Translocation Of Protein Kinase C Research Articles

Protein kinase C (PKC) inhibitor Calphostin C activates PKC in a light-dependent manner at high concentrations via the production of singlet oxygen

Calphostin C (Cal-C) is a protein kinase C (PKC) inhibitor that binds to its C1 domain. The aim of the present study was to elucidate the action of Cal-C in addition to PKC inhibition. First, we confirmed that Cal-C at low concentrations (<200 nM) inhibit phorbol ester-induced PKC translocation and G-protein-coupled receptor (GPCR)-mediated PKC activation. Cal-C at higher concentrations (>2 μM) increased intracellular calcium ion concentrations ([Ca2+]i) in a concentration-dependent manner. The origin of this increase is the mobilization of the endoplasmic reticulum (ER), which does not involve GPCR or ryanodine receptors. Cal-C at high concentrations also cause structural changes in the ER, such as the formation of vacuoles and aggregates, and calcium leakage from the ER. At 2 μM, Cal-C translocated a calcium-sensitive PKCα. Studies using a C-kinase activity reporter and a myristoylated alanine-rich protein kinase C substrate fused with green fluorescent protein (GFP) have also revealed that Cal-C at high concentrations activate PKC in living cells. Additionally, the PKC-activating effects of Cal-C were light-dependent. Finally, studies using Si-DMA, an indicator of singlet oxygen, showed that Cal-C at high concentrations generated singlet oxygen, causing structural changes in the ER and leakage of calcium into the cytosol, which triggered PKC activation. This study confirms the novel action of Cal-C, solely considered a PKC inhibitor. Cal-C acted as a PKC inhibitor at low concentrations and a PKC activator at high concentrations by generating singlet oxygen in a light-dependent manner, suggesting that Cal-C can be used in photodynamic therapy.

Identification of protein kinase C domains involved in its translocation induced by propofol

Propofol is widely used for general anesthesia and sedation; however, the mechanisms of its anesthetic and adverse effects are not fully understood. We have previously shown that propofol activates protein kinase C (PKC) and induces its translocation in a subtype-specific manner. The purpose of this study was to identify the PKC domains involved in propofol-induced PKC translocation. The regulatory domains of PKC consist of C1 and C2 domains, and the C1 domain is subdivided into the C1A and C1B subdomains. Mutant PKCα and PKCδ with each domain deleted were fused with green fluorescent protein (GFP) and expressed in HeLa cells. Propofol-induced PKC translocation was observed by time-lapse imaging using a fluorescence microscope. The results showed that persistent propofol-induced PKC translocation to the plasma membrane was abolished by the deletion of both C1 and C2 domains in PKCα and by the deletion of the C1B domain in PKCδ. Therefore, propofol-induced PKC translocation involves the C1 and C2 domains of PKCα and the C1B domain of PKCδ. We also found that treatment with calphostin C, a C1 domain inhibitor, abolished propofol-induced PKCδ translocation. In addition, calphostin C inhibited the propofol-induced phosphorylation of endothelial nitric oxide synthase (eNOS). These results suggest that it may be possible to modulate the exertion of propofol effects by regulating the PKC domains involved in propofol-induced PKC translocation.

Oxidative stress induced by NOX2 contributes to neuropathic pain via plasma membrane translocation of PKC\u03b5 in rat dorsal root ganglion neurons

