scholarly journals A role for protein kinase C subtypes α and ε in phorbol-ester-enhanced K+- and carbachol-evoked noradrenaline release from the human neuroblastoma SH-SY5Y

1994 ◽  
Vol 297 (2) ◽  
pp. 407-413 ◽  
Author(s):  
N A Turner ◽  
M G Rumsby ◽  
J H Walker ◽  
F A McMorris ◽  
S G Ball ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Phorbol Ester ◽  
Prolonged Exposure ◽  
Human Neuroblastoma ◽  
Kinase C ◽  
Pkc Epsilon ◽  
Pkc Zeta ◽  
Pkc Alpha ◽  
Na Release

Protein kinase C (PKC) consists of a family of closely related subtypes which differ in their localization and activation properties. Our previous studies have suggested a role for PKC in the regulation of noradrenaline (NA) release from the human neuroblastoma SH-SY5Y. Here we have used two approaches to characterize the PKC subtypes present in SH-SY5Y cells. Firstly, the PCR was used to show that SH-SY5Y cells contain mRNA encoding PKC subtypes alpha, beta, gamma, delta, epsilon and zeta. Secondly, immunoblotting showed that SH-SY5Y cells express PKC subtypes alpha, epsilon and zeta at the protein level. Prolonged (48 h) exposure of cells to the phorbol ester phorbol 12-myristate 13-acetate (PMA; 100 nM) resulted in a marked decrease in the amounts of PKC-alpha and PKC-epsilon, with no change in levels of PKC-zeta. Prolonged PMA treatment had no significant effect on K(+)-evoked NA release from SH-SY5Y cells, whereas carbachol-evoked release was increased 2.2-fold. However, prolonged exposure to PMA completely inhibited the ability of acute (12 min) PMA treatment to enhance both K(+)- and carbachol-evoked NA release. The specific PKC inhibitor RO 31-7459 (10 microM) was found to inhibit K(+)- and carbachol-evoked release by 27% and 68% respectively. RO 31-7549 also completely inhibited the ability of acute PMA treatment to enhance release. These data suggest that PKC-alpha and/or PKC-epsilon play an essential role in the regulation of PMA-enhanced K(+)- and carbachol-evoked NA release in SH-SY5Y cells.

scholarly journals Selective down-regulation of protein kinase c-ε by carcinogens does not prevent stimulation of phospholipase D by phorbol ester and platelet-derived growth factor

1994 ◽  
Vol 300 (3) ◽  
pp. 751-756 ◽  
Author(s):  
Z Kiss ◽  
W H Anderson
Keyword(s):  
Protein Kinase C ◽  
Growth Factor ◽  
Protein Kinase ◽  
Phospholipase D ◽  
Platelet Derived Growth Factor ◽  
Kinase C ◽  
Pkc Epsilon ◽  
Pkc Zeta ◽  
Fibroblast Line ◽  
Pkc Alpha

It is well established that activators of protein kinase C (PKC) also enhance the activity of phospholipase D (PLD), and that this regulatory mechanism is altered in transformed cells. Here we used the C3H/10T1/2 mouse embryo fibroblast line, a cellular model for the study of carcinogenesis, to examine possible effects of carcinogens on the PKC isoenzyme pattern and on the regulation of PLD by the PKC activators phorbol 12-myristate 13-acetate (PMA) and platelet-derived growth factor (PDGF). Treatment of these fibroblasts with 0.5 microgram/ml 7,12-dimethyl-benz[a]anthracene or benzo[a]pyrene for 24 h greatly decreased (> 80%) the amount of immunoreactive PKC-epsilon. Of the remaining three isoenzymes identified, carcinogens alone had no effect on the cellular status of PKC-alpha and PKC-delta, although they appeared to promote slightly PMA-induced membrane translocation of the cytosolic forms of these isoenzymes in exponentially growing cells. Carcinogens and/or PMA had no effects on the cellular content or distribution of PKC-zeta. Chronic (24 h) treatments with carcinogens resulted in increased or decreased release of [14C]ethanolamine or [14C]choline from the appropriate prelabelled phospholipids, respectively. However, carcinogens failed to block the stimulatory effects of PMA and PDGF on the hydrolysis of phosphatidylethanolamine and phosphatidylcholine or on the synthesis of phosphatidylethanol mediated by PLD. These data indicate that in fibroblasts PKC-epsilon is not a major regulator of PLD activity.

