Protein Kinase C-Mediated Desmin Phosphorylation is Related to Myofibril Disarray in Cardiomyopathic Hamster Heart1

The cardiomyopathic (CM) Syrian golden hamster (strain UM-X7.1) exhibits a hereditary cardiomyopathy, which causes premature death resulting from congestive heart failure. The CM animals show extensive cardiac myofibril disarray and myocardial calcium overload. The present study has been undertaken to examine the role of desmin phosphorylation in myofibril disarray observed in CM hearts. The data from skinned myofibril protein phosphorylation assays have shown that desmin can be phosphorylated by protein kinase C (PKC). There is no significant difference in the content of desmin between CM and control hamster hearts. However, the desmin from CM hearts has a higher phosphorylation level than that of the normal hearts. Furthermore, we have examined the distribution of desmin and myofibril organization with immunofluorescent microscopy and immunogold electron microscopy in cultured cardiac myocytes after treatment with the PKC-activating phorbol ester, 12-O-tetradecanylphorbol-13-acetate (TPA). When the cultured normal hamster cardiac cells are treated with TPA, desmin filaments are disassembled and the myofibrils become disarrayed. The myofibril disarray closely mimics that observed in untreated CM cultures. These results suggest that disassembly of desmin filaments, which could be caused by PKC-mediated phosphorylation, may be a factor in myofibril disarray in cardiomyopathic cells and that the intermediate filament protein, desmin, plays an Important role in maintaining myofibril alignment in cardiac cells.

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Use of a PCR-based method to characterize protein kinase C isoform expression in cardiac cells

AJP Cell Physiology ◽

10.1152/ajpcell.1993.264.5.c1350 ◽

1993 ◽

Vol 264 (5) ◽

pp. C1350-C1359 ◽

Cited By ~ 47

Author(s):

T. A. Kohout ◽

T. B. Rogers

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Cardiac Cells ◽

Rt Pcr ◽

Kinase C ◽

Pkc Zeta ◽

Pcr Products ◽

Isoform Expression ◽

Novel Isoforms ◽

Single Reaction

Molecular cloning has identified at least nine unique isozymes of protein kinase C (PKC) designated alpha, beta I, beta II, gamma, delta, epsilon, zeta, and eta/L, with the recent addition of the theta-isoform. Previous attempts to characterize PKC isoform expression in heart have been limited by low levels of protein and perhaps by the presence of novel isoforms. Thus to critically examine the diversity of PKC expression in cardiac cells, we developed a reverse transcriptase-polymerase chain reaction (RT-PCR) approach that would amplify regions of the target cDNA of all the PKC isozymes in a single reaction. Degenerate oligonucleotide primers were designed to recognize sequences in the conserved regions of the PKC sequence motif: the cysteine-rich and the ATP-binding regions. Amplification of target PKC cDNA sequences resulted in PCR products with unique sizes and restriction digestion properties. The system was validated by identifying PCR products that correspond to all of the PKC isoform transcripts, except PKC-zeta, in a single reaction with cDNA derived from hippocampus. Cardiac cDNA was RT-PCR amplified, and the products were analyzed by a combination of restriction mapping and DNA sequencing that revealed the presence of only the alpha, delta, epsilon, and eta isoforms in adult rat cardiac myocytes and cultured neonatal ventricular myocytes. A unique nondegenerate primer pair was synthesized to recognize PKC-zeta cDNA. Results with these primers show that PKC-zeta is present in both cardiac myocyte preparations as well. The RT-PCR method developed here is an efficient approach that is broadly useful to examine PKC expression in many tissues, including the identification of potentially novel isoforms.(ABSTRACT TRUNCATED AT 250 WORDS)

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Modulation of Na + Current Inactivation by Stimulation of Protein Kinase C in Cardiac Cells

Circulation Research ◽

10.1161/01.res.81.3.380 ◽

1997 ◽

Vol 81 (3) ◽

pp. 380-386 ◽

Cited By ~ 20

Author(s):

Cheryl L. Watson ◽

Michael R. Gold

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Cardiac Cells ◽

Na Current ◽

Kinase C ◽

Stimulation Of

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Alpha 1-adrenoceptor and purinoceptor agonists modulate Na-H antiport in single cardiac cells