BackgroundNicotinamide adenine dinucleotide phosphate oxidase 2 (NOX2)-induced oxidative stress, including the production of reactive oxygen species (ROS) and hydrogen peroxide, plays a pivotal role in neuropathic pain. Although the activation and plasma membrane translocation of protein kinase C (PKC) isoforms in dorsal root ganglion (DRG) neurons have been implicated in multiple pain models, the interactions between NOX2-induced oxidative stress and PKC remain unknown.MethodsA spared nerve injury (SNI) model was established in adult male rats. Pharmacologic intervention and AAV-shRNA were applied locally to DRGs. Pain behavior was evaluated by Von Frey tests. Western blotting and immunohistochemistry were performed to examine the underlying mechanisms. The excitability of DRG neurons was recorded by whole-cell patch clamping.ResultsSNI induced persistent NOX2 upregulation in DRGs for up to 2 weeks and increased the excitability of DRG neurons, and these effects were suppressed by local application of gp91-tat (a NOX2-blocking peptide) or NOX2-shRNA to DRGs. Of note, the SNI-induced upregulated expression of PKCε but not PKC was decreased by gp91-tat in DRGs. Mechanical allodynia and DRG excitability were increased by ψεRACK (a PKCε activator) and reduced by εV1-2 (a PKCε-specific inhibitor). Importantly, εV1-2 failed to inhibit SNI-induced NOX2 upregulation. Moreover, the SNI-induced increase in PKCε protein expression in both the plasma membrane and cytosol in DRGs was attenuated by gp91-tat pretreatment, and the enhanced translocation of PKCε was recapitulated by H2O2 administration. SNI-induced upregulation of PKCε was blunted by phenyl-N-tert-butylnitrone (PBN, an ROS scavenger) and the hydrogen peroxide catalyst catalase. Furthermore, εV1-2 attenuated the mechanical allodynia induced by H2O2ConclusionsNOX2-induced oxidative stress promotes the sensitization of DRGs and persistent pain by increasing the plasma membrane translocation of PKCε.

Function-Oriented Synthesis: Design, Synthesis, and Evaluation of Highly Simplified Bryostatin Analogues.

Using a function-oriented synthesis strategy, we designed, synthesized, and evaluated the simplest bryostatin 1 analogues reported to date, in which bryostatin's A- and B-rings are replaced by a glutarate linker. These analogues, one without and one with a C26-methyl group, exhibit remarkably different protein kinase C (PKC) isoform affinities. The former exhibited bryostatin-like binding to several PKC isoforms with Ki's < 5 nM, while the latter exhibited PKC affinities that were up to ∼180-fold less potent. The analogue with bryostatin-like PKC affinities also exhibited bryostatin-like PKC translocation kinetics in vitro, indicating rapid cell permeation and engagement of its PKC target. This study exemplifies the power of function-oriented synthesis in reducing structural complexity by activity-informed design, thus enhancing synthetic accessibility, while still maintaining function (biological activity), collectively providing new leads for addressing the growing list of therapeutic indications exhibited by PKC modulators.

Combinatorial molecule screening identified a novel diterpene and the BET inhibitor CPI-203 as differentiation inducers of primary acute myeloid leukemia cells.

Combination treatment has proven effective for patients with acute promyelocytic leukemia, exemplifying the importance of therapy targeting multiple components of oncogenic regulation for a successful outcome. However, recent studies have shown that the mutational complexity of acute myeloid leukemia (AML) precludes the translation of molecular targeting into clinical success. Here, as a complement to genetic profiling, we used unbiased, combinatorial in vitro drug screening to identify pathways that drive AML and to develop personalized combinatorial treatments. First, we screened 513 natural compounds on primary AML cells and identified a novel diterpene (H4) that preferentially induced differentiation of FLT3 wild-type AML, while FLT3-ITD/mutations conferred resistance. The samples responding to H4, displayed increased expression of myeloid markers, a clear decrease in the nuclear-cytoplasmic ratio and the potential of re-activation of the monocytic transcriptional program reducing leukemia propagation in vivo. By combinatorial screening using H4 and molecules with defined targets, we demonstrated that H4 induces differentiation by the activation of the protein kinase C (PKC) signaling pathway, and in line with this, activates PKC phosphorylation and translocation of PKC to the cell membrane. Furthermore, the combinatorial screening identified a bromo- and extra-terminal domain (BET) inhibitor that could further improve H4-dependent leukemic differentiation in FLT3 wild-type monocytic AML. These findings illustrate the value of an unbiased, multiplex screening platform for developing combinatorial therapeutic approaches for AML.

Synthesis and evaluation of designed PKC modulators for enhanced cancer immunotherapy

Bryostatin 1 is a marine natural product under investigation for HIV/AIDS eradication, the treatment of neurological disorders, and enhanced CAR T/NK cell immunotherapy. Despite its promising activity, bryostatin 1 is neither evolved nor optimized for the treatment of human disease. Here we report the design, synthesis, and biological evaluation of several close-in analogs of bryostatin 1. Using a function-oriented synthesis approach, we synthesize a series of bryostatin analogs designed to maintain affinity for bryostatin’s target protein kinase C (PKC) while enabling exploration of their divergent biological functions. Our late-stage diversification strategy provides efficient access to a library of bryostatin analogs, which per our design retain affinity for PKC but exhibit variable PKC translocation kinetics. We further demonstrate that select analogs potently increase cell surface expression of CD22, a promising CAR T cell target for the treatment of leukemias, highlighting the clinical potential of bryostatin analogs for enhancing targeted immunotherapies.