scholarly journals Tissue and cellular distribution of the extended family of protein kinase C isoenzymes

1992 ◽  
Vol 117 (1) ◽  
pp. 121-133 ◽  
Author(s):  
WC Wetsel ◽  
WA Khan ◽  
I Merchenthaler ◽  
H Rivera ◽  
AE Halpern ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Western Blot ◽  
Extended Family ◽  
Calcium Dependent ◽  
Kinase C ◽  
Pkc Epsilon ◽  
Pkc Zeta ◽  
Gamma Peak ◽  
Pkc Alpha

Polyclonal isoenzyme-specific antisera were developed against four calcium-independent protein kinase C (PKC) isoenzymes (delta, epsilon, epsilon', and zeta) as well as the calcium-dependent isoforms (alpha, beta I, beta II, and gamma). These antisera showed high specificities, high titers, and high binding affinities (3-370 nM) for the peptide antigens to which they were raised. Each antiserum detected a species of the predicted molecular weight by Western blot that could be blocked with the immunizing peptide. PKC was sequentially purified from rat brain, and the calcium-dependent forms were finally resolved by hydroxyapatite chromatography. Peak I reacted exclusively with antisera to PKC gamma, peak II with PKC beta I and -beta II, and peak III with PKC alpha. These same fractions, however, were devoid of immunoreactivity for the calcium-independent isoenzymes. The PKC isoenzymes demonstrated a distinctive tissue distribution when evaluated by Western blot and immunocytochemistry. PCK delta was present in brain, heart, spleen, lung, liver, ovary, pancreas, and adrenal tissues. PKC epsilon was present in brain, kidney, and pancreas, whereas PKC epsilon' was present predominantly in brain. PKC zeta was present in most tissues, particularly the lung, brain, and liver. Both PKC delta and PKC zeta showed some heterogeneity of size among the different tissues. PKC alpha was present in all organs and tissues examined. PKC beta I and -beta II were present in greatest amount in brain and spleen. Although the brain contained the most PKC gamma immunoreactivity, some immunostaining was also seen in adrenal tissue. These studies provide the first evidence of selective organ and tissue distributions of the calcium-independent PKC isoenzymes.

scholarly journals Phosphatidic acid activation of protein kinase C-ζ overexpressed in COS cells: comparison with other protein kinase C isotypes and other acidic lipids

1994 ◽  
Vol 304 (3) ◽  
pp. 1001-1008 ◽  
Author(s):  
C Limatola ◽  
D Schaap ◽  
W H Moolenaar ◽  
W J van Blitterswijk
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Phosphatidic Acid ◽  
Acid Activation ◽  
Cos Cells ◽  
Kinase C ◽  
Pkc Epsilon ◽  
Pkc Zeta ◽  
Pkc Alpha ◽  
Acidic Lipids

