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.

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 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.

Phorbol ester-stimulated exocytosis in lacrimal gland: PKC might not be the sole effector

1993 ◽  
Vol 264 (4) ◽  
pp. C1045-C1050 ◽  
Author(s):  
D. Zoukhri ◽  
C. Sergheraert ◽  
B. Rossignol
Keyword(s):  
Phorbol Ester ◽  
Lacrimal Gland ◽  
Inhibitory Effect ◽  
Complete Inhibition ◽  
Pkc Delta ◽  
Kinase C ◽  
Pkc Epsilon ◽  
Diethylaminoethyl Cellulose ◽  
Half Maximal Effective Concentration ◽  
Pkc Alpha

In this work we show that, although both phorbol 12-myristate 13-acetate (PMA) and 4 beta-phorbol 12,13-dibutyrate (PdBu) stimulate the protein discharge in the rat lacrimal gland with the same half-maximal effective concentration (EC50 approximately 2 x 10(-7) M), PdBu is more efficient in eliciting this response compared with PMA. We also show that sphingosine and chelerythrine have no inhibitory effect on the protein discharge stimulated by PMA or PdBu at concentrations up to 2 x 10(-4) and 3 x 10(-5) M, respectively. With staurosporine, a complete inhibition could not be obtained even at 1 microM. However, only with trifluoperazine (TFP) we obtained a complete inhibition of the PMA-induced protein discharge at 10(-4) M TFP. On the other hand, we show that three diacylglycerol-permeant analogues (1-oleoyl-2-acetyl-sn-glycerol, 1,2-dioctanoyl-sn-glycerol, and 1,2-didecanoyl-sn-glycerol) do not stimulate protein discharge. In a previous report from our laboratory (30), we showed that the rat lacrimal gland expresses the alpha-isoform of protein kinase C (PKC). In this study, using specific antibodies directed against the newly identified isoforms of PKC, we show on a diethylaminoethyl-cellulose fraction that, besides PKC-alpha, the rat lacrimal gland expresses PKC-epsilon, as previously suggested by Dartt et al. (11), and PKC-delta. Our results question the direct implication of PKC activity as a sole effector of the phorbol ester-stimulated protein secretion in the rat lacrimal gland.

Interferons block protein kinase C-dependent but not-independent activation of Raf-1 and mitogen-activated protein kinases and mitogenesis in NIH 3T3 cells

1994 ◽  
Vol 14 (12) ◽  
pp. 8018-8027
Author(s):  
J Xu ◽  
S Rockow ◽  
S Kim ◽  
W Xiong ◽  
W Li
Keyword(s):  
Protein Kinase C ◽  
Growth Factor ◽  
Protein Kinase ◽  
Phorbol Ester ◽  
Myristate Acetate ◽  
Mitogen Activated Protein Kinases ◽  
Pkc Delta ◽  
Kinase C ◽  
Mitogen Activated Protein ◽  
Nih 3T3

Interferons (IFNs) exert antiproliferative effects on many types of cells. The underlying molecular mechanism, however, is unclear. One possibility is that IFNs block growth factor-induced mitogenic signaling, which involves activation of Ras/Raf-1/MEK/mitogen-activated protein kinase. We have tested this hypothesis by using HER14 cells (NIH 3T3 cell expressing both platelet-derived growth factor [PDGF] and epidermal growth factor [EGF] receptors) as a model system. Our studies showed that IFNs (alpha/beta and gamma) blocked PDGF-and phorbol ester- but not EGF-stimulated DNA synthesis and cell proliferation. While the ligand-stimulated receptor tyrosine phosphorylation and interaction with downstream signaling molecules, such as GRB2, were not affected, IFNs specifically blocked PDGF- and phorbol ester- but not EGF-stimulated activation of Raf-1, mitogen-activated protein kinases, and tyrosine phosphorylation of an unidentified 34-kDa protein. This inhibition could be detected as early as 5 min after IFN treatments and was insensitive to cycloheximide, indicating that de novo protein synthesis is not required. The IFN-induced inhibition acted upstream of Raf-1 kinase and downstream of diacyl glycerol/phorbol ester, suggesting that protein kinase C (PKC) is the potential primary target. Consistently, downregulation of PKC by chronic phorbol myristate acetate treatment or inhibition of PKC by H7 and staurosporine blocked PDGF- and phorbol myristate acetate- but not EGF-induced signaling and DNA synthesis. Moreover, incubating cells with antisense oligodeoxyribonucleotides of PKC delta eliminated production of PKC delta protein and specifically blocked PDGF- but not EGF-stimulated mitogenesis in these cells. Thus, these studies have elucidated a major difference in the early events of EGF-and PDGF-stimulated signal transduction and, more importantly, revealed a novel mechanism by which IFNs may execute their antiproliferative function.

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.

Reactive Oxygen Species Precede the ε Isoform of Protein Kinase C in the Anesthetic Preconditioning Signaling Cascade

2003 ◽  
Vol 99 (2) ◽  
pp. 421-428 ◽  
Author(s):  
Enis Novalija ◽  
Leo G. Kevin ◽  
Amadou K.S. Camara ◽  
Zeljko J. Bosnjak ◽  
John P. Kampine ◽  
...  
Keyword(s):  
Reactive Oxygen Species ◽  
Protein Kinase C ◽  
Signaling Cascade ◽  
Oxygen Species ◽  
Anesthetic Preconditioning ◽  
Pkc Delta ◽  
Kinase C ◽  
Pkc Epsilon ◽  
Ros Formation ◽  
Pkc Activation

Background Protein kinase C (PKC) and reactive oxygen species (ROS) are known to have a role in anesthetic preconditioning (APC). Cardiac preconditioning by triggers other than volatile anesthetics, such as opioids or brief ischemia, is known to be isoform selective, but the isoform required for APC is not known. The authors aimed to identify the PKC isoform that is involved in APC and to elucidate the relative positions of PKC activation and ROS formation in the APC signaling cascade. Methods Isolated guinea pig hearts were subjected to 30 min of ischemia and 120 min of reperfusion. Before ischemia, hearts were either untreated or treated with sevoflurane (APC) in the absence or presence of the nonspecific PKC inhibitor chelerythrine, the PKC-delta inhibitor PP101, or the PKC-epsilon inhibitor PP149. Spectrofluorometry and the fluorescent probes dihydroethidium were used to measure intracellular ROS, and effluent dityrosine as used to measure extracellular ROS release. Results Previous sevoflurane exposure protected the heart against ischemia-reperfusion injury, as previously described. Chelerythrine or PP149 abolished protection, but PP101 did not. ROS formation was observed during sevoflurane exposure and was not altered by any of the PKC inhibitors. Conclusions APC is mediated by PKC-epsilon but not by PKC-delta. Furthermore, PKC activation probably occurs downstream of ROS generation in the APC signaling cascade.

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.

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