Integrating virtual screening and biochemical experimental approach to identify potential anti-cancer agents from drug databank.

Chemical libraries constitute a reservoir of pharmacophoric molecules to identify potent anti-cancer agents. Virtual screening of heterocyclic compound library in conjugation with the agonist-competition assay, toxicity-carcinogenicity analysis, and string-based structural searches enabled us to identify several drugs as potential anti-cancer agents targeting protein kinase C (PKC) as a target. Molecular modeling study indicates that Cinnarizine fits well within the PKC C2 domain and exhibits extensive interaction with the protein residues. Molecular dynamics simulation of PKC-Cinnarizine complex at different temperatures (300, 325, 350, 375, and 400[Formula: see text]K) confirms that Cinnarizine fits nicely into the C2 domain and forms a stable complex. The drug Cinnarizine was found to bind PKC with a dissociation constant Kd of [Formula: see text]M. The breast cancer cells stimulated with Cinnarizine causes translocation of PKC-[Formula: see text] to the plasma membrane as revealed by immunoblotting and immunofluorescence studies. Cinnarizine also dose dependently reduced the viability of MDAMB-231 and MCF-7 breast cancer cells with an IC[Formula: see text] of [Formula: see text] and [Formula: see text]g/mL, respectively. It is due to the disturbance of cell cycle of breast cancer cells with reduction of S-phase and accumulation of cells in G1-phase. It disturbs mitochondrial membrane potentials to release cytochrome C into the cytosol and activates caspase-3 to induce apoptosis in cancer cells. The cell death was due to induction of apoptosis involving mitochondrial pathway. Hence, the current study has assigned an additional role to Cinnarizine as an activator of PKC and potentials of the approach to identify new molecules for anti-cancer therapy. Thus, in silico screening along with biochemical experimentation is a robust approach to assign additional roles to the drugs present in the databank for anti-cancer therapy.

Biochemical Studies and Virtual Screening of Phytochemical Reservoir from Northeastern Indian Plants to Identify Anti-Cancer Agents

In the present study, medicinal phytochemicals found in plants of north-eastern India were virtually screened against the C1b regulatory domain of Protein Kinase C (PKC), an important enzyme in cancer cell-biology. Amongst the few top hits, the molecules Gallic acid (GA) and Epigallocatechin gallate (EGCG) were identified through an agonist competition assay. Both phytochemical are non-toxic and non-mutagenic. An in vitro PKC binding assay confirmed that GA and EGCG were strong ligands of PKC. Both the phytochemicals formed numerous hydrogen bonding and hydrophobic interactions with the amino acids lining the ligand binding pocket of the C1b domain of PKC. They were able to induce translocation of PKC to the plasma membrane in MDAMB-231 breast cancer cells. Furthermore, GA and EGCG dose-dependently reduced the viability of breast cancer cells by arresting the cell-cycle. Eventually, mitochondrial pathway of apoptosis was identified as the mechanism of loss of breast cancer cellular viability. Thus, our study has revealed that tea phytochemicals GA and EGCG exhibit anti-cancer activity by acting as strong ligands of PKC. This study encourages the further identification of newer novel and even more potent such PKC-directed phytochemicals from natural herbs & beverages as a part of herbal anti-cancer therapy.

Intracellular Ca2+ is an essential factor for cell damage induced by unsaturated carbonyl compounds

The unsaturated carbonyl compounds are known as the environmental pollutants. Acrolein (ACR) and methyl vinyl ketone (MVK) are representative unsaturated carbonyl compounds. ACR is contained in smoke, automobile exhaust, industrial waste, and several foods. MVK is widely used as the industrial chemical. Although ACR and MVK are highly toxic, the molecular mechanism for their cytotoxicity has been unclear. We have previously reported that ACR and MVK are major cytotoxic compounds in the gas phase of cigarette smoke, and protein kinase C (PKC) inhibitor and NADPH oxidases inhibitor partially rescued cells from ACR- or MVK-induced cell death (Noya etal., Toxicology, 314, 1-10, 2013). PKC translocation, which is hallmark for PKC activation, and cell damage were induced by treatment of cultured cells with ACR or MVK. Intracellular Ca2+ chelator completely suppressed ACR- or MVK-induced PKC translocation to the cell membrane and cell damage, while extracellular Ca2+ chelator had no effects on ACR- and MVK-induced cytotoxicity. These results suggest that intracellular Ca2+ is an essential factor for cell damage caused by both PKC-dependent and PKC-independent pathways, and mobilization of Ca2+ from intracellular Ca2+ stores is induced by ACR or MVK.

