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The Journal of Biological Chemistry May 1995The Caenorhabditis elegans Unc-13 protein is a novel member of the phorbol ester receptor family having a single cysteine-rich region with high homology to those present...
Characterization of the cysteine-rich region of the Caenorhabditis elegans protein Unc-13 as a high affinity phorbol ester receptor. Analysis of ligand-binding interactions, lipid cofactor requirements, and inhibitor sensitivity.
The Caenorhabditis elegans Unc-13 protein is a novel member of the phorbol ester receptor family having a single cysteine-rich region with high homology to those present in protein kinase C (PKC) isozymes and the chimaerins. We expressed the cysteine-rich region of Unc-13 in Escherichia coli and quantitatively analyzed its interactions with phorbol esters and related analogs, its phospholipid requirements, and its inhibitor sensitivity. [3H]Phorbol 12,13-dibutyrate [3H]PDBu bound with high affinity to the cysteine-rich region of Unc-13 (Kd = 1.3 +/- 0.2 nM). This affinity is similar to that of other single cysteine-rich regions from PKC isozymes as well as n-chimaerin. As also described for PKC isozymes and n-chimaerin, Unc-13 bound diacylglycerol with an affinity about 2 orders of magnitude weaker than [3H]PDBu. Structure-activity analysis revealed significant but modest differences between recombinant cysteine-rich regions of Unc-13 and PKC delta. In addition, Unc-13 required slightly higher concentrations of phospholipid for reconstitution of [3H]PDBu binding. Calphostin C, a compound described as a selective inhibitor of PKC, was also able to inhibit [3H]PDBu binding to Unc-13, suggesting that this inhibitor is not able to distinguish between different classes of phorbol ester receptors. In conclusion, although our results revealed some differences in ligand and lipid cofactor sensitivities, Unc-13 represents a high affinity cellular target for the phorbol esters as well as for the lipid second messenger diacylglycerol, at least in C. elegans. The use of phorbol esters or some "specific" antagonists of PKC does not distinguish between cellular pathways involving different PKC isozymes or novel phorbol ester receptors such as n-chimaerin or Unc-13.
Topics: Amino Acid Sequence; Animals; Base Sequence; Bryostatins; Caenorhabditis elegans; Caenorhabditis elegans Proteins; Carrier Proteins; Cysteine; DNA Primers; Helminth Proteins; Lactones; Ligands; Macrolides; Molecular Sequence Data; Naphthalenes; Phorbol 12,13-Dibutyrate; Phospholipids; Polycyclic Compounds; Protein Kinase C; Receptors, Drug; Recombinant Proteins
PubMed: 7537738
DOI: 10.1074/jbc.270.18.10777 -
Genes and Immunity Jul 2016Linker for activation of T cells (LAT) is a raft-associated, transmembrane adapter protein critical for T-cell development and function. LAT expression is transiently...
Linker for activation of T cells (LAT) is a raft-associated, transmembrane adapter protein critical for T-cell development and function. LAT expression is transiently upregulated upon T-cell receptor (TCR) engagement, but molecular mechanisms conveying TCR signaling to enhanced LAT transcription are not fully understood. Here we found that a Jurkat subline J.CaM2, initially characterized as LAT deficient, conditionally re-expressed LAT upon the treatment with a protein kinase C activator, phorbol 12-myristate 13-acetate (PMA). We took advantage of the above observation for studying cis-elements and trans-acting factors contributing to the activation-induced expression of LAT. We identified a LAT gene region spanning nucleotide position -14 to +357 relative to the ATG start codon as containing novel cis-regulatory elements that were able to promote PMA-induced reporter transcription in the absence of the core LAT promoter. Interestingly, a point mutation in LAT intron 1, identified in J.CaM2 cells, downmodulated LAT promoter activity by 50%. Mithramycin A, a selective Sp1 DNA-binding inhibitor, abolished LAT expression upon PMA treatment as did calcium ionophore ionomycin (Iono) and valproic acid (VPA), widely used as an anti-epileptic drug. Our data introduce J.CaM2 cells as a model for dissecting drivers and blockers of activation induced expression of LAT.