Phosphatidic acid (PA) is produced rapidly in agonist-stimulated cells, but the physiological function of this PA is unknown. We have examined the effects of PA on distinct isoforms of protein kinase C (PKC) using a new cell-free assay system. Addition of PA to cytosol from COS cells overexpressing PKC-alpha, -epsilon or -zeta differentially-activated all three isotypes, as shown by PKC autophosphorylation, and prominent phosphorylation of multiple endogenous substrates. In the absence of Ca2+, the diacylglycerol-insensitive zeta-isotype of PKC was most strongly activated by both PA and bisPA, a newly identified product of activated phospholipase D, with each lipid inducing its own profile of protein phosphorylation. BisPA was also a strong activator of PKC-epsilon, but a weak activator of PKC-alpha. Ca2+, at > or = 0.1 microM, inhibited PA and bisPA activation of PKC-zeta, but did not affect PKC-epsilon activation. In contrast, PKC-alpha was strongly activated by PA only in the presence of Ca2+. BisPA-induced phosphorylations mediated by PKC-zeta could be mimicked in part by other acidic phospholipids and unsaturated fatty acids. PA activation of PKC-zeta was unique in that PA not only stimulated PKC-zeta-mediated phosphorylation of distinctive substrates, but also caused an upward shift in electrophoretic mobility of PKC-zeta, which was not observed with other acidic lipids or with PKC-alpha or -epsilon. We have presented evidence that this mobility shift is not caused by PKC-zeta autophosphorylation, but it coincides with physical binding of PA to PKC-zeta. These results suggest that in cells stimulated under conditions where intracellular Ca2+ is at (or has returned to) basal level, PA may be a physiological activator of PKC-zeta.

Expression of protein kinase C isoforms during cardiac ventricular development

1995 ◽  
Vol 269 (3) ◽  
pp. H1087-H1097 ◽  
Author(s):  
A. Clerk ◽  
M. A. Bogoyevitch ◽  
S. J. Fuller ◽  
A. Lazou ◽  
P. J. Parker ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Total Protein ◽  
Particulate Fraction ◽  
Pkc Delta ◽  
Kinase C ◽  
Pkc Epsilon ◽  
Pkc Zeta ◽  
Rat Hearts ◽  
Pkc Alpha

The expression of protein kinase C (PKC) isoforms (PKC-alpha, PKC-beta 1, PKC-delta, PKC-epsilon, and PKC-zeta) was studied by immunoblotting in whole ventricles of rat hearts during postnatal development (1-26 days) and in the adult. PKC-alpha, PKC-beta 1, PKC-delta, PKC-epsilon, and PKC-zeta were detected in ventricles of 1-day-old rats, although PKC-alpha and PKC-beta 1 were only barely detectable. All isoforms were rapidly downregulated during development, with abundances relative to total protein declining in the adult to < 25% of 1-day-old values. PKC-beta 1 was not detectable in adult ventricles. The specific activity of PKC was also downregulated. The rat ventricular myocyte becomes amitotic soon after birth but continues to grow, increasing its protein content 40- to 50-fold between the neonate and the 300-g adult. An important question is thus whether the amount of PKC per myocyte is downregulated. With the use of isolated cells, immunoblotting showed that the contents per myocyte of PKC-alpha and PKC-epsilon increased approximately 10-fold between the neonatal and adult stages. In rat ventricles, the rank of association with the particulate fraction was PKC-delta > PKC-epsilon > PKC-zeta. Association of these isoforms with the particulate fraction was less in the adult than in the neonate. In primary cultures of ventricular myocytes prepared from neonatal rat hearts, 1 microM 12-O-tetradecanoylphorbol-13-acetate (TPA) elicited translocation of PKC-alpha, PKC-delta, and PKC-epsilon from the soluble to the particulate fraction in < 1 min, after which time no further translocation was observed. Prolonged exposure (16 h) of myocytes to 1 microM TPA caused essentially complete downregulation of these isoforms, although downregulation of PKC-epsilon was slower than for PKC-delta. In contrast, PKC-zeta was neither translocated nor downregulated by 1 microM TPA. Immunoblotting of human ventricular samples also revealed downregulation of PKC relative to total protein during fetal/postnatal development.

scholarly journals Protein kinase C group B members PKC-δ, -ɛ, -ζ and PKC-L(η). Comparison of properties of recombinant proteins in vitro and in vivo

1992 ◽  
Vol 283 (3) ◽  
pp. 781-787 ◽  
Author(s):  
M Liyanage ◽  
D Frith ◽  
E Livneh ◽  
S Stabel
Keyword(s):  
Protein Kinase C ◽  
Recombinant Proteins ◽  
Phorbol Ester ◽  
Insect Cells ◽  
Pkc Delta ◽  
Kinase C ◽  
Pkc Epsilon ◽  
Pkc Zeta ◽  
Group A ◽  
Group B

