SEARCH:   Advanced

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Rights and Permissions Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Gubina, E. Articles by Mufson, R.A. Search for Related Content PubMed PubMed Citation Articles by Gubina, E. Articles by Mufson, R.A. Related Collections Hematopoiesis and Stem Cells Social Bookmarking                   What's this?  Previous Article  |  Table of Contents  |  Next Article  Blood, Vol. 91 No. 3 (February 1), 1998: pp. 823-829 Overexpression of Protein Kinase C Isoform but not  in Human Interleukin-3-Dependent Cells Suppresses Apoptosis and Induces bcl-2 Expression By E. Gubina, M.S. Rinaudo, Z. Szallasi, P.M. Blumberg, and R.A. Mufson From the Department of Immunology, Holland Laboratory/American Red Cross, Rockville, MD; Laboratory of Cellular Carcinogenesis and Tumor Promotion, National Cancer Institute, Bethesda, MD; and Department of Pharmacology, Uniformed Services University of the Health Sciences, Bethesda, MD.     ABSTRACT Abstract Introduction Methods Results Discussion References Hematopoietic progenitor cells die by apoptosis after removal of the appropriate colony-stimulating factor (CSF). Recent pharmacologic data have implicated protein kinase C (PKC) in the suppression of apoptosis in interleukin-3 (IL-3) and granulocyte-macrophage (GM)-CSF-dependent human myeloid cells. Because IL-3 and GM-CSF induce increases in diacylglycerol without mobilizing intracellular Ca++, it seemed that one of the novel Ca++ independent isoforms of PKC was involved. We report here that overexpression of PKC in factor-dependent human TF-1 cells extends cell survival in the absence of cytokine. Overexpression of PKC does not have this effect. By 72 to 96 hours after cytokine withdrawal, the PKC transfectants remain distributed in all phases of the cell cycle, as shown by fluorescence-activated cell sorting (FACS) analysis, while little intact cellular DNA is detectable in vector or PKC transfectants. PKC induces bcl-2 protein expression fivefold to sixfold over the levels in empty vector transfectants, whereas the levels in PKC transfectants are similar to those in vector controls.     INTRODUCTION Abstract Introduction Methods Results Discussion References THE INTERLEUKIN-3 (IL-3) receptor initiates the signals for survival and proliferation in primitive hematopoietic progenitor cells and certain multipotential leukemias in vitro.1,2 Ligand engagement of the receptor activates Src and Janus family tyrosine kinases associated with the  subunit of the receptor.3-6 Downstream of tyrosine phosphorylation the p21 Ras, Raf-1, and mitogen-activated protein kinases (MAPK) signal transduction cascade is activated.7-11 This cascade has been closely related to the induction of cell proliferation by IL-3 and granulocyte-macrophage colony-stimulating factor (GM-CSF). In addition, however, IL-3-induced tyrosine phosphorylation also causes activation of a phosphatidylcholine-dependent phospholipase C, increased levels of sn-1',2' diacylglycerol, and extended activation of protein kinase C (PKC) in hematopoietic cells, as well as in NIH 3T3 cells expressing a reconstituted human IL-3 receptor.12,13 The role of PKC in IL-3 receptor signal transduction has remained unclear. Recent pharmacologic studies, however, have implicated the activation of PKC as part of a signal transduction cascade required for the suppression of apoptosis in IL-3/GM-CSF-dependent human myeloid cells. Inhibitors of PKC, but not inhibitors of cyclic nucleotide-dependent kinases, drive human IL-3-dependent cell lines MO7E and TF-1 into apoptosis even in the presence of IL-3.14,15 In addition, IL-3 induction of the antiapoptotic gene bcl-2 requires PKC in TF-1 cells.15 Conversely, inhibition of PKC by structurally unrelated inhibitors reduces bcl-2 levels in TF-1 cells even in the presence of IL-3. Further, downregulation of PKC by extended incubation in phorbol ester reduces bcl-2 levels in TF-1 cells and drives these cells into apoptosis in the absence or presence of IL-3. Although in many cell types, activation of PKC by phorbol ester suppresses apoptosis in other cells such as murine thymocytes and human Burkitt lymphoma cells, phorbol ester can actually induce apoptosis as it does in TF-1 cells.16,17 Thus, whether particular isoforms are rapidly activated or downregulated may be cell type-specific and determine whether PKC suppresses or induces apoptosis. PKC is a multigene family of enzymes encoded by different mRNA species.18,19 The family can be divided into three subgroups: (1) the classic isoforms that require Ca+2 and diacylglycerol for activation, (2) the novel isoforms that require diacylglycerol, but not Ca+2, and (3) the atypical enzymes that require neither Ca+2 nor diacylglycerol. Growing evidence suggests that individual isoforms subserve distinct physiologic functions.18,19 It is likely that one of the novel isoforms of PKC is implicated in IL-3 activation because IL-3 induces diacylglycerol from phosphatidylcholine hydrolysis without elevating intracellular free Ca+2 in human IL-3-dependent cells.12 The , , , and  isoforms comprise the novel isoform subgroup. We have focused on the isoform because previous work has shown that this isoform is activated by GM-CSF in association with phosphatidylcholine hydrolysis in TF-1 cells.20 In this study, we have used the cDNAs for PKC  and  transfected into the human IL-3-dependent cell line TF-1 to investigate the role of specific novel isoforms in cell survival after cytokine withdrawal. We have chosen the  isoform as a control for  because  and  have been shown to have opposite effects on myeloid cell physiology.21 We report that overexpression of the  isoform extends the survival of TF-1 cells in the absence of IL-3 . The  isoform transfectants also showed significantly increased levels of bcl-2 protein. Transfection of the  isoform had neither effect. Thus, a specific novel isoform of PKC may be required for the suppression of apoptosis through the IL-3 receptor.     