Protein kinase C-epsilon is the likely mediator of mucin exocytosis in human colonic cell lines

1997 ◽  
Vol 272 (1) ◽  
pp. G31-G37 ◽  
Author(s):  
D. H. Hong ◽  
J. F. Forstner ◽  
G. G. Forstner
Keyword(s):  
Protein Kinase C ◽  
Cell Line ◽  
Cell Lines ◽  
Mucin Secretion ◽  
T84 Cells ◽  
Kinase C ◽  
Pkc Epsilon ◽  
Pkc Alpha ◽  
Colonic Cells ◽  
Ht 29

The phorbol ester, phorbol 12-myristate 13-acetate (PMA), induces mucin secretion in the colonic tumor cell line T84 in a Ca(2+)-independent manner. To determine whether a specific protein kinase C (PKC) isoform is involved in colonic cells, we compared PMA-dependent mucin secretion by three human colonic tumor cell lines (T84, HT-29/A1, and LS 180) with the expression of PKC isoforms alpha, beta, delta, epsilon, and zeta, previously identified in human colon (L. A. Davidson, Y. H. Jiang, J. D. Derr, H. Aukema, J. R. Lupton, and R. S. Chapkin. Arch. Biochem. Biophys. 312:547-553, 1994). In each cell line PMA (10(-7) M) caused mucin secretion within 30 min. PMA-dependent mucin secretion was three to four times greater from HT-29/A1 and T84 cells than from LS 180 cells. All three-cell lines contained mRNA for PKC-alpha, PKC-epsilon, and PKC-zeta but not PKC-beta or -delta. Each cell line also expressed PKC-alpha, -epsilon, and -zeta protein. PKC-epsilon expression (mRNA and protein) was three to four times greater in HT-29/A1 and T84 cells than in LS 180 cells, correlating with PMA-responsive mucin secretion, whereas all cell lines contained similar levels of PKC-alpha mRNA and protein. When cells were stimulated by PMA, only PKC-epsilon was translocated from cytosol to membrane fractions early enough to stimulate mucin secretion. Because PKC-epsilon is also a Ca(2+)-independent isoform, it is likely to mediate mucin exocytosis in colonic cells.

Enzastaurin (LY317615), an Oral Protein Kinase C β Inhibitor, Induces Apoptosis in Multiple Myeloma Cell Lines.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1577-1577
Author(s):  
Mujahid A. Rizvi ◽  
Kulsoom Ghias ◽  
Katharine M. Davies ◽  
Chunguang Ma ◽  
Nancy L. Krett ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Cell Line ◽  
Cell Lines ◽  
Cancer Cell Line ◽  
Colon Cancer Cell Line ◽  
Myeloma Cell ◽  
Akt Pathway ◽  
Kinase C

Abstract The protein kinase C (PKC) family consists of 11 serine/threonine protein kinase isoforms that are involved with cell proliferation and differentiation, gene transcription, and tumor-induced angiogenesis. The PKC pathway has been shown to play a role in the regulation of cell growth in several hematologic malignancies. However, in multiple myeloma (MM), the role of the PKC pathway has not been extensively studied. Enzastaurin (LY317615), an acyclic bisindolylmaleimide, is an oral inhibitor of the PKC β isozyme. Enzastaurin has been reported to induce apoptosis and suppress proliferation in the HCT116 colon cancer cell line by inhibiting the AKT pathway. The objective of this study was to assess the efficacy of Enzastaurin in inducing apoptosis in MM cell lines and to investigate possible mechanisms of apoptosis. A spectrum of MM cell lines, with unique characteristics (dexamethasone-sensitive, dexamethasone-resistant, chemotherapy-sensitive, chemotherapy-resistant) were treated with Enzastaurin. There is evidence of cell death in all cell lines at clinically significant concentrations (1–3μM) after 72 hours of treatment. The dexamethasone-sensitive MM1.S cell line was used to further assess the effect of Enzastaurin in the presence of dexamethasone, insulin-like growth factor-1 (IGF-1), and IL-6. IGF-1 and IL-6 are potent growth factors for MM and have been observed to blunt the cytotoxic activity of dexamethasone. Dexamethasone and Enzastaurin appear to have an additive effect in the induction of apoptosis. In addition, while IGF-1 slightly decreases the effect of Enzastaurin, IL-6 has no effect. Enzastaurin treatment also induces a decrease in GSK3β and AKTSerine473 phosphorylation. GSK3β phosphorylation is thought to be a reliable pharmacodynamic marker for Enzastaurin activity. In conclusion, these data indicate that Enzastaurin induces apoptosis in MM cells and suppression of the AKT pathway may be one of the mechanisms by which Enzastaurin exerts its anti-myeloma activity.