Of the recently identified protein kinase C (PKC) types of group B (delta, epsilon, zeta, eta, PKC-L), only PKC-epsilon has been characterized in great detail. In order to compare the regulatory and catalytic properties of these new kinases, we have expressed PKC-delta, -epsilon, -zeta and PKC-L as recombinant proteins from their cDNAs in insect cells via baculovirus vectors and in mammalian COS-1 cells. After expression in insect cells, phorbol ester binding and kinase activities of the group B enzymes were compared with the respective activities of a member of group A, PKC-gamma. Although PKC-delta and PKC-L(eta) bind phorbol ester to a similar or the same extent as PKC-gamma, they show a distinctively different behaviour towards conventional PKC substrates such as histone, myelin basic protein, protamine and protamine sulphate, suggesting either that phorbol esters are not able to fully activate these enzymes or that their substrate specificities are very different from those of the group A enzymes. PKC-zeta, a polypeptide of 80 kDa, does not bind phorbol ester and does not phosphorylate these substrates to a significant extent. Consistent with their ability to bind phorbol ester, recombinant PKC-delta and PKC-epsilon are down-regulated in COS cells by prolonged treatment with phorbol ester, whereas PKC-zeta protein levels remain unaltered.

scholarly journals Phosphorylation of p90 and p52 in response to phorbol-esters in Swiss/3T3 cells overexpressing protein kinase C-alpha.

1992 ◽  
Vol 3 (9) ◽  
pp. 1049-1056 ◽  
Author(s):  
H Eldar ◽  
E Livneh
Keyword(s):  
Protein Kinase C ◽  
Growth Factor ◽  
Protein Kinase ◽  
Cell Lines ◽  
Phorbol Ester ◽  
Phorbol Esters ◽  
Tumor Promotion ◽  
3T3 Cells ◽  
Kinase C ◽  
Pkc Alpha

Cell lines stably overexpressing protein kinase C (PKC)-alpha were previously described by us. These cell lines were generated by the introduction of the full length cDNA coding for PKC-alpha into Swiss/3T3 cells. Here we show that activation of PKC-alpha by phorbol-esters induced in these cells specific phosphorylation of two cellular proteins p90 and p52. Phosphorylation of p80 (MARCKS protein), previously identified as a substrate for PKC, was also enhanced. Phosphorylated p90 and p52 proteins were associated with particulate membrane-enriched fractions and were extractable with the use of nonionic detergents. Time course analysis of phorbol-ester induced phosphorylation of p90 and p52 revealed maximal stimulation of phosphorylation after 15-30 min. Phosphamino acid analysis showed that phosphorylation of p90 and p52 occurred mainly on serine residues. Phosphorylation of p52 was also on threonine residues. Whereas, phorbol ester activation induced phosphorylation of both p90 and p52, the mitogens platelet-derived growth factor (PDGF) and fibroblast growth factor (FGF) enhanced phosphorylation of p90, but not p52. Thus, our studies showed the involvement of PKC-alpha in the regulation of p90 and p52 phosphorylation and provided direct evidence for the role of PKC-alpha in cellular signaling by PDGF and FGF. Moreover, the fact that phosphorylation of p52 was specific to phorbol ester activation may suggest its involvement in tumor promotion. Characterization of p90 and p52 will enable us to reveal the phosphorylation cascade activated downstream to PKC-alpha and to determine their role in mitogenic signaling and tumor promotion.

scholarly journals Nerve growth factor activates calcium-insensitive protein kinase C-ɛ in PC-12 rat pheochromocytoma cells

1993 ◽  
Vol 295 (3) ◽  
pp. 767-772 ◽  
Author(s):  
M Ohmichi ◽  
G Zhu ◽  
A R Saltiel
Keyword(s):  
Protein Kinase C ◽  
Nerve Growth Factor ◽  
Growth Factor ◽  
Protein Kinase ◽  
Kinase Activity ◽  
Pheochromocytoma Cells ◽  
Pc 12 Cells ◽  
Kinase C ◽  
Pkc Epsilon ◽  
Pkc Alpha

