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Kathryn E Meier - One of the best experts on this subject based on the ideXlab platform.
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Effects of protein kinase C activators on Phorbol ester-sensitive and -resistant EL4 thymoma cells.
Carcinogenesis, 1997Co-Authors: Heather M. Sansbury, April E. Wisehart-johnson, Sandra Fulwood, Chen Qi, Kathryn E MeierAbstract:Phorbol ester-sensitive EL4 murine thymoma cells respond to Phorbol 12-myristate 13-acetate with activation of ERK mitogen-activated protein kinases, synthesis of interleukin-2, and death, whereas Phorbol ester-resistant variants of this cell line do not exhibit these responses. Additional aspects of the resistant phenotype were examined, using a newly-established resistant cell line. Phorbol ester induced morphological changes, ERK activation, calcium-dependent activation of the c-Jun N-terminal kinase (JNK), interleukin-2 synthesis, and growth inhibition in sensitive but not resistant cells. A series of protein kinase C activators caused membrane translocation of protein kinase C's (PKCs) α,η, and θ in both cell lines. While PKCη was expressed at higher levels in sensitive than in resistant cells, overexpression of PKCη did not restore Phorbol ester-induced ERK activation to resistant cells. In sensitive cells, PKC activators had similar effects on cell viability and ERK activation, but differed in their abilities to induce JNK activation and interleukin-2 synthesis. PD 098059, an inhibitor of the mitogen activated protein (MAP)/ERK kinase kinase MEK, partially inhibited ERK activation and completely blocked Phorbol ester-induced cell death in sensitive cells. Thus MEK and/or ERK activation, but not JNK activation or interleukin-2 synthesis, appears to be required for Phorbol ester-induced toxicity. Alterations in Phorbol ester response pathways, rather than altered expression of PKC isoforms, appear to confer Phorbol ester resistance to EL4 cells.
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Synergistic Effects of Insulin and Phorbol Ester on Mitogen-activated Protein Kinase in Rat-1 HIR Cells
Journal of Biological Chemistry, 1996Co-Authors: Stewart M Knoepp, April E. Wisehart-johnson, Cynthia D. Bradshaw, Krishna Murthy ELLA, Maria G. Buse, Kathryn E MeierAbstract:Abstract Regulation of the activity of the extracellular signal regulated kinase (ERK) mitogen-activated protein kinases was examined in Rat-1 HIR, a fibroblast cell line overexpressing the human insulin receptor. Insulin or Phorbol ester induced partial activations of ERKs, while a combination of insulin and Phorbol ester resulted in a synergistic activation. Preincubation with Phorbol ester increased the subsequent response to insulin. Phorbol ester did not enhance tyrosine phosphorylation of the insulin receptor. Insulin did not enhance activation of phospholipase D in response to Phorbol ester. Lysophosphatidic acid also acted synergistically with insulin to induce ERK activation. Lysophosphatidic acid alone had little effect on ERK, and did not activate phospholipase D. The combination of Phorbol ester and insulin maintained tyrosine phosphorylation of focal adhesion kinase, while insulin alone decreased its tyrosine phosphorylation. Phorbol ester induced phosphorylation of Shc on serine/threonine, while insulin induced tyrosine phosphorylation of Shc and Shc-Grb2 binding. These results suggest that full activation of ERKs in fibroblasts can require the cooperation of at least two signaling pathways, one of which may result from a protein kinase C-dependent phosphorylation of effectors regulating ERK activation. In this manner, Phorbol esters may enhance mitogenic signals initiated by growth factor receptors.
