Protein Kinase C Subtypes Research Articles

Effects of exercise and bryostatin-1 on functional recovery and posttranslational modification in the perilesional cortex after cerebral infarction.

Strokes can cause a variety of sequelae, such as paralysis, particularly in the early stages after stroke onset. Rehabilitation therapy atthis time often provides some degree of paralysis recovery. Neuroplasticity in the peri-infarcted cerebral cortex induced by exercise training may contribute to recovery of paralysis after cerebral infarction. However, the molecular mechanism of this process remains unclear. This study focused on brain protein kinase C (PKC), which is speculated to be involved in neuroplasticity. We evaluated the functional recovery of cerebral infarction model rats, by using rotarod test after running wheel training and with/without administration of bryostatin, a PKC activator. In addition, the expression of phosphorylated and unphosphorylated PKC subtypes, glycogen synthase kinase 3β (GSK3β), and collapsin response-mediator proteins 2 (CRMP2) were analyzed by Western blotting. In the rotarod test, bryostatin administration alone had no effect on gait duration, but the combination of training and this drug significantly prolonged gait duration compared with training alone. In protein expression analysis, the combination of training and bryostatin significantly increased phosphorylation of PKCα and PKCε isoforms, increased phosphorylation of GSK3β, which acts downstream of PKC, and decreased phosphorylation of CRMP2. The effect of bryostatin in combination with training appears to be mediated via PKC phosphorylation, with effects on functional recovery occurring through the downstream regulation of GSK3β and CRMP2 phosphorylation.

Abundant oleoyl-lysophosphatidylethanolamine in brain stimulates neurite outgrowth and protects against glutamate toxicity in cultured cortical neurons.

Lysophosphatidylethanolamines (LPEs) are bioactive lysophospholipids that have been suggested to play important roles in several biological processes. We performed a quantitative analysis of LPE species and showed their composition in mouse brain. We examined the roles of oleoyl-LPE (18:1 LPE), which is one of the abundant LPE species in brain. In cultured cortical neurons, application of 18:1 LPE-stimulated neurite outgrowth. The effect of 18:1 LPE on neurite outgrowth was inhibited by Gq/11 inhibitor YM-254890, phospholipase C (PLC) inhibitor U73122, protein kinase C (PKC) inhibitor Go6983 or mitogen-activated protein kinase (MAPK) inhibitor U0126. Additionally, 18:1 LPE increased the phosphorylation of MAPK/extracellular signal-regulated kinase 1/2. These results suggest that the action of 18:1 LPE on neurite outgrowth is mediated by the Gq/11/PLC/PKC/MAPK pathway. Moreover, we found that application of 18:1 LPE protects neurons from glutamate-induced excitotoxicity. This effect of 18:1 LPE was suppressed by PKC inhibitor Go6983. These results suggest that 18:1 LPE protects neurons from glutamate toxicity via PKC inhibitor Go6983-sensitive PKC subtype. Collectively, our results demonstrated that 18:1 LPE stimulates neurite outgrowth and protects against glutamate toxicity in cultured cortical neurons. Our findings provide insights into the physiological or pathological roles of 18:1 LPE in the brain.

Targeting Protein Kinase C in Glioblastoma Treatment.

Glioblastoma (GBM) is the most frequent and aggressive primary brain tumor and is associated with a poor prognosis. Despite the use of combined treatment approaches, recurrence is almost inevitable and survival longer than 14 or 15 months after diagnosis is low. It is therefore necessary to identify new therapeutic targets to fight GBM progression and recurrence. Some publications have pointed out the role of glioma stem cells (GSCs) as the origin of GBM. These cells, with characteristics of neural stem cells (NSC) present in physiological neurogenic niches, have been proposed as being responsible for the high resistance of GBM to current treatments such as temozolomide (TMZ). The protein Kinase C (PKC) family members play an essential role in transducing signals related with cell cycle entrance, differentiation and apoptosis in NSC and participate in distinct signaling cascades that determine NSC and GSC dynamics. Thus, PKC could be a suitable druggable target to treat recurrent GBM. Clinical trials have tested the efficacy of PKCβ inhibitors, and preclinical studies have focused on other PKC isozymes. Here, we discuss the idea that other PKC isozymes may also be involved in GBM progression and that the development of a new generation of effective drugs should consider the balance between the activation of different PKC subtypes.

