scholarly journals Protein kinase C epsilon delays latency until anoxic depolarization through arc expression and GluR2 internalization

2017 ◽  
Vol 37 (12) ◽  
pp. 3774-3788 ◽  
Author(s):  
Charles H Cohan ◽  
Holly M Stradecki-Cohan ◽  
Kahlilia C Morris-Blanco ◽  
Nathalie Khoury ◽  
Kevin B Koronowski ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Learning And Memory ◽  
Global Cerebral Ischemia ◽  
Mrna Levels ◽  
Dependent Manner ◽  
Kinase C ◽  
Anoxic Depolarization ◽  
Protein Kinase C Epsilon

Global cerebral ischemia is a debilitating injury that damages the CA1 region of the hippocampus, an area important for learning and memory. Protein kinase C epsilon (PKCɛ) activation is a critical component of many neuroprotective treatments. The ability of PKCɛ activation to regulate AMPA receptors (AMPARs) remains unexplored despite the role of AMPARs in excitotoxicity after brain ischemia. We determined that PKCɛ activation increased expression of a protein linked to learning and memory, activity-regulated cytoskeleton-associated protein (arc). Also, arc is necessary for neuroprotection and confers protection through decreasing AMPAR currents via GluR2 internalization. In vivo, activation of PKCɛ increased arc expression through a BDNF/TrkB pathway, and decreased GluR2 mRNA levels. In hippocampal cultured slices, PKCɛ activation decreased AMPAR current amplitudes in an arc- and GluR2-dependent manner. Additionally, PKCɛ activation triggered an arc- and GluR2 internalization-dependent delay in latency until anoxic depolarization. Inhibiting arc also blocked PKCɛ-mediated neuroprotection against lethal oxygen and glucose deprivation. These data characterize a novel PKCɛ-dependent mechanism that for the first time defines a role for arc and AMPAR internalization in conferring neuroprotection.

Endotoxin-Induced Tissue Factor in Human Monocytes is Dependent upon Protein Kinase C Activation

1993 ◽  
Vol 70 (05) ◽  
pp. 800-806 ◽  
Author(s):  
C Ternisien ◽  
M Ramani ◽  
V Ollivier ◽  
F Khechai ◽  
T Vu ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Tissue Factor ◽  
Factor Ix ◽  
Transcriptional Level ◽  
Mrna Levels ◽  
Dependent Manner ◽  
Human Monocytes ◽  
Kinase C ◽  
Pkc Activation

SummaryTissue factor (TF) is a transmembrane receptor which, in association with factors VII and Vila, activates factor IX and X, thereby activating the coagulation protease cascades. In response to bacterial lipopolysaccharide (LPS) monocytes transcribe, synthesize and express TF on their surface. We investigated whether LPS-induced TF in human monocytes is mediated by protein kinase C (PKC) activation. The PKC agonists phorbol 12- myristate 13-acetate (PMA) and phorbol 12, 13 dibutyrate (PdBu) were both potent inducers of TF in human monocytes, whereas 4 alpha-12, 13 didecanoate (4 a-Pdd) had no such effect. Both LPS- and PMA-induced TF activity were inhibited, in a concentration dependent manner, by three different PKC inhibitors: H7, staurosporine and calphostin C. TF antigen determination confirmed that LPS-induced cell-surface TF protein levels decreased in parallel to TF functional activity under staurosporine treatment. Moreover, Northern blot analysis of total RNA from LPS- or PMA-stimulated monocytes showed a concentration-dependent decrease in TF mRNA levels in response to H7 and staurosporine. The decay rate of LPS-induced TF mRNA evaluated after the arrest of transcription by actinomycin D was not affected by the addition of staurosporine, suggesting that its inhibitory effect occurred at a transcriptional level. We conclude that LPS-induced production of TF and its mRNA by human monocytes are dependent on PKC activation.

scholarly journals Post-transcriptional regulation of apolipoprotein E expression in mouse macrophages by phorbol ester

1993 ◽  
Vol 292 (1) ◽  
pp. 105-111 ◽  
Author(s):  
L Dory
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Apolipoprotein E ◽  
Phorbol Ester ◽  
Mrna Levels ◽  
Dependent Manner ◽  
Kinase C ◽  
Protein Kinase C Activity ◽  
Mouse Macrophages ◽  
Apoe Expression

