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Kathryn E Meier - One of the best experts on this subject based on the ideXlab platform.

  • Effects of protein kinase C activators on Phorbol Ester-sensitive and -resistant EL4 thymoma cells.
    Carcinogenesis, 1997
    Co-Authors: Heather M. Sansbury, April E. Wisehart-johnson, Sandra Fulwood, Chen Qi, Kathryn E Meier
    Abstract:

    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.

  • Synergistic Effects of Insulin and Phorbol Ester on Mitogen-activated Protein Kinase in Rat-1 HIR Cells
    Journal of Biological Chemistry, 1996
    Co-Authors: Stewart M Knoepp, April E. Wisehart-johnson, Cynthia D. Bradshaw, Krishna Murthy ELLA, Maria G. Buse, Kathryn E Meier
    Abstract:

    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.

  • 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, 1993
    Co-Authors: Katrina C Gause, Karen A Licciardi, Marsha J Peterson, Miwako K Homma, Rony Seger, Edwin G. Krebs, Kathryn E Meier
    Abstract:

    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.

Peter M. Blumberg - One of the best experts on this subject based on the ideXlab platform.

  • abstract 3928 structural basis for the failure of the c1 domain of rasgrp2 to bind Phorbol Ester
    Cancer Research, 2015
    Co-Authors: Agnes Czikora, Noemi Kedei, Nancy E. Lewin, Daniel J Lundberg, Peter M. Blumberg
    Abstract:

    C1 domains represent the recognition motif for the second messenger diacylglycerol (DAG) and for the Phorbol Esters in protein kinase C, RasGRP, and several other families of signaling proteins. In addition to such typical C1 domains, atypical C1 domains have been identified which possess varying levels of sequence and structural homology with the typical C1 domains but fail to bind DAG or Phorbol Ester. Among RasGRP family members, RasGRP1 and RasGRP3 possess typical C1 domains, whereas the C1 domain of RasGRP2 stands out as being atypical. In order to better understand the structural constraints for ligand binding, we have analyzed the basis for the failure of the C1 domain of RasGRP2 to bind ligands. Using site-directed mutagenesis, we have identified four critical amino acid residues responsible for the lack of Phorbol Ester sensitivity. We found that the C1 domain of RasGRP2 had weak binding affinity (Kd = 2890 ± 240 nM) in vitro for [3H]Phorbol 12,13-dibutyrate ([3H]PDBu). Replacing all four of critical amino acid residues (Asn7, Ser8, Ala19 and Ile21) with the corresponding residues (Thr7, Tyr8, Gly19 and Leu21, respectively) of RasGRP1 resulted in potent [3H]PDBu binding affinity (Kd = 1.47 ± 0.03 nM) and translocation in response to PMA in the LNCaP cell line. The mutant C1 domains incorporating one to three of these critical residues showed intermediate binding and translocation properties. Of the four substitutions, S8Y made the greatest contribution. Phospholipid requirements for [3H]PDBu binding to the mutant C1 domains were determined; at constant total phospholipid, the requirement for the proportion of phosphatidylserine in phosphatidylserine : phosphatidylcholine mixtures decreased in going from the single S8Y mutant to the quadruple mutant. Binding activity for DAG was also restored in the mutant C1 domains, in parallel with that for Phorbol Ester. The full length RasGRP2 protein containing the mutated C1 domains likewise showed strong Phorbol Ester binding, albeit modestly weaker than that of the purified C1 domain (Kd = 8.2 ± 1.1 nM for the full length protein containing all four mutations) and displayed translocation in the LNCaP (human prostate adenocarcinoma) cells in response to Phorbol Ester. RasGRP2 is a guanyl exchange factor for Rap1. A Rap1 GTPase pull-down assay was used to compare the guanine nucleotide exchange activity of wild type and mutated full length RasGRP2 in human embryonic kidney 293 (HEK 293) cells in the presence or absence of Phorbol Ester. These cells were transfected with either the wild type or the mutant RasGRP2. Consistent with the ability of Phorbol Ester to induce translocation of the full length RasGRP2 with the mutated C1 domain, Phorbol Ester enhanced the ability of the mutated RasGRP2 to activate Rap1. Citation Format: Agnes Czikora, Daniel J. Lundberg, Nancy E. Lewin, Noemi Kedei, Peter M. Blumberg. Structural basis for the failure of the C1 domain of RasGRP2 to bind Phorbol Ester. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3928. doi:10.1158/1538-7445.AM2015-3928