498-P: Involvement of Phosphorylation of Beta-1 Integrin in Renin Production and Blood Pressure Regulation in Diabetic Nephropathy

Diabetic nephropathy (DN) and hypertension exacerbate each other, but many aspects of the mechanisms between these two pathologies remain unclear. The juxtaglomerular cells (JGC) secrete renin and trigger the renin-angiotensin-aldosterone system involved in blood pressure regulation. Our previous immunohistochemical analysis showed JGC-specific phosphorylation of β1 integrin (Itg), a cellular matrix adhesion factor. Quantitative analysis indicated the negative correlation between the phosphorylated β1 (pβ1) Itg and renin expression in JGC. To analyze the details of this relationship, As4.1, a cell-line of JGC was used. As protein kinase C (PKC) subtype epsilon is a putative kinase of the β1 Itg in JGC, PKC activator (phorbol 12-myristate 13-acetate, PMA) and PKC inhibitor (bisindolylmaleimide I, BIM-1) as well as TGF β1 which is known to stimulate a pathway to phosphorylate β1 Itg in JGC were applied to the culture of As4.1 cells. After 24 hours, expression of the mRNA levels of renin was gained by BIM-1 but suppressed by PMA or TGF β1. In the course of the cell culture with PMA, the mRNA levels of renin was increased till 90 minutes after application. However, it decreased subsequently, ultimately to 10% of the baseline level. Additional application of PMA at 6, 12 and 24 hours after the first application of PMA did not reproduce the temporal increase of renin expression. Time course of the cell culture with TGF β1 showed continuous decrease of the mRNA levels of renin. As PMA has broad spectrum for PKC subtypes, it was estimated that some activated PKC subtypes promoted temporal increase of renin expression in the early phase, however, later changes of cell state produced by pβ1 Itg suppressed the renin expression irreversibly. Our results confirmed a negative correlation between pβ1 Itg and renin expression in JGC cell-line, suggesting a link between β1 Itg and the regulatory system that controls renin production and secretion. Disclosure K. Sawada: None. M. Toyoda: Speaker's Bureau; Self; Boehringer Ingelheim Pharmaceuticals, Inc., Daiichi Sankyo Company, Limited, Eli Lilly and Company, Kyowa Hakko Kirin Co., Ltd., Medtronic Japan Co., Ltd., Mitsubishi Tanabe Pharma Corporation, Novo Nordisk Inc., Sanofi, Sumitomo Dainippon Pharma Co., Ltd., Takeda Pharmaceutical Company Limited. N. Saito: None. M. Kimura: None. H. Moriya: None. M. Fukagawa: Research Support; Self; Kyowa Hakko Kirin Co., Ltd. Speaker's Bureau; Self; Bayer Yakuhin, Ltd., Kissei Pharmaceutical Co., Ltd., Ono Pharmaceutical Co., Ltd., Torii Pharmaceutical Co., Ltd.

Yessotoxin activates cell death pathways independent of Protein Kinase C in K-562 human leukemic cell line

Protein Kinase C (PKC) is a group of enzymes involved in pro-survival or pro-apoptotic events depending on the cellular model. Moreover, Yessotoxin (YTX) modulates its expression and activates different cell death pathways. In K-562 tumor cell line, YTX induces apoptosis and autophagy after 24 and 48h of incubation, respectively, and the toxin carries out its action through the phosphodiesterase 4A (PDE4A). Therefore, the levels of two subtypes of PKC, conventional (cPKC) and δ isotype of novel PKC (PKCδ) were studied at these times after YTX incubation. Also their involvement in the cell death activated by the toxin and their relationship with PDE4A was checked. The expression of cPKC and PKCδ in cytosol, plasma membrane and nucleus was studied in normal and PDE4A-silenced cells. Furthermore, cell viability of normal cells, as well as cPKC-, PKCδ- and PDE4A-silenced cells was tested by Lactate Dehydrogenase (LDH) assay. As a result, PKCδ showed a key role in K-562 cell survive, since without this protein, K-562 cell decreased their viability. Furthermore, modulation of PKCs by YTX treatment was observed, however, the changes in the expression of these proteins are independent of cell death activated by the toxin. In addition, the modulation of PKCs detected is PDE4A-dependent, since the silencing of this protein change PKC expression pattern.

