Phorbol Dibutyrate Binding Research Articles

Identification of the activator binding residues in protein kinase C theta C1B domain

PKCtheta is a Ser/ Thr kinase, which is critically involved in immunity. Diacylglycerol (DAG) and phorbol esters (PE) bind to C1A and C1B domain to activate nPKC and cPKC. Selective PKCtheta inhibition may manage autoimmune disorders. Design of PKCtheta inhibitor targeting C1 domain requires the knowledge of C1 domain structure and its activator binding residues. In this direction we solved crystal structure of PKCtheta C1B and identified possible activator binding residues by molecular docking of a library of DAG and PE analogs into PKCtheta C1B as receptor. Five residues were identified, which are capable of binding to DAG/ PE analogs. To determine the role of these residues in activator binding Y239A, T243A, W253G, L255G and Q258 in isolated C1B domain were designed, expressed and purified from E. coli and their binding affinity (Kd/Ki) for Phorbol 12, 13‐Dibutyrate (PDBu) and Dioctanoylglycerol (DOG) were measured by radioactive PDBu binding assay. All the mutants showed significantly reduced binding affinity for both PDBu and DOG whereas Q258 showed the maximum reduction than the wild type (WT). Same mutations in full length PKCtheta were expressed in HEK293 cells and showed reduced Phorbol 12‐Myristate 13‐Acetate and DOG induced membrane translocation compared to WT. Our studies indicated that these residues are important for activator binding and will aid in future design of the PKCtheta inhibitor.

Distribution of Protein Kinase C in the Hippocampus of the Gerbil and Rat: Autoradiographic Analysis by [3H]Phorbol 12,13-Dibutyrate

In-vitro quantitative receptor autoradiography with [3H]phorbol 12,13-dibutyrate (PDBu) was used to determine the affinity constant (Kd) and the maximum number of receptor sites (Bmax) for protein kinase C (PKC) in subregions of the gerbil hippocampus, and to compare the distribution of [3H]PDBu binding sites in the gerbil hippocampus with that in the rat hippocampus. The Kd and Bmax values in the subregions of the gerbil hippocampus were estimated at 2.6-3.8 nM and 2.38-2.54 pmol (mg tissue)-1, respectively. The distribution of hippocampus [3H]PDBu binding sites was uniform in the gerbil but not in the rat. The [3H]PDBu binding activities in the strata oriens of the CA1 and CA3 subfields and the molecular layer of the dentate gyrus in the rat hippocampus were significantly higher than in the gerbil hippocampus. However, binding activity in the stratum lacunosum-moleculare of the rat CA1 subfield was statistically lower. These data demonstrate a difference in the distribution of [3H]PDBu binding activity in the hippocampus between the gerbil and rat.

Properties of a resiniferatoxin-stimulated, calcium inhibited but phosphatidylserine-dependent kinase, which is distinct from protein kinase C isotypes alpha, beta 1, gamma, delta, epsilon and eta.

We have separated a resiniferatoxin-stimulated histone-kinase activity from human neutrophils, elicited mouse macrophages and murine alveolar macrophages by hydroxyapatite chromatography. The assay conditions for resiniferatoxin kinase were optimized as part of this study and in the presence of phosphatidylserine but absence of Ca2+ the Ka for histone IIIs phosphorylation by resiniferatoxin was calculated as 16 nM. Using a phosphate gradient of 20-500 mM, peaks of protein kinase C activity could be washed from the hydroxyapatite column in 300 nM phosphate and resiniferatoxin kinase recovered in 500 mM phosphate. At the optimum concentration of 160 nM, the ability of resiniferatoxin to induce enzyme activity was compared with a range of phorbol esters all at the same concentration. These related compounds failed to activate resiniferatoxin kinase although they have previously been shown to activate protein kinase C isotypes. Similarly sn-1,2,-dioleoylglycerol and the potent irritant capsaicin at 30 microM failed to activate the kinase. A Scatchard analysis of [3H] phorbol dibutyrate binding produced a linear plot (Kd 41.6 nM; Bmax 11.6 fmol unit-1) and binding was inhibited by resiniferatoxin and 12-O-tetradecanoylphorbol-13-acetate (TPA), with resiniferatoxin 700 times more potent than TPA in this respect. A radiolabelled resiniferatoxin binding assay was also used to demonstrate specific binding of [3H]resiniferatoxin which could be inhibited by unlabelled compound. Resiniferatoxin kinase activity was shown to be distinct from the protein kinase C isotypes alpha, beta 1, gamma, delta and epsilon by means of immunological analysis and from the eta isotype, because that isotype was not stimulated by resiniferatoxin but was stimulated by TPA when a pseudosubstrate was used. In addition the resiniferatoxin-stimulated activity was inhibited in-vitro by the addition of Ca2+ (Ki 0.1-0.5 nM free Ca2+). Further purification of resiniferatoxin kinase by Superose chromatography indicated a major activity fraction of about 70-90 kDa. Thus resiniferatoxin kinase, isolated from human and mouse inflammatory cells is distinct from the known isotypes of protein kinase C and is a major resiniferatoxin receptor.