  • Comparison of transcriptional response to Phorbol Ester, bryostatin 1, and bryostatin analogs in LNCaP and U937 cancer cell lines provides insight into their differential mechanism of action.
    Biochemical Pharmacology, 2012
    Co-Authors: Noemi Kedei, Andrea Telek, Aleksandra M. Michalowski, Matthew B. Kraft, Yam B. Poudel, Arnab Rudra, Mark E. Petersen, Gary E Keck, Wei Li, Peter M. Blumberg
    Abstract:

    Abstract Bryostatin 1, like the Phorbol Esters, binds to and activates protein kinase C (PKC) but paradoxically antagonizes many but not all Phorbol Ester responses. Previously, we have compared patterns of biological response to bryostatin 1, Phorbol Ester, and the bryostatin 1 derivative Merle 23 in two human cancer cell lines, LNCaP and U937. Bryostatin 1 fails to induce a typical Phorbol Ester biological response in either cell line, whereas Merle 23 resembles Phorbol Ester in the U937 cells and bryostatin 1 in the LNCaP cells. Here, we have compared the pattern of their transcriptional response in both cell lines. We examined by qPCR the transcriptional response as a function of dose and time for a series of genes regulated by PKCs. In both cell lines bryostatin 1 differed primarily from Phorbol Ester in having a shorter duration of transcriptional modulation. This was not due to bryostatin 1 instability, since bryostatin 1 suppressed the Phorbol Ester response. In both cell lines Merle 23 induced a pattern of transcription largely like that of Phorbol Ester although with a modest reduction at later times in the LNCaP cells, suggesting that the difference in biological response of the two cell lines to Merle 23 lies downstream of this transcriptional regulation. For a series of bryostatins and analogs which ranged from bryostatin 1-like to Phorbol Ester-like in activity on the U937 cells, the duration of transcriptional response correlated with the pattern of biological activity, suggesting that this may provide a robust platform for structure activity analysis.

  • the bryostatin 1 a ring acetate is not the critical determinant for antagonism of Phorbol Ester induced biological responses
    Organic Letters, 2009
    Co-Authors: Gary E Keck, Noemi Kedei, Matthew B. Kraft, Wei Li, Nancy E. Lewin, Peter M. Blumberg
    Abstract:

    The contribution of the A-ring C7 acetate to the function of bryostatin 1 has been investigated through synthesis and biological evaluation of an analogue incorporating this feature into the bryopyran core structure. No enhanced binding affinity for protein kinase C (PKC) was observed, relative to previously characterized analogues lacking the C7 acetate. Functional assays showed biological responses characteristic of those induced by the Phorbol Ester PMA and distinctly different from those observed with bryostatin 1.

  • Stoichiometric binding of diacylglycerol to the Phorbol Ester receptor
    Journal of Cellular Biochemistry, 2004
    Co-Authors: Bernhard König, Patricia A. Dinitto, Peter M. Blumberg
    Abstract:

    : The major Phorbol Ester receptor is the Ca++-activated, phospholipid-dependent protein kinase C. Diacylglycerol stimulates protein kinase C in a fashion similar to the Phorbol Esters. Likewise, it inhibits Phorbol Ester binding competitively. Both results suggest that diacylglycerol is the/an endogenous Phorbol Ester analogue. Alternatively, the diacylglycerol might simply be acting to modify the phospholipid environment of the protein. If diacylglycerol were indeed functioning as an analogue, it should interact with the receptor stoichiometrically. This interaction can be quantitated by measuring the perturbation in apparent diacylglycerol binding affinity as a function of the ratio of diacylglycerol to receptor. We report here that 1,2-dioleoylglycerol interacts with the receptor with the predicted stoichiometry.