Protein Kinase C Mediated Extraembryonic Endoderm Differentiation of Human Embryonic Stem Cells

Unlike mouse embryonic stem cells (ESCs), which are closely related to the inner cell mass, human ESCs appear to be more closely related to the later primitive ectoderm. For example, human ESCs and primitive ectoderm share a common epithelial morphology, growth factor requirements, and the potential to differentiate to all three embryonic germ layers. However, it has previously been shown that human ESCs can also differentiate to cells expressing markers of trophoblast, an extraembryonic lineage formed before the formation of primitive ectoderm. Here, we show that phorbol ester 12-O-tetradecanoylphorbol 13-acetate causes human ESCs to undergo an epithelial mesenchymal transition and to differentiate into cells expressing markers of parietal endoderm, another extraembryonic lineage. We further confirmed that this differentiation is through the activation of protein kinase C (PKC) pathway and demonstrated that a particular PKC subtype, PKC-δ, is most responsible for this transition.

Involvement of kappa opioid receptors in the formalin-induced inhibition of analgesic tolerance to morphine via suppression of conventional protein kinase C activation

Repeated morphine treatment results in a decreased analgesic effect or the development of analgesic tolerance. However, we reported that some inflammatory chronic pain may inhibit morphine tolerance via kappa opioid receptor (KOR) activation. In this study, we further investigated the role of KOR in the inhibition of morphine tolerance in a chronic pain condition with a focus on the regulation of protein kinase C (PKC) activity. Chronic pain was induced by formalin treatment into the dorsal part of the left hind paws of mice. The analgesic effect of morphine was measured by the tail flick method. We analysed the protein expression of PKC and its activity, and G-protein activity of mu opioid receptor (MOR) under repeated morphine treatment with or without formalin treatment. We found that conventional subtypes of PKC (cPKC) were up-regulated by repeated morphine treatment. Also, antisense oligonucleotide (AS-ODN) targeting cPKC completely suppressed the development of morphine tolerance. The disappearance of the repeated morphine-induced up-regulation of cPKC was completely reversed by treatment with AS-ODN targeting KOR. In addition, AS-ODN targeting KOR significantly reversed the chronic pain-induced down-regulation of PKC activity or up-regulation of MOR [(35)S]GTPgammaS binding activity after repeated morphine treatment. These results indicate that KOR plays an important role in the inhibition of repeated morphine-induced cPKC up-regulation under chronic pain condition. Furthermore, this may result in the increase of MOR activity and in the inhibition of morphine tolerance under chronic pain condition.

Agonist-dependent μ-opioid receptor signaling can lead to heterologous desensitization

Desensitization of the µ-opioid receptor (MOR) has been implicated as an important regulatory process in the development of tolerance to opiates. Monitoring the release of intracellular Ca 2+ ([Ca 2+] i), we reported that [D-Ala 2, N-Me-Phe 4, Gly 5-ol]-enkephalin (DAMGO)-induced receptor desensitization requires receptor phosphorylation and recruitment of β-arrestins (βArrs), while morphine-induced receptor desensitization does not. In current studies, we established that morphine-induced MOR desensitization is protein kinase C (PKC)-dependent. By using RNA interference techniques and subtype specific inhibitors, PKCε was shown to be the PKC subtype activated by morphine and the subtype responsible for morphine-induced desensitization. In contrast, DAMGO did not increase PKCε activity and DAMGO-induced MOR desensitization was not affected by modulating PKCε activity. Among the various proteins within the receptor signaling complex, Gαi2 was phosphorylated by morphine-activated PKCε. Moreover, mutating three putative PKC phosphorylation sites, Ser 44, Ser 144 and Ser 302 on Gαi2 to Ala attenuated morphine-induced, but not DAMGO-induced desensitization. In addition, pretreatment with morphine desensitized cannabinoid receptor CB1 agonist WIN 55212-2-induced [Ca 2+] i release, and this desensitization could be reversed by pretreating the cells with PKCε inhibitor or overexpressing Gαi2 with the putative PKC phosphorylation sites mutated. Thus, depending on the agonist, activation of MOR could lead to heterologous desensitization and probable crosstalk between MOR and other Gαi-coupled receptors, such as the CB1.