MATERIALS AND METHODS Abstract Introduction Methods Results Discussion References Preparation of PKC cDNA expressing plasmids.   The cDNA for PKC was a full-length cDNA cloned from a mouse myeloid tumor cell line ABPL-.22 The full-length PKC cDNA was obtained by screening a mouse brain cDNA library as previously described by Mischak et al.23 The cDNA for PKC was subcloned by blunt end ligation into the Bam H1 site of the vector pMTH.23 The  cDNA was subcloned into a pMTH vector modified to add a 12 amino acid PKC epitope tag to the PKC as previously described.24,25 Expression of the  isoform from pMTH produces enzymatically active protein,23 as does the epitope tagged  isoform.26 The Xho I and MluI sites in the epitope tagging pMTH vector were used to ensure undirectionality.24 The epitope tag consists of the amino acids KGFSYFGEDLMD that are derived from the carboxy terminal end of  isoform. The epitope tag is specifically designed to be recognized by a commercially available polyclonal antibody made against amino acids 726-737 in the carboxy terminus of PKC (GIBCO-BRL, Gaithersburg, MD). The antibody was purified by the manufacturer using affinity chromatography with immobilized peptide and is specific for this epitope in PKC. Transfection and cell selection.   Human IL-3-dependent TF-1 cells were transfected using Lipofectamine Reagent in OptiMEM medium (GIBCO-BRL, Gaithersburg, MD). In each transfection, 2 µg of DNA was used per 12 µL of lipofectamine reagent, and transfections were incubated for 5 hours at 37°C. After transfection, 3 × 106 cells in 0.8 mL of OptiMEM containing 5 ng/mL of recombinant human IL-3 (BioSource Inc, Camarilllo, CA) were plated in each well of a 24-well tissue culture plate. Cells were selected with 600 µg/mL of active G418 (Geneticin from GIBCO-BRL) in the presence of 5 ng/mL of IL-3 for 4 weeks. At the end of the selection, individual colonies were isolated from the bottoms of the wells and expanded into cell lines. Determination of cell survival and apoptosis.   Cell survival was determined by trypan blue exclusion and enumeration of viable cells using a hemocytometer. Apoptotic cells were also determined in inhibitor studies using an enzyme-linked immunosorbent assay (ELISA) (Boehringer Mannheim Inc, Indianapolis, IN) that employed sandwich antibodies recognizing histones and DNA. This assay allowed the quantitation of soluble nucleosomes in cell lysates. Cell cycle analysis by flow cytometry.   Cells were washed and suspended in 1 mL of 70% ethanol at 4°C overnight. After incubation, cells were again washed in phosphate-buffered saline and resuspended in this solution containing 10 µg/mL RNase. Cells were then incubated at 37°C for 0.5 hours. At the end of the incubation, propidium iodide was added to a concentration of 50 µg/mL. The stained cells were then analyzed for DNA content by flow cytometry on a Becton-Dickinson FACScan flow cytometer. Immunoblot analysis of cellular protein.   For immunoblots, cells were lysed by incubation in the cold with a buffer containing 1% Nonidet P-40 (NP-40), 50 mmol/L HEPES, pH 7.4, 100 mmol/L NaF, 10 mmol/L Na phenylphosphate, 2 mmol/L phenylmethylsulfonyl fluoride, 10 µg/mL leupeptin, and 2 µg/mL aprotinin. Protein concentration was measured by BCA Protein Assay (Pierce Inc, Rockford, IL), and exactly the same amount of protein was loaded for each lane (15 to 30 µg) in a given experiment. Proteins were separated by polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes for immunoblotting with appropriate antibodies. Immunoreactive bands were visualized by chemiluminescence (ECL, Amersham, Arlington Heights, IL). Densitometric analysis.   Densitometry was performed by digitizing chemiluminescent images on a flat bed scanner and analyzing the integrated optical densities of the digitized bands using the One-D Gel Scan Program from Scanalytics, Inc (Bilerica MA).     RESULTS Abstract Introduction Methods Results Discussion References Characterization of PKC isoforms in TF-1 cells.   We began our analysis by examining the spectrum of PKC isoforms present in TF-1 cells (Fig 1). Aliquots of cell lysates were immunoblotted with a set of commercially available isoform specific antibodies (Transduction Laboratories, Lexington, KY). Among the classical isoforms,  and  were detected, but not . Of the novel isoforms , , and  were detected. The  isoform may be present, but a useful antibody was not available. Finally, we also detected the atypical isoforms , , and . View larger version (17K): [in this window] [in a new window]   Fig 1. Immunoblot of PKC isoforms present in TF-1 cells. Exponentially growing TF-1 cells were lysed and proteins were separated by electrophoresis through 10% sodium dodecyl sulfate (SDS) polyacrylamide gels. Separated proteins were analyzed by immunoblotting with a series of commercially available isoform specific antibodies (Transduction Laboratories). The isoforms were visualized by chemiluminescence and exposure to x-ray film. Transfection and overexpression of PKC  and  isoforms in TF-1 cells.   