  • Phospholipid and Ca++ dependency of Phorbol Ester receptors
    Journal of Cellular Biochemistry, 2004
    Co-Authors: Bernhard König, Patricia A. Di Nitto, Peter M. Blumberg
    Abstract:

    The phospholipid and Ca++ dependency of a partially purified Phorbol Ester apo-receptor from the soluble fraction of mouse brain homogenates was studied. This apo-receptor is believed to be identical with the Ca++ and phospholipid-dependent protein kinase C. Binding of Phorbol Esters to the receptor/kinase C was shown to be entirely dependent on phospholipids. The negatively charged phospholipids phosphatidylserine, phosphatidylinositol, and phosphatidic acid all fully reconstituted binding. The neutral phospholipids were inactive. Among active phospholipids and mixtures of phospholipids, substantial differences ( > 100-fold) were observed in the amounts required to achieve reconstitution. Although Ca++ was not required for reconstitution of binding activity, it dramatically (up to 100-fold) increased the potency of phospholipids for reconstitution. The phospholipids not only permitted reconstitution of the apo-receptor but also played a major role in determining the binding characteristics of the complex. The KD values of [3H]Phorbol 12,13–dibutyrate were in the range of 0.8 nM for the complex with phosphatidylserine to 30 nM for the complex with diolcoyl-phosphatidic acid. Like the binding affinity, the stimulation of protein kinase C activity by Phorbol Esters was dependent on the phospholipid into which the receptor/kinase C was reconstituted. The importance of the lipid domain for controlling the receptor/kinase C activity and for modulation of cellular sensitivity to Phorbol Esters is discussed.

Marcelo G Kazanietz - One of the best experts on this subject based on the ideXlab platform.

  • Phorbol Ester-induced apoptosis and senescence in cancer cell models.
    Methods in Enzymology, 2008
    Co-Authors: Liqing Xiao, M. Cecilia Caino, Vivian A. Von Burstin, José Luis Oliva, Marcelo G Kazanietz
    Abstract:

    Abstract Protein kinase C (PKC) isozymes catalyze the phosphorylation of substrates that play key roles in the control in proliferation, differentiation, and survival. Treatment of cells with Phorbol Esters, activators of classical and novel PKC isozymes, leads to a plethora of responses in a strict cell‐type–dependent specific manner. Interestingly, a few cell models undergo apoptosis in response to Phorbol Ester stimulation, including androgen‐dependent prostate cancer cells. This effect involves the autocrine secretion of death factors and activation of the extrinsic apoptotic cascade. We have recently found that in other models, such as lung cancer cells, Phorbol Esters lead to irreversible growth arrest and senescence. This chapter describes the methods we use to assess these Phorbol Ester responses in cancer cell models, focusing on apoptosis and senescence.

  • novel nonkinase Phorbol Ester receptors the c1 domain connection
    Molecular Pharmacology, 2002
    Co-Authors: Marcelo G Kazanietz
    Abstract:

    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.

  • Phorbol Esters and Related Analogs Regulate the Subcellular Localization of β2-Chimaerin, a Non-protein Kinase C Phorbol Ester Receptor
    Journal of Biological Chemistry, 2001
    Co-Authors: Maria J. Caloca, Hongbin Wang, Andrew S. Delemos, Shaomeng Wang, Marcelo G Kazanietz
    Abstract:

    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.