PKC412, a Small Molecule Tyrosine Kinase Inhibitor, Regulates Survival and Proliferation Pathways in Waldenström Macroglobulinemia

Background: Waldenström's Macroglobulinemia (WM) is an incurable lymphoplasmacytic lymphoma with limited options of therapy. Protein kinase C (PKC) is a multigene family that that plays an important role in the regulation of cell growth and tumor progression. PKC412, a derivate of staurosporine, is a competitive inhibitor of ATP binding to PKC subtypes. This PKC inhibitor was used to examine its role in WM.Methods: WM cell lines (BCWM1 and WSU-WM) and IgM secreting low-grade lymphoma cell lines (MEC1, RL) were used. Bone marrow primary CD19+ cells and bone marrow stromal cells (BMSC) were obtained from patients with WM after informed consent. Cytotoxicity and DNA synthesis were measured by MTT assay and thymidine uptake assay. Cell signaling of HSP response, VEGF signaling and AKT pathways were determined by Western Blot analysis. Cell cycle and apoptosis analysis were obtained through flow cytometry.Results: We showed that PKC412 induced cytotoxicity in WM cell lines tested (IC50 of 1uM) with no cytotoxic effect on healthy donor peripheral blood mononuclear cells at concentrations as high as 5uM. Cell proliferation and apoptosis analysis showed similar results, in a time independent manner. A G2/M arrest was observed upon cell cycle analysis, suggesting a possible mechanism for apoptosis. Similarly, the Akt pathway was investigated and was found to show a decrease in its activity in a dose dependent manner. Finally, PKC412 regulated VEGF levels in WM samples indicating an anti-angiogenic activity.Conclusion: PKC412 has a significant effect on the survival and proliferation of WM cells, including regulation of angiogenesis. These studies provide the framework for clinical evaluation of this novel PKC inhibitor in Waldenström Macroglobulinemia.

The regulation of glycine transporter GLYT1 is mainly mediated by protein kinase Cα in C6 glioma cells

Glycine has been shown to possess important functions as a bidirectional neurotransmitter. At synaptic clefts, the concentration of glycine is tightly regulated by the uptake of glycine released from nerve terminals into glial cells by the transporter GLYT1. It has been recently demonstrated that protein kinase C (PKC) mediates the downregulation of GLYT1 activity in several cell systems. However, it remains to be elucidated which subtypes of PKC might be important in the regulation of GLYT1 activity. In this study, we attempted to make clear the mechanism of the phorbol 12-myristate 13-acetate (PMA)-suppressed uptake of glycine in C6 glioma cells which have the native expression of GLYT1. In C6 cells, the expression of PKCα, PKCδ, and PKCɛ of the PMA-activated subtypes was detected. The PMA-suppressed action was fully reversed by the removal of both extracellular and intracellular Ca 2+. Furthermore, the inhibitory effects of PMA or thymeleatoxin (THX), which is a selective activator of conventional PKC (cPKC), were blocked by the downregulation of all PKCs expressed in C6 cells by long-term incubation with THX, or pretreatment with GF109203X or Gö6983, which are broad inhibitors of PKC, or Gö6976, a selective inhibitor of cPKC. On the other hand, treatment of C6 cells with ingenol, a selective activator of novel PKCs, especially PKCδ and PKCɛ, did not affect the transport of glycine. Silencing of PKCδ expression by using RNA interference or pretreatment with the inhibitor peptide for PKCɛ had no effect on the PMA-suppressed uptake of glycine. Together, these results suggest PKCα to be a crucial factor in the regulation of glycine transport in C6 cells.

The Protein Kinase C (PKC) Family of Proteins in Cytokine Signaling in Hematopoiesis

The members of the protein kinase C (PKC) family of proteins play important roles in signaling for various growth factors, cytokines, and hormones. Extensive work over the years has led to the identification of three major groups of PKC isoforms. These include the classic PKCs (PKCalpha, PKCbeta(I), PKCbeta(II), PKCgamma), the novel PKCs (PKCdelta, PKCepsilon, PKCeta, PKCmu, PKCtheta), and the atypical PKCs (PKCzeta, PKCiota/lambda). All these PKC subtypes have been shown to participate in the generation of signals for important cellular processes and to mediate diverse and, in some cases, opposing biologic responses. There is emerging evidence that these kinases also play key functional roles in the regulation of cell growth, apoptosis, and differentiation of hematopoietic cells. In this review, both the engagement of the various PKC members in cytokine and growth factor signaling and their role in the regulation of hematopoiesis are discussed.