Phorbol ester-mediated differentiation of THP-1 cells (a human monocytic cell line) into mature macrophages is associated with a transcriptional induction of apolipoprotein E (apoE) expression [Auwerx, Deeb, Brunzell, Peng and Chait (1988) Biochemistry 27, 2651-2655]. Endotoxin, on the other hand, which may also act through activation of protein kinase C, is a potent inhibitor of apoE expression in mouse macrophages [Werb and Chin (1983) J. Biol. Chem. 258, 10642-10648]. The present experiments examine the effect of phorbol ester, an activator of protein kinase C, on the apoE expression in mouse thioglycollate-elicited peritoneal macrophages. Phorbol ester inhibits apoE expression in a specific, time- and dose-dependent manner. A 75% inhibition in the rate of apoE secretion, but not that of total protein, was observed following a 4.5 h incubation with 160 nM phorbol ester, although nearly full inhibition was obtained with 40 nM. The changes in apoE secretion were paralleled by similar changes in apoE synthesis, indicating synthesis as the primary site of action. The decreased rates of apoE synthesis are shown not to be due to increased apoE degradation. The profound inhibition of apoE synthesis was not accompanied by significant changes in apoE mRNA levels at any concentration of phorbol ester (up to 16 microM), or length of treatment (up to 24 h), suggesting a post-transcriptional locus of regulation of apoE expression. Although the early changes in apoE synthesis correlate with increased microsomal protein kinase C activity, the suppression of apoE expression persists even during conditions of nearly complete (> 95%) loss of protein kinase C activity, suggesting that the direct or indirect effect of protein kinase C on apoE expression is mediated by a stable phosphorylated protein, or that the observed effects are mediated through a protein kinase C species that is not readily downregulated by phorbol esters. The presented studies clearly demonstrate the potential importance of the translational regulation of apoE expression through the protein kinase C signal transduction pathway.

14-3-3 Isotypes facilitate coupling of protein kinase C-ζ to Raf-1: negative regulation by 14-3-3 phosphorylation

2000 ◽  
Vol 345 (2) ◽  
pp. 297-306 ◽  
Author(s):  
Paulus C. J. VAN DER HOEVEN ◽  
José C. M. VAN DER WAL ◽  
Paula RUURS ◽  
Marc C. M. VAN DIJK ◽  
Wim J. VAN BLITTERSWIJK
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Complex Formation ◽  
Dependent Manner ◽  
Cos Cells ◽  
Kinase C ◽  
Pkc Ζ ◽  
Co Precipitation

14-3-3 Proteins may function as adapters or scaffold in signal-transduction pathways. We found previously that protein kinase C-ζ (PKC-ζ) can phosphorylate and activate Raf-1 in a signalling complex [van Dijk, Hilkmann and van Blitterswijk (1997) Biochem. J. 325, 303-307]. We report now that PKC-ζ-Raf-1 interaction is mediated by 14-3-3 proteins in vitro and in vivo. Co-immunoprecipitation experiments in COS cells revealed that complex formation between PKC-ζ and Raf-1 is mediated strongly by the 14-3-3β and -θ isotypes, but not by 14-3-3ζ. Far-Western blotting revealed that 14-3-3 binds PKC-ζ directly at its regulatory domain, where a S186A mutation in a putative 14-3-3-binding domain strongly reduced the binding and the complex formation with 14-3-3β and Raf-1. Treatment of PKC-ζ with lambda protein phosphatase also reduced its binding to 14-3-3β in vitro. Preincubation of an immobilized Raf-1 construct with 14-3-3β facilitated PKC-ζ binding. Together, the results suggest that 14-3-3 binds both PKC-ζ (at phospho-Ser-186) and Raf-1 in a ternary complex. Complex formation was much stronger with a kinase-inactive PKC-ζ mutant than with wild-type PKC-ζ, supporting the idea that kinase activity leads to complex dissociation. 14-3-3β and -θ were substrates for PKC-ζ, whereas 14-3-3ζ was not. Phosphorylation of 14-3-3β by PKC-ζ negatively regulated their physical association. 14-3-3β with its putative PKC-ζ phosphorylation sites mutated enhanced co-precipitation between PKC-ζ and Raf-1, suggesting that phosphorylation of 14-3-3 by PKC-ζ weakens the complex in vivo. We conclude that 14-3-3 facilitates coupling of PKC-ζ to Raf-1 in an isotype-specific and phosphorylation-dependent manner. We suggest that 14-3-3 is a transient mediator of Raf-1 phosphorylation and activation by PKC-ζ.

scholarly journals The Central Role of Protein Kinase C Epsilon in Cyanide Cardiotoxicity and Its Treatment

