Role of Protein Kinase C α in Nerve Growth Factor-Induced Arachidonic Acid Release from PC12 Cells

Abstract: Nerve growth factor (NGF) increases arachidonic acid (AA) release by PC12 pheochromocytoma cells. To explore the role of protein kinase C (PKC) in this action of NGF, PKC was down-regulated by long-term treatment of the cells with phorbol 12-myristate 13-acetate (PMA). Such prolonged exposure to PMA (1 µ M ) resulted in the inhibition of NGF-induced AA release. Moreover, pretreatment of PC12 cells with the protein kinase inhibitor staurosporine or with calphostin C, a specific inhibitor of PKC, also blocks the increase of AA release induced by NGF. These data, as well as that PMA alone can induce AA release in PC12 cells, suggest that PKC is necessary for NGF-induced AA release. Immunoblot analysis of whole cell lysates by using antibodies against various PKC isoforms revealed that our PC12 cells contained PKCs α, δ, ε, and ζ. PMA down-regulation depleted PKCs α, δ, and ε, and partially depleted ζ. To see which isoform was involved in NGF-induced AA release, an isoform-specific PKC inhibitor was used. GO 6976, a compound that inhibits PKCs α and β specifically, blocked NGF-induced AA release. In addition, thymeleatoxin, a specific activator of PKCs α, β, and γ, induced AA release from PC12 cells in amounts comparable with those seen with NGF. Taken together, these data suggest that PKC α plays a role in NGF-induced AA release.     

Pentylenetetrazole-Induced Chemoshock Affects Protein Kinase C and Substrate Proteins in Mouse Brain

Abstract: Protein kinase C (PKC) activity, western blot analysis of PKCα, β, γ, ε, and ζ by isozyme-specific antibodies, and in vitro phosphorylation of endogenous substrate proteins were studied in the mice brain after pentyl-enetetrazole-induced chemoshock. The PKC isozymes and endogenous substrates in the crude cytosolic and membrane fractions were partially purified by DE-52 columns eluted with buffer A containing 100 or 200 m M KCI. This method consistently separates cytosolic and membrane proteins and various PKC isoforms. The 100 m M KCI eluates from DE-52 columns contain more PKC α and β in both cytosol and membrane than the 200 m M KCI eluates, whereas PKCγ, ε, and ζappear in equal amounts in these two eluates. The kinase activity assayed by phosphorylation of exogenous histone was increased in the chemoshocked mice in both the cytosol and membrane of 200 m M KCI eluates. In further analysis by immunoblotting, this increased activity was found to be due to the increase in content of PKC7 isozyme. As for novel-type ε and ζ isozymes, they were not altered in the chemoshocked mice. From autoradiography, the endogenous substrate 17-kDa neurogranin, which was shown below 21 kDa, was mostly eluted by 100 m M KCI from the DE-52 column, whereas 43-kDa neuromodulin, which was also demonstrated in sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, only appeared in the 200 m M KCI eluates. The in vitro phosphorylation of neuromodulin was found to be increased in the chemoshocked mice. Therefore, the increased phosphorylation of neuromodulin and increased content of the PKCγ isoform were involved in the pentylenetetrazole-induced chemoshock.     

Protein kinase C in the human heart: differential regulation of the isoforms in aortic stenosis or dilated cardiomyopathy

Objectives Protein kinase C (PKC) is a central enzyme in the regulation of growth and hypertrophy. Little was known on PKC isoform regulation in human heart. Goal of this study was to characterize the isoforms of protein kinase C in human heart, their changes during ontogenesis, and their regulation in myocardial hypertrophy and heart failure. Methods In left ventricular and atrial samples from adults with end-stage dilated cardiomyopathy (DCM), from adults with severe aortic stenosis (AS), from small infants undergoing repair of ventricular septal defects, and from healthy organ donors (CO), activity of protein kinase C and the expression of its isozymes were examined. Results In the adult human heart, the isoforms PKC-α, PCK-β, PKC-δ, PKC-ε, PKC-λ/-ι, and PKC-ζ were detected both on protein and on mRNA level. All isozymes are subjected to downregulation during ontogenesis. No evidence, however, exists for an isoform shift from infancy to adulthood. DCM leads to a pronounced upregulation of PKC-β. Severe left ventricular hypertrophy in AS, however, recruits a distinct isoform pattern, i.e., isoforms PKC-α, PKC-δ, PKC-ε, PKC-λ/-ι, and PKC-ζ are upregulated, whereas PKC-β is not changed under this condition. Conclusion This work gives evidence for a differential recruitment of human PKC isoforms in various forms of myocardial hypertrophy and heart failure. Gregor Simonis and Steffen K. Briem contributed equally to this work.     