Proteinkinase C Isoformen in Hornhautepithel und -endothel

Protein kinase C (PKC) plays a key role in cell metabolism. Three subgroups and 12 isoforms have been isolated so far, catalysing specific functions in cell metabolism. The demonstration of PKC subtypes in corneal tissue has been inconsistent. The aim of this study was to verify the expression of several PKC subgroups and isoforms in human and bovine corneal epithelial and endothelial cells. PKC subgroups and isoforms were studied using polyclonal antibodies. Antibodies to PKC-alpha, -delta, -epsilon and -zeta, representing all three PKC subgroups, bound in human and bovine corneal epithelium and endothelium. No binding was found for antibodies to PKC-beta2. For the first time the presence of all three PKC subgroups was demonstrated in human and bovine corneal epithelial and endothelial cells. Further studies are needed to show the role of these subgroups in cellular functions such as cell proliferation and differentiation.

Parthenolide sensitizes ultraviolet (UV)-B-induced apoptosis via protein kinase C-dependent pathways

Parthenolide (PN) is the principal sesquiterpene lactone in feverfew (Tanacetum parthenium) with proven anti-inflammatory properties. We have previously reported that PN possesses strong anticancer activity in ultraviolet B (UVB)-induced skin cancer in SKH-1 hairless mice. In order to further understand the mechanism(s) involved in the anticancer activity of PN, we investigated the role of protein kinase C (PKC) in the sensitization activity of PN on UVB-induced apoptosis. Several subtypes of PKC have been reported to be involved in UVB-induced signaling cascade with both pro- and anti-apoptotic activities. Here we focused on two isoforms of PKC: novel PKCdelta and atypical PKCzeta. In JB6 murine epidermal cells, UVB induces the membrane translocations of both PKCs, and PN pre-treatment enhances the membrane translocation of PKCdelta, but inhibits the translocation of PKCzeta. Similar results were also detected when the activities of these PKCs were tested with the PKC kinase assay. Moreover, pre-treatment with a specific PKCdelta inhibitor, rotterlin, completely diminishes the sensitization effect of PN on UVB-induced apoptosis. When cells were transiently transfected with dominant negative PKCdelta or wild-type PKCzeta, the sensitization effect of PN on UVB-induced apoptosis was also drastically reduced. Further mechanistic study revealed that PKCzeta, but not PKCdelta, is required for UVB-induced p38 MAPK activation and PN is likely to act through PKCzeta to suppress p38 activation in UVB-treated JB6 cells. In conclusion, we demonstrated that PN sensitizes UVB-induced apoptosis via PKC-dependent pathways.

Evidence that protein kinase Cα interacts with and regulates the glial glutamate transporter GLT‐1

Many of the sodium-dependent neurotransmitter transporters are rapidly (within minutes) regulated by protein kinase C (PKC), with changes in activity being correlated with changes in transporter trafficking to or from the plasma membrane. Our recent studies suggest that one of the classical subtypes of PKC, PKCalpha, may selectively mediate redistribution of the neuronal glutamate transporter, excitatory amino acid carrier (EAAC)1, and show that PKCalpha can be co-immunoprecipitated with EAAC1. When the glial glutamate transporter GLT-1a is transfected into C6 glioma cells, this transporter is internalized in response to activation of PKC, but the PKC subtype involved in this regulation is unknown. In the present study, expression of the phorbol ester-activated subtypes of PKC was examined in C6 glioma transfected with GLT-1. Of the classical subtypes, only PKCalpha was detected, and of the non-classical subtypes, PKCdelta and PKCepsilon were detected. In this system, phorbol ester-dependent internalization of GLT-1 was blocked by a general inhibitor of PKCs (bisindolylmaleimide II) and by concentrations of Gö6976 that selectively block classical PKCs, but not by an inhibitor of PKCdelta (rottlerin). PKCalpha immunoreactivity was found in GLT-1 immunoprecipitates obtained from transfected C6 cells and from crude rat brain synaptosomes, a milieu that better mimics in vivo conditions. The amount of PKCalpha in both types of immunoprecipitate was modestly increased by phorbol ester, and this increase was blocked by a PKC antagonist. These studies suggest that PKCalpha may be required for the regulated redistribution of GLT-1.