  • Pharmacology of the receptors for the Phorbol Ester tumor promoters: Multiple receptors with different biochemical properties
    Biochemical Pharmacology, 2000
    Co-Authors: Marcelo G Kazanietz, Maria J. Caloca, Pilar Eroles, Teruhiko Fujii, Maria Laura Garcia-bermejo, Muredach P. Reilly, Hongbin Wang
    Abstract:

    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.

  • eyes wide shut protein kinase c isozymes are not the only receptors for the Phorbol Ester tumor promoters
    Molecular Carcinogenesis, 2000
    Co-Authors: Marcelo G Kazanietz
    Abstract:

    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.

Noemi Kedei - One of the best experts on this subject based on the ideXlab platform.

  • structural basis for the failure of the c1 domain of ras guanine nucleotide releasing protein 2 rasgrp2 to bind Phorbol Ester with high affinity
    Journal of Biological Chemistry, 2016
    Co-Authors: Agnes Czikora, Noemi Kedei, Nancy E. Lewin, Daniel J Lundberg, Adelle Abramovitz, Megan L Peach, Xiaoling Zhou, Raymond C Merritt, Elizabeth A Craft, Derek C Braun
    Abstract:

    Abstract The C1 domain represents the recognition module for diacylglycerol and Phorbol Esters in protein kinase C, Ras guanine nucleotide releasing protein (RasGRP), and related proteins. RasGRP2 is exceptional in that its C1 domain has very weak binding affinity (Kd = 2890 ± 240 nm for [3H]Phorbol 12,13-dibutyrate. We have identified four amino acid residues responsible for this lack of sensitivity. Replacing Asn7, Ser8, Ala19, and Ile21 with the corresponding residues from RasGRP1/3 (Thr7, Tyr8, Gly19, and Leu21, respectively) conferred potent binding affinity (Kd = 1.47 ± 0.03 nm) in vitro and membrane translocation in response to Phorbol 12-myristate 13-acetate in LNCaP cells. Mutant C1 domains incorporating one to three of the four residues showed intermediate behavior with S8Y making the greatest contribution. Binding activity for diacylglycerol was restored in parallel. The requirement for anionic phospholipid for [3H]Phorbol 12,13-dibutyrate binding was determined; it decreased in going from the single S8Y mutant to the quadruple mutant. The full-length RasGRP2 protein with the mutated C1 domains also showed strong Phorbol Ester binding, albeit modestly weaker than that of the C1 domain alone (Kd = 8.2 ± 1.1 nm for the full-length protein containing all four mutations), and displayed translocation in response to Phorbol Ester. RasGRP2 is a guanyl exchange factor for Rap1. Consistent with the ability of Phorbol Ester to induce translocation of the full-length RasGRP2 with the mutated C1 domain, Phorbol Ester enhanced the ability of the mutated RasGRP2 to activate Rap1. Modeling confirmed that the four mutations helped the binding cleft maintain a stable conformation.

  • abstract 3928 structural basis for the failure of the c1 domain of rasgrp2 to bind Phorbol Ester
    Cancer Research, 2015
    Co-Authors: Agnes Czikora, Noemi Kedei, Nancy E. Lewin, Daniel J Lundberg, Peter M. Blumberg
    Abstract:

    C1 domains represent the recognition motif for the second messenger diacylglycerol (DAG) and for the Phorbol Esters in protein kinase C, RasGRP, and several other families of signaling proteins. In addition to such typical C1 domains, atypical C1 domains have been identified which possess varying levels of sequence and structural homology with the typical C1 domains but fail to bind DAG or Phorbol Ester. Among RasGRP family members, RasGRP1 and RasGRP3 possess typical C1 domains, whereas the C1 domain of RasGRP2 stands out as being atypical. In order to better understand the structural constraints for ligand binding, we have analyzed the basis for the failure of the C1 domain of RasGRP2 to bind ligands. Using site-directed mutagenesis, we have identified four critical amino acid residues responsible for the lack of Phorbol Ester sensitivity. We found that the C1 domain of RasGRP2 had weak binding affinity (Kd = 2890 ± 240 nM) in vitro for [3H]Phorbol 12,13-dibutyrate ([3H]PDBu). Replacing all four of critical amino acid residues (Asn7, Ser8, Ala19 and Ile21) with the corresponding residues (Thr7, Tyr8, Gly19 and Leu21, respectively) of RasGRP1 resulted in potent [3H]PDBu binding affinity (Kd = 1.47 ± 0.03 nM) and translocation in response to PMA in the LNCaP cell line. The mutant C1 domains incorporating one to three of these critical residues showed intermediate binding and translocation properties. Of the four substitutions, S8Y made the greatest contribution. Phospholipid requirements for [3H]PDBu binding to the mutant C1 domains were determined; at constant total phospholipid, the requirement for the proportion of phosphatidylserine in phosphatidylserine : phosphatidylcholine mixtures decreased in going from the single S8Y mutant to the quadruple mutant. Binding activity for DAG was also restored in the mutant C1 domains, in parallel with that for Phorbol Ester. The full length RasGRP2 protein containing the mutated C1 domains likewise showed strong Phorbol Ester binding, albeit modestly weaker than that of the purified C1 domain (Kd = 8.2 ± 1.1 nM for the full length protein containing all four mutations) and displayed translocation in the LNCaP (human prostate adenocarcinoma) cells in response to Phorbol Ester. RasGRP2 is a guanyl exchange factor for Rap1. A Rap1 GTPase pull-down assay was used to compare the guanine nucleotide exchange activity of wild type and mutated full length RasGRP2 in human embryonic kidney 293 (HEK 293) cells in the presence or absence of Phorbol Ester. These cells were transfected with either the wild type or the mutant RasGRP2. Consistent with the ability of Phorbol Ester to induce translocation of the full length RasGRP2 with the mutated C1 domain, Phorbol Ester enhanced the ability of the mutated RasGRP2 to activate Rap1. Citation Format: Agnes Czikora, Daniel J. Lundberg, Nancy E. Lewin, Noemi Kedei, Peter M. Blumberg. Structural basis for the failure of the C1 domain of RasGRP2 to bind Phorbol Ester. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3928. doi:10.1158/1538-7445.AM2015-3928

  • Comparison of transcriptional response to Phorbol Ester, bryostatin 1, and bryostatin analogs in LNCaP and U937 cancer cell lines provides insight into their differential mechanism of action.
    Biochemical Pharmacology, 2012
    Co-Authors: Noemi Kedei, Andrea Telek, Aleksandra M. Michalowski, Matthew B. Kraft, Yam B. Poudel, Arnab Rudra, Mark E. Petersen, Gary E Keck, Wei Li, Peter M. Blumberg
    Abstract:

    Abstract Bryostatin 1, like the Phorbol Esters, binds to and activates protein kinase C (PKC) but paradoxically antagonizes many but not all Phorbol Ester responses. Previously, we have compared patterns of biological response to bryostatin 1, Phorbol Ester, and the bryostatin 1 derivative Merle 23 in two human cancer cell lines, LNCaP and U937. Bryostatin 1 fails to induce a typical Phorbol Ester biological response in either cell line, whereas Merle 23 resembles Phorbol Ester in the U937 cells and bryostatin 1 in the LNCaP cells. Here, we have compared the pattern of their transcriptional response in both cell lines. We examined by qPCR the transcriptional response as a function of dose and time for a series of genes regulated by PKCs. In both cell lines bryostatin 1 differed primarily from Phorbol Ester in having a shorter duration of transcriptional modulation. This was not due to bryostatin 1 instability, since bryostatin 1 suppressed the Phorbol Ester response. In both cell lines Merle 23 induced a pattern of transcription largely like that of Phorbol Ester although with a modest reduction at later times in the LNCaP cells, suggesting that the difference in biological response of the two cell lines to Merle 23 lies downstream of this transcriptional regulation. For a series of bryostatins and analogs which ranged from bryostatin 1-like to Phorbol Ester-like in activity on the U937 cells, the duration of transcriptional response correlated with the pattern of biological activity, suggesting that this may provide a robust platform for structure activity analysis.