Topics: Adaptor Proteins, Signal Transducing; Cell Culture Techniques; Humans; Introns; Ionomycin; Jurkat Cells; Membrane Proteins; Phorbol Esters; Plicamycin; Point Mutation; Promoter Regions, Genetic; Sp1 Transcription Factor; Transcriptional Activation; Valproic Acid
PubMed: 27278128
DOI: 10.1038/gene.2016.25 -
FEBS Letters Feb 1988The ability of the tumor-promoting phorbol ester 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA) to induce protein kinase C (PKC) translocation and lysosomal...
The ability of the tumor-promoting phorbol ester 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA) to induce protein kinase C (PKC) translocation and lysosomal enzyme release was examined in skin fibroblasts from normal subjects and from patients with cystic fibrosis (CF). As compared to normal fibroblasts, those CF exhibited: (i) an increased sensitivity to the effect of PMA on the disappearance of PKC from cytosolic fractions as well as a greater and earlier recovery, in the membrane fraction, of the PKC activity lost in the cytosolic fraction; (ii) an earlier response to PMA for its effect on beta-N-acetylglucosaminidase release. In contrast, the inactive phorbol ester 4 alpha-phorbol 12,13-didecanoate (4 alpha PDD) proved ineffective in inducing PKC translocation and beta-N-acetylglucosaminidase release in both normal and CF fibroblasts. The data suggest a defect in the regulation of PKC activity in CF fibroblasts, which may lead to altered secretion.
Topics: Acetylglucosaminidase; Cystic Fibrosis; Dose-Response Relationship, Drug; Fibroblasts; Humans; Lysosomes; Phorbol Esters; Protein Kinase C; Tetradecanoylphorbol Acetate; Time Factors
PubMed: 3345834
DOI: 10.1016/0014-5793(88)80818-4 -
The Journal of Experimental Medicine Apr 1996Intercellular adhesion molecule (ICAM) 1/CD54 plays an important role in T cell dependent B cell activation and for function of B lymphocytes as antigen-presenting...
Intercellular adhesion molecule (ICAM) 1/CD54 plays an important role in T cell dependent B cell activation and for function of B lymphocytes as antigen-presenting cells. ICAM-1 expression is upregulated as a consequence of B lymphocyte antigen receptor (BCR) signaling, thereby serving to render antigen-stimulated B cells more receptive to T cell-mediated costimulatory signals. We have investigated BCR-induced expression of the Icam-1 gene in primary B cells and B cell lines and have found it to be dependent on BCR-induced expression of the transcription factor EGR1. Icam-1 transcription, induced by BCR cross-linking or bypassing the BCR with phorbol ester, is absent in a B cell line in which the EGR1-encoding gene (egr-1) is methylated and not expressed. A potential EGR1-binding site was located at -701 bp upstream of the murine Icam-1 gene transcription start site and shown by electrophoretic mobility shift assay to bind to murine EGR1. Mutation of this site in the context of 1.1 kb of the Icam-1 promoter significantly abrogated transcriptional induction by phorbol ester and anti-mu stimulation in primary B cells. A direct effect of EGR1 on the Icam-1 promoter is suggested by the ability of EGR1 expressed from an SV40-driven expression vector transactivate the wild-type Icam-1 promoter, whereas mutation of the EGR1 mutation of the EGR1 binding motif at -701 bp markedly compromises this induction. These data identify EGR1 as a signaling intermediate in BCR-stimulated B cell functional responses, specifically linking BCR signal transduction to induction of the Icam-1 gene. Furthermore, similar findings for BCR-induced CD44 gene induction (Maltzman, J.S., J.A. Carman, and J.G. Monroe. 1996. Role of EGR1 in regulation of stimulus-dependent CD44 transcription in B lymphocytes. Mol. Cell. Biol. In press) suggest that EGR1 may be an important signaling molecule for regulating levels of migration and adhesion molecules during humoral immune responses.
Topics: Animals; B-Lymphocytes; Base Sequence; Binding Sites; Clone Cells; Consensus Sequence; DNA-Binding Proteins; Early Growth Response Protein 1; Immediate-Early Proteins; Intercellular Adhesion Molecule-1; Mice; Mice, Inbred BALB C; Molecular Sequence Data; Phorbol Esters; Promoter Regions, Genetic; Protein Binding; Receptors, Antigen; Transcription Factors; Transcription, Genetic; Transcriptional Activation
PubMed: 8666932
DOI: 10.1084/jem.183.4.1747 -
The Journal of Biological Chemistry Aug 1989The human multipotential hematopoietic cell line K562 expresses fibronectin receptor (FNR) subunits of 160 kDa (alpha chain) and 120 kDa (beta chain). Treatment with...