Stolonidiol: Synthesis, Target Identification, and Mechanism for Choline Acetyltransferase Activation.

Stolonidiol, a marine natural product, has been reported to potentiate the activity of choline acetyltransferase (ChAT), the enzyme that produces the neurotransmitter acetylcholine. Here we report the total synthesis of stolonidiol starting from (R)-(+)-limonene. To identify the mechanism by which ChAT activity is increased, we sought to identify the biological target of stolonidiol. We show that stolonidiol binds to the phorbol ester binding site of protein kinase C (PKC), induces translocation of PKC to the cell membrane, and activates kinase activity. Furthermore, we confirmed the increase in ChAT activity observed upon treatment of cells with stolonidiol and show that this effect is mediated by PKC. Collectively, our data strongly suggest that PKC activation by stolonidiol is responsible for the resulting potentiation of ChAT activity.

α-Cyperone Inhibits PMA-Induced EPCR Shedding through PKC Pathway.

α-Cyperone, a sesquiterpene compound represents 25.23% of the total oil and is the most abundant compound in Cyperus rotundus oil. Endothelial cell protein C receptor (EPCR) is a main member in protein C (PC) anti-coagulation system. EPCR could be shed from cell surface, and is mediated by tumor necrosis factor-α converting enzyme (TACE). Nothing that EPCR is a marker of vascular barrier integrity in vascular inflammatory disease and takes part in systemic inflammatory disease. In this study, we investigated whether α-cyperone could inhibit EPCR shedding. To observe the effect, we investigated this issue by detection the effect of α-cyperone on phorbol-12-myristate 13-acetate (PMA)-induced EPCR shedding in human umbilical vein endothelial cells (HUVECs). The cells were pretreated with α-cyperone for 12 h, and then stimulated by PMA for 1 h. The solute EPCR (sEPCR) and expression of membrane EPCR (mEPCR) were measured by enzyme-linked immunosorbent assay (ELISA) and Western blot. The mRNA, protein level and activity of TACE were tested by quantitative (q)RT-PCR, Western blot and InnoZyme TACE activity assay kit. Furthermore, we measured the protein level of mitogen-activated protein kinase (MAPK) signaling and protein kinase C (PKC) pathway under this condition by Western blot. The results showed that α-cyperone could suppress PMA-induced EPCR shedding through inhibiting the expression and activity of TACE. In addition, α-cyperone could inhibit PKC translocation, but not have an effect on phosphorylation of c-Jun N-terminal kinase (JNK), p38 and extracellular regulated protein kinases (ERK) 1/2. Given these results, α-cyperone inhibits PMA-induced EPCR shedding through PKC pathway, which will provide an experimental basis for further research on α-cyperone.

Implication of protein kinase C of the left intermediate medial mesopallium in memory impairments induced by early prenatal morphine exposure in one-day old chicks

Previously we reported that prenatal morphine exposure during embryonic days 5–8 can cause cognitive deficits of one-trial passive avoidance learning (PAL) in one-day old chicks. Because protein kinase C (PKC) has been associated with memory capacity, we investigated the effects of prenatal morphine exposure on PKC isoforms expression in the left intermediate medial mesopallium (IMM) of chick brain at a time when memory tests were performed at 30, 120 and 360min respectively following training in PAL paradigm. We found that the level of PKCα in the membrane fractions in left IMM was decreased but that in the cytosol fractions showed a increased trend in prenatally morphine-exposed chicks with impaired long-term memory (120 and 360min). Moreover, the translocation of PKC δ from cytosol to membrane in left IMM was shown in prenatal morphine group which had significantly impaired long-term memory at 360min after training. Furthermore, there were no statistical differences between the two groups regarding the expressions of PKCα and PKC δ in the membrane fraction, although their levels in the cytosol fraction of prenatal morphine group which showed impaired intermediate-term memory at 30min after training, were quite different from that of prenatal saline group. Taken together, these results indicate that PKCα and PKC δ in the left IMM are differentially involved in the impairments of long-term memory induced by prenatal morphine exposure. Neither PKCα nor PKC δ in left IMM may be associated with the disruption of intermediate-term memory of chicks prenatally exposed to morphine.