We used the cDNAs for the murine  and  isoforms of PKC cloned into the mammalian cell expression vector pMTH.22,23,25 At the amino acid level, these murine cDNAs code for proteins that are 90% and 99% homologous to their respective human counterparts.27,28 Both the human and murine isoforms of each type have the same in vitro substrate specificity. The pMTH vector contains a divalent cation inducible metallothionine promoter driving the PKC genes. Expression of the protein from this vector is thus zinc ion inducible. The PKC isoform containing plasmids or empty vector were transfected by lipofection into the multifactor-dependent human CD34+ cell line TF-1 cultured in IL-3. Stable cell lines overexpressing each isoform were selected in medium containing IL-3 and the antibiotic G418. Figure 2 shows that several cell lines were isolated that overexpressed PKC or PKC even in the absence of zinc induction. The ability of 70 µm Zn++ to induce PKC expression clearly established that the overexpression is from the pMTH plasmid driven by metallothionine promoter and not due to cell line variation resulting from G418 selection. View larger version (67K): [in this window] [in a new window]   Fig 2. Expression of PKC isoforms in selected TF-1 transfectants. TF-1 cells were transfected with appropriate plasmids containing either PKC or  cDNA inserts. Transfectants were selected in the antibiotic G418. Isoform overexpression was determined either with (+) or without (-) induction with 70 µmol/L zinc acetate for 18 hours. Enzyme overexpression was detected by immunoblotting after proteins were separated by electrophoresis in a 10% SDS polyacrylamide gel. Results from immunoblots of isoform transfected cells probed with affinity purified rabbit polyclonal antibodies against murine PKC and cross-reacting with the human protein (GIBCO/BRL) are shown in (A), and results from  transfected cells probed with antipeptide monoclonal antibodies recognizing both murine and human PKC (Transduction Laboratories) are shown in (B). Levels of overexpression were determined by digitizing ECL film images on the blots and analyzing the images with the 1D Gel Scan program (Scanalytics). Levels of PKC overexpression between and  transfectants cannot be compared in this figure because it is a composite of immunoblots from two different experiments. Figure 2 is a composite of immunoblots from different experiments, and therefore the levels of protein in the  and  overexpressers cannot be directly compared. We have therefore performed an experiment in which the levels in the overexpressers can be compared in the same immunoblot. In this experiment, we have also asked whether PKC and PKC overexpression are independent of IL-3 in these cells. To do this, we have taken advantage of the epitope tag and identified both isoforms in the same blot with an antibody recognizing the  epitope tag. Densitometric analysis of the cell lines in Fig 3 showed that the levels of overexpression were not significantly different between the  and  overexpressers. In addition, the overexpression of the  and  PKC isoforms was not decreased by withdrawing IL-3 from the cells for up to 40 hours. Comparison of the levels of overexpression to the basal level of these isoforms demonstrated that the  and  cell lines were overexpressing their respective isoforms eight times the basal level in vector transfected cell lines (Fig 2). The vector containing cell lines, which were transfected with an empty vector, served as controls in all subsequent experiments. View larger version (80K): [in this window] [in a new window]   Fig 3. Effect of IL-3 deprivation on PKC overexpression in and  TF-1 transfectants. Cell lines overexpressing PKC or PKC were grown in the absence or presence of IL-3 (10 ng/mL) for 40 hours. At the end of incubation, lysates were prepared and equal amounts of protein were analyzed by electrophoresis in the same polyacrylamide gel. Immunoblot analysis was performed with an isoform specific polyclonal antibody that recognized the 12 amino acid epitope. Thus both PKC and PKC with the epitope tag could be detected in the same immunoblot. Relative levels of and  overexpression were directly compared in the immunoblot by digitizing the immunoblot image and analyzing with the 1D Gel Scan program (Scanalytics). PKC overexpression and the suppression of apoptosis.   We have previously shown that when TF-1 cells growing in IL-3 are deprived of cytokine they die by apoptosis and their nuclear DNA is degraded to oligonucleosome size fragments. The effect of PKC isoform overexpression on cell survival was therefore determined by culturing the cells in serum containing medium lacking only IL-3 and scoring viable surviving cells with time after deprivation. By 72 hours of cytokine deprivation, only 1% to 2% of vector or PKC transfected cells remain viable; however, among the cell lines transfected with PKC 60% to 80% of the deprived cells remain viable (Fig 4A). Even after 120 hours of IL-3 deprivation when no cells remain viable in the PKC or vector transfected cultures, 30% of the PKC transfected cells remain viable. We examined the morphology of the transfectants cultured in IL-3 or after IL-3 deprivation for 72 hours (Fig 4B). The vector and PKC cells are completely apoptotic at this time. Most of the PKC cells are intact and normal appearing, although there are morphologic differences between PKC cells growing in IL-3 and deprived of IL-3. Conditioned medium from PKC cells did not effect the survival of vector transfectants or wild-type TF-1 cells. Thus, PKC is acting similarly to a cell survival gene like bcl-2 and is not producing an autocrine phenotype. View larger version (21K): [in this window] [in a new window]   View larger version (84K): [in this window] [in a new window]   Fig 4. Survival of vector and PKC TF-1 cell transfectants after IL-3 deprivation. Cells were deprived of IL-3 for increasing periods of time and cell viability was determined by vital dye exclusion and enumeration of viable cells by light microscopy. (A) Transfectants represented are vector (-, ), PKC1 (-), PKC10 (-), PKC8 (-), and PKC25 (-). The percentage of viable cells is expressed in relation to the viable cell count at the beginning of the experiment. The experiment was repeated three times with similar results each time. (B) Photomicrographs of representative transfectants after 72 hours in the presence (+) or absence (-) of IL-3. Cell cycle analysis of TF-1 transfectants.   