The human multipotential hematopoietic cell line K562 expresses fibronectin receptor (FNR) subunits of 160 kDa (alpha chain) and 120 kDa (beta chain). Treatment with 12-O-tetradecanoylphorbol 13-acetate (TPA) led to reduced binding of K562 to immobilized fibronectin (FN), although treated cells expressed 10-fold more cell surface FNR than untreated cells. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis confirmed this and showed altered electrophoretic mobilities of FNR subunits from TPA-treated cells. TPA treatment affected N-linked glycosylation, as tunicamycin treatment of K562 cells abolished differences in FNR mobility. Sialidase treatment of FNR immunoprecipitates minimized and sialidase treatment of intact cells eliminated these mobility differences between subunits from control and TPA-treated cells. Reduced sialylation of FNR from TPA-treated cells was further demonstrated by chromatography with bead-coupled lectins and by the greater negative charge of untreated K562 FNR subunits in two-dimensional isoelectric focusing-polyacrylamide gel electrophoresis. A relationship between reduced FNR sialylation and reduced FN binding was suggested by adhesion assays of sialidase-treated K562 which showed that desialylation of cell surface FNR was associated with decreased cell adhesion. Thus, TPA treatment reduces the function, increases the expression, and alters the structure of K562 FNR, and these changes appear to involve FNR sialylation.
Topics: Cell Adhesion; Cell Line; Chromatography, Affinity; Electrophoresis, Polyacrylamide Gel; Fibronectins; Glycosylation; Humans; Kinetics; Leukemia, Erythroblastic, Acute; Neuraminidase; Phorbol Esters; Receptors, Fibronectin; Receptors, Immunologic; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured
PubMed: 2526813
DOI: No ID Found -
Journal of Biomolecular Structure &... 2023C1 domains are lipid-binding structural units of about 50 residues. Typical C1 domains associate with the plasma membrane and bind to diacylglycerol/phorbol ester during...
C1 domains are lipid-binding structural units of about 50 residues. Typical C1 domains associate with the plasma membrane and bind to diacylglycerol/phorbol ester during the activation of the proteins containing these domains. Although the overall structure of the C1 domains are similar, there are differences in their primary sequence and in the orientation of the ligand/lipid binding residues. To gain structural insights into the ligand/lipid binding, we performed molecular docking of phorbol 13-acetate into the C1 domain and 1.0 μs molecular dynamics simulation on the C1 domain-ligand-lipid ternary system for PKCθ C1A, PKCδ C1B, PKCβII C1B, PKCθ C1B, Munc13-1 C1, and βII-Chimaerin C1. We divided these C1 domains into three types based on the orientations of Gln-27 and Trp/Tyr-22. In type 1, Trp/Tyr-22 is outside and Gln-27 is inside the ligand binding pocket. In type 2, both Trp/Tyr-22 and Gln-27 are outside the ligand binding pocket, and in type 3, Trp/Tyr-22 is inside and Gln-27 is outside the pocket. The type 1 C1 domains showed higher ligand binding and higher membrane binding with a shorter distance between the C1 domain and the membrane than the type 2 and type 3. For ligand binding, Pro-11 plays a major role in the type 1 and 2, and Gly-23 in the type 1 and type 3 C1 domains. This study elucidates the role of Gln-27, Trp-22, Pro-11 and Gly-23 in ligand/lipid binding in typical C1 domains and bears significance in developing selective modulators of C1 domain-containing proteins.Communicated by Ramaswamy H. Sarma.
Topics: Molecular Dynamics Simulation; Molecular Docking Simulation; Protein Structure, Tertiary; Ligands; Protein Binding; Binding Sites; Phorbol Esters; Lipids
PubMed: 36602779
DOI: 10.1080/07391102.2022.2163699 -
The Journal of Biological Chemistry Mar 1994Using a cell line stably transfected with the rat follitropin (FSH) receptor cDNA we demonstrate that the FSH receptor becomes phosphorylated when cells are exposed to...