Involvement of γ protein kinase C in estrogen‐induced neuroprotection against focal brain ischemia through G protein‐coupled estrogen receptor

The neuroprotective effects of estrogen were studied in the ischemic model mice by 90 min transient unilateral middle cerebral artery occlusion (MCAO) followed by 22.5 h reperfusion. The total infarct size in C57BL/6 female mice after MCAO and reperfusion was significantly smaller than that in male mice. Intraperitoneal injection of estrogen after the start of reperfusion significantly reduced the infarct volume in the male mice. However, no significant gender difference was found in total infarct size in gamma protein kinase C (PKC)-knockout mice, suggesting that the neuroprotective effects of estrogen are due to the activation of a specific subtype of PKC, gammaPKC, a neuron-specific PKC subtype, in the brain. We demonstrated that exogenous estrogen-induced neuroprotection was attenuated in gammaPKC-knockout mice. Immunocytochemical study showed that gammaPKC was translocated to nerve fiber-like structures when observed shortly after MCAO and reperfusion. We also visualized the rapid and reversible translocation of gammaPKC-GFP (green fluorescent protein) by estrogen stimulation in living CHO-K1 cells. These results suggest that the activation of gammaPKC through the G-protein-coupled estrogen receptors on the plasma membrane is involved in the estrogen-induced neuroprotection against focal brain ischemia.

Two different pathways link G-protein-coupled receptors with tyrosine kinases for the modulation of growth and survival in human hematopoietic progenitor cells

The G-protein-coupled receptor agonists CXCL12 (SDF-1, a chemokine) and thrombin showed opposite effects on growth and survival of multipotent and erythroid human hematopoietic progenitor cells. CXCL12 promoted growth in multipotent cells by activating the RhoA–Rho kinase pathway. Its effect was largely blocked by Y-27632, a specific inhibitor of Rho kinase, and by clostridial toxin B, a specific inhibitor of Rho family proteins. Rho activation required a G i-mediated stimulation of tyrosine kinases, which was blocked by PP2 and tyrphostin AG 490, inhibitors of Src and Jak type kinases, respectively. By contrast, in erythroid cells, inhibitors of Src family and c-Abl tyrosine kinases (tyrphostin AG 82, PP2, imatinib) enhanced protein kinase C (PKC)-dependent cell growth and antagonized thrombin-promoted apoptosis by specifically stimulating PKCβ activity. The PKC activating phorbol ester PMA (a growth factor in erythroid cells) induced the activation of Lyn and c-Abl tyrosine kinases, thus establishing a feedback inhibition of PKCβ. Hence, developmental stage-specific crosstalk between PKC subtypes and tyrosine kinases appear to determine whether growth and survival of hematopoietic cells are promoted or inhibited by G-protein-coupled receptor agonists.

Role of specific protein kinase C isoforms in modulation of β 1- and β 2-adrenergic receptors

The function of β-adrenergic receptor (βAR) is modulated by the activity status of α 1-adrenergic receptors (α 1ARs) via molecular crosstalk, and this becomes evident when measuring cardiac contractile responses to adrenergic stimulation. The molecular mechanism underlying this crosstalk is unknown. We have previously demonstrated that overexpression of α 1B-adrenergic receptor (α 1BAR) in transgenic mice leads to a marked desensitization of βAR-mediated adenylyl cyclase stimulation which is correlated with increased levels of activated protein kinase C (PKC) β, δ and ε [J. Mol. Cell. Cardiol. 30 (1998) 1827]. Therefore, we wished to determine which PKC isoforms play a role in heterologous βAR desensitization and also which isoforms of the βAR were the molecular target(s) for PKC. In experiments using constitutively activated PKC expression constructs transfected into HEK 293 cells also expressing the β 2AR, constitutively active (CA)-PKC overexpression was first confirmed by immunoblots using specific anti-PKC antibodies. We then demonstrated that the different PKC subtypes lead to a decreased maximal cAMP accumulation following isoproterenol stimulation with a rank order of PKCα≥PKCζ>PKCε>PKCβII. However, a much more dramatic desensitization of adenylyl cyclase stimulation was observed in cells co-transfected with different PKC isoforms and β 1AR. Further, the modulation of β 1AR by PKC isoforms had a different rank order than for the β 2AR: PKCβII>PKCα>PKCε>PKCζ. PKC-mediated desensitization was reduced by mutating consensus cAMP-dependent protein kinase (PKA)/PKC sites in the third intracellular loop and/or the carboxy-terminal tail of either receptor. Our results demonstrate therefore that the β 1AR is the most likely molecular target for PKC-mediated heterologous desensitization in the mammalian heart and that modulation of adrenergic receptor activity in any given cell type will depend on the complement of PKC isoforms present.