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.1993.264.2.h310 ◽

1993 ◽

Vol 264 (2) ◽

pp. H310-H319 ◽

Cited By ~ 3

Author(s):

M. Puceat ◽

O. Clement-Chomienne ◽

A. Terzic ◽

G. Vassort

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Cardiac Cells ◽

Extracellular Medium ◽

Beta Adrenoceptor ◽

Kinase C ◽

Intracellular Ca ◽

Acid Load ◽

Ethanesulfonic Acid ◽

Stimulation Of

We investigated the effects of an alpha 1-adrenoceptor (phenylephrine) and a purinoceptor agonist (ATP), both of which accelerate the phosphoinositide turnover, on the Na-H antiport activity of rat single cardiac cells using the pH-sensitive fluorescent indicator seminaphthorhodafluor-1 (SNARF-1). Both phenylephrine, in the presence of a beta-adrenoceptor blocker, and ATP enhanced the ability of the cell to regulate its intracellular pH (pHi) after an imposed acid load. This effect was observed in HCO3-free N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) and prevented by Na-H antiport inhibitors ethylisopropylamiloride (EIPA) or amiloride. Similar results were obtained when cells were bathed in an acidic extracellular medium. Hence, the alpha 1-adrenoceptor and purinoceptor agonists activate the Na-H antiport even when it is partially inhibited by extracellular protons. To further evaluate the effects of the two neurohormones, the rate of proton efflux was estimated as a function of the magnitude of the imposed acid load. The results indicate that the agonist-induced modulation of the Na-H antiport is caused by an acceleration of its exchange activity and by a shift of its dependence on pHi toward more alkaline pH values. The agonist-mediated stimulation of the antiport was also observed in partially depolarized cells and was not dependent on intracellular Ca. Phorbol 12-myristate 13-acetate was not able to reproduce the effects of the agonists on the Na-H antiport. Conversely, the inhibitors of protein kinase C did not prevent the activation of the antiport by the neurohormones. Thus our data suggest that neither a Ca-calmodulin-dependent kinase nor protein kinase C is responsible for the alpha 1-adrenoceptor- and purinoceptor-mediated stimulation of the antiport.

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Different effects of intracellular Ca and protein kinase C on cardiac T and L Ca currents

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.1991.261.2.h364 ◽

1991 ◽

Vol 261 (2) ◽

pp. H364-H379 ◽

Cited By ~ 14

Author(s):

G. N. Tseng ◽

P. A. Boyden

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Time Course ◽

Kinase Inhibitor ◽

Cardiac Cells ◽

Kinase C ◽

Ventricular Cells ◽

Intracellular Messengers ◽

Intracellular Ca ◽

Ca Currents

We studied the effects of changing the intracellular Ca level ([Ca]i) and activating protein kinase C on the cardiac T and L Ca channels in single canine ventricular and Purkinje cells. Lowering [Ca]i increased the L current but decreased the T current, whereas elevating [Ca]i caused opposite changes. In ventricular cells, isoproterenol (1 microM) increased the amplitude of not only the L but also the T currents; the latter effect probably was secondary to a rise in [Ca]i following the augmentation of the L current. 12-O-tetradecanoylphorbol-13-acetate (TPA, 100 nM) decreased the T current but first increased and then decreased the L current. The TPA effects on the T and L currents were not mimicked by a phorbol ester that does not activate PKC (4 alpha-phorbol 12,13-didecanoate) and were prevented by a protein kinase inhibitor (H-8), confirming the involvement of PKC activity in these modulatory processes. We conclude that elevating [Ca]i and activating PKC have opposite effects on the T and L Ca currents in canine cardiac cells. The extent and time course of the changes in these two intracellular messengers will most likely determine the effects on the two cardiac Ca currents of neurotransmitters and hormones that can activate phospholipase C.