Application of surface plasmon resonance imaging to monitoring G protein-coupled receptor signaling and its modulation in a heterologous expression system.

BackgroundG protein-coupled receptors (GPCRs) are ubiquitous surface proteins mediating various biological responses and thus, important targets for therapeutic drugs. GPCRs individually produce their own signaling as well as modulate the signaling of other GPCRs. Real-time observation of GPCR signaling and modulation in living cells is key to molecular study of biological responses and pharmaceutical development. However, fluorescence imaging, the technique widely used for this purpose, requires a fluorescent dye which may inhibit biological responses or a fluorescent-tagged target protein created through time-consuming genetic manipulation. In this study, we applied two-dimensional surface plasmon resonance (SPR) imaging to monitoring the translocation of protein kinase C (PKC), a major GPCR-coupled signaling molecule in the widely used HEK293 cell lines and examined whether the signaling of, and, modulation between heterologously expressed GPCRs can be measured without fluorescent labeling.ResultsWe cultured HEK293 cells on the gold-plated slide glass and evoked SPR at the interface between the cell’s plasma membrane and the gold surface with incident light. The translocation of activated native PKC to the plasma membrane is expected to alter the incident angle-SPR extent relation, and this could be detected as a change in the intensity of light reflection from the specimen illuminated at a fixed incident angle. Direct activation of PKC with 12-O-tetradecanoylphorbol-13-acetate increased the reflection intensity. This increase indeed reported PKC translocation because it was reduced by a pre-treatment with bisindolylmaleimide-1, a PKC inhibitor. We further applied this technique to a stable HEK293 cell line heterologously expressing the GPCRs type-1 metabotropic glutamate receptor (mGluR1) and adenosine A1 receptor (A1R). (RS)-3,5-dihydroxyphenylglycine, a mGluR1 agonist, increased the reflection intensity, and the PKC inhibitor reduced this increase. A pre-treatment with (R)-N6-phenylisopropyladenosine, an A1R-selective agonist suppressed mGluR1-mediated reflection increase. These results suggest that our technique can detect PKC translocation initiated by ligand binding to mGluR1 and its modulation by A1R.ConclusionsSPR imaging turned out to be utilizable for monitoring GPCR-mediated PKC translocation and its modulation by a different GPCR in a heterologous expression system. This technique provides a powerful yet easy-to-use tool for molecular study of biological responses and pharmaceutical development.

Phorbol Myristate Acetate Suppresses Breast Cancer Cell Growth via Down‐regulation of P‐Rex1 Expression

Phosphatidylinositol‐3,4,5‐trisphosphate‐dependent Rac exchange factor 1 (P‐Rex1) is highly overexpressed in estrogen receptor‐positive and ErbB2‐overexpressing human luminal breast tumors. The present study was to investigate the role and mechanism underlying phorbol‐12‐myristate‐13‐acetate (PMA), a protein kinase C (PKC) activator, down‐regulation of P‐Rex1 in breast cancer cells. P‐Rex1 mRNA and protein expression were determined by real‐time PCR and western blot analysis, respectively. Cell growth was analyzed by counting cells number and 5‐bromo‐2′‐deoxyuridine (BrdU) cell proliferation assay. The translocation of PKC was examined by immunofluorescence staining. Our results showed that treatment with PMA caused a time‐ and concentration‐dependent decrease in P‐Rex1 protein levels in breast cancer MCF‐7 and BT‐474 cells with a maximum reduction of 87.2 ± 1.1 and 57.0 ± 8.6%, respectively, at a concentration of 10 ng/ml. Interestingly, either PMA treatment or silence of P‐Rex1 by siRNA inhibits MCF‐7 and BT‐474 cell growth by 60–80%, which can be attenuated by restoring P‐Rex1 expression. MCF‐7 and BT‐474 cells express PMA‐sensitive conventional (α and β) and novel (δ, ɛ and η) PKC isoforms. Immunofluorescence staining analysis of breast cancer cells showed translocation of PKCs from cytosol to the plasma membrane and/or nucleus, an indication of their activation upon PMA stimulation. The PKC general inhibitor Gö6983, but not the conventional PKC isoform inhibitor Gö6976, blocked PMA inhibition of P‐Rex1 expression and breast cancer cell growth. Expression of a constitutively active PKCɛ mutant suppressed P‐Rex1 expression and breast cancer cell growth, which was blocked by Gö6983 treatment. In contrast, expression of wild‐type PKCɛ, wild‐type or constitutively active PKCδ and PKCη had no effect. Together, our data suggest that PMA suppresses breast cancer cell growth via P‐Rex1 down‐regulation and PKCɛ activation plays a key role in this regulation. Our study may provide a novel strategy for development of chemotherapeutic agents for P‐Rex1‐overexpressing breast cancer patients that develop resistance to anti‐estrogen and/or anti‐ErbB2 therapies.Support or Funding InformationSupported by grants from NIH (5P20GM103489 and R01HL116849), Nebraska State LB595 research program and the National Natural Science Foundation of China (31100595 and 31300683).