Nicotinic Agonists, Phorbol Esters, and Growth Factors Activate Two Extracellular Signal-Regulated Kinases, ERK1 and ERK2, in Bovine Chromaffin Cells

Treatment of bovine chromaffin cells with nicotinic agonists, phorbol esters, and growth factors increases protein kinase activity toward microtubule-associated protein-2 and myelin basic protein (MBP) in vitro. To characterize the kinases that are activated by these agents, we separated chromaffin cell proteins by electrophoresis in sodium dodecyl sulfate-polyacrylamide gels into which MBP had been incorporated, allowed the proteins to renature, and then assayed MBP kinase activity by incubating the gels with [gamma-32P]ATP. Chromaffin cells contain a family of kinases that phosphorylate MBP in vitro. Two of these kinases, of M(r) 46,000 and 42,000 (PK46 and PK42), were activated by treatment of the cells with dimethylphenylpiperazinium (DMPP), phorbol 12,13-dibutyrate (PDBu), or insulin-like growth factor I (IGF-I). Activation of PK46 and PK42 by DMPP was dependent on extracellular Ca2+, whereas the effects of PDBu and IGF-I were Ca2+ independent. Down-regulation of protein kinase C by incubation of the cells with PDBu abolished the activation of PK46 and PK42 by DMPP, PDBu, and IGF-I. Staurosporine, a protein kinase C inhibitor, prevented the activation of PK46 and PK42 by DMPP and PDBu but did not block the activation of these kinases by IGF-I. Immunoblotting experiments with antiphosphotyrosine (anti-PTyr) antibodies demonstrated that agents that increased the kinase activities of PK46 and PK42 also increased the apparent PTyr content of M(r) 46,000 and 42,000 proteins. PK46 and PK42 comigrated with proteins that reacted with antibodies against extracellular signal-regulated kinases (ERKs). Thus, PK46 and PK42 appear to be the bovine homologues of ERK1 and ERK2.(ABSTRACT TRUNCATED AT 250 WORDS)     

α, βI, βII, δ, and ε Protein Kinase C Isoforms and Compound Activity in the Sciatic Nerve of Normal and Diabetic Rats

Abstract: Defective protein kinase C (PKC) has been implicated in impaired Na⁺,K⁺-ATPase activity in the sciatic nerve of streptozotocin-induced diabetic rats. In the present study, α, βI, βII, γ, δ, and ε isoform-specific antibodies were used in parallel to the measurement of compound PKC activity for the characterization of PKC distribution and isoform expression in sciatic nerves of normal and diabetic rats. To distinguish isoform expression between the axonal and glial compartments, PKC isoforms were evaluated in nerves subjected to Wallerian degeneration and in a pure primary Schwann cell culture. α, βI, βII, δ, and ε but no γ isoforms were detected in sciatic nerve. Similar immunoreactivity was observed in degenerated nerves 3–4 days after transection except for diminished βI and ε species; in Schwann cell cultures, only α, βII, δ, and ε were detected. In normal nerves, two-thirds of PKC compound activity was found in the cytosol and 50% of total enzyme activity translocated to the Na⁺,K⁺-ATPase-enriched membrane fraction with phorbol myristate acetate. Similar redistribution patterns were observed for the immunoreactivity of all isoforms with the exception of δ, which did not translocate to the membrane with phorbol myristate acetate. No abnormality in compound PKC activity, in the immunoreactive intensity, or in the distribution of PKC isoforms could be detected in rat sciatic nerve after 6–12 weeks of diabetes. Thus, defective activation rather than decreased intrinsic PKC activity may occur in diabetic neuropathy.     