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effects of Phorbol ester on mitogen activated protein kinase kinase activity in wild type and Phorbol ester resistant el4 thymoma cells
Journal of Biological Chemistry, 1993Co-Authors: Katrina C Gause, Karen A Licciardi, Marsha J Peterson, Miwako K Homma, Rony Seger, Edwin G. Krebs, Kathryn E MeierAbstract:Abstract Phorbol ester-sensitive and -resistant EL4 thymoma cell lines differ in their ability to activate mitogen-activated protein kinase (MAPK) in response to Phorbol ester. Treatment of wild-type EL4 cells with Phorbol ester results in the rapid activations of MAPK and pp90rsk kinase, a substrate for MAPK, while neither kinase is activated in response to Phorbol ester in variant EL4 cells. This study examines the activation of MAPK kinase (MAPKK), an activator of MAPK, in wild-type and variant EL4 cells. Phosphorylation of a 40-kDa substrate, identified as MAPK, was observed following in vitro phosphorylation reactions using cytosolic extracts or Mono Q column fractions prepared from Phorbol ester-treated wild-type EL4 cells. MAPKK activity coeluted with a portion of the inactive MAPK upon Mono Q anion-exchange chromatography, permitting detection of the MAPKK activity in fractions containing both kinases. This MAPKK activity was present in Phorbol ester-treated wild-type cells, but not in Phorbol ester-treated variant cells or in untreated wild-type or variant cells. The MAPKK from wild-type cells was able to activate MAPK prepared from either wild-type or variant cells. MAPKK activity could be stimulated in both wildtype and variant EL4 cells in response to treatment of cells with okadaic acid. These results indicate that the failure of variant EL4 cells to activate MAP kinase in response to Phorbol ester is due to a failure to activate MAPKK. Therefore, the step that confers Phorbol ester resistance to variant EL4 cells lies between the activation of protein kinase C and the activation of MAPKK.
Marcelo G Kazanietz - One of the best experts on this subject based on the ideXlab platform.
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novel nonkinase Phorbol ester receptors the c1 domain connection
Molecular Pharmacology, 2002Co-Authors: Marcelo G KazanietzAbstract:In recent years, there have been great advances in our understanding of the pharmacology and biology of the receptors for the Phorbol ester tumor promoters and the second messenger diacylglycerol (DAG). The traditional view of protein kinase C (PKC) as the sole receptor for the Phorbol esters has been challenged with the discovery of proteins unrelated to PKC that bind Phorbol esters with high affinity, suggesting a high degree of complexity in the signaling pathways activated by DAG. These novel “nonkinase” Phorbol ester receptors include chimaerins (a family of Rac GTPase activating proteins), RasGRPs (exchange factors for Ras/Rap1), and Munc13 isoforms (scaffolding proteins involved in exocytosis). In all cases, Phorbol ester binding occurs at the single C1 domain present in these proteins and, as in PKC isozymes, ligand binding is a phospholipid-dependent event. Moreover, the novel Phorbol ester receptors are also subject to subcellular redistribution or “translocation” by Phorbol esters, leading to their association to different effector and/or regulatory molecules. Clearly, the use of Phorbol esters as specific activators of PKC in cellular models is questionable. Alternative pharmacological and molecular approaches are therefore needed to dissect the involvement of each receptor class as a mediator of Phorbol ester/DAG responses.