Figure 5 shows a cell cycle analysis from TF-1 transfectants deprived of IL-3 using propidium iodide staining and flow cytometry. In all of the cell lines, small peaks of hypodiploid apoptotic cells can be detected around channel 100 on the fluorescence axis, even while the cells are growing in IL-3. In the presence of IL-3, all the cell lines show a 2N (G0) peak at channel 200 and a 4N (G2/M) peak around channel 800. Cells falling between 200 and 800 are in S phase. By 48 hours after cytokine deprivation, there is a marked reduction in the G2/M phase cells in the vector transfected or PKC transfected cells, while the PKC transfectants continue to show cells in all phases of the cell cycle. At 72 hours of deprivation, the G1 peak is beginning to disappear in the PKC and vector cell lines. The G2/M peak is also diminished in these cells. In the PKC cultures, however, cells remain distributed in all phases of the cell cycle at this time. By 96 hours after deprivation, no intact DNA is visible in the vector and PKC transfected cells, while intact cycling cells are still stainable in the PKC culture. Although only results from PKC10 and PKC25 are shown, flow cytometry analysis of PKC1, and PKC8 transfectants showed the same results. To confirm that the PKC transfectants were surviving due to PKC isoform overexpression, these cells were treated with the specific PKC inhibitor GF 109203X.29 This inhibitor drove both the  transfectant and vector control cells into apoptosis confirming that their survival was dependent on PKC (data not shown). View larger version (29K): [in this window] [in a new window]   Fig 5. Determination of cell cycle distribution of representative TF-1 transfectants in the presence or absence of IL-3. Cells were stained with propidium iodide at the indicated times and analyzed for cell cycle distribution by flow cytometry. The 2N and 4N DNA containing cells are in the 400 and 800 fluorescence channels, respectively. The percentage of cells with subdiploid DNA content is indicated in each panel. Similar profiles were obtained from the PKC1 and PKC8 transfectants. For all cell lines, 104 cells were counted for each analysis, and the experiments were performed twice with similar results each time. Effect of PKC overexpression on bcl-2 induction.   In our previous studies, we determined that PKC appeared to be part of the IL-3 receptor signal transduction pathway that modulated expression of the cell survival gene bcl-2. We therefore determined whether PKC overexpression altered bcl-2 protein levels in TF-1 cells. Lysates from cells growing in IL-3 or deprived of IL-3 for increasing periods of time were prepared and analyzed for bcl-2 expression by immunoblotting using a monoclonal antibody that specifically recognizes the 26 kD human bcl-2 protein. Figure 6 shows the results from a representative experiment. In three separate experiments, the bcl-2 levels in the cell lines PKC1, and 10 were fivefold to sevenfold greater than in vector transfectants, while PKC cells had levels only 1.5 to 2.0 times that in vector transfectants. At 38 hours after IL-3 deprivation, the level of bcl-2 in vector transfected cells had decreased to 58% ± 15% (mean ± standard error of mean [SEM]) of the control levels, while the levels of bcl-2 in PKC 10 and 1 cells remained induced at 5.5 ± .6 and 6.8 ± .8 (mean ± SEM) times the control level in vector transfectants, respectively. The PKC cells in the absence of IL-3 showed an approximate 70% decrease in bcl-2 levels in these experiments (Fig 6). View larger version (32K): [in this window] [in a new window]   Fig 6. The bcl-2 protein content of TF-1 transfectants. Cell lysates were prepared from vector or PKC isoform overexpressing cells grown in the presence of IL-3 (+IL-3) or deprived of IL-3 (-IL-3) for 40 hours. Proteins in cell lysates were separated by electrophoresis in 16% SDS polyacrylamide gel and analyzed by immunoblotting using an antihuman bcl-2 murine monoclonal antibody obtained from Santa Cruz Biotechnology Inc (Santa Cruz, CA). Similar results were obtained in three different experiments.     DISCUSSION Abstract Introduction Methods Results Discussion References The activation of PKC by the IL-3 receptor has been observed in both murine and human hematopoietic cell types; however the physiologic role of this serine/threonine kinase in IL-3 receptor signal transduction has been unclear. The studies presented here implicate one novel isoform of PKC in the suppression of apoptosis after IL-3 deprivation in TF-1 cells. Interestingly, the  isoform was more effective than the  isoform in suppressing apoptosis. A role for the  isoform in GM-CSF signaling has also been observed previously.20 GM-CSF and other cytokines induce a metabolic burst in target cells, and this can be detected by microphysiometry as an increase in proton efflux from the cells. Extended treatment of TF-1 cells with PKC antisense oligonucleotides specifically reduced levels of this isoform in these cells. This reduction in the  isoform was accompanied by an 80% decrease in the GM-CSF-induced proton efflux.20 GM-CSF, IL-3, and IL-5 all share the same receptor  subunit, and thus PKC is probably part of an important signal transduction cascade linked to the suppression of apoptosis by this family of cytokines.30-33 We cannot exclude the possibility that other classic or novel isoforms of PKC may also contribute to signaling suppression of apoptosis through the IL-3 receptor. Work is in progress to compare the overexpression of other classic and novel PKC isoforms on suppression of apoptosis due to cytokine withdrawal. Although other investigators have overexpressed the  or  isoforms in IL-3-dependent murine hematopoietic cells, no changes in IL-3 dependence in the absence or presence of phorbol ester were observed.19 These investigators did not, however, carefully assess cell