A novel role for sphingolipid metabolism in oxidant-mediated skeletal muscle fatigue. Focus on “Sphingomyelinase stimulates oxidant signaling to weaken skeletal muscle and promote fatigue”

sphingolipids are a chemically diverse group of biological lipids typified by a sphingoid base backbone derived from condensation of a fatty acyl-CoA to an amino acid (typically, but not always, serine) ([24][1], [27][2]). These lipids occur throughout eukaryotic species, thus reflecting their

The relationship between the metabolism of sphingomyelin species and the hemolysis of sheep erythrocytes induced by Clostridium perfringens α-toxin

Clostridium perfringens alpha-toxin induces the hemolysis of sheep erythrocytes by activating the metabolism of sphingomyelin (SM) via a GTP binding protein in membranes. alpha-Toxin stimulated the formation of 15-N-nervonoyl sphingosine (C24:1-ceramide), which was identified by positive ion fast atom bombardment-MS and 1H-NMR spectroscopy. C24:1-ceramide stimulated the toxin-induced hemolysis of saponin-pretreated sheep erythrocytes and increased the production of sphingosine 1-phosphate (S1P) in the cells, but N-lignoceroyl sphingosine did not. These events elicited by the toxin in the presence of C24:1-ceramide were significantly attenuated by treatment with dihydrosphingosine, a sphingosine kinase inhibitor. TLC showed that the level of C24:1-ceramide was highest among the ceramides with an unsaturated bond in the fatty acyl chain in the detergent-resistant membranes (DRMs). The toxin specifically bound to DRMs rich in cholesterol, resulting in the hydrolysis of N-nervonoic sphingomyelin (C24:1-SM) in DRMs. Treatment of the cells with pertussis toxin (PT) inhibited the alpha-toxin-induced formation of C24:1-ceramide from C24:1-SM in DRMs and hemolysis, indicating that endogenous sphingomyelinase, which hydrolyzes C24:1-SM to C24:1-ceramide, is controlled by PT-sensitive GTP binding protein in membranes. These results show that the toxin-induced metabolism of C24:1-SM to S1P in DRMs plays an important role in the toxin-induced hemolysis of sheep erythrocytes.

Role of the Diacylglycerol Kinase α-Conserved Domains in Membrane Targeting in Intact T Cells

Diacylglycerol kinase (DGK) phosphorylates diacylglycerol to phosphatidic acid, modifying the cellular levels of these two lipid mediators. Ten DGK isoforms, grouped into five subtypes, are found in higher organisms. All contain a conserved C-terminal domain and at least two cysteine-rich motifs of unknown function. DGKalpha is a type I enzyme that acts as a negative modulator of diacylglycerol-based signals during T cell activation. Here we studied the functional role of the DGKalpha domains using mutational analysis to investigate membrane binding in intact cells. We show that the two atypical C1 domains are essential for plasma membrane targeting of the protein in intact cells but unnecessary for catalytic activity. We also identify the C-terminal sequence of the protein as essential for membrane binding in a phosphatidic acid-dependent manner. Finally we demonstrate that, in the absence of the calcium binding domain, receptor-dependent translocation of the truncated protein is regulated by phosphorylation of Tyr(335). This functional study provides new insight into the role of the so-called conserved domains of this lipid kinase family and demonstrates the existence of additional domains that confer specific plasma membrane localization to this particular isoform.