Using a cell line stably transfected with the rat follitropin (FSH) receptor cDNA we demonstrate that the FSH receptor becomes phosphorylated when cells are exposed to FSH. Since binding of FSH to its receptor results in an increase in cAMP and inositol phosphate accumulation, we examined the potential involvement of protein kinase A and C in mediating receptor phosphorylation. Stimulation of protein kinase A does not appear to be necessary because hFSH-induced receptor phosphorylation was minimally impaired in a cell line that overexpresses cAMP phosphodiesterase. Moreover, stimulation of the protein kinase A pathway with other agonists result in minimal phosphorylation of the FSH receptor. Stimulation of the protein kinase C with a phorbol ester did result in an increase in receptor phosphorylation, and down-regulation of the protein kinase C decreased, but did not abolish, the FSH-induced receptor phosphorylation. The possible impact of phosphorylation on the functions of the receptor was examined by testing if conditions that lead to phosphorylation decrease the ability of FSH to stimulate cAMP synthesis. Our data show that as with the addition of FSH, addition of a phorbol ester also results in a decrease in the ability of FSH to stimulate cAMP synthesis.
Topics: Adenylyl Cyclases; Animals; Cell Line; Cyclic AMP; Follicle Stimulating Hormone; Humans; Inositol Phosphates; Phorbol Esters; Phosphoproteins; Phosphorylation; Phosphoserine; Phosphothreonine; Protein Kinase C; Rats; Receptors, FSH; Recombinant Proteins; Second Messenger Systems; Signal Transduction; Tetradecanoylphorbol Acetate; Time Factors
PubMed: 8132609
DOI: No ID Found -
The Journal of Biological Chemistry Dec 1985Esterase 1, a well-characterized mouse plasma protein of unknown function, has activity against a wide range of ester substrates including beta-alanine nitrophenyl...
Esterase 1, a well-characterized mouse plasma protein of unknown function, has activity against a wide range of ester substrates including beta-alanine nitrophenyl esters and 17 beta-esters of estradiol. In this article, we report that esterase 1 is also responsible for a majority of the phorbol-12-ester hydrolase activity in mouse plasma. Incubation of homogeneous esterase 1 with 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA) at either 4 or 37 degrees C for up to 18 h yielded phorbol 13 alpha-acetate as the only hydrolysis product. Specific polyclonal antibodies to esterase 1 inhibited 95% of PMA hydrolysis by a purified esterase 1 preparation and 65% of PMA hydrolysis by mouse plasma. Perfused mouse liver homogenates contain two distinct phorbol diester hydrolases with apparent molecular masses of 65 kDa and 56 kDa, respectively. The 65-kDa protein appears to be immunologically identical to the plasma enzyme, while the 56-kDa protein, found in liver but not in plasma, is immunologically distinct. Phorbol 12-myristate, phorbol 12,13-dibutyrate, and PMA were found to be competitive inhibitors of the beta-alanine-nitrophenyl esterase activity of esterase 1 with Ki values of approximately 7 microM. Phorbol 13-acetate and phorbol itself were less effective with Ki values of 37 and 140 microM, respectively. Sodium salts of valeric and myristic acids did not inhibit at 10 microM. The above results indicate that efficient substrate binding requires a phorbol 12-ester. Similar results were obtained with estradiol 17 beta-valerate which is a better substrate for esterase 1 than is PMA. Our results strongly suggest that esterase 1 and a recently described phorbol ester hydrolase isolated from mouse serum (Saito, M., and Egawa, K. (1984) J. Biol. Chem. 259, 5821-5826) are the same and are immunologically and kinetically distinct from the 56-kDa phorbol 12-ester hydrolase in mouse liver.
Topics: Animals; Antibody Specificity; Carboxylesterase; Carboxylic Ester Hydrolases; Chromatography, High Pressure Liquid; Cross Reactions; Estradiol; Kinetics; Liver; Mice; Phorbol 12,13-Dibutyrate; Phorbol Esters; Phorbols; Tetradecanoylphorbol Acetate
PubMed: 3864781
DOI: No ID Found -
Biochimica Et Biophysica Acta Aug 1997Phorbol esters such as 12-O-tetradecanoylphorbol-13-acetate (TPA) activate protein kinase C and have been previously shown to down-regulate surfactant proteins SP-A and...