survival after IL-3 deprivation. Our data, in TF-1 cells, confirm that PKC overexpression does not abrogate the factor dependence of these cells, but that this isoform acts like a cell survival gene. Overexpression of the classic cell survival gene bcl-2 in factor-dependent hematopoietic cells does not create factor independent cell lines, but only suppresses apoptosis for an increased period of time when the transfectants are deprived of IL-3.34 Thus, it is likely that PKC is part of the IL-3 receptor signal transduction cascade that modulates expression of bcl-2 and/or other survival genes. In the present studies, we have shown that PKC transfectants have levels of bcl-2 protein that are five to seven times greater than those in vector transfected cells even after removal of IL-3. The levels of bcl-2 protein in PKC transfectants is elevated 1.5 to twofold in the presence of IL-3 compared with vector transfectants, but falls dramatically after cytokine removal. Thus, PKC induction of bcl-2 may contribute substantially to the survival of these cells. This observation is consistent with recent data showing that overexpression of bcl-2 in TF-1 cells increases survival in the absence of GM-CSF.35 This does not exclude the possibility that PKC may also induce other genes that contribute to TF-1 cell survival. The different biologic effects of PKC and  in TF-1 cells have also been observed in other cell types. In NIH 3T3 cells, overexpression of the  isoform causes these cells to acquire an anchorage independent phenotype. The  isoform, however, does not induce this phenotype.23 In rat basophils, isoform , but not , was found to be involved in signaling antigen-induced c-jun mRNA accumulation.36 Perhaps most significantly in the human cell line U937, the  isoform appears to be involved in the induction of apoptosis. Ionizing radiation induces apoptosis in these cells and this is associated with the proteolytic cleavage and activation of PKC, probably by a protease from the IL-1 converting enzyme family.37,38 Overexpression of bcl-2 blocks both the onset of apoptosis and the proteolytic cleavage of isoform  in these cells. In contrast, when the catalytically active proteolytic fragment of the  isoform is overexpressed in HeLa or 3T3 cells, it induces the morphologic characteristics of apoptosis.38 Thus, the  isoform is associated with activation of apoptosis. Differential phosphorylation of substrates in vivo has yet to be demonstrated for the different isotypes, although differential phosphorylation has been demonstrated with purified proteins in vitro.18,19 The signal transduction cascades regulating bcl-2 expression in lymphoid or myeloid cells are poorly understood. Recent work has shown, however, that the bcl-2 promoter contains a cyclic adenosine monophosphate response element (CRE).39 Mutation of this site resulted in loss of CREB binding and the loss of functional activity of the bcl-2 promoter in transient transfection assays. Further, in the immature Ramos B-cell line, bcl-2 expression is increased following short-term phorbol ester treatment, and the increased expression is dependent on the CRE site.39 Phorbol ester stimulation also resulted in increased phosphorylation of CREB on serine 133, and this phosphorylation is mediated by PKC rather than by protein kinase A. Interestingly, IL-3 also induces phosphorylation of CREB at serine 133, although the kinase responsible for this phosphorylation has not been identified.40 We are currently investigating the relationship of PKC to IL-3-induced CREB 133 phosphorylation and the regulation of bcl-2 gene expression.     FOOTNOTES    Submitted March 27, 1997; accepted October 3, 1997.    Supported by United States Public Health Service Grant No. CA53609-06.    Address reprint requests to R.A. Mufson, PhD, Department of Immunology, Holland Laboratory/American Red Cross, 15601 Crabbs Branch Way, Rockville, MD 20855.    The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. section 1734 solely to indicate this fact.     REFERENCES Abstract Introduction Methods Results Discussion References 1. Williams GT, Smith CA, Spooncer E, Dexter TM, Taylor DR: Hematopoietic colony stimulating factors promote cell survival by suppressing apoptosis. Nature 343:76, 1990[Medline] [Order article via Infotrieve] 2. Koury MJ, Bondurant MC: Erythropoietin retards DNA breakdown and prevents programmed death in erythroid progenitor cells. Science 248:378, 1990[Abstract/Free Full Text] 3. Rao P, Mufson RA: A membrane proximal domain of the human interleukin-3 receptor c subunit that signals DNA synthesis in NIH3T3 cells specifically binds a complex of Src and Janus family kinases and phosphatidylinositol 3-kinase. J Biol Chem 270:6688, 1994 4. Anderson SM, Jorgensen B: Activation of src related kinases by IL-3. J Immunol 155:1660, 1995[Abstract] 5. Ihle JN, Witthuhn BA, Quelle FW, Yamamoto K, Thierfelder WE, Kreider B, Silvennoinen O: Signaling by the cytokine receptor superfamily. Trends Biochem Sci 19:222, 1994[Medline] [Order article via Infotrieve] 6. Quelle FW, Sato N, Witthuhn BA, Inhorn RC, Eder M, Miyajima A, Griffin JD, Ihle JN: JAK2 associates with the betac chain of the receptor for granulocyte-macrophage colony-stimulating factor, and its activation requires the membrane-proximal region. Mol Cell Biol 14:4335, 1994[Abstract/Free Full Text] 7. Spangler R, Bailey SC, Sytkowski AJ: Erythropoietin increases c-myc mRNA by a protein kinase C dependent pathway. J Biol Chem 266:681, 1991[Abstract/Free Full Text] 8. Carroll MP, Clark-Lewis I, Rapp UR, May WS: Interleukin-3 and granulocyte macrophage colony stimulating factor mediate rapid phosphorylation and activation of cytosotic c-Raf. J Biol Chem 265:19812, 1990[Abstract/Free Full Text] 9. Turner B, Rapp U, App H, Greene M, Dobashi K, Reed J: Interleukin 2 induces tyrosine phosphorylation and activation of p72-74 Raf-1 kinase in a T-cell line. Proc Natl Acad Sci USA 88:1227, 1991[Abstract/Free Full Text] 10. Carroll MP, Spivak JL, McMahon M, Weich N, Rapp UR, May WS: Erythropoietin induces Raf-1 activation and Raf-1 is required for erythropoietin mediated proliferation. J Biol Chem 266:14964, 1991[Abstract/Free Full Text] 11. Okuda A, Sanghera JS, Pelech SL, Kanakura Y, Hallek M, Griffin JD, Drucker BJ: Granulocyte-macrophage colony-stimulating factor, interleukin-3, and steel factor induce rapid tyrosine phosphorylation of p42 and p44 MAP kinase. Blood 79:2880, 1992[Abstract/Free Full Text] 12. Rao P, Mufson RA: Human interleukin-3 induces activation of phosphatidylcholine specific phospholipase C and translocation of protein kinase C.  