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 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.

scholarly journals 1029-180 Impact of protein kinase C-epsilon for myocardial hypertrophy after chronic pressure overload: In-vivo study in protein kinase C-epsilon knockout mice

2004 ◽  
Vol 43 (5) ◽  
pp. A456
Author(s):  
Gunnar G Klein ◽  
Dagmar Oppermann ◽  
Arnd Schaefer ◽  
Martin Fuchs ◽  
Denise Hilfiker ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Knockout Mice ◽  
Myocardial Hypertrophy ◽  
Pressure Overload ◽  
In Vivo Study ◽  
Kinase C ◽  
Protein Kinase C Epsilon

Apoptin Induced Tumour Specific Killing Via Protein Kinase C Isoforms: A Potential Therapeutic Strategy In Plasma Cell Dyscrasias

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 5039-5039
Author(s):  
Jie Jiang ◽  
Daryl Cole ◽  
Nigel Westwood ◽  
Lee Macpherson ◽  
Farzin Farzaneh ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Death ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Cell Line ◽  
Phosphorylation Sites ◽  
Normal Cells ◽  
Myeloma Cells ◽  
Kinase C

Abstract Abstract 5039 There is mounting evidence that malignant cells have an intrinsic ability to prevent apoptosis. In the present study we provide evidence that the ectopic expression of Apoptin can restore the failing apoptosis program in myeloma cells via protein kinase C b (PKCb) and overcome intrinsic or acquired resistance to cell death. Apoptin (VP3), a chicken anemia virus (CAV)-derived protein has been shown to possess tumor specific cytotoxicity; its expression induces apoptosis in human tumor and transformed cells but there is little or no cytotoxic effect in normal human cells or cell lines derived from different tissues including peripheral blood mononuclear cells, fibroblast and epithelial cells. Several studies have shown that the tumor specific killing of Apoptin correlates with its phosphorylation and its subcellular localization. In cancer cells, Apoptin is localized in the nucleus and is phosphorylated on threonine108 by an as yet unknown kinase, whereas in normal cells Apoptin is detected in the cytoplasm and is essentially unphosphorylated. We developed a lentiviral vector encoding a GFP-Apoptin fusion gene (LV-GFP-AP), which delivers the Apoptin gene efficiently to haematopoietic cells. Apoptin significantly and selectively killed a number of leukemia cell lines including K562, HL60, U937, KG1 and NB4. In particular, the dexamethasone resistant multiple myeloma cell line MM1.R and the dexamethasone sensitive cell line MM1.S were efficiently killed by Apoptin. In contrast normal CD34+ cells were not killed and maintained their differentiation potential in multilineage colony formation assays. In addition, we showed that the dexamethasone resistant MM1.R cells were considerably more susceptible to Apoptin induced cell death than the parental matched MM1.S cells. This correlated with increased phosphorylation and activation of the Apoptin protein in MM1.R cells. Expression profiling of MM1.R and MM1.S cells identified a number of differentially expressed kinases. PKCb was over-expressed 9 fold in MM1.R cells and we showed, by immunoprecipitation and in vivo kinase studies, that this kinase was responsible for Apoptin phosphorylation. Analysis of the Apoptin amino acid sequence for potential phosphorylation sites indicated seven putative phosphorylation sites corresponding to the PKC kinase consensus motifs (S/TXK/R or S/TXXK/R). These sites included Thr-108, which has been previously shown to be phosphorylated in tumor cells, but not in normal cells. In vitro studies showed that recombinant Apoptin protein was phosphorylated by recombinant GST-PKCb protein at the Thr-108 site. Addition of a PKCb specific inhibitor resulted in diminished Apoptin phosphorylation whilst an unrelated inhibitor had no such effect. Furthermore, shRNA knockdown or drug mediated inhibition of PKCb in vivo significantly reduced Apoptin phosphorylation. Finally, we found that Apoptin mediated cell death proceeded via the up-regulation of PKCb, activation of caspase-9/3, cleavage of the PKCd catalytic domain and down-regulation of MERTK and AKT protein kinases. Collectively these results demonstrate a novel pathway for Apoptin activation involving PKCb and PKCd. Our results show that Apoptin is able to effectively eliminate multiple myeloma cells which have become resistant to dexamethasone. In addition, this study has led to the identification of tumor specific cellular targets such as PKCb, whose modulation by shRNAs and small molecule drugs can induce strong anti-myeloma effects. Importantly, the evidence from our data suggests that protein kinase C inhibitors may have an important therapeutic role in plasma cell neoplasia. Disclosures: No relevant conflicts of interest to declare.