Phorbol esters such as 12-O-tetradecanoylphorbol-13-acetate (TPA) activate protein kinase C and have been previously shown to down-regulate surfactant proteins SP-A and SP-B in H441 adenocarcinoma cells. We used H441 cells and human fetal lung to further study the mechanism of TPA action and to examine physiologic relevance. In H441 cells, TPA (10 nM) treatment for 24 h decreased SP-A mRNA content to approximately 5% of control cells, with half-maximal effect at approximately 0.5 nM, and reduced SP-A gene transcription rate to 28% of control after 8 h exposure. In cells cultured in the presence of dexamethasone, which increases the low basal level of SP-B expression, TPA decreased both SP-B mRNA content (approximately 8% of control) and rate of transcription (7% of control). In cultured human fetal lung explants, TPA decreased SP-A and SP-B protein and mRNA in a time- and dose-dependent fashion, with half-maximal effect on mRNAs at approximately 3 nM and approximately 50% inhibition after 24 h of exposure, and similarly reduced SP-A and SP-B gene transcription (approximately 55% of control at 8-24 h). We conclude that TPA acts primarily at the level of gene transcription to down-regulate both SP-A and SP-B in H441 and fetal lung cells, and we speculate that inflammatory and other agents that act through PKC may modulate expression of the surfactant proteins and alter surfactant function in vivo.
Topics: Culture Techniques; Down-Regulation; Gene Expression; Humans; Lung; Phorbol Esters; Proteolipids; Pulmonary Surfactant-Associated Protein A; Pulmonary Surfactant-Associated Proteins; Pulmonary Surfactants; RNA, Messenger; Transcription, Genetic; Tumor Cells, Cultured
PubMed: 9294011
DOI: 10.1016/s0167-4781(97)00070-5 -
The Journal of Biological Chemistry Feb 1989Differences between the influences of phorbol esters (such as 4 beta-12-O-tetradecanoylphorbol 13-acetate) and of fatty acids (such as oleic acid) on the synthesis and...
Differences between the influences of phorbol esters (such as 4 beta-12-O-tetradecanoylphorbol 13-acetate) and of fatty acids (such as oleic acid) on the synthesis and turnover of phosphatidylcholine (PtdCho) and other phospholipids have been studied in glioma (C6), neuroblastoma (N1E-115), and hybrid (NG108-15) cells in culture using [methyl-3H]choline, [32P]Pi, [1,2-14C]ethanolamine, or 1-14C-labeled fatty acids as lipid precursors. 100-500 microM oleic acid stimulated PtdCho synthesis 3- to 5-fold in all three cell lines, but had little influence on chase of choline label following a 24-h pulse. Phorbol ester (50-200 nM) stimulated PtdCho synthesis 1.5- to 3-fold in C6 cells, was without effect in N1E-115 cells, and had intermediate effects on NG108-15 cells. Phorbol ester stimulated both uptake of extracellular choline and synthesis of PtdCho, whereas fatty acid stimulated only synthesis. Release of radioactivity from 24-h pulse-labeled PtdCho to the medium was enhanced by phorbol ester in C6 cells. Incorporation of [32P]Pi, primarily into PtdCho, was stimulated, whereas utilization of [1,2-14C]ethanolamine or 1-14C-fatty acid was little altered by phorbol ester. C6 cells "down-regulated" with phorbol ester lost the stimulatory response of subsequent treatment with phorbol esters on PtdCho synthesis, but the response to fatty acid was enhanced. Fatty acid had little influence on the relative binding of phorbol ester or "translocation" of phorbol ester binding sites. Accordingly, metabolism of phospholipids in these cultured cells of neural origin is markedly influenced by cell type, phospholipid class, condition of incubation medium, and nature of stimulator. Phorbol esters and fatty acids appear to enhance phospholipid synthesis and turnover by distinct intracellular mechanisms.
Topics: Animals; Cell Line; Choline; Ethanolamine; Ethanolamines; Fatty Acids; Glioma; Hybrid Cells; Kinetics; Neuroblastoma; Phosphatidylcholines; Phospholipids; Subcellular Fractions; Tetradecanoylphorbol Acetate
PubMed: 2914928
DOI: No ID Found