2019 ◽  
Vol 171 (1) ◽  
pp. 247-257 ◽  
Author(s):  
Joseph Y Cheung ◽  
Salim Merali ◽  
JuFang Wang ◽  
Xue-Qian Zhang ◽  
Jianliang Song ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Cardiac Contractility ◽  
Ionic Currents ◽  
Ion Fluxes ◽  
Consensus Sequences ◽  
Kinase C ◽  
Protein Kinase C Epsilon ◽  
Life Threatening

Abstract In adult mouse myocytes, brief exposure to sodium cyanide (CN) in the presence of glucose does not decrease ATP levels, yet produces profound reduction in contractility, intracellular Ca2+ concentration ([Ca2+]i) transient and L-type Ca2+ current (ICa) amplitudes. We analyzed proteomes from myocytes exposed to CN, focusing on ionic currents associated with excitation-contraction coupling. CN induced phosphorylation of α1c subunit of L-type Ca2+ channel and α2 subunit of Na+-K+-ATPase. Methylene blue (MB), a CN antidote that we previously reported to ameliorate CN-induced reduction in contraction, [Ca2+]i transient and ICa amplitudes, was able to reverse this phosphorylation. CN decreased Na+-K+-ATPase current contributed by α2 but not α1 subunit, an effect that was also counteracted by MB. Peptide consensus sequences suggested CN-induced phosphorylation was mediated by protein kinase C epsilon (PKCε). Indeed, CN stimulated PKC kinase activity and induced PKCε membrane translocation, effects that were prevented by MB. Pretreatment with myristoylated PKCε translocation activator or inhibitor peptides mimicked and inhibited the effects of CN on ICa and myocyte contraction, respectively. We conclude that CN activates PKCε, which phosphorylates L-type Ca2+ channel and Na+-K+-ATPase, resulting in depressed cardiac contractility. We hypothesize that this inhibition of ion fluxes represents a novel mechanism by which the cardiomyocyte reduces its ATP demand (decreased ion fluxes and contractility), diminishes ATP turnover and preserves cell viability. However, this cellular protective effect translates into life-threatening cardiogenic shock in vivo, thereby creating a profound disconnect between survival mechanisms at the cardiomyocyte level from those at the level of the whole organism.

Abstract TP267: Protein Kinase C Epsilon-activation Mediates Ischemic Neuroprotection by Activating an Activity-regulated Cytoskeleton-associated Protein-dependent Mechanism

Stroke ◽  
2017 ◽  
Vol 48 (suppl_1) ◽  
Author(s):  
Charles H Cohan ◽  
Holly M Stradecki ◽  
Kahlilia C Morris-Blanco ◽  
Nathalie Khoury ◽  
Kevin B Koronowski ◽  
...  
Keyword(s):  
Cell Death ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Glucose Deprivation ◽  
T Test ◽  
Kinase C ◽  
Protein Kinase C Epsilon ◽  
Cultured Slices ◽  
Dependent Mechanism

Introduction: Cerebral ischemia can trigger cell death in the CA1 region of the hippocampus, an area important for memory. Protein kinase C epsilon (PKCε) activation prior to ischemia protects the CA1 from injury by modulating neurotransmission. The underlying mechanism needs further study. Hypothesis: PKCε-induced neuroprotection increases latency until anoxic depolarization (AD) through an activity-regulated cytoskeleton-associated protein (arc)-dependent mechanism of regulating AMPAR currents. Results: In vivo activation of PKCε by the PKCε-activator ψε-Receptor of Activated C Kinase (ψεRACK) increased BDNF 5.99 +/- 0.11 fold and TrkB phosphorylation levels 2.94 +/- 0.32 fold (enhancers of arc protein levels) (n = 4, p < 0.005, t-test). Arc mRNA and protein was increased 143.97 +/-7.68 % and 1.91 +/- 0.22 fold (n = 9, p < 0.005, t-test). Inhibition of arc using antisense oligodeoxynucleotides (arc AS ODNs) in cultured slices blocked PKCε-mediated neuroprotection against lethal oxygen and glucose deprivation (OGD) from 35.91 +/- 5.97 to 74.93 +/- 4.24 % cell death (n = 6, p < 0.005, ANOVA, Bonferroni). ΨεRACK decreased AMPAR-mediated mEPSC amplitude to 12.75 +/- 0.35 pA from 14.80 +/- 0.39 pA (n = 20, p < 0.01, ANOVA, Bonferroni). This effect was arc-dependent. Additionally, ψεRACK treatment increased latency until AD from 29.27 +/- 3.6 min 50.77 +/- 5.08 min (n = 13, p < 0.01, ANOVA, Bonferroni). This increase was arc-dependent, and required AMPAR internalization. Inhibiting internalization reduced AD latency from 54.5 +/- 8.40 min to 22.3 + 5.17 min (n = 6, p <0.005, t-test). Conclusion: Arc expression is necessary for neuroprotection afforded by PKCε-activation, modulates excitatory synaptic strength, and increases latency until AD. Methods: Western blot: Proteins (40 μg) were separated on a 12% SDS-PAGE gel. Immunofluorescence : 30 μm sections were incubated with 1:500 NeuN and 1:50 arc in PBS with 0.8% triton. Cultured slices preparation : sections from P9-11 rats were plated on inserts and cultured for 14 days. PI measurements of cell death : Slices were incubated in medium with 2 μg/mL PI. mEPSC measurements : Whole-cell voltage clamp was performed. AD: OGD, perfusate was switched to glucose free media, and bubbled with a 95% N 2 and 5% CO 2 gas.

