stimulation.

CPKC-dependent Sequestration of Membrane-recycling Components in a Subset of Recycling Endosomes

In addition to the classical role of protein kinase C (PKC) as a mediator of transmembrane signals initiated at the plasma membrane, there is also significant evidence to suggest that a more sustained PKC activity is necessary for a variety of long term cellular responses. To date, the subcellular localization of PKC during sustained activation has not been extensively studied. We report here that long term activation of PKC (1 h) leads to the selective translocation of classical PKC isoenzymes, alpha and betaII, to a juxtanuclear compartment. Juxtanuclear translocation of PKC required an intact C1 and C2 domain, and occurred in a microtubule-dependent manner. This juxtanuclear compartment was localized close to the Golgi complex but displayed no overlap with Golgi markers, and was resistant to dispersal with Golgi disrupting agents, brefeldin A and nocodazole. Further characterization revealed that PKCalpha and betaII translocated to a compartment that colocalized with the small GTPase, rab11, which is a marker for the subset of recycling endosomes concentrated around the microtubule-organizing center/centrosome. Analysis of the functional consequence of cPKC translocation on membrane recycling demonstrated a cPKC-dependent sequestration of transferrin, a marker of membrane recycling, in the cPKC compartment. These results identify a novel site for cPKC translocation and define a novel function for the sustained activation of PKCalpha and betaII in regulation of recycling components.

Intracytoplasmic domains of MHC class II molecules are essential for lipid-raft-dependent signaling.

In addition to their role in antigen presentation, major histocompatibility complex (MHC) class II molecules have been widely described as signaling proteins in diverse antigen-presenting cells (APCs) including B cells and dendritic cells. By contrast, little is known of the signaling function of MHC class II molecules expressed in solid tumors. We describe the functional organization and signaling ability of I-Ak expressed in a sarcoma, and report the recruitment of I-Ak to lipid rafts after MHC class II engagement. Lipid raft integrity was required for I-Ak-mediated reorganization of the actin cytoskeleton and translocation of protein kinase C-alpha(PKC-alpha) to the precise site of stimulation via I-Ak. Truncation of the intracytoplasmic domains of I-Ak did not perturb I-Ak recruitment to lipid rafts but abrogated PKC-alpha translocation and actin rearrangement. PKC-alpha was detected in lipid microdomains and enrichment of activated PKC-alphain lipid rafts was induced by I-Ak signaling. Ordering of the molecular events following engagement of the MHC class II molecules revealed that I-Ak recruitment to lipid rafts precedes signaling. This is consistent with the absence of a requirement for the intracytoplasmic tails for localization to lipid rafts. These data reveal that lipid-rich microdomains play a key role in MHC class II-mediated signaling in a solid tumor.

The catalytic domain limits the translocation of protein kinase C alpha in response to increases in Ca2+ and diacylglycerol.

Translocation of protein kinase C (PKC) alpha, beta II, delta and epsilon fused to enhanced green fluorescent protein (EGFP) was studied in living neuroblastoma cells by confocal microscopy. Exposure to carbachol elicited transient translocation of PKC alpha-EGFP and beta II-EGFP in most of the cells, PKC delta-EGFP in a few cells and induced sustained translocation of PKC epsilon-EGFP. To monitor levels of Ca(2+) and diacylglycerol and the translocation of PKC in the same cell, the Ca(2+)-sensitive C2 domain, diacylglycerol-sensitive C1 domains and full-length PKC were fused to red, cyan and yellow fluorescent proteins respectively. PKC alpha was translocated a few seconds after the C2 domain, which represents an increase in Ca(2+). This delay was insensitive to removal of the pseudosubstrate in PKC alpha, but the isolated regulatory domain translocated simultaneously with the C2 domain. Translocation of PKC epsilon coincided with the increase in diacylglycerol. Ionomycin induced translocation of PKC alpha and the C2 domain, whereas 1,2-dioctanoylglycerol caused translocation of the C1 domains and PKC epsilon, but not PKC alpha. Experiments with individual C1 domains showed that treatment with carbachol or phorbol 12,13-dibutyrate elicited translocation of PKC alpha C1a, PKC epsilon C1a and PKC epsilon C1b, whereas PKC alpha C1b was largely insensitive to these agents. In contrast with full-length PKC alpha, the regulatory domain of PKC alpha and pseudosubstrate-devoid PKC alpha responded to the carbachol-stimulated increase in diacylglycerol.

Dependence of Gonadotropin-releasing Hormone-induced Neuronal MAPK Signaling on Epidermal Growth Factor Receptor Transactivation

The hypothalamic decapeptide, gonadotropin-releasing hormone (GnRH), utilizes multiple signaling pathways to activate extracellularly regulated mitogen-activated protein kinases (ERK1/2) in normal and immortalized pituitary gonadotrophs and transfected cells expressing the GnRH receptor. In immortalized hypothalamic GnRH neurons (GT1-7 cells), which also express GnRH receptors, GnRH, epidermal growth factor (EGF), and phorbol 12-myristate 13-acetate (PMA) caused marked phosphorylation of ERK1/2. This action of GnRH and PMA, but not that of EGF, was primarily dependent on activation of protein kinase C (PKC), and the ERK1/2 responses to all three agents were abolished by the selective EGF receptor kinase inhibitor, AG1478. Consistent with this, both GnRH and EGF increased tyrosine phosphorylation of the EGF receptor. GnRH and PMA, but not EGF, caused rapid phosphorylation of the proline-rich tyrosine kinase, Pyk2, at Tyr(402). This was reduced by Ca(2+) chelation and inhibition of PKC, but not by AG1478. GnRH stimulation caused translocation of PKC alpha and -epsilon to the cell membrane and enhanced the association of Src with PKC alpha and PKC epsilon, Pyk2, and the EGF receptor. The Src inhibitor, PP2, the C-terminal Src kinase (Csk), and dominant-negative Pyk2 attenuated ERK1/2 activation by GnRH and PMA but not by EGF. These findings indicate that Src and Pyk2 act upstream of the EGF receptor to mediate its transactivation, which is essential for GnRH-induced ERK1/2 phosphorylation in hypothalamic GnRH neurons.