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Phorbol Esters and Related Analogs Regulate the Subcellular Localization of β2-Chimaerin, a Non-protein Kinase C Phorbol Ester Receptor
Journal of Biological Chemistry, 2001Co-Authors: Maria J. Caloca, Andrew S. Delemos, Shaomeng Wang, Hongbin Wang, Marcelo G KazanietzAbstract:Abstract The novel Phorbol ester receptor β2-chimaerin is a Rac-GAP protein possessing a single copy of the C1 domain, a 50-amino acid motif initially identified in protein kinase C (PKC) isozymes that is involved in Phorbol ester and diacylglycerol binding. We have previously shown that, like PKCs, β2-chimaerin binds Phorbol esters with high affinity in a phospholipid-dependent manner (Caloca, M. J., Fernandez, M. N., Lewin, N. E., Ching, D., Modali, R., Blumberg, P. M., and Kazanietz, M. G. (1997) J. Biol. Chem. 272, 26488–26496). In this paper we report that like PKC isozymes, β2-chimaerin is translocated by Phorbol esters from the cytosolic to particulate fraction. Phorbol esters also induce translocation of α1 (n)- and β1-chimaerins, suggesting common regulatory mechanisms for all chimaerin isoforms. The subcellular redistribution of β2-chimaerin by Phorbol esters is entirely dependent on the C1 domain, as revealed by deletional analysis and site-directed mutagenesis. Interestingly, β2-chimaerin translocates to the Golgi apparatus after Phorbol ester treatment, as revealed by co-staining with the Golgi marker BODIPY-TR-ceramide. Structure relationship analysis of translocation using a series of PKC ligands revealed substantial differences between translocation of β2-chimaerin and PKCα. Strikingly, the mezerein analog thymeleatoxin is not able to translocate β2-chimaerin, although it very efficiently translocates PKCα. Phorbol esters also promote the association of β2-chimaerin with Rac in cells. These data suggest that chimaerins can be positionally regulated by Phorbol esters and that each Phorbol ester receptor class has distinct pharmacological properties and targeting mechanisms. The identification of selective ligands for each Phorbol ester receptor class represents an important step in dissecting their specific cellular functions.
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Pharmacology of the receptors for the Phorbol ester tumor promoters: Multiple receptors with different biochemical properties
Biochemical Pharmacology, 2000Co-Authors: Marcelo G Kazanietz, Maria J. Caloca, Pilar Eroles, Teruhiko Fujii, Maria Laura Garcia-bermejo, Muredach P Reilly, Hongbin WangAbstract:Abstract The Phorbol ester tumor promoters and related analogs are widely used as potent activators of protein kinase C (PKC). The Phorbol esters mimic the action of the lipid second messenger diacylglycerol (DAG). The aim of this commentary is to highlight a series of important and controversial concepts in the pharmacology and regulation of Phorbol ester receptors. First, Phorbol ester analogs have marked differences in their biological properties. This may be related to a differential regulation of PKC isozymes by distinct analogs. Moreover, it seems that marked differences exist in the ligand recognition properties of the C1 domains, the Phorbol ester/DAG binding sites in PKC isozymes. Second, an emerging theme that we discuss here is that Phorbol esters also target receptors unrelated to PKC isozymes, a concept that has been largely ignored. These novel receptors lacking kinase activity include chimaerins (a family of Rac-GTPase-activating proteins), RasGRP (a Ras exchange factor), and Unc-13/Munc-13 (a family of proteins involved in exocytosis). Unlike the classical and novel PKCs, these “non-kinase” Phorbol ester receptors possess a single copy of the C1 domain. Interestingly, each receptor class has unique pharmacological properties and biochemical regulation. Lastly, it is well established that Phorbol esters and related analogs can translocate each receptor to different intracellular compartments. The differential pharmacological properties of the Phorbol ester receptors can be exploited to generate specific agonists and antagonists that will be helpful tools to dissect their cellular function.
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eyes wide shut protein kinase c isozymes are not the only receptors for the Phorbol ester tumor promoters
Molecular Carcinogenesis, 2000Co-Authors: Marcelo G KazanietzAbstract:In addition to the well-characterized interaction with classical and novel protein kinase C (PKC) isozymes, the Phorbol ester tumor promoters bind to other receptors lacking kinase activity. Among these novel Phorbol ester receptors, two families of proteins may play a role in the regulation of cell growth and malignant transformation: chimaerins and ras guanyl–releasing protein (ras-GRP). These proteins possess a single copy of the C1 domain that is involved in binding of Phorbol esters and the lipid second messenger diacylglycerol. Four isoforms of chimaerins (α1-, α2-, β1-, and β2-chimaerins) have been isolated to-date, all of them possessing GTPase-activating protein activity for Rac, a small GTP-binding protein that controls actin cytoskeleton organization, cell-cycle progression, adhesion, and migration. Ras-GRP is a guanine nucleotide exchange factor for ras and promotes malignant transformation in fibroblasts in a Phorbol ester–dependent manner. The C1 domain in Ras-GRP may, therefore, have a dominant role in Ras-GRP activation and is essential for Phorbol ester–dependent activation of downstream effectors of ras, i.e., the mitogen-activated protein kinase cascade. Thus, a novel concept emerges in which Phorbol esters may exert cellular responses through pathways not involving Phorbol ester–responsive PKC isozymes. The discovery of “nonPKC” Phorbol ester receptors adds an additional level of complexity to the understanding of Phorbol ester effects and the molecular mechanisms of carcinogenesis. Mol. Carcinog. 28:5–11, 2000. © 2000 Wiley-Liss, Inc.