Cancer Res 54:777, 1994[Abstract/Free Full Text] 13. Rao P, Kitamura T, Miyajima A, Mufson RA: Human IL-3 receptor signaling: Rapid induction of phosphatidylcholine hydrolysis is independent of protein kinase C but dependent on tyrosine phosphorylation in transfected NIH 3T3. J Immunol 154:1664, 1995[Abstract] 14. Rajotte D, Haddad P, Haman A, Cragoe EJ, Hoang T: Role of protein kinase C and the Na+/H+ antiporter in suppression of apoptosis by granulocyte-macrophage colony stimulating factor and interleukin-3. J Biol Chem 267:9980, 1992[Abstract/Free Full Text] 15. Rinaudo M, Su K, Falk LA, Haldar S, Mufson, RA: Human interleukin-3 receptor modulates bcl-2 mRNA and protein levels through protein kinase C in TF-1 cells. Blood 86:80, 1995 16. Harutoshi K, Takushi T, Cachexia O, Jun M, Ishimura Y: Activation of a suicide process of thymocytes through DNA fragmentation by calcium ionophores and phorbol esters. J Immunol 143:1790, 1989[Abstract] 17. Haddock H, Ishii H, Gobe, G: Epstein-Barr virus infection is associated with increased apoptosis in untreated and phorbol ester-treated human Burkitt's lymphoma (AW-Ramos) cells. Biochem Biophys Res Commun 192:1415, 1993 18. Dekker L, Palmer R, Parker R: The protein kinase C and protein kinase C related gene families. Curr Opin Struct Biol 5:396, 1995[Medline] [Order article via Infotrieve] 19. Dekker LV, Parker PJ: Protein Kinase Ca question of specificity. Trends Biochem Sci 19:73, 1994[Medline] [Order article via Infotrieve] 20. Baxter GT, Miller D, Kuo RC, Wada G, Owicki J: Protein kinase C epsilon is induced in granulocyte-macrophage colony stimulating factor signal transduction: Evidence from microphysiometry and antisense oligonucleotide experiments. Biochemistry 31:10950, 1992[Medline] [Order article via Infotrieve] 21. Mischak H, Pierce JH, Goodnight JA, Kazanietz MG, Blumberg PM, Mushinski JF: Phorbol ester induced myeloid differentiation is mediated by protein kinase C alpha and delta but not by beta II, epsilon, zeta, and eta. J Biol Chem 268:20110, 1993[Abstract/Free Full Text] 22. Mischak H, Bodenteich A, Kokn W, Goodnight JA, Hoper F, Mushinski JF: Mouse protein kinase C-, the major isoform expressed in mouse hematopoietic cells: Sequence of the cDNA, expression pattern and characterization of the protein. Biochemistry 30:7925, 1991[Medline] [Order article via Infotrieve] 23. Mischak H, Goodnight JA, Kolch W, Martiny-Baron G, Schechtele C, Kazanietz MG, Blumberg PM, Pierce JH, Mushinski JF: Overexpression of protein kinase c- and - in NIH 3T3 cells induces opposite effects on growth, morphology, anchorage dependence, and tumorigenicity. J Biol Chem 268:6090, 1993[Abstract/Free Full Text] 24. Szallasi Z, Denning MF, Chang EY, Rivera J, Yuspa SH, Lehel C, Olah Z, Anderson WB, Blumberg PM: Development of tyrosine phosphorylation sites: Application to PKC  phosphorylated upon activation of the high affinity receptor for IgE in rat basophilic leukemia cells. Biochem Biophys Res Commun 216:103, 1995[Medline] [Order article via Infotrieve] 25. Olah Z, Lehel C, Jakab G, Anderson WB: A cloning and epsilon-epitope tagging insert for the expression of polymerase chain reaction generated cDNA fragments in Escherichia coli and mammalian cells. Anal Biochem 221:94, 1994[Medline] [Order article via Infotrieve] 26. Acs P, Bogi K, Lorenzo PF, Marquez AM, Biro T, Szallasi Z, Blumberg PM: The catalytic domain of protein kinase C chimeras modulates the affinity and targeting of phorbol ester-induced translocation. J Biol Chem 272:22148,1997 27. Basta P, Strickland MB, Holmes W, Loomis C, Ballas L, Burns D: Sequence and expression of human protein kinase C-epsilon. Biochem Biophys Acta 1132:154, 1992[Medline] [Order article via Infotrieve] 28. Aris J, Basta P, Holmes W, Ballas L, Moonmaw C, Rankl N, Blobel G, Loomis C, Burns D: Molecular and biochemical characterization of a recombinant human PKC delta family member. Biochem Biophys Acta 1174:171, 1993[Medline] [Order article via Infotrieve] 29. Toullec D, Pianetti P, Coste H, Bellevergue P, Grand-Perret T, Ajakane M, Brudet V, Boissin P, Boursier E, Loriolle F, Duhamel L, Charon D, Kirilovsky J: The bisindolylmaleimide GF 109203X is a potent and selective inhibitor of protein kinase C.  J Biol Chem 266:15771, 1991[Abstract/Free Full Text] 30. Kinoshita T, Yokota T, Arai KI, Miyajima A: Regulation of bcl-2 expression by oncogenic ras protein in hematopoietic cells. Oncogene 10:2207, 1995[Medline] [Order article via Infotrieve] 31. Kinoshita T, Yokota T, Arai K, Miyajima A: Suppression of apoptotic cell death in hematopoietic cells by signaling through the IL-3/GM-CSF receptors. EMBO J 14:266, 1995[Medline] [Order article via Infotrieve] 32. O'Farrell A-M, Masatoshi L, Mui A, Miyajima A: Signaling pathways activated in a unique mast cell line where interleukin-3 supports survival and stem cell factor is required for a proliferative response. Blood 87:3655, 1994[Abstract/Free Full Text] 33. Miyajima A, Kitamura T, Harada N, Yokota T, Arai KI: Cytokine receptors and signal transduction. Annu Rev Immunol 10:295, 1992[Medline] [Order article via Infotrieve] 