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.

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 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 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.

A fully automated capillary western system for absolute quantitation of endogenous PKC proteins: An approach to correlate protein quantity with function.

2012 ◽  
Vol 30 (30_suppl) ◽  
pp. 31-31
Author(s):  
Jin-Qiu Chen ◽  
Madeleine Heldman ◽  
Michelle Herrmann ◽  
Noemi Kedei ◽  
Peter Blumberg ◽  
...  
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Relative Abundance ◽  
U937 Cells ◽  
Bryostatin 1 ◽  
Absolute Quantitation ◽  
Pkc Delta ◽  
Pkc Epsilon ◽  
Pkc Isoforms ◽  
Pkc Alpha

31 Background: The human prostate cell line LNCaP and the human myelocytic leukemia cell line U937 differ dramatically in their responses to the two protein kinase C (PKC) targeted ligands phorbol 12-myristate 13-acetate (PMA) and bryostatin 1 and show complex differences in the patterns of transcriptional responses that they induce. Quantitation of relative abundance of individual PKC isoforms in the two cell lines may help to link the downstream effects of the two compounds to these isoforms. Methods: Simple Western is a capillary-based automated Western system recently developed by ProteinSimple. All steps following sample preparation are fully automated in the Simple Western system, including sample loading, size-based protein separation, immunoprobing, washing, detection and data analysis. Simple Western is gel-free and blot-free, uses less amount of samples, and produces highly quantitative, reproducible information that cannot be generated using regular Western assays. Using the Simple Western system, we developed a method for absolute quantitation of endogenous proteins in cell lysates and quantified PKC isoforms in LNCaP and U937 cells. Results: PKC isoforms were measured at levels of picogram or sub-picogram per nanogram cell lysate. PKC delta was identified as the dominant PKC isoforms in both cell lines. In LNCaP cells, PKC delta expression is ~20-fold higher than PKC alpha, ~40-fold higher than PKC epsilon, and at least 20-fold higher than PKC beta. In U937 cells, PKC delta expression is similar to PKC beta, at least 200 fold higher than PKC alpha, and ~50-fold higher than PKC epsilon. Conclusions: The Simple Western system, with its high-quality data quantitation and excellent assay reproducibility, allowed us to detect both the relative abundance of the PKC isoforms and their absolute quantitation in the tested cells. It circumvents the problem that antibodies of different affinities for different proteins yield a misleading impression of relative abundance and it provides an approach to accurately correlate protein quantities with their function.

scholarly journals Overexpression of protein kinase C in HT29 colon cancer cells causes growth inhibition and tumor suppression.

1990 ◽  
Vol 10 (9) ◽  
pp. 4650-4657 ◽  
Author(s):  
P M Choi ◽  
K M Tchou-Wong ◽  
I B Weinstein
Keyword(s):  
Colon Cancer ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Cell Line ◽  
Cell Lines ◽  
Control Cell ◽  
Tumor Promoter ◽  
Vector System ◽  
Human Colon Cancer ◽  
Kinase C

By using a retrovirus-derived vector system, we generated derivatives of the human colon cancer cell line HT29 that stably overexpress a full-length cDNA encoding the beta 1 isoform of rat protein kinase C (PKC). Two of these cell lines, PKC6 and PKC7, displayed an 11- to 15-fold increase in PKC activity when compared with the C1 control cell line that carries the vector lacking the PKC cDNA insert. Both of the overexpresser cell lines exhibited striking alterations in morphology when exposed to the tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA). Following exposure to TPA, PKC6 and PKC7 cells displayed increased doubling time, decreased saturation density, and loss of anchorage-independent growth in soft agar; but these effects were not seen with the C1 cells. Also, in contrast to the control cells, the PKC-overproducing cells failed to display evidence of differentiation, as measured by alkaline phosphatase activity, when exposed to sodium butyrate. In addition, the PKC-overexpresser cells displayed decreased tumorigenicity in nude mice, even in the absence of treatment with TPA. These results provide the first direct evidence that PKC can inhibit tumor cell growth. Thus, in some tumors, PKC might act as a growth-suppressor gene.

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.

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