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β2-Chimaerin Is a High Affinity Receptor for the Phorbol Ester Tumor Promoters
Journal of Biological Chemistry, 1997Co-Authors: Maria J. Caloca, Nieves Fernandez, Nancy E. Lewin, Dixie Ching, Rama Modali, Peter M Blumberg, Marcelo G KazanietzAbstract:Abstract β2-chimaerin, a member of the GTPase-activating proteins for the small GTP-binding protein p21Rac, possesses a single cysteine-rich domain with high homology to those implicated in Phorbol ester and diacylglycerol binding in protein kinase C (PKC) isozymes. We have expressed β2-chimaerin in Sf9 insect cells using the baculovirus expression system and determined that, like PKCs, β2-chimaerin binds Phorbol esters with high affinity in the presence of phosphatidylserine as a cofactor. Scatchard plot analysis using the radioligand [3H]Phorbol 12,13-dibutyrate revealed a dissociation constant of 1.9 ± 0.2 nm for β2-chimaerin. Likewise, β2-chimaerin is a high affinity receptor for the bryostatins, a class of atypical PKC activators. A detailed comparison of structure-activity relations using several Phorbol ester analogs revealed striking differences in binding recognition between β2-chimaerin and PKCα. Although the diacylglycerol 1-oleoyl-2-acetylglycerol binds with similar potency to both β2-chimaerin and PKCα, the mezerein analog thymeleatoxin has 56-fold less affinity for binding to β2-chimaerin. To establish whether β2-chimaerin responds to Phorbol esters in cellular systems, we overexpressed β2-chimaerin in COS-7 cells and monitored its subcellular distribution after Phorbol ester treatment. Interestingly, as described previously for PKC isozymes, β2-chimaerin translocates from cytosolic to particulate fractions as a consequence of Phorbol ester treatment. Our results demonstrate that β2-chimaerin is a novel target for the Phorbol ester tumor promoters. The expansion of the family of Phorbol ester receptors strongly suggests a potential for the “non-kinase” receptors as cellular mediators of the Phorbol ester responses.
April E. Wisehart-johnson - One of the best experts on this subject based on the ideXlab platform.
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Effects of protein kinase C activators on Phorbol ester-sensitive and -resistant EL4 thymoma cells.
Carcinogenesis, 1997Co-Authors: Heather M. Sansbury, April E. Wisehart-johnson, Sandra Fulwood, Chen Qi, Kathryn E MeierAbstract:Phorbol ester-sensitive EL4 murine thymoma cells respond to Phorbol 12-myristate 13-acetate with activation of ERK mitogen-activated protein kinases, synthesis of interleukin-2, and death, whereas Phorbol ester-resistant variants of this cell line do not exhibit these responses. Additional aspects of the resistant phenotype were examined, using a newly-established resistant cell line. Phorbol ester induced morphological changes, ERK activation, calcium-dependent activation of the c-Jun N-terminal kinase (JNK), interleukin-2 synthesis, and growth inhibition in sensitive but not resistant cells. A series of protein kinase C activators caused membrane translocation of protein kinase C's (PKCs) α,η, and θ in both cell lines. While PKCη was expressed at higher levels in sensitive than in resistant cells, overexpression of PKCη did not restore Phorbol ester-induced ERK activation to resistant cells. In sensitive cells, PKC activators had similar effects on cell viability and ERK activation, but differed in their abilities to induce JNK activation and interleukin-2 synthesis. PD 098059, an inhibitor of the mitogen activated protein (MAP)/ERK kinase kinase MEK, partially inhibited ERK activation and completely blocked Phorbol ester-induced cell death in sensitive cells. Thus MEK and/or ERK activation, but not JNK activation or interleukin-2 synthesis, appears to be required for Phorbol ester-induced toxicity. Alterations in Phorbol ester response pathways, rather than altered expression of PKC isoforms, appear to confer Phorbol ester resistance to EL4 cells.