Protein kinase C (PKC) family members were examined in PC-12 rat pheochromocytoma cells to evaluate their role in the action of nerve growth factor (NGF). Immunoblot analysis of whole cell lysates using antibodies against various PKC isoforms revealed that PC-12 cells contained PKC-alpha, -delta, -epsilon and zeta. Assay of the protein kinase activity in these different anti-PKC immunoprecipitates demonstrated that NGF stimulated the kinase activity of PKC-epsilon, but not PKC-alpha, -delta and -zeta. Both histone phosphorylation and autophosphorylation of PKC-epsilon were increased by treatment of PC-12 cells with NGF. This increased phosphorylation observed in vitro is rapid, occurring maximally at 2.5 min and declining thereafter. Moreover, this effect of NGF is dose-dependent over physiological concentrations of the growth factor. Although the mechanistic basis for this specificity in PKC activation is not clear, NGF acutely stimulated the production of diacylglycerol without causing corresponding changes in intracellular Ca2+ concentrations. These results suggest that NGF may selectively stimulate the Ca(2+)-insensitive epsilon isoform of PKC by a phosphatidylinositol-independent mechanism.

scholarly journals Yeast phenotype classifies mammalian protein kinase C cDNA mutants.

1993 ◽  
Vol 13 (8) ◽  
pp. 4728-4735 ◽  
Author(s):  
H Riedel ◽  
L Su ◽  
H Hansen
Keyword(s):  
Protein Kinase C ◽  
Catalytic Activity ◽  
Protein Kinase ◽  
Phorbol Ester ◽  
Colony Size ◽  
Receptor Protein ◽  
Amino Terminal ◽  
Kinase C ◽  
Carboxyl Terminal ◽  
Pkc Alpha

The phorbol ester receptor protein kinase C (PKC) gene family encodes essential mediators of eukaryotic cellular signals. Molecular dissection of their mechanisms of action has been limited in part by the lack of random mutagenesis approaches and by the complexity of signaling pathways in mammalian cells which involve multiple PKC isoforms. Here we present a rapid screen which permits the quantification of mammalian PKC activity phenotypically in the yeast Saccharomyces cerevisiae. Bovine PKC alpha cDNA is functionally expressed in S. cerevisiae. This results in a phorbol ester response: a fourfold increase in the cell doubling time and a substantial decrease in yeast colony size on agar plates. We have expressed pools of bovine PKC alpha cDNAs mutagenized by Bal 31 deletion of internal, amino-terminal, or carboxyl-terminal sequences and have identified three classes of mutants on the basis of their distinct yeast phenotypes. Representatives of each class were analyzed. An internal deletion of amino acids (aa) 172 to 225 displayed ligand-dependent but reduced catalytic activity, an amino-terminal truncation of aa 1 to 153 displayed elevated and ligand-independent activity, and a carboxyl-terminal 26-aa truncation (aa 647 to 672) lacked activity under any conditions. Additional mutations confirmed the distinct functional characteristics of these classes. Our data show that deletion of the V1 and C1 regions results in elevated basal catalytic activity which is still Ca2+ responsive. Internal deletions in the V2 and C2 regions do not abolish phorbol ester or Ca2+ regulation of PKC activity, suggesting that most of the C2 domain is not essential for phorbol ester stimulation and most of the regulatory domain is dispensable for Ca2+ regulation of PKC activity. These distinct activities od the PKC mutants correlate with a specific and proportional yeast phenotype and are quantified on agar plates by yeast colony size. This provides a phenotypic screen which is suitable to identity rare, randomly altered but active mammalian PKC mutants. It quantifies their catalytic and biological activities in response to PKC activators or inhibitors for a systematic mapping of PKC structure and function or PKC-drug interaction.

Do adult rat ventricular myocytes express protein kinase C-alpha?