scholarly journals gp140v-fms molecules expressed at the surface of cells transformed by the McDonough strain of feline sarcoma virus are phosphorylated in tyrosine and serine.

1986 ◽  
Vol 6 (12) ◽  
pp. 4745-4748 ◽  
Author(s):  
T Tamura ◽  
E Simon ◽  
H Niemann ◽  
G T Snoek ◽  
H Bauer
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Specific Inhibitor ◽  
Dependent Manner ◽  
Sodium Orthovanadate ◽  
Transmembrane Glycoprotein ◽  
Kinase C ◽  
Sarcoma Virus

Cells transformed by the McDonough strain of feline sarcoma virus express at their surface a v-fms-specific transmembrane glycoprotein designated gp140v-fms. By labeling with 32Pi, gp140v-fms was shown to be phosphorylated 30-fold more in serine residues than were the cytosolic v-fms polypeptides gp180gag-fms and gp120v-fms. By using the phosphotyrosine phosphatase-specific inhibitor sodium orthovanadate, an additional tyrosine phosphorylation was observed in vivo, again involving predominantly gp140v-fms. In vitro studies showed that the v-fms proteins were phosphorylated by protein kinase C in a calcium- and phosphatidylserine-dependent manner.

scholarly journals Protein Kinase C Epsilon Regulates Mitochondrial Pools of Nampt and NAD Following Resveratrol and Ischemic Preconditioning in the Rat Cortex

2014 ◽  
Vol 34 (6) ◽  
pp. 1024-1032 ◽  
Author(s):  
Kahlilia C Morris-Blanco ◽  
Charles H Cohan ◽  
Jake T Neumann ◽  
Thomas J Sick ◽  
Miguel A Perez-Pinzon
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Ischemic Preconditioning ◽  
Nicotinamide Adenine Dinucleotide ◽  
Nicotinamide Adenine ◽  
Dependent Manner ◽  
Nicotinamide Phosphoribosyltransferase ◽  
Resveratrol Treatment ◽  
Kinase C ◽  
Protein Kinase C Epsilon

Preserving mitochondrial pools of nicotinamide adenine dinucleotide (NAD) or nicotinamide phosphoribosyltransferase (Nampt), an enzyme involved in NAD production, maintains mitochondrial function and confers neuroprotection after ischemic stress. However, the mechanisms involved in regulating mitochondrial-localized Nampt or NAD have not been defined. In this study, we investigated the roles of protein kinase C epsilon (PKCε) and AMP-activated protein kinase (AMPK) in regulating mitochondrial pools of Nampt and NAD after resveratrol or ischemic preconditioning (IPC) in the cortex and in primary neuronal-glial cortical cultures. Using the specific PKCε agonist ψεRACK, we found that PKCε induced robust activation of AMPK in vitro and in vivo and that AMPK was required for PKCε-mediated ischemic neuroprotection. In purified mitochondrial fractions, PKCε enhanced Nampt levels in an AMPK-dependent manner and was required for increased mitochondrial Nampt after IPC or resveratrol treatment. Analysis of intrinsic NAD autofluorescence using two-photon microscopy revealed that PKCε modulated NAD in the mitochondrial fraction. Further assessments of mitochondrial NAD concentrations showed that PKCε has a key role in regulating the mitochondrial NAD+/nicotinamide adenine dinucleotide reduced (NADH) ratio after IPC and resveratrol treatment in an AMPK- and Nampt-dependent manner. These findings indicate that PKCε is critical to increase or maintain mitochondrial Nampt and NAD after pathways of ischemic neuroprotection in the brain.