  • the bryostatin 1 a ring acetate is not the critical determinant for antagonism of Phorbol Ester induced biological responses
    Organic Letters, 2009
    Co-Authors: Gary E Keck, Noemi Kedei, Matthew B. Kraft, Wei Li, Nancy E. Lewin, Peter M. Blumberg
    Abstract:

    The contribution of the A-ring C7 acetate to the function of bryostatin 1 has been investigated through synthesis and biological evaluation of an analogue incorporating this feature into the bryopyran core structure. No enhanced binding affinity for protein kinase C (PKC) was observed, relative to previously characterized analogues lacking the C7 acetate. Functional assays showed biological responses characteristic of those induced by the Phorbol Ester PMA and distinctly different from those observed with bryostatin 1.

Nancy E. Lewin - One of the best experts on this subject based on the ideXlab platform.

  • structural basis for the failure of the c1 domain of ras guanine nucleotide releasing protein 2 rasgrp2 to bind Phorbol Ester with high affinity
    Journal of Biological Chemistry, 2016
    Co-Authors: Agnes Czikora, Noemi Kedei, Nancy E. Lewin, Daniel J Lundberg, Adelle Abramovitz, Megan L Peach, Xiaoling Zhou, Raymond C Merritt, Elizabeth A Craft, Derek C Braun
    Abstract:

    Abstract The C1 domain represents the recognition module for diacylglycerol and Phorbol Esters in protein kinase C, Ras guanine nucleotide releasing protein (RasGRP), and related proteins. RasGRP2 is exceptional in that its C1 domain has very weak binding affinity (Kd = 2890 ± 240 nm for [3H]Phorbol 12,13-dibutyrate. We have identified four amino acid residues responsible for this lack of sensitivity. Replacing Asn7, Ser8, Ala19, and Ile21 with the corresponding residues from RasGRP1/3 (Thr7, Tyr8, Gly19, and Leu21, respectively) conferred potent binding affinity (Kd = 1.47 ± 0.03 nm) in vitro and membrane translocation in response to Phorbol 12-myristate 13-acetate in LNCaP cells. Mutant C1 domains incorporating one to three of the four residues showed intermediate behavior with S8Y making the greatest contribution. Binding activity for diacylglycerol was restored in parallel. The requirement for anionic phospholipid for [3H]Phorbol 12,13-dibutyrate binding was determined; it decreased in going from the single S8Y mutant to the quadruple mutant. The full-length RasGRP2 protein with the mutated C1 domains also showed strong Phorbol Ester binding, albeit modestly weaker than that of the C1 domain alone (Kd = 8.2 ± 1.1 nm for the full-length protein containing all four mutations), and displayed translocation in response to Phorbol Ester. RasGRP2 is a guanyl exchange factor for Rap1. Consistent with the ability of Phorbol Ester to induce translocation of the full-length RasGRP2 with the mutated C1 domain, Phorbol Ester enhanced the ability of the mutated RasGRP2 to activate Rap1. Modeling confirmed that the four mutations helped the binding cleft maintain a stable conformation.

