scholarly journals Isoenzyme specificity of bisindolylmaleimides, selective inhibitors of protein kinase C

1993 ◽  
Vol 294 (2) ◽  
pp. 335-337 ◽  
Author(s):  
S E Wilkinson ◽  
P J Parker ◽  
J S Nixon
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Cell Types ◽  
Side Chain ◽  
Conformationally Restricted ◽  
Kinase C ◽  
Pkc Epsilon ◽  
Wide Range ◽  
Pkc Alpha ◽  
Different Cell Types

The protein kinase C (PKC) family of isoenzymes is believed to mediate a wide range of signal-transduction pathways in many different cell types. A series of bisindolylmaleimides have been evaluated as inhibitors of members of the conventional PKC family (PKCs-alpha, -beta, -gamma) and of a representative of the new, Ca(2+)-independent, PKC family, PKC-epsilon. In contrast with the indolocarbazole staurosporine, all the bisindolylmaleimides investigated showed slight selectivity for PKC-alpha over the other isoenzymes examined. In addition, bisindolylmaleimides bearing a conformationally restricted side-chain were less active as inhibitors of PKC-epsilon. Most noticeable of these was Ro 32-0432, which showed a 10-fold selectivity for PKC-alpha and a 4-fold selectivity for PKC-beta I over PKC-epsilon.

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.

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.

scholarly journals Induction of Ca2+/calmodulin-stimulated cyclic AMP phosphodiesterase (PDE1) activity in Chinese hamster ovary cells (CHO) by phorbol 12-myristate 13-acetate and by the selective overexpression of protein kinase C isoforms

1995 ◽  
Vol 310 (3) ◽  
pp. 975-982 ◽  
Author(s):  
S Spence ◽  
G Rena ◽  
G Sweeney ◽  
M D Houslay
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Cyclic Amp ◽  
Cho Cells ◽  
Chinese Hamster Ovary Cells ◽  
Rt Pcr ◽  
Degenerate Primers ◽  
Kinase C ◽  
Pkc Epsilon ◽  
Pkc Alpha

The cAMP phosphodiesterase (PDE) activity of CHO cells was unaffected by the addition of Ca2+ +calmodulin (CaM), indicating the absence of any PDE1 (Ca2+/CaM-stimulated PDE) activity. Treatment with the tumour promoting phorbol ester phorbol 12-myristate 13-acetate (PMA) led to the rapid transient induction of PDE1 activity which attained a maximum value after about 13 h before slowly decreasing. Such induction was attenuated by actinomycin D. PCR primers were designed to hybridize with two regions identified as being characteristic of PDE1 forms found in various species and predicted to amplify a 601 bp fragment. RT-PCR using degenerate primers allowed an approx. 600 bp fragment to be amplified from RNA preparations of rat brain but not from CHO cells unless they had been treated with PMA. CHO cells transfected to overexpress protein kinase C (PKC)-alpha and PKC-epsilon, but not those transfected to overexpress PKC-beta I or PKC-gamma, exhibited a twofold higher PDE activity. They also expressed a PDE1 activity, with Ca2+/CaM effecting a 1.8-2.8-fold increase in total PDE activity. RT-PCR, with PDE1-specific primers, identified an approx. 600 bp product in CHO cells transfected to overexpress PKC-alpha and PKC-epsilon, but not in those overexpressing PKC-beta I or PKC-gamma. Treatment of PKC-alpha transfected cells with PMA caused a rapid, albeit transient, increase in PDE1 activity, which reached a maximum some 1 h after PMA challenge, before returning to resting levels some 2 h later. The residual isobutylmethylxanthine (IBMX)-insensitive PDE activity was dramatically reduced (approx. 4-fold) in the PKC-gamma transfectants, suggesting that the activity of the cyclic AMP-specific IBMX-insensitive PDE7 activity was selectively reduced by overexpression of this particular PKC isoform. These data identify a novel point of ‘cross-talk’ between the lipid and cyclic AMP signalling systems where the action of specific PKC isoforms is shown to cause the induction of Ca2+/CaM-stimulated PDE (PDE1) activity. It is suggested that this protein kinase C-mediated process might involve regulation of PDE1 gene expression by the AP-1 (fos/jun) system.

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 The regulatory domain of protein kinase C-ε restricts the catalytic-domain-specificity

1991 ◽  
Vol 276 (1) ◽  
pp. 257-260 ◽  
Author(s):  
C Pears ◽  
D Schaap ◽  
P J Parker
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Catalytic Domain ◽  
Structural Basis ◽  
Regulatory Domain ◽  
Substrate Selection ◽  
Kinase C ◽  
Pkc Epsilon ◽  
Pkc Alpha

Protein kinase C (PKC) consists of a family of closely related enzymes that can be divided into two subfamilies (alpha, beta and gamma and delta, epsilon and zeta) on the basis of primary sequence. Functional differences have also been described; thus PKC-alpha, PKC-beta and PKC-gamma readily phosphorylate histone IIIS in vitro, whereas PKC-epsilon will not employ this substrate efficiently. We have previously demonstrated, however, that proteolytic cleavage of PKC-epsilon generates a constitutive kinase activity that is an efficient histone IIIS kinase [Schaap, Hsuan, Totty & Parker (1990) Eur. J. Biochem. 191, 431-435]. In order to investigate the structural basis for this switch in specificity, we have constructed a chimaeric protein containing the regulatory domain of PKC-epsilon fused to the catalytic domain of PKC-gamma. When this is expressed in COS1 cells the chimaeric kinase shows a substrate-specificity similar to that of PKC-epsilon rather than to that of PKC-gamma. This demonstrates a role for the regulatory domain in substrate selection of PKC-epsilon.

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 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.

scholarly journals Effects of insulin on the translocation of protein kinase C-θ and other protein kinase C isoforms in rat skeletal muscles

1995 ◽  
Vol 308 (1) ◽  
pp. 177-180 ◽  
Author(s):  
K Yamada ◽  
A Avignon ◽  
M L Standaert ◽  
D R Cooper ◽  
B Spencer ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Skeletal Muscles ◽  
Phorbol Esters ◽  
Kinase C ◽  
Pkc Epsilon ◽  
Pkc Isoforms ◽  
Pkc Alpha

Protein kinase C (PKC)-theta is a newly recognized major PKC isoform in skeletal muscle. In this study we found that insulin provoked rapid biphasic increases in membrane-associated immunoreactive PKC-theta, as well as PKC-alpha, PKC-beta and PKC-epsilon, in rat soleus muscles incubated in vitro. Effects of insulin on PKC isoforms in the soleus were comparable in magnitude with those of phorbol esters. Increases in membrane-associated PKC-theta, PKC-alpha, PKC-beta and PKC-epsilon were also observed in rat gastrocnemius muscles after insulin treatment in vivo. Our findings suggest that PKC-theta, like other diacylglycerol-sensitive PKC isoforms (alpha, beta and epsilon), may play a role in insulin action in skeletal muscles.

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