34. Nunez G, London L, Hockenberry D, Alexander M, McKearn JP, Korsmeyer SJ: Deregulated bcl-2 gene expression selectively prolongs survival of growth factor deprived hematopoietic cell lines. J Immunol 144:3602, 1990[Abstract] 35. Ito T, Hotta T: Overexpression of bcl-2 suppresses apoptotic cell death of the human leukemic cell line TF-1. Nippon Rinsho 54:1815, 1996[Medline] [Order article via Infotrieve] 36. Razin E, Szallasi Z, Kazaniets MG, Blumberg PM, Rivera J: Protein Kinase C-beta and C-epsilon link the mast cell high affinity receptor for IgE to the expression of c-fos and c-jun. Proc Natl Acad Sci USA 91:7722, 1994[Abstract/Free Full Text] 37. Emoto Y, Manome G, Meinhardt H, Kisaki S, Kharbanda M, Robertson M, Ghayur T, Wong W-H, Kamer R, Weichselbaum R, Kufe D: Proteolytic activation of protein kinase C delta by an ICE-like portease in apoptotic cells. EMBO J 14:6148, 1995[Medline] [Order article via Infotrieve] 38. Emoto Y, Kisaki H, Manome Y, Kharbanda S, Kufe D: Activation of protein kinase C delta in human myeloid leukemia cells treated with 1-beta-D arabinofuranosylcytosine. Blood 87:1990, 1996[Abstract/Free Full Text] 39. Wilson BE, Mochon E, Boxer LM: Production of bcl-2 expression by phosphorylated CREB proteins during B-cell activation and rescue from apoptosis Mol Cell Biol 16:5546, 1996 40. Lee HJ, Mignacca RC, Sakamoto KM: Transcriptional activation of egr-1 by granulocyte-macrophage colony stimulating factor but not interleukin-3 requires phosphorylation of cyclic AMP response element binding protein (CREB) in serine 33.  J Biol Chem 270:15979, 1995[Abstract/Free Full Text] © 1998 by The American Society of Hematology.   0006-4971/98/91-0036$3.00/0 CiteULike    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this? This article has been cited by other articles: T. W. Day, C.-H. Wu, and A. R. Safa Etoposide Induces Protein Kinase C{delta}- and Caspase-3-Dependent Apoptosis in Neuroblastoma Cancer Cells Mol. Pharmacol., September 1, 2009; 76(3): 632 - 640. [Abstract] [Full Text] [PDF] W. Koh, K. Sachidanandam, A. N. Stratman, A. Sacharidou, A. M. Mayo, E. A. Murphy, D. A. Cheresh, and G. E. Davis Formation of endothelial lumens requires a coordinated PKC{epsilon}-, Src-, Pak- and Raf-kinase-dependent signaling cascade downstream of Cdc42 activation J. Cell Sci., June 1, 2009; 122(11): 1812 - 1822. [Abstract] [Full Text] [PDF] R. Steinberg, O. A. Harari, E. A. Lidington, J. J. Boyle, M. Nohadani, A. M. Samarel, M. Ohba, D. O. Haskard, and J. C. Mason A Protein Kinase C{epsilon}-Anti-apoptotic Kinase Signaling Complex Protects Human Vascular Endothelial Cells against Apoptosis through Induction of Bcl-2 J. Biol. Chem., November 2, 2007; 282(44): 32288 - 32297. [Abstract] [Full Text] [PDF] H. Ardehali Signaling Mechanisms in Ischemic Preconditioning: Interaction of PKC{epsilon} and MitoKATP in the Inner Membrane of Mitochondria Circ. Res., October 13, 2006; 99(8): 798 - 800. [Full Text] [PDF] R. Mangat, T. Singal, N. S. Dhalla, and P. S. Tappia Inhibition of phospholipase C-{gamma}1 augments the decrease in cardiomyocyte viability by H2O2 Am J Physiol Heart Circ Physiol, August 1, 2006; 291(2): H854 - H860. [Abstract] [Full Text] [PDF] P. Mirandola, G. Gobbi, C. Ponti, I. Sponzilli, L. Cocco, and M. Vitale PKC{epsilon} controls protection against TRAIL in erythroid progenitors Blood, January 15, 2006; 107(2): 508 - 513. [Abstract] [Full Text] [PDF] F. Lallemend, S. Hadjab, G. Hans, G. Moonen, P. P. Lefebvre, and B. Malgrange Activation of protein kinase C{beta}I constitutes a new neurotrophic pathway for deafferented spiral ganglion neurons J. Cell Sci., October 1, 2005; 118(19): 4511 - 4525. [Abstract] [Full Text] [PDF] S. M. L. Tse, D. Mason, R. J. Botelho, B. Chiu, M. Reyland, K. Hanada, R. D. Inman, and S. Grinstein Accumulation of Diacylglycerol in the Chlamydia Inclusion Vacuole: POSSIBLE ROLE IN THE INHIBITION OF HOST CELL APOPTOSIS J. Biol. Chem., July 1, 2005; 280(26): 25210 - 25215. [Abstract] [Full Text] [PDF] T. Kajimoto, Y. Shirai, N. Sakai, T. Yamamoto, H. Matsuzaki, U. Kikkawa, and N. Saito Ceramide-induced Apoptosis by Translocation, Phosphorylation, and Activation of Protein Kinase C{delta} in the Golgi Complex J. Biol. Chem., March 26, 2004; 279(13): 12668 - 12676. [Abstract] [Full Text] [PDF] A. D. Friedman, D. Nimbalkar, and F. W. Quelle Erythropoietin Receptors Associate with a Ubiquitin Ligase, p33RUL, and Require Its Activity for Erythropoietin-induced Proliferation J. Biol. Chem., July 11, 2003; 278(29): 26851 - 26861. [Abstract] [Full Text] [PDF] L. Ding, H. Wang, W. Lang, and L. Xiao Protein Kinase C-epsilon Promotes Survival of Lung Cancer Cells by Suppressing Apoptosis through Dysregulation of the Mitochondrial Caspase Pathway J. Biol. Chem., September 13, 2002; 277(38): 35305 - 35313. [Abstract] [Full Text] [PDF] A. Goerke, N. Sakai, E. Gutjahr, W. A. Schlapkohl, J. F. Mushinski, H. Haller, W. Kolch, N. Saito, and H. Mischak Induction of Apoptosis by Protein Kinase Cdelta Is Independent of Its Kinase Activity J. Biol. Chem., August 23, 2002; 277(35): 32054 - 32062. [Abstract] [Full Text] [PDF] Y. Levites, T. Amit, M. B. H. Youdim, and S. Mandel Involvement of Protein Kinase C Activation and Cell Survival/ Cell Cycle Genes in Green Tea Polyphenol (-)-Epigallocatechin 3-Gallate Neuroprotective Action J. Biol. Chem., August 16, 2002; 277(34): 30574 - 30580. [Abstract] [Full Text] [PDF] M. Gomez-Angelats and J. A. Cidlowski Invited Review: Cell Volume Control and Signal Transduction in Apoptosis Toxicol Pathol, August 1, 2002; 30(5): 541 - 551. [Abstract] [PDF] A. Zrachia, M. Dobroslav, M. Blass, G. Kazimirsky, I. Kronfeld, P. M. Blumberg, D. Kobiler, S. Lustig, and C. Brodie Infection of Glioma Cells with Sindbis Virus Induces Selective Activation and Tyrosine Phosphorylation of Protein Kinase C delta . IMPLICATIONS FOR SINDBIS VIRUS-INDUCED APOPTOSIS J. Biol. Chem., June 21, 2002; 277(26): 23693 - 23701. [Abstract] [Full Text] [PDF] M. L. Garcia-Bermejo, F. C. Leskow, T. Fujii, Q. Wang, P. M. Blumberg, M. Ohba, T. Kuroki, K.