2,3,7,8-tetrachlorodibenzo-p-dioxin exposure influence the expression of glutamate transporter GLT-1 in C6 glioma cells via the Ca(2+) /protein kinase C pathway.

The widespread environmental contaminant, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), is considered one of the most toxic dioxin-like compounds. Although epidemiological studies have shown that TCDD exposure is linked to some neurological and neurophysiological disorders, the underlying mechanism of TCDD-mediated neurotoxicity has remained unclear. Astrocytes are the most abundant cells in the nervous systems, and are recognized as the important mediators of normal brain functions as well as neurological, neurodevelopmental and neurodegenerative brain diseases. In this study, we investigated the role of TCDD in regulating the expression of glutamate transporter GLT-1 in astrocytes. TCDD, at concentrations of 0.1-100 nm, had no significantly harmful effect on the viability of C6 glioma cells. However, the expression of GLT-1 in C6 glioma cells was downregulated in a dose- and time-dependent manner. TCDD also caused activation of protein kinase C (PKC), as TCDD induced translocation of the PKC from the cytoplasm or perinuclear to the membrane. The translocation of PKC was inhibited by one Ca(2+) blocker, nifedipine, suggesting that the effects are triggered by the initial elevated intracellular concentration of free Ca(2+) . Finally, we showed that inhibition of the PKC activity reverses the TCDD-triggered reduction of GLT-1. In summary, our results suggested that TCDD exposure could downregulate the expression of GLT-1 in C6 via Ca(2+) /PKC pathway. The downregulation of GLT-1 might participate in TCDD-mediated neurotoxicity. Copyright © 2016 John Wiley & Sons, Ltd.

Lysophosphatidic acid upregulates connective tissue growth factor expression in osteoblasts through the GPCR/PKC and PKA pathways.

Lysophosphatidic acid (LPA) is an efficient, bioactive phospholipid involved in various biological processes. In this study, LPA-induced connective tissue growth factor (CTGF/CCN2) expression and the underlying mechanisms were investigated using the MC3T3-E1 cell line. The MC3T3-E1 cells were stimulated with an inhibitor of LPA receptors, an activator and inhibitor of protein kinase C (PKC) and protein kinase A (PKA) for indicated periods of time. RT-qPCR and western blot analyses were used to measure the expression levels of CCN2. Immunofluorescence staining was used to observe the translocation of PKC. The mRNA expression level of CCN2 was increased following stimulation of the cells with LPA; LPA transiently induced the mRNA expression of CCN2; maximum expression levels were observed 2 h following stimulation with LPA. This increase was accompanied by CCN2 protein synthesis. LPA receptor1/3 was inhibited by Ki16425, a specific inhibitor of LPA1/3; as a result, the LPA-induced increase in CCN2 expression was abrogated. LPA also induced the membrane translocation of PKC and enhanced PKC activity in the osteoblasts. Pre-treatment of the osteoblasts with staurosporine prevented the increase in CCN2 expression by induced by LPA, and the activation of PKC by phorbol 12-myristate 13-acetate (PMA) enhanced CCN2 expression, indicating that the PKC pathway is involved in the LPA-induced increase in CCN2 expression. The interference of PKA signaling also led to the induction of CCN2 expresion by LPA. These data indicate that LPA increases CCN2 expression through the activation of PKC and PKA. Thus, the regulatory functions of the PKC and PKA pathways are implicated in the LPA-induced increase in CCN2 expression.