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Phosphorylation of GAP-43 (growth-associated protein of 43 kDa) by conventional, novel and atypical isotypes of the protein kinase C gene family: differences between oligopeptide and polypeptide phosphorylation

Biochemical Journal ◽

10.1042/bj3170219 ◽

1996 ◽

Vol 317 (1) ◽

pp. 219-224 ◽

Cited By ~ 28

Author(s):

Silke A. OEHRLEIN ◽

Peter J. PARKER ◽

Thomas HERGET

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Phosphorylation Site ◽

Mammalian Brain ◽

Major Protein ◽

Calmodulin Binding ◽

Kinase C ◽

Significant Difference ◽

Complete Protein

GAP-43 (growth-associated protein of 43 kDa; also known as neuromodulin, P-57, B-50 and F-1) is a neuronal calmodulin binding protein and a major protein kinase C (PKC) substrate in mammalian brain. Here we describe the phosphorylation by and the site specificity of different PKC isotypes. The conventional PKC β1 and the novel PKCs Δ and ϵ effectively phosphorylated recombinant GAP-43 in vitro; atypical PKC ζ did not. The Km values (between 0.6 and 2.3 μM) were very low, demonstrating a high-affinity interaction between kinase and substrate. All PKC isotypes were shown to phosphorylate serine-41 in GAP-43. When using a 19-amino-acid oligopeptide based on the GAP-43 phosphorylation site as substrate, there was a significant difference compared with polypeptide phosphorylation. The Vmax values of PKC β1 and PKC ϵ were much higher for this oligopeptide than for the complete protein (up to 10-fold); in contrast, their apparent affinities for the peptide were much lower (up to 100-fold) than for the intact GAP-43 polypeptide. Furthermore, phosphorylation of the GAP-43 oligopeptide by PKC β1 was more sensitive to a catalytic-site inhibitor than was phosphorylation of intact GAP-43. These results suggest that there are multiple sites of interaction between GAP-43 and PKC.

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Protein Kinase C Downregulation upon Rapamycin Treatment Attenuates Neuroinflammation and Mitochondrial Disease

Innovation in Aging ◽

10.1093/geroni/igaa057.3281 ◽

2020 ◽

Vol 4 (Supplement_1) ◽

pp. 889-890

Author(s):

Anthony Grillo ◽

Alessandro Bitto ◽

Matt Kaeberlein

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Critical Role ◽

Specific Inhibitor ◽

Mitochondrial Diseases ◽

Leigh Syndrome ◽

Premature Death ◽

Kinase C ◽

Pkc Β ◽

Pkc Inhibitors

Abstract Mitochondrial dysfunction causes many poorly understood diseases, such as Leigh Syndrome, that are often caused by dysfunctions in proteins involved in the electron transport chain. My lab previously reported mTOR is pathologically involved in the neurodegenerative phenotype and premature death of mice missing the Complex I subunit Ndufs4 (Ndufs4-/- mice). We discovered treatment with rapamycin extends lifespan, reduces neuroinflammation, and attenuates the neurodegenerative phenotype in these mice, although the mechanisms remain unclear. Rapamycin-treated Ndufs4-/- mice exhibited decreased activation of the mTORC1 pathway. It also deactivated the mTORC2 pathway. We observed that phosphorylation of the canonical protein kinase C (PKC) isoforms (PKC-α, -β, and -γ) decreased more than any other kinases, leading us to hypothesize its deactivation contributes to the observed lifespan extension. To test this, we treated Ndufs4-/- mice with three different PKC inhibitors: the pan-PKC inhibitors GO6983 and GF109203X, and the PKC-β specific inhibitor ruboxistaurin. Similar to rapamycin, all three drugs were able to significantly delay the onset of neurological symptoms (i.e. clasping) and increase survival. We also observed that PKC-β inhibition reduced skin inflammation to suppress the hair loss phenotype displayed by Ndufs4-/- mice at weaning. We further discovered PKC-β inhibition reduces neuroinflammation by deactivating the NF-kB inflammatory pathway. These results suggest that mTORC2 may play a critical role in the etiology of mitochondrial diseases such as Leigh Syndrome.

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Filamin A is required for vimentin-mediated cell adhesion and spreading

AJP Cell Physiology ◽

10.1152/ajpcell.00323.2009 ◽

2010 ◽

Vol 298 (2) ◽

pp. C221-C236 ◽

Cited By ~ 60

Author(s):

Hugh Kim ◽

Fumihiko Nakamura ◽

Wilson Lee ◽

Yulia Shifrin ◽

Pamela Arora ◽

...