Phorbol Ester-Enhanced Noradrenaline Secretion Correlates with the Presence and Activity of Protein Kinase C-α in Human SH-SY5Y Neuroblastoma Cells

Abstract: The effect of inhibition and down-regulation of protein kinase C (PKC) subtypes α, ε, and ζ on noradrenaline (NA) secretion from human SH-SY5Y neuroblastoma cells was investigated. The PKC inhibitor Ro 31-7549 inhibited carbachol-evoked NA release (IC₅₀ 0.6 µ M ) but not 100 m M K⁺-evoked release. In addition, Ro 31-7549 inhibited the enhancement of carbachol- and K⁺-evoked release after pretreatment with 12- O -tetradecanoylphorbol 13-acetate (TPA; 100 n M ) for 8 min, with IC₅₀ values of 0.7 and 2.4 µ M , respectively. Immunoblotting studies showed that prolonged exposure (48 h) of SH-SY5Y cells to phorbol 12,13-dibutyrate (PDBu) or bryostatin-1 caused down-regulation of PKC-α and PKC-ε but not PKC-ζ. Under these conditions, the acute TPA enhancement of NA release was inhibited. Moreover, the inhibition of TPA-enhanced secretion was also apparent after only 2-h exposure to either PDBu or bryostatin-1, conditions that caused down-regulation of PKC-α, but not PKC-ε or ζ. The PKC inhibitor Gö-6976 (2 µ M ), which has been shown to inhibit selectively PKC-α and β in vitro, also inhibited the TPA enhancement of carbachol- and K⁺-evoked NA release by >50%. These data suggest that in SH-SY5Y cells, the ability of TPA to enhance carbachol- and K⁺-evoked NA secretion is due to activation of PKC-\ga.     

Programmed Cell Death in Cortical Chick Embryo Astrocytes Is Associated with Activation of Protein Kinase PK60 and Ceramide Formation

Abstract: Embryonic astrocytes respond readily to serine/threonine kinase regulation in terms of cytoskeleton assembly, mitotic activity, and cell fate. We now present evidence that these responses include apoptosis. Staurosporine induced apoptosis in astrocyte cultures derived from chick embryo cerebral hemispheres, as assayed both by immunocytochemical detection of new 3-hydroxy DNA ends and production of 200-bp DNA fragment laddering. Staurosporine treatment also resulted in the prolonged (>24 h) activation of a 60-kDa serine/threonine protein kinase (PK60), increased ceramide formation (fourfold after 24 h), increased glutamine synthetase activity, and significant apoptosis (40%) after 24 h. PK60 was shown to be cytoskeleton associated and its activity, as measured by phosphorylation of myelin basic protein, was rapid, increased for up to 3 h, and was stable for at least 24 h. Other protein kinase C inhibitors, H8, sphingosine, calphostin C, or the protein kinase A inhibitor KT5720 did not induce either PK60 activation or apoptosis. The dose-dependent increase in [³H]palmitate labeling of ceramide and a specific decrease in labeling of its precursor sphingomyelin were not blocked by the biosynthetic inhibitor fumonisin β1 but were increased (in a dose-dependent manner) by the coaddition of the ceramidase inhibitor oleoylethanolamine. Exogenous addition of C2-ceramide induced apoptosis but did not activate PK60. These results suggest that apoptosis in embryonic astrocytes involves pathways similar to those described in other cell types and that the activation of PK60 and formation of ceramide are early events in the pathway.     