Isoform-specific dynamic translocation of PKC by α1-adrenoceptor stimulation in live cells

Protein kinase C (PKC) plays key roles in the regulation of signal transduction and cellular function in various cell types. At least ten PKC isoforms have been identified and intracellular localization and trafficking of these individual isoforms are important for regulation of enzyme activity and substrate specificity. PKC can be activated downstream of Gq-protein coupled receptor (GqPCR) signaling and translocate to various cellular compartments including plasma membrane (PM). Recent reports suggested that different types of GqPCRs would activate different PKC isoforms (classic, novel and atypical PKCs) with different trafficking patterns. However, the knowledge of isoform-specific activation of PKC by each GqPCR is limited. α1-Adrenoceptor (α1-AR) is one of the GqPCRs highly expressed in the cardiovascular system. In this study, we examined the isoform-specific dynamic translocation of PKC in living HEK293T cells by α1-AR stimulation (α1-ARS). Rat PKCα, βI, βII, δ, ε and ζ fused with GFP at C-term were co-transfected with human α1A-AR into HEK293T cells. The isoform-specific dynamic translocation of PKC in living HEK293T cells by α1-ARS using phenylephrine was measured by confocal microscopy. Before stimulation, GFP-PKCs were localized at cytosolic region. α1-ARS strongly and rapidly translocated a classical PKC (cPKC), PKCα, (<30 s) to PM, with PKCα returning diffusively into the cytosol within 5 min. α1-ARS rapidly translocated other cPKCs, PKCβI and PKCβII, to the PM (<30 s), with sustained membrane localization. One novel PKC (nPKC), PKCε, but not another nPKC, PKCδ, was translocated by α1-AR stimulation to the PM (<30 s) and its membrane localization was also sustained. Finally, α1-AR stimulation did not cause a diacylglycerol-insensitive atypical PKC, PKCζ translocation. Our data suggest that PKCα, β and ε activation may underlie physiological and pathophysiological responses of α1-AR signaling for the phosphorylation of membrane-associated substrates including ion-channel and transporter proteins in the cardiovascular system.

Protein Kinase C (PKC)ζ Pseudosubstrate Inhibitor Peptide Promiscuously Binds PKC Family Isoforms and Disrupts Conventional PKC Targeting and Translocation.

PKMζ is generated via an alternative transcriptional start site in the atypical protein kinase C (PKC)ζ isoform, which removes N-terminal regulatory elements, including the inhibitory pseudosubstrate domain, consequently rendering the kinase constitutively active. Persistent PKMζ activity has been proposed as a molecular mechanism for the long-term maintenance of synaptic plasticity underlying some forms of memory. Many studies supporting a role for PKMζ in synaptic plasticity and memory have relied on the PKCζ pseudosubstrate-derived ζ-inhibitory peptide (ZIP). However, recent studies have demonstrated that ZIP-induced impairments to synaptic plasticity and memory occur even in the absence of PKCζ, suggesting that ZIP exerts its actions via additional cellular targets. In this study, we demonstrated that ZIP interacts with conventional and novel PKC, in addition to atypical PKC isoforms. Moreover, when brain abundance of each PKC isoform and affinity for ZIP are taken into account, the signaling capacity of ZIP-responsive pools of conventional and novel PKCs may match or exceed that for atypical PKCs. Pseudosubstrate-derived peptides, like ZIP, are thought to exert their cellular action primarily by inhibiting PKC catalytic activity; however, the ZIP-sensitive catalytic core of PKC is known to participate in the enzyme's subcellular targeting, suggesting an additional mode of ZIP action. Indeed, we have demonstrated that ZIP potently disrupts PKCα interaction with the PKC-targeting protein A-kinase anchoring protein (AKAP) 79 and interferes with ionomycin-induced translocation of conventional PKC to the plasma membrane. Thus, ZIP exhibits broad-spectrum action toward the PKC family of enzymes, and this action may contribute to its unique ability to impair memory.

Common mechanisms of Alzheimer's disease and ischemic stroke: the role of protein kinase C in the progression of age-related neurodegeneration.