Ceramide-induced Apoptosis by Translocation, Phosphorylation, and Activation of Protein Kinase Cδ in the Golgi Complex

Protein kinase C (PKC), a Ca(2+)/phospholipid-dependent protein kinase, is known as a key enzyme in various cellular responses, including apoptosis. However, the functional role of PKC in apoptosis has not been clarified. In this study, we focused on the involvement of PKCdelta in ceramide-induced apoptosis in HeLa cells and examined the importance of spatiotemporal activation of the specific PKC subtype in apoptotic events. Ceramide-induced apoptosis was inhibited by the PKCdelta-specific inhibitor rottlerin and also was blocked by knockdown of endogenous PKCdelta expression using small interfering RNA. Ceramide induced the translocation of PKCdelta to the Golgi complex and the concomitant activation of PKCdelta via phosphorylation of Tyr(311) and Tyr(332) in the hinge region of the enzyme. Unphosphorylatable PKCdelta (mutants Y311F and Y332F) could translocate to the Golgi complex in response to ceramide, suggesting that tyrosine phosphorylation is not necessary for translocation. However, ceramide failed to activate PKCdelta lacking the C1B domain, which did not translocate to the Golgi complex, but could be activated by tyrosine phosphorylation. These findings suggest that ceramide translocates PKCdelta to the Golgi complex and that PKCdelta is activated by tyrosine phosphorylation in the compartment. Furthermore, we utilized species-specific knockdown of PKCdelta by small interfering RNA to study the significance of phosphorylation of Tyr(311) and Tyr(332) in PKCdelta for ceramide-induced apoptosis and found that phosphorylation of Tyr(311) and Tyr(332) is indispensable for ceramide-induced apoptosis. We demonstrate here that the targeting mechanism of PKCdelta, dual regulation of both its activation and translocation to the Golgi complex, is critical for the ceramide-induced apoptotic event.

Protein kinase C: its role in activity-dependent Purkinje cell dendritic development and plasticity.

The cerebellum is a central organ in the control of motor learning and performance. In this respect, the cellular plasticity model systems of multiple climbing fiber elimination and long-term depression have been intensively studied. The signalling pathways involved in these plastic changes are now well understood on a molecular level and protein kinase C (PKC) activity appears to be crucially involved in both processes. Furthermore, as shown in recent studies, Purkinje cell dendritic development also critically depends on the activity of PKC. Thereby, the Ca(2+)-dependent PKC subtypes, activated by synaptic inputs through metabotropic glutamate receptors, trigger functional changes as well as long-term anatomical maturation of the Purkinje cell dendritic tree during cerebellar development at different time levels. This review summarizes these findings and forwards the hypothesis of a link between the functional mechanisms underlying LTD and the differentiation of Purkinje cell dendrites.

Phorbol Myristate Acetate-Dependent Interaction of Protein Kinase Cα and the Neuronal Glutamate Transporter EAAC1

Sodium-dependent transporters clear extracellular glutamate in the mammalian CNS. Activation of protein kinase C (PKC) rapidly increases the activity of the neuronal glutamate transporter EAAC1 (excitatory amino acid carrier-1). This effect is associated with redistribution of EAAC1 to the cell membrane and appears to be dependent on a particular PKC subtype, PKCalpha. In the present study, we sought to determine whether this specificity for regulation of EAAC1 is associated with the formation of EAAC1-PKCalpha complexes. In C6 glioma cells, activation of PKC with phorbol 12-myristate 13-acetate (PMA) induced formation of EAAC1-PKCalpha complexes but did not induce formation of complexes with PKCdelta, a PKC not thought to regulate EAAC1. Formation of these complexes was blocked by inhibitors of PKC. Confocal microscopy revealed that PMA caused EAAC1 and PKCalpha to colocalize in clusters at or near the cell surface. The EAAC1-PKCalpha complexes were also observed in rat brain synaptosomes, demonstrating that this interaction is not restricted to C6 cells. These data demonstrate that EAAC1 and PKCalpha interact in a PKC-dependent manner that is associated with EAAC1 redistribution. Although PKC activation has been implicated in the regulation of many different neurotransmitter transporters, this study provides the first example of an interaction between a neurotransmitter transporter and PKC. PKCalpha also forms complexes with GluR2 (glutamate receptor subunit 2) and causes a reduction in the levels of GluR2-containing AMPA receptors at the plasma membrane. Together, these data suggest that PKCalpha may simultaneously trigger the redistribution of EAAC1 and glutamate receptors.