Differential Expression and Subcellular Localization of Protein Kinase C α, β, γ, δ, and ɛ Isoforms in SH-SY5Y Neuroblastoma Cells: Modifications During Differentiation

Abstract: A decrease in protein kinase C activity caused either by treatment with inhibitors, such as staurosporine or H-7, or by prolonged exposure to phorbol diesters has been proposed to be involved in the early events of SH-SY5Y neuroblastoma cell differentiation. Because eight distinct isoforms of protein kinase C with discrete subcellular and tissue distributions have been described, we determined which isoforms are present in SH-SY5Y cells and studied their modifications during differentiation. The α, β, δ, and ɛ isoforms were present in SH-SY5Y cells, as well as in rat brain. Protein kinase C-α and -β₁ were the most abundant isoforms in SH-SY5Y cells, and immunoreactive protein kinase C-δ and -ɛ were present in much smaller amounts than in rat brain. Subcellular fractionation and immunocytochemistry demonstrated that all four isoforms are distributed bimodally in the cytoplasm and the membranes. Immunocytochemical analysis showed that the α isoform is associated predominantly with the plasma membrane and the processes extended during treatment with 12-tetradecanoyl-13-acetyl-β-phorbol or staurosporine, and that protein kinase C-ɛ is predominantly membrane-bound. Its localization did not change during differentiation. Western blots of total SH-SY5Y cell extracts and of subcellular fractions probed with isoform-specific polyclonal antibodies showed that when SH-SY5Y cells acquired a morphologically differentiated phenotype, protein kinase C-α and -ɛ decreased, and protein kinase C-β₁, did not change. These data suggest distinct roles for the different protein kinase C isoforms during neuronal differentiation, as well as possible involvement of protein kinase α and ɛ in neuritogenesis.     

Chronic Sodium Valproate Selectively Decreases Protein Kinase C α and ε In Vitro

Abstract: Valproic acid (VPA) is a fatty acid antiepileptic with demonstrated antimanic properties, but the molecular mechanism or mechanisms underlying its therapeutic efficacy remain to be elucidated. In view of the increasing evidence demonstrating effects of the first-line antimanic drug, lithium, on protein kinase C (PKC), we investigated the effects of VPA on various aspects of this enzyme. Chronic exposure (6–7 days) of rat C6 glioma cells to "therapeutic" concentrations (0.6 m M ) of VPA resulted in decreased PKC activity in both membrane and cytosolic fractions and increased the cytosol/membrane ratio of PKC activity. Western blot analysis revealed isozyme-selective decreases in the levels of PKC α and ε (but not δ or ζ) in both the membrane and cytosolic fractions after chronic VPA exposure; VPA added to reaction mixtures did not alter PKC activity or ³H-phorbol ester binding. Together, these data suggest that chronic VPA indirectly lowers the levels of specific isozymes of PKC in C6 cells. Given the pivotal role of PKC in regulating neuronal signal transduction and modulating intracellular cross-talk between neurotransmitter systems, the specific decreases in PKC α and ε may play a role in the antimanic effects of VPA.     

Modulation of ζ-Protein Kinase C by Cyclic AMP in PC12 Cells Occurs Through Phosphorylation by Protein Kinase A

Abstract: Although cyclic AMP (cAMP) has been reported to cross talk with the protein kinase C (PKC) system, effects of elevated intracellular cAMP on the activities of specific PKC isoforms have not been studied. We report findings from a permeabilized cell assay that was used to examine changes in the activity of the atypical PKC isoforms brought about by exposure of PC12 cells to agents that elevate intracellular cAMP. We found that increases in intracellular cAMP led to rapid stimulation of atypical PKC activity, 40–70% above control, for a sustained period of time, a response that occurred independent of the phorbol 12-myristate 13-acetate (PMA)-sensitive PKC isoforms. Changes in intracellular cAMP levels resulted in a dose-dependent redistribution of ζ-PKC to the cytoplasm with a concomitant increase in the phosphorylation state of the enzyme. Incubation of purified ζ-PKC with increasing concentrations of PKA likewise caused a twofold increase in the phosphorylation state of ζ-PKC. In contrast to the positive effect that PKA-mediated phosphorylation had on the activity of ζ-PKC, the enzyme displayed reduced binding to ras when phosphorylated. Taken together, these findings are consistent with the hypothesis that protein phosphorylation of PKC acts as a positive effector of its enzyme activity and may serve as a negative modulator for interaction with other proteins.     