1997 ◽  
Vol 272 (5) ◽  
pp. H2485-H2491 ◽  
Author(s):  
V. Rybin ◽  
S. F. Steinberg
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Ventricular Myocytes ◽  
Chromatographic Purification ◽  
Adult Rat ◽  
Pkc Delta ◽  
Kinase C ◽  
Pkc Epsilon ◽  
Pkc Isoforms ◽  
Pkc Alpha

Although calcium-insensitive protein kinase C (PKC) isoforms (PKC-epsilon and PKC-delta) are consistently detected in adult ventricular myocytes, the evidence that adult ventricular myocytes also express calcium-sensitive PKC-alpha is inconsistent. The current study used four different anti-PKC-alpha-antibodies to resolve some of the uncertainties regarding the immunodetection of PKC-alpha in adult ventricular myocytes. Three of the antibodies used in this study barely (GIBCO-BRL) or rather faintly (Transduction Laboratories and Seikagaku America) recognize PKC-alpha in crude preparations from adult ventricular myocytes. Although each of these antibodies recognizes a prominent 80-kDa band, which is similar in size to PKC-alpha, this represents nonspecific immunoreactivity and should not be confused with PKC-alpha. This conclusion is based on peptide-blocking experiments (GIBCO-BRL), the absence of the requisite sensitivity to calcium- and phorbol 12-myristate 13-acetate-induced translocation (Seikagaku America and Transduction Laboratories), and/or the failure to copurify with PKC-alpha on DEAE-Sephacel chromatography. Nevertheless, an antibody from Upstate Biotechnology clearly recognizes PKC-alpha and not other unrelated nonspecific immunoreactive species in crude preparations from adult ventricular myocytes. Each of the antisera used in this study could detect PKC-alpha immunoreactivity following chromatographic purification of the samples to enrich for PKC-alpha and remove nonspecific immunoreactive proteins. These results suggest that PKC-alpha is expressed by adult ventricular myocytes and argue that differences in the sensitivity and/or specificity of available antisera contribute to at least some of the confusion regarding PKC-alpha expression in adult ventricular myocytes.

Epsilon-isoenzyme of protein kinase C induces a Ca(2+)-independent contraction in vascular smooth muscle

1996 ◽  
Vol 271 (2) ◽  
pp. C589-C594 ◽  
Author(s):  
A. Horowitz ◽  
O. Clement-Chomienne ◽  
M. P. Walsh ◽  
K. G. Morgan
Keyword(s):  
Smooth Muscle ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Kinase Inhibitor ◽  
Force Response ◽  
Inhibitory Peptide ◽  
Aortic Smooth Muscle ◽  
Kinase C ◽  
Pkc Epsilon ◽  
Pkc Zeta

We provide here the first direct evidence for in situ functional specificity of protein kinase C (PKC)-epsilon as a regulator of smooth muscle contractility. PKC is known to cause a Ca(2+)-independent contraction of ferret aortic smooth muscle, and the expression of two Ca(2+)-independent PKC isoenzymes, epsilon and zeta, has been demonstrated in this tissue. To test directly the hypothesis that one of these isoenzymes regulates contractility, constitutively active forms of PKC-epsilon and PKC-zeta were applied to saponin-permeabilized single ferret aortic smooth muscle cells. PKC-zeta caused no significant force response, but PKC-epsilon induced contraction of a magnitude (105 +/- 8 micrograms) similar to that produced by phenylephrine (110 +/- 10 micrograms), a relatively selective alpha 1-adrenergic agonist that triggers a PKC-dependent contraction. The PKC-epsilon-induced contraction was reversed by the PKC pseudosubstrate inhibitory peptide, PKC19-31. The myosin light chain kinase inhibitor 1-(5-chloronaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine (ML-9) did not affect the force response of PKC-epsilon-activated cells, suggesting that PKC-epsilon may induce this contraction solely via thin filament disinhibition. In support of this conclusion, calponin and caldesmon were shown to be good in vitro substrates of PKC-epsilon but not of PKC-zeta.

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