Ischemic stroke and Alzheimer's disease (AD), despite being distinct disease entities, share numerous pathophysiological mechanisms such as those mediated by inflammation, immune exhaustion, and neurovascular unit compromise. An important shared mechanistic link is acute and chronic changes in protein kinase C (PKC) activity. PKC isoforms have widespread functions important for memory, blood-brain barrier maintenance, and injury repair that change as the body ages. Disease states accelerate PKC functional modifications. Mutated forms of PKC can contribute to neurodegeneration and cognitive decline. In some cases the PKC isoforms are still functional but are not successfully translocated to appropriate locations within the cell. The deficits in proper PKC translocation worsen stroke outcome and amyloid-β toxicity. Cross talk between the innate immune system and PKC pathways contribute to the vascular status within the aging brain. Unfortunately, comorbidities such as diabetes, obesity, and hypertension disrupt normal communication between the two systems. The focus of this review is to highlight what is known about PKC function, how isoforms of PKC change with age, and what additional alterations are consequences of stroke and AD. The goal is to highlight future therapeutic targets that can be applied to both the treatment and prevention of neurologic disease. Although the pathology of ischemic stroke and AD are different, the similarity in PKC responses warrants further investigation, especially as PKC-dependent events may serve as an important connection linking age-related brain injury.

Abstract 5315: Quantitative proteomic assessment of differential ligand responses downstream of protein kinase C activation

Abstract Bryostatin 1 (bryo 1) is an anticancer agent currently in multiple clinical trials with a unique mechanism of action. Whereas bryo 1 binds to and activates protein kinase C (PKC) like the phorbol esters, it paradoxically antagonizes many but not all phorbol ester responses. In LNCaP prostate cancer cells the typical phorbol ester PMA induces TNFalpha secretion and apoptosis while bryo 1 fails to do so and antagonizes the PMA effect when used in combination. The mechanisms responsible for the unique effect of bryo 1 are still unknown. We have shown previously, using transcription factor analysis of microarray data from LNCaP cells, that multiple transcription factor families are involved in the dramatic transcriptional response to PMA and bryo 1 (3131 genes for PMA and 1569 genes for bryo 1). Further, analysis of 45 signaling pathways using reporter assays identified significant changes in 7 pathways induced both by PMA and bryo 1 at 6 hours. To further investigate the altered pathways, the novel Simple Western capillary immunoassay system (ProteinSimple) was used to analyze protein level changes following PKC activation. Simple Western system performs automated western analysis in capillaries using nanograms of lysate and provides digital quantitation with good sensitivity and excellent reproducibility. The system runs at a through-put of 96 sample / target combinations in a single overnight run. We analyzed 35 signals in total lysates (for detection of changes in protein levels and phosphorylation) and in nuclear extracts (for detection of translocation) from LNCaP cells treated for 60 min and 6 hrs. Changes downstream of PKC activation were measured, including PKC/PKD1 phosphorylation, activation of multiple members of the MEK/ERK pathway and levels of transcription factors of the NFKB, AP1, Stat and EGR families. Simple Western results correlated very well with conventional western analysis. The analysis validated the changes described previously using other methods on activation of MEK/ERK pathway and the changes in PKC phosphorylation, translocation and degradation. Bryo1 is different from PMA in failing to induce phosphorylation of PKCdelta at Tyr311 and the translocation of PKCalpha, delta, epsilon, and PKD1 to the nuclear fraction. Bryo1 is similar to PMA in inducing many of the early responses detected at 60 min (pPKCdeltaSer299, pMEK, pERK1/2, pPKD1 in total lysates, decrease in STAT3 levels etc.) but it fails to sustain the induced responses or to induce the late responses detected at 6 hours (pNFκBp65, increase in Fra1 and FosB levels, degradation of cMyc, translocation of cRel and RelB etc.). Using Simple Western system, we developed an approach for precise and accurate measurement of differential PMA and bryo 1 signaling in PKC activation. Further investigation needs to be performed to identify the mechanism/s responsible for the early termination of bryo 1 induced responses. Citation Format: Jin-Qiu Chen, Noemi Kedei, Michelle A. Herrmann, Peter M. Blumberg. Quantitative proteomic assessment of differential ligand responses downstream of protein kinase C activation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5315. doi:10.1158/1538-7445.AM2014-5315