Selective Activation of Phospholipase Cγ1 and Distinct Protein Kinase C Subspecies in Intracellular Signaling by Hepatocyte Growth Factor/Scatter Factor in Primary Cultured Rat Neocortical Cells

Abstract: Hepatocyte growth factor/scatter factor (HGF) was recently reported to function as a neurotrophic factor in the CNS. To investigate the intracellular signal pathways after activation of the HGF receptor c-Met in primary cultured rat neocortical cells, in vitro kinase assays were performed. HGF stimulation enhances the phosphorylation of endogenous 80- and 45-kDa substrates. Studies with protein kinase inhibitors and phorbol 12-myristate 13-acetate showed that protein kinase C (PKC) is activated intracellularly. The 80-kDa protein was identified to be the major PKC substrate MARCKS. Although four PKC subspecies, PKCα, PKCε, PKCγ, and PKCλ, were expressed in the cells, only PKCα, PKCε, and PKCγ were selectively translocated in the plasma membrane after HGF stimulation. As expected from these three PKC subspecies, phosphorylation of phospholipase Cγ1 (PLCγ1) but not phosphatidylinositol 3-kinase was enhanced, although the stimulation of brain-derived neurotrophic factor induced phosphorylation of phosphatidylinositol 3-kinase. In contrast to the neocortical cells, HGF did not enhance phosphorylation of PLCγ1 in primary astrocytes. We also found that activated PKC(s) served as a major mitogen-activated protein kinase activator in this pathway. These findings suggest that HGF exerts neurotrophic effects through selective phosphorylation of PLCγ1 and activation of distinct PKC subspecies in neocortical cells, most likely neurons.     

Different GABAB-Mediated Effects on Protein Kinase C Activity and Immunoreactivity in Neonatal and Adult Rat Hippocampal Slices

Abstract: The effects of GABA on protein kinase C (PKC) were investigated in rat hippocampal slices at various postnatal ages [postnatal day (P) 1-P60]. At P4, GABA (300 µ M ) induced a rapid (in 1–2 min) 40–50% increase of PKC activity in the membrane fraction and a decrease in the cytosol. These effects were mediated by GABA_B receptors because (a) they were neither blocked by 10 µ M bicuculline nor reproduced by 10 µ M isoguvacine and (b) they were mimicked by the GABA_B agonist baclofen (3–30 µ M ), an effect fully antagonized by the GABA_B antagonist 2-hydroxysaclofen (10 µ M ). A baclofen-induced increased PKC activity in the membrane fraction was only present during the early postnatal period (P1–P14); it was associated with a translocation from the cytosol to the membrane of the immunoreactivity of some PKC isoforms (α-, β-, and ε-PKCs). In contrast, after P21, PKC activity and α-, β-, ε-, and γ-PKC immunoreactivities were decreased by baclofen in the membrane fraction and increased in the cytosol. These results suggest that the stimulation of GABA_B receptors differentially modulates PKC activity via distinct second messenger pathways in developing and mature hippocampi.     

Staurosporine Induces Astrocytic Phenotypes and Differential Expression of Specific PKC Isoforms in C6 Glial Cells

Abstract: In this study we examined the effects of staurosporine, a potent inhibitor of protein kinase C (PKC), on the differentiation of C6 glial cells and on the expression and cellular distribution of specific PKC isoforms. Staurosporine reduced cell proliferation and induced distinctive changes in the morphological appearance of the cells to that characteristic of cells exhibiting astrocytic phenotypes. The differentiative effect of staurosporine was further indicated by the increased expression of two proteins related to astrocytic phenotypes, glial fibrillary acidic protein (GFAP) and glutamine synthetase. Thus, staurosporine induced a dose-dependent increase both in GFAP immunoreactivity and in the activity and protein levels of glutamine synthetase. Staurosporine also induced a decrease in the expression of PKC-β₂ and an increase in that of PKC-γ. In addition, it induced translocation of PKC-ε from the membrane to the cytosol, whereas no differences were observed in the distribution of the other PKC isoforms. The results of our study indicate that staurosporine induced astrocytic phenotypes in glial cells and that changes in the expression and cellular distribution of these PKC isoforms may be related to astrocytic differentiation.     