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Synergistic Effects of Insulin and Phorbol Ester on Mitogen-activated Protein Kinase in Rat-1 HIR Cells
Journal of Biological Chemistry, 1996Co-Authors: Stewart M Knoepp, April E. Wisehart-johnson, Cynthia D. Bradshaw, Krishna Murthy ELLA, Maria G. Buse, Kathryn E MeierAbstract:Abstract Regulation of the activity of the extracellular signal regulated kinase (ERK) mitogen-activated protein kinases was examined in Rat-1 HIR, a fibroblast cell line overexpressing the human insulin receptor. Insulin or Phorbol ester induced partial activations of ERKs, while a combination of insulin and Phorbol ester resulted in a synergistic activation. Preincubation with Phorbol ester increased the subsequent response to insulin. Phorbol ester did not enhance tyrosine phosphorylation of the insulin receptor. Insulin did not enhance activation of phospholipase D in response to Phorbol ester. Lysophosphatidic acid also acted synergistically with insulin to induce ERK activation. Lysophosphatidic acid alone had little effect on ERK, and did not activate phospholipase D. The combination of Phorbol ester and insulin maintained tyrosine phosphorylation of focal adhesion kinase, while insulin alone decreased its tyrosine phosphorylation. Phorbol ester induced phosphorylation of Shc on serine/threonine, while insulin induced tyrosine phosphorylation of Shc and Shc-Grb2 binding. These results suggest that full activation of ERKs in fibroblasts can require the cooperation of at least two signaling pathways, one of which may result from a protein kinase C-dependent phosphorylation of effectors regulating ERK activation. In this manner, Phorbol esters may enhance mitogenic signals initiated by growth factor receptors.
Heather M. Sansbury - One of the best experts on this subject based on the ideXlab platform.
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Effects of protein kinase C activators on Phorbol ester-sensitive and -resistant EL4 thymoma cells.
Carcinogenesis, 1997Co-Authors: Heather M. Sansbury, April E. Wisehart-johnson, Sandra Fulwood, Chen Qi, Kathryn E MeierAbstract:Phorbol ester-sensitive EL4 murine thymoma cells respond to Phorbol 12-myristate 13-acetate with activation of ERK mitogen-activated protein kinases, synthesis of interleukin-2, and death, whereas Phorbol ester-resistant variants of this cell line do not exhibit these responses. Additional aspects of the resistant phenotype were examined, using a newly-established resistant cell line. Phorbol ester induced morphological changes, ERK activation, calcium-dependent activation of the c-Jun N-terminal kinase (JNK), interleukin-2 synthesis, and growth inhibition in sensitive but not resistant cells. A series of protein kinase C activators caused membrane translocation of protein kinase C's (PKCs) α,η, and θ in both cell lines. While PKCη was expressed at higher levels in sensitive than in resistant cells, overexpression of PKCη did not restore Phorbol ester-induced ERK activation to resistant cells. In sensitive cells, PKC activators had similar effects on cell viability and ERK activation, but differed in their abilities to induce JNK activation and interleukin-2 synthesis. PD 098059, an inhibitor of the mitogen activated protein (MAP)/ERK kinase kinase MEK, partially inhibited ERK activation and completely blocked Phorbol ester-induced cell death in sensitive cells. Thus MEK and/or ERK activation, but not JNK activation or interleukin-2 synthesis, appears to be required for Phorbol ester-induced toxicity. Alterations in Phorbol ester response pathways, rather than altered expression of PKC isoforms, appear to confer Phorbol ester resistance to EL4 cells.