-C. Han, J. Lee, V. E. Marquez, et al. Diacylglycerol (DAG)-lactones, a New Class of Protein Kinase C (PKC) Agonists, Induce Apoptosis in LNCaP Prostate Cancer Cells by Selective Activation of PKCalpha J. Biol. Chem., January 4, 2002; 277(1): 645 - 655. [Abstract] [Full Text] M. Blass, I. Kronfeld, G. Kazimirsky, P. M. Blumberg, and C. Brodie Tyrosine Phosphorylation of Protein Kinase C{delta} Is Essential for Its Apoptotic Effect in Response to Etoposide Mol. Cell. Biol., January 1, 2002; 22(1): 182 - 195. [Abstract] [Full Text] [PDF] E. Gubina, X. Luo, E. Kwon, K. Sakamoto, Y. F. Shi, and R. A. Mufson {beta}c Cytokine Receptor-Induced Stimulation of cAMP Response Element Binding Protein Phosphorylation Requires Protein Kinase C In Myeloid Cells: A Novel Cytokine Signal Transduction Cascade J. Immunol., October 15, 2001; 167(8): 4303 - 4310. [Abstract] [Full Text] [PDF] O. Sordet, C. Rebe, I. Leroy, J.-M. Bruey, C. Garrido, C. Miguet, G. Lizard, S. Plenchette, L. Corcos, and E. Solary Mitochondria-targeting drugs arsenic trioxide and lonidamine bypass the resistance of TPA-differentiated leukemic cells to apoptosis Blood, June 15, 2001; 97(12): 3931 - 3940. [Abstract] [Full Text] [PDF] N. Shiroshita, M. Musashi, K. Sakurada, K. Kimura, Y. Tsuda, S. Ota, H. Iwasaki, T. Miyazaki, T. Kato, H. Miyazaki, et al. Involvement of Protein Kinase C-epsilon in Signal Transduction of Thrombopoietin in Enhancement of Interleukin-3-Dependent Proliferation of Primitive Hematopoietic Progenitors J. Pharmacol. Exp. Ther., June 1, 2001; 297(3): 868 - 875. [Abstract] [Full Text] C.-Y. Chen, P. Juo, J. S. Liou, C.-Q. Li, Q. Yu, J. Blenis, and D. V. Faller The Recruitment of Fas-associated Death Domain/Caspase-8 in Ras-induced Apoptosis Cell Growth Differ., June 1, 2001; 12(6): 297 - 306. [Abstract] [Full Text] [PDF] P. Maher How Protein Kinase C Activation Protects Nerve Cells from Oxidative Stress-Induced Cell Death J. Neurosci., May 1, 2001; 21(9): 2929 - 2938. [Abstract] [Full Text] [PDF] R. Yu, S. Mandlekar, T.-H. Tan, and A.-N. T. Kong Activation of p38 and c-Jun N-terminal Kinase Pathways and Induction of Apoptosis by Chelerythrine Do Not Require Inhibition of Protein Kinase C J. Biol. Chem., March 24, 2000; 275(13): 9612 - 9619. [Abstract] [Full Text] [PDF] T. Fujii, M. L. Garcia-Bermejo, J. L. Bernabo, J. Caamano, M. Ohba, T. Kuroki, L. Li, S. H. Yuspa, and M. G. Kazanietz Involvement of Protein Kinase C delta (PKCdelta ) in Phorbol Ester-induced Apoptosis in LNCaP Prostate Cancer Cells. LACK OF PROTEOLYTIC CLEAVAGE OF PKCdelta J. Biol. Chem., March 10, 2000; 275(11): 7574 - 7582. [Abstract] [Full Text] [PDF] P. J. Reddig, N. E. Dreckschmidt, J. Zou, S. E. Bourguignon, T. D. Oberley, and A. K. Verma Transgenic Mice Overexpressing Protein Kinase C{{epsilon}} in Their Epidermis Exhibit Reduced Papilloma Burden but Enhanced Carcinoma Formation after Tumor Promotion Cancer Res., February 1, 2000; 60(3): 595 - 602. [Abstract] [Full Text] Y. Tan, H. Ruan, M. R. Demeter, and M. J. Comb p90RSK Blocks Bad-mediated Cell Death via a Protein Kinase C-dependent Pathway J. Biol. Chem., December 3, 1999; 274(49): 34859 - 34867. [Abstract] [Full Text] [PDF] J. A. Knauf, R. Elisei, D. Mochly-Rosen, T. Liron, X.-N. Chen, R. Gonsky, J. R. Korenberg, and J. A. Fagin Involvement of Protein Kinase Cepsilon (PKCepsilon ) in Thyroid Cell Death. A TRUNCATED CHIMERIC PKCepsilon CLONED FROM A THYROID CANCER CELL LINE PROTECTS THYROID CELLS FROM APOPTOSIS J. Biol. Chem., August 13, 1999; 274(33): 23414 - 23425. [Abstract] [Full Text] [PDF] M. Haslauer, K. Baltensperger, and H. Porzig Erythropoietin- and Stem Cell Factor-Induced DNA Synthesis in Normal Human Erythroid Progenitor Cells Requires Activation of Protein Kinase Calpha and Is Strongly Inhibited by Thrombin Blood, July 1, 1999; 94(1): 114 - 126. [Abstract] [Full Text] [PDF] C.-Y. Chen and D. V. Faller Selective Inhibition of Protein Kinase C Isozymes by Fas Ligation J. Biol. Chem., May 28, 1999; 274(22): 15320 - 15328. [Abstract] [Full Text] [PDF] C.-D. Jun, C.-D. Oh, H.-J. Kwak, H.-O. Pae, J.-C. Yoo, B.-M. Choi, J.-S. Chun, R.-K. Park, and H.-T. Chung Overexpression of Protein Kinase C Isoforms Protects RAW 264.7 Macrophages from Nitric Oxide-Induced Apoptosis: Involvement of c-Jun N-Terminal Kinase/Stress-Activated Protein Kinase, p38 Kinase, and CPP-32 Protease Pathways J. Immunol., March 15, 1999; 162(6): 3395 - 3401. [Abstract] [Full Text] [PDF] G. C. Mayne and A. W. Murray Evidence That Protein Kinase Cepsilon Mediates Phorbol Ester Inhibition of Calphostin C- and Tumor Necrosis Factor-alpha -induced Apoptosis in U937 Histiocytic Lymphoma Cells J. Biol. Chem., September 11, 1998; 273(37): 24115 - 24121. [Abstract] [Full Text] [PDF] F. K. Racke, D. Wang, Z. Zaidi, J. Kelley, J. Visvader, J.-W. Soh, and A. N. Goldfarb A Potential Role for Protein Kinase C-epsilon in Regulating Megakaryocytic Lineage Commitment J. Biol. Chem., January 5, 2001; 276(1): 522 - 528. [Abstract] [Full Text] [PDF] Y. Shizukuda, M. E. Reyland, and P. M. Buttrick Protein kinase C-delta modulates apoptosis induced by hyperglycemia in adult ventricular myocytes Am J Physiol Heart Circ Physiol, May 1, 2002; 282(5): H1625 - H1634. [Abstract] [Full Text] [PDF] This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Rights and Permissions Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Gubina, E. Articles by Mufson, R.A. Search for Related Content PubMed PubMed Citation Articles by Gubina, E. Articles by Mufson, R.A. Related Collections Hematopoiesis and Stem Cells Social Bookmarking                   What's this?

	Copyright © 1998 by American Society of Hematology         Online ISSN: 1528-0020