Ca2+-Dependent and Ca2+-Independent Protein Kinase C Changes in the Brains of Patients with Alzheimer's Disease

Abstract: We examined protein kinase C (PKC) activity in Ca²⁺-dependent PKC (Ca²⁺-dependent PKC activities) and Ca²⁺-independent PKC (Ca²⁺-independent PKC activities) assay conditions in brains from Alzheimer's disease (AD) patients and age-matched controls. In cytosolic and membranous fractions, Ca²⁺-dependent and Ca²⁺-independent PKC activities were significantly lower in AD brain than in control brain. In particular, reduction of Ca²⁺-independent PKC activity in the membranous fraction of AD brain was most enhanced when cardiolipin, the optimal stimulator of PKC-ε, was used in the assay; whereas Ca²⁺-independent PKC activity stimulated by phosphatidylinositol, the optimal stimulator of PKC-δ, was not significantly reduced in AD. Further studies on the protein levels of Ca²⁺-independent PKC-δ, PKC-ε, and PKC-ζ in AD brain revealed reduction of the PKC-ε level in both cytosolic and membranous fractions, although PKC-δ and PKC-ζ levels were not changed. These findings indicated that Ca²⁺-dependent and Ca²⁺-independent PKC are changed in AD, and that among Ca²⁺-independent PKC isozymes, the alteration of PKC-ε is a specific event in AD brain, suggesting its crucial role in AD pathophysiology.     

Protein Kinase C Activation Potentiates the Rapid Secretion of the Amyloid Precursor Protein from Rat Cortical Synaptosomes

Abstract : In this study we have used the presynaptic-rich rat cerebrocortical synaptosomal preparation to investigate the proteolytic cleavage of the amyloid precursor protein (AβPP) by the α-secretase pathway within the βA4 domain to generate a soluble secreted N-terminal fragment (AβPP_s). AβPP was detected in crude cortical synaptosomal membranes, although at a lower density than that observed in whole-tissue homogenates. Protein kinase C (PKC) activation induced a translocation of the conventional PKC isoform β1 and novel PKCε from cytosol to membrane fractions, but there was no alteration in the proportion of AβPP associated with the Tritonsoluble and -insoluble fractions. AβPP_s was constitutively secreted from cortical synaptosomes, with this secretion being enhanced significantly by the direct activation of PKC with phorbol ester. The PKC-induced secretion of AβPP_s was only partially blocked by the PKC inhibitor GF109203X (2.5 μ M ), whereas the phosphorylation of the myristoylated alanine-rich C kinase substrate (MARCKS) protein was significantly inhibited by GF109203X. The differential sensitivities of the MARCKS phosphorylation and AβPP_s secretion to GF109203X may imply that different PKC isoforms are involved in these two events in the synaptosomal system. These findings strongly suggest that the α-secretase activity leading to the secretion of AβPP_s can occur at the level of the presynaptic terminal.     

Phosphorylation by Protein Kinase C of Annexin 2 in Chromaffin Cells Stimulated by Nicotine

Abstract: Annexin 2 phosphorylated in vitro by protein kinase C has been shown to restore partially catecholamine secretion in streptolysin O-permeabilized chromaffin cells depleted of their protein kinase C activity. This result suggested a phosphorylation of annexin 2 in stimulated cells. Nicotine stimulation induced an increase of ³²P incorporation in annexin 2 heavy chain concomitant with catecholamine release. This incorporation results from phosphorylation by protein kinase C because (a) serine was the only phosphorylated residue, (b) ³²P incorporation was inhibited by the protein kinase inhibitors H7, GF 109203X, and staurosporine, and (c) activators of this enzyme, 12- O -tetradecanoylphorbol 13-acetate and 1,2-dioctanoylglycerate, increased the incorporation of radioactivity. The phosphorylated heavy chain had an electrophoretic mobility lower than that of the unmodified one, thus allowing determination of the fraction of phosphorylated protein. In the resting state, a significant fraction of annexin 2 heavy chain was phosphorylated, and nicotine stimulation resulted in an activation of both phosphorylation and dephosphorylation. Phosphorylation was largely increased in the presence of okadaic acid, indicating the involvement of type 1 and 2A phosphatases.     