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.

Time dependent protein kinase C epsilon activation during desflurane-induced pharmacological preconditioning in the rat heart in vivo

2004 ◽  
Vol 21 (Supplement 32) ◽  
pp. 52-53
Author(s):  
O. Toma ◽  
N. C. Weber ◽  
J. I. Wolter ◽  
B. Preckel ◽  
W. Schlack
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Rat Heart ◽  
Time Dependent ◽  
Dependent Protein Kinase ◽  
Kinase C ◽  
Protein Kinase C Epsilon ◽  
Dependent Protein ◽  
Pharmacological Preconditioning

In vitro and in vivo suppression of growth of rat liver epithelial tumor cells by antisense oligonucleotide against protein kinase C-alpha

2000 ◽  
Vol 33 (4) ◽  
pp. 601-608 ◽  
Author(s):  
Shwu-Bin Lin ◽  
Li-Ching Wu ◽  
Siao-Ling Huang ◽  
Hui-Lun Hsu ◽  
Sung-Hwa Hsieh ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Tumor Cells ◽  
Rat Liver ◽  
Antisense Oligonucleotide ◽  
Epithelial Tumor ◽  
Kinase C ◽  
Epithelial Tumor Cells

Chronic treatment by the calcium channel blocker ± PN 200-110 in the rat counteracts the stimulations of pituitary weight, prolactin release and pituitary C-kinase activity induced by a chronic estradiol treatment, in vivo

1990 ◽  
Vol 122 (3) ◽  
pp. 403-408
Author(s):  
Ph. Touraine ◽  
P. Birman ◽  
F. Bai-Grenier ◽  
C. Dubray ◽  
F. Peillon ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Calcium Channel ◽  
Calcium Channel Blocker ◽  
Channel Blocker ◽  
Plasma Prolactin ◽  
Estradiol Treatment ◽  
Kinase C ◽  
Protein Kinase C Activity

Abstract In order to investigate whether a calcium channel blocker could modulate the protein kinase C activity in normal and estradiol pretreated rat pituitary, female Wistar rats were treated or not (controls) with ± PN 200-110 (3 mg · kg−1 · day−1, sc) for 8 days or with estradiol cervical implants for 8 or 15 days, alone or in combination with PN 200-110 the last 8 days. Estradiol treatment induced a significant increase in plasma prolactin levels and pituitary weight. PN 200-110 administered to normal rats did not modify these parameters, whereas it reduced the effects of the 15 days estradiol treatment on prolactin levels (53.1 ± 4.9 vs 95.0 ±9.1 μg/l, p<0.0001) and pituitary weight (19.9 ± 0.4 vs 23.0 ± 0.6 mg, p <0.001), to values statistically comparable to those measured after 8 days of estradiol treatment. PN 200-110 alone did not induce any change in protein kinase C activity as compared with controls. In contrast, PN 200-110 treatment significantly counteracted the large increase in soluble activity and the decrease in the particulate one induced by estradiol between day 8 and day 15. We conclude that PN 200-110 opposed the stimulatory effects of chronic in vivo estradiol treatment on plasma prolactin levels and pituitary weight and that this regulation was related to a concomitant modulation of the protein kinase C activity.

A Study of Protein Kinase C Isozyme Distribution in Relation to Bcl-2 Expression during Apoptosis of Epithelial Cells in Vivo

1993 ◽  
Vol 207 (1) ◽  
pp. 68-73 ◽  
Author(s):  
Kirstine A. Knox ◽  
Gerald D. Johnson ◽  
John Gordon
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Epithelial Cells ◽  
Kinase C
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