  • abstract 3928 structural basis for the failure of the c1 domain of rasgrp2 to bind Phorbol Ester
    Cancer Research, 2015
    Co-Authors: Agnes Czikora, Noemi Kedei, Nancy E. Lewin, Daniel J Lundberg, Peter M. Blumberg
    Abstract:

    C1 domains represent the recognition motif for the second messenger diacylglycerol (DAG) and for the Phorbol Esters in protein kinase C, RasGRP, and several other families of signaling proteins. In addition to such typical C1 domains, atypical C1 domains have been identified which possess varying levels of sequence and structural homology with the typical C1 domains but fail to bind DAG or Phorbol Ester. Among RasGRP family members, RasGRP1 and RasGRP3 possess typical C1 domains, whereas the C1 domain of RasGRP2 stands out as being atypical. In order to better understand the structural constraints for ligand binding, we have analyzed the basis for the failure of the C1 domain of RasGRP2 to bind ligands. Using site-directed mutagenesis, we have identified four critical amino acid residues responsible for the lack of Phorbol Ester sensitivity. We found that the C1 domain of RasGRP2 had weak binding affinity (Kd = 2890 ± 240 nM) in vitro for [3H]Phorbol 12,13-dibutyrate ([3H]PDBu). Replacing all four of critical amino acid residues (Asn7, Ser8, Ala19 and Ile21) with the corresponding residues (Thr7, Tyr8, Gly19 and Leu21, respectively) of RasGRP1 resulted in potent [3H]PDBu binding affinity (Kd = 1.47 ± 0.03 nM) and translocation in response to PMA in the LNCaP cell line. The mutant C1 domains incorporating one to three of these critical residues showed intermediate binding and translocation properties. Of the four substitutions, S8Y made the greatest contribution. Phospholipid requirements for [3H]PDBu binding to the mutant C1 domains were determined; at constant total phospholipid, the requirement for the proportion of phosphatidylserine in phosphatidylserine : phosphatidylcholine mixtures decreased in going from the single S8Y mutant to the quadruple mutant. Binding activity for DAG was also restored in the mutant C1 domains, in parallel with that for Phorbol Ester. The full length RasGRP2 protein containing the mutated C1 domains likewise showed strong Phorbol Ester binding, albeit modestly weaker than that of the purified C1 domain (Kd = 8.2 ± 1.1 nM for the full length protein containing all four mutations) and displayed translocation in the LNCaP (human prostate adenocarcinoma) cells in response to Phorbol Ester. RasGRP2 is a guanyl exchange factor for Rap1. A Rap1 GTPase pull-down assay was used to compare the guanine nucleotide exchange activity of wild type and mutated full length RasGRP2 in human embryonic kidney 293 (HEK 293) cells in the presence or absence of Phorbol Ester. These cells were transfected with either the wild type or the mutant RasGRP2. Consistent with the ability of Phorbol Ester to induce translocation of the full length RasGRP2 with the mutated C1 domain, Phorbol Ester enhanced the ability of the mutated RasGRP2 to activate Rap1. Citation Format: Agnes Czikora, Daniel J. Lundberg, Nancy E. Lewin, Noemi Kedei, Peter M. Blumberg. Structural basis for the failure of the C1 domain of RasGRP2 to bind Phorbol Ester. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3928. doi:10.1158/1538-7445.AM2015-3928

  • the bryostatin 1 a ring acetate is not the critical determinant for antagonism of Phorbol Ester induced biological responses
    Organic Letters, 2009
    Co-Authors: Gary E Keck, Noemi Kedei, Matthew B. Kraft, Wei Li, Nancy E. Lewin, Peter M. Blumberg
    Abstract:

    The contribution of the A-ring C7 acetate to the function of bryostatin 1 has been investigated through synthesis and biological evaluation of an analogue incorporating this feature into the bryopyran core structure. No enhanced binding affinity for protein kinase C (PKC) was observed, relative to previously characterized analogues lacking the C7 acetate. Functional assays showed biological responses characteristic of those induced by the Phorbol Ester PMA and distinctly different from those observed with bryostatin 1.

  • β2-Chimaerin Is a High Affinity Receptor for the Phorbol Ester Tumor Promoters
    Journal of Biological Chemistry, 1997
    Co-Authors: Maria J. Caloca, Peter M. Blumberg, Nieves Fernandez, Nancy E. Lewin, Dixie Ching, Rama Modali, Marcelo G Kazanietz
    Abstract:

    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.