CHRONIC PROPRANOLOL TREATMENT CAUSES DESENSITIZATION OF THE STEROIDOGENIC RESPONSE IN TESTICULAR INTERSTITIAL CELLS BUT DOES NOT ALTER PROTEIN KINASE C

We investigated effects of chronic propranolol treatment on the secretory response of rat testicular interstitial cells (testosterone secretion) to subsequent in vitro stimulation with activators of protein kinase-C (PK-C) (L-propranolol, phorbol 12, 13-dibutyrate (PDBu), LHRH) or activators of protein kinase A (PK-A), (hCG or dibutyryl cAMP (dbcAMP)). We determined [3H]PDBu binding and PK-C activity in these cells. Treatment of rats with propranolol (Inderal 500 mg/L of water for 5 weeks) reduced by 48%, 50% and 29% the L-propranolol-, LHRH- or PDBu-induced testosterone secretion, respectively, when compared to cells from controls. This desensitization in testosterone secretion in vitro was also present when the testicular interstitial cells were stimulated with hCG or dbcAMP (secretion decreased by 65%/57%, respectively, when compared to cells from control rats). Challenging the cells originated from rats that received propranolol chronically with the addition in vitro of propranolol resulted in an additional reduction of the hCG/dbcAMP-stimulated testosterone secretion. Chronic propranolol-induced desensitization was not associated with a loss in [3H]PDBu binding or a decrease in PK-C activity. Chronic propranolol-induced desensitization can be uncoupled from down-regulation of protein kinase C. The effector responsible for the desensitization could be distal to the protein kinase C and protein kinase A.

Odor increases [ 3H]phorbol dibutyrate binding to protein kinase C in olfactory structures of rat brain. Effect of entorhinal cortex lesion

Since protein kinase C (PKC) is known to be activated in the olfactory bulb and in several limbic areas related to odor processing, we determined whether an olfactory stimulus was able to modulate the activity of PKC in animals with bilateral entorhinal cortex lesion. The translocation of PKC from the cytosol to the membrane was studied using the phorbol ester 12,13-dibutyrate ([ 3H]PDBu) binding in control and bilateral entorhinal cortex (EC) lesioned rats. The lesion of EC per se did not significantly affect [ 3H]PDBu binding in any of the brain structures analyzed, while odor stimulation induced it in both control and EC-lesioned groups in the external plexiform layer of the olfactory bulb. In contrast, an odor-induced increase of [ 3H]PDBu binding in internal glomerular layer of the olfactory bulb was only observed in EC lesioned animals. Similar results were obtained in the piriform cortex. In both CA1 and CA3 hippocampal subfields, odor stimulation induced an increase of [ 3H]PDBu binding in both control and EC-lesioned animals, the increase being potentiated only in CA1 of lesioned rats. The dentate gyrus and the amygdala exhibited a similar pattern of [ 3H]PDBu binding, showing a significant increase exclusively in EC-lesioned animals after odor stimulation. The results strongly suggest that the EC plays a key role in odor processing. PKC appears to play an important role in responding to the activation of lipid second messengers, which have been described to be involved in the processing of odor stimuli in several structures of the olfactory pathway.

Decursin and PDBu: Two PKC activators distinctively acting in the megakaryocytic differentiation of K562 human erythroleukemia cells

Protein kinase C (PKC) plays an important role in the proliferation and differentiation of various cell types including normal and leukemic hematopoietic cells. Phorbol 12,13-dibutyrate (PDBu) induces the megakaryocytic differentiation of K562 human erythroleukemia cells through PKC activation. Decursin, a pyranocoumarin from Angelica gigas, exhibits the cytotoxic effects on various human cancer cell lines and in vitro PKC activation. We report here the differences between two PKC activators, tumor-suppressing decursin and tumor-promoting PDBu, in their actions on the megakaryocytic differentiation of K562 cells. First of all, decursin inhibited PDBu-induced bleb formation in K562 cells. Decursin also inhibited the PDBu-induced megakaryocytic differentiation of K562 cells that is characterized by an increase in substrate adhesion, the secretion of granulocyte/macrophage colony stimulating factor (GM-CSF) and interleukin-6 (IL-6), and the surface expression of integrin β3. The binding of PDBu to PKC was competitively inhibited by decursin. Decursin induced the more rapid down-regulation of PKC α and βII isozymes than that induced by PDBu in K562 cells. Unlike PDBu, decursin promoted the translocation of PKC α and βII to the nuclear membrane. Decursin-induced faster down-regulation and nuclear translocation of PKC α and βII were not affected by the presence of PDBu. All these results indicate that decursin and phorbol ester are PKC activators distinctively acting in megakaryocytic differentiation and PKC modulation in K562 leukemia cells.

Tumor promoter binding of the protein kinase C C1 homology domain peptides of RasGRPs, chimaerins, and Unc13s

Recent investigations discovered nonkinase-type phorbol ester receptors, RasGRPs, chimaerins, and Unc13s. Phorbol ester binding occurs at