Tumor-promoting phorbol esters induce angiogenesis in vivo

1988 ◽  
Vol 254 (2) ◽  
pp. C318-C322 ◽  
Author(s):  
P. B. Morris ◽  
T. Hida ◽  
P. J. Blackshear ◽  
G. K. Klintworth ◽  
J. L. Swain
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Tumor Growth ◽  
Phorbol Esters ◽  
Dependent Manner ◽  
Tumor Promoters ◽  
Kinase C ◽  
Support Tumor Growth ◽  
Activate Protein Kinase

It has been hypothesized that tumor growth is dependent on the concomitant growth of its vascular supply, and thus agents that stimulate angiogenesis may help support tumor growth. Phorbol esters are potent tumor promoters that induce a variety of biochemical effects in cells, including activation of protein kinase C. The specific mechanisms responsible for tumor promotion by phorbol esters are unknown. The objective of this study was to determine whether the tumor-promoting phorbol esters can induce vascular growth. Phorbol esters were tested for their ability to stimulate angiogenesis in vivo using the chick chorioallantoic membrane and rabbit cornea assays. The active tumor promoters 12-O-tetradecanoyl phorbol-13-acetate and phorbol 12,13-didecanoate, which activate protein kinase C, were found to stimulate angiogenesis in a dose-dependent manner. In contrast, 4 alpha-phorbol 12,13-didecanoate, which is inactive as a tumor promoter and does not activate protein kinase C, did not stimulate angiogenesis. Phorbol esters may be indirect angiogenic factors, since no mitogenic effect on bovine capillary endothelial cells in culture could be detected. The results demonstrate that the tumor-promoting activity of phorbol esters may, in part, be secondary to stimulation of neovascularization to support tumor growth and suggest a role for the activation of protein kinase C in this process.

scholarly journals Insulin increases mRNA levels of protein kinase C-α and -β in rat adipocytes and protein kinase C-α, -β and -θ in rat skeletal muscle

1995 ◽  
Vol 308 (1) ◽  
pp. 181-187 ◽  
Author(s):  
A Avignon ◽  
M L Standaert ◽  
K Yamada ◽  
H Mischak ◽  
B Spencer ◽  
...  
Keyword(s):  
Adipose Tissue ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Mrna Levels ◽  
Rat Adipocytes ◽  
Kinase C ◽  
Pkc Epsilon ◽  
Pkc Alpha

Effects of insulin of levels of mRNA encoding protein kinase C (PKC)-alpha, PKC-beta, PKC-epsilon and PKC-theta were examined by ribonuclease protection assay in primary cultures of rat adipocytes in vitro, and in rat adipose tissue and gastrocnemius muscle in vivo. In all cases, insulin increased the levels of PKC-alpha mRNA and PKC-beta mRNA, and, in muscle, insulin also increased the level of PKC-theta mRNA. PKC-epsilon mRNA levels, on the other hand, were not altered significantly. Insulin also stimulated the apparent translocation of PKC-alpha, -beta, -epsilon and -theta, to the membrane fractions of adipocytes, adipose tissue and gastrocnemius muscles, and, in some instances, total PKC levels were diminished, e.g. PKC-alpha and PKC-beta in cultured adipocytes in vitro and/or whole adipose tissue in vivo, and PKC-alpha and PKC-theta in the gastrocnemius muscle. Thus, insulin-induced increases in PKC mRNA may have been partly compensatory in nature to restore PKC levels following translocation and proteolytic losses. However, much more severe depletion of PKC-alpha and PKC-beta by phorbol ester treatment in cultured rat adipocytes in vitro resulted in, if anything, smaller increases in PKC-alpha mRNA and PKC-beta mRNA, and it therefore appears that insulin effects on PKC mRNA levels were not simply due to decreases in respective PKC levels. In addition, effects of insulin, particularly on PKC-beta mRNA, could not be attributed to increased glucose metabolism, which alone decreased PKC-beta mRNA in cultured adipocytes in vitro. We conclude that insulin-induced translocation and degradation of PKC-alpha, PKC-beta and PKC-theta are attended by selective increases in their mRNAs. This mechanism of increasing mRNA may be important in maintaining PKC levels during the continued action of insulin.

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