Keyword(s):

Protein Kinase C ◽

Cell Adhesion ◽

Protein Kinase ◽

Cell Spreading ◽

Intermediate Filament Protein ◽

Β1 Integrin ◽

Filamin A ◽

Integrin Activation ◽

Hek 293 ◽

Kinase C

Cell adhesion and spreading are regulated by complex interactions involving the cytoskeleton and extracellular matrix proteins. We examined the interaction of the intermediate filament protein vimentin with the actin cross-linking protein filamin A in regulation of spreading in HEK-293 and 3T3 cells. Filamin A and vimentin-expressing cells were well spread on collagen and exhibited numerous cell extensions enriched with filamin A and vimentin. By contrast, cells treated with small interfering RNA (siRNA) to knock down filamin A or vimentin were poorly spread; both of these cell populations exhibited >50% reductions of cell adhesion, cell surface β1 integrin expression, and β1 integrin activation. Knockdown of filamin A reduced vimentin phosphorylation and blocked recruitment of vimentin to cell extensions, whereas knockdown of filamin and/or vimentin inhibited the formation of cell extensions. Reduced vimentin phosphorylation, cell spreading, and β1 integrin surface expression, and activation were phenocopied in cells treated with the protein kinase C inhibitor bisindolylmaleimide; cell spreading was also reduced by siRNA knockdown of protein kinase C-ε. By immunoprecipitation of cell lysates and by pull-down assays using purified proteins, we found an association between filamin A and vimentin. Filamin A also associated with protein kinase C-ε, which was enriched in cell extensions. These data indicate that filamin A associates with vimentin and to protein kinase C-ε, thereby enabling vimentin phosphorylation, which is important for β1 integrin activation and cell spreading on collagen.

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In vivo α-adrenergic responses and troponin I phosphorylation: anesthesia interactions

Journal of Applied Physiology ◽

10.1152/japplphysiol.00959.2004 ◽

2005 ◽

Vol 98 (4) ◽

pp. 1163-1170 ◽

Cited By ~ 8

Author(s):

Guy A. MacGowan ◽

Jennifer Rager ◽

Sanjeev G. Shroff ◽

Michael A. Mathier

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Transgenic Mice ◽

Troponin I ◽

Contractile Dysfunction ◽

Wild Type ◽

Adrenergic Stimulation ◽

Kinase C ◽

Significant Difference

The mechanisms by which α-adrenergic stimulation of the heart in vivo can cause contractile dysfunction are not well understood. We hypothesized that α-adrenergic-mediated contractile dysfunction is mediated through protein kinase C phosphorylation of troponin I, which in in vitro experiments has been shown to reduce actomyosin Mg-ATPase activity. We studied pressure-volume loops in transgenic mice expressing mutant troponin I lacking protein kinase C phosphorylation sites and hypothesized altered responses to phenylephrine. As anesthesia agents can produce markedly different effects on contractility, we studied two agents: avertin and α-chloralose-urethane. With α-chloralose-urethane, at baseline, there were no contractile abnormalities in the troponin I mutants. Phenylephrine produced a 50% reduction in end-systolic elastance in wild-type controls, although a 9% increase in troponin I mutants ( P < 0.05). Avertin was associated with reduced contractility compared with α-chloralose-urethane. Avertin anesthesia, at baseline, produced a reduction in end-systolic elastance by 31% in the troponin I mutants compared with wild-type ( P < 0.05), and this resulted in further marked systolic and diastolic dysfunction with phenylephrine in the troponin I mutants. Dobutamine produced no significant difference in the contractile phenotype of the transgenic mice with either anesthetic regimen. In conclusion, these data (α-chloralose-urethane) demonstrate that α-adrenergic-mediated force reduction is mediated through troponin I protein kinase C phosphorylation. β-Adrenergic responses are not mediated through this pathway. Altering the myofilament force-calcium relationship may result in in vivo increased sensitivity to negative inotropy. Thus choice of a negative inotropic anesthetic agent (avertin) with phenylephrine can lead to profound contractile dysfunction.

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