Nociceptin/Orphanin FQ Activates Mitogen-Activated Protein Kinase in Chinese Hamster Ovary Cells Expressing Opioid Receptor-Like Receptor

Abstract: The effect of nociceptin/orphanin FQ (N/OFQ), an endogenous ligand for the newly identified opioid receptor-like (ORL₁) receptor, on mitogen-activated protein kinase (MAPK) was investigated in Chinese hamster ovary cells stably expressing ORL₁ receptor. N/OFQ rapidly stimulated phosphorylation and activity of MAPK (p42 and p44 isoforms) in a concentration-dependent manner. The p42 isoform was preferentially activated by N/OFQ. Maximal activation (5.4 ± 1.2-fold of basal for p42 isoform) was achieved after a 1-min exposure of cells to 100 nM N/OFQ. The activation was blocked completely by pretreatment with pertussis toxin, but was not reversed by naloxone. U-73122, a phospholipase C-specific inhibitor, significantly inhibited phospholipase C activity, as well as MAPK activation stimulated by N/OFQ. Furthermore, N/OFQ-stimulated MAPK activation was suppressed by a protein kinase C-specific inhibitor, chelerythrine. The results demonstrate that N/OFQ can effectively stimulate MAPK by the activation of ORL₁ receptor and pertussis toxin-sensitive G proteins, and that phospholipase C, as well as protein kinase C, is critically involved in these processes.     

Activation of Protein Kinase C-α and Translocation of the Myristoylated Alanine-Rich C-Kinase Substrate Correlate with Phorbol Ester-Enhanced Noradrenaline Release from SH-SY5Y Human Neuroblastoma Cells

Abstract: The aim of this study was to investigate the mechanism by which short-term pretreatment with the phorbol ester 12- O -tetradecanoylphorbol 13-acetate (TPA; 100 n M ) enhances noradrenaline (NA) release from the human neuroblastoma cell line SH-SY5Y. Subcellular fractionation and immunocytochemical studies demonstrated that an 8-min TPA treatment caused translocation of the α-subtype of protein kinase C (PKC) from the cytosol to the plasma membrane. In contrast, TPA altered the distribution of PKC-ε from cytosolic and membrane-associated to cytoskeleton- and membrane-associated TPA had no effect on the cytosolic location of PKC-ζ. Subcellular fractionation studies also showed that the myristoylated alanine-rich C-kinase substrate (MARCKS), a major neuronal PKC substrate that has been implicated in the mechanism of neurotransmitter release, translocated from membranes to cytosol in response to an 8-min TPA treatment. Under these conditions the level of phosphorylation of MARCKS increased threefold. The ability of TPA to enhance NA release and to cause the translocation and phosphorylation of MARCKS was inhibited by the PKC inhibitor Ro 31-8220 (10 µ M ). Selective down-regulation of PKC subtypes by prolonged exposure to phorbol 12,13-dibutyrate (100 n M ) attenuated the TPA-induced enhancement of NA release and the translocation of MARCKS over an interval similar to that of down-regulation of PKC-α (but not -ε or -ζ). Thus, we have demonstrated a strong correlation between the translocation of MARCKS and the enhancement of NA release from SH-SY5Y cells due to the TPA-induced activation of PKC-α.     

Staurosporine: An Effective Inhibitor for Ca2+/Calmodulin-Dependent Protein Kinase II

We investigated the effect of staurosporine on Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) purified from rat brain. (a) Staurosporine (10-100 nM) inhibited the activity of CaM kinase II. The half-maximal and maximal inhibitory concentrations were 20 and 100 nM, respectively. (b) The inhibition with staurosporine was of the noncompetitive type with respect to ATP, calmodulin, and phosphate acceptor (beta-casein). (c) Staurosporine suppressed the auto-phosphorylation of alpha- and beta-subunits of CaM kinase II at concentrations similar to those at which the enzyme activity was inhibited. (d) Staurosporine also attenuated the Ca2+/calmodulin-independent activity of the autophosphorylated CaM kinase II. These results suggest that staurosporine inhibits CaM kinase II by interacting with the catalytic domain, distinct from the ATP-binding site or substrate-binding site, of the enzyme and that staurosporine is an effective inhibitor for CaM kinase II in the cell system.