Sarwat Sultana - One of the best experts on this subject based on the ideXlab platform.
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glycyrrhizin exhibits potential chemopreventive activity on 12 o tetradecanoyl Phorbol 13 acetate induced cutaneous oxidative stress and tumor promotion in swiss albino mice
Journal of Enzyme Inhibition and Medicinal Chemistry, 2007Co-Authors: Sahar Rahman, Sarwat SultanaAbstract:Glycyrrhizin and its aglycone, glycyrrhetic acid has been found useful for various therapeutic purposes. Glycyrrhizin has been shown to possess many physiological functions like anti-inflammatory activity, detoxification and inhibition of carcinogenic promoters. 12-O-Tetradecanoyl Phorbol-13-acetate (TPA), a well-known phorbal ester is known for its tumor promotion activity. The induction of inflammation in skin mediated by TPA is believed to be governed by cyclooxygenase (COX), lipoxygenase and ornithine decarboxylase (ODC). These markers of inflammatory responses are important for skin tumor promotion. In our present study, we studied the chemopreventive effect of glycyrrhizin on TPA (20 nmol/0.2 mL acetone/animal, topically)-induced oxidative stress and hyperproliferation markers in skin. TPA enhanced lipid peroxidation with reduction in the level of catalase, glutathione, glutathione peroxidase, glutathione reductase and glutathione-s-transferase. TPA treatment also enhanced ODC activity and [3H] thym...
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tephrosia purpurea alleviates Phorbol ester induced tumor promotion response in murine skin
Pharmacological Research, 2001Co-Authors: Mohammad Saleem, Salahuddin Ahmed, Aftab Alam, Sarwat SultanaAbstract:Abstract In recent years, considerable emphasis has been placed on identifying new cancer chemopreventive agents, which could be useful for the human population. Tephrosia purpurea has been shown to possess significant activity against hepatotoxicity, pharmacological and physiological disorders. Earlier we showed that Tephrosia purpurea inhibits benzoyl peroxide-mediated cutaneous oxidative stress and toxicity. In the present study, we therefore assessed the effect of Tephrosia purpurea on 12-O-tetradecanoyl phorbal-13-acetate (TPA; a well-known Phorbol ester) induced cutaneous oxidative stress and toxicity in murine skin. The pre-treatment of Swiss albino mice with Tephrosia purpurea prior to application of croton oil (Phorbol ester) resulted in a dose-dependent inhibition of cutaneous carcinogenesis. Skin tumor initiation was achieved by a single topical application of 7,12-dimethyl benz(a)anthracene (DMBA) (25 μ g per animal per 0.2 ml acetone) to mice. Ten days later tumor promotion was started by twice weekly topical application of croton oil (0.5% per animal per 0.2 ml acetone, v /v). Topical application of Tephrosia purpurea 1 h prior to each application of croton oil (Phorbol ester) resulted in a significant protection against cutaneous carcinogenesis in a dose-dependent manner. The animals pre-treated with Tephrosia purpurea showed a decrease in both tumor incidence and tumor yield as compared to the croton oil (Phorbol ester)-treated control group. In addition, a significant reduction in TPA-mediated induction in cutaneous ornithine decarboxylase (ODC) activity and [ 3 H]thymidine incorporation was also observed in animals pre-treated with a topical application of Tephrosia purpurea . The effect of topical application of Tephrosia purpurea on TPA-mediated depletion in the level of enzymatic and non-enzymatic molecules in skin was also evaluated and it was observed that topical application of Tephrosia purpurea prior to TPA resulted in the significant recovery of TPA-mediated depletion in the level of these molecules, namely glutathione, glutathione S-transferase, glutathione reductase and catalase. From these data we suggest that Tephrosia purpurea can abrogate the tumor-promoting effect of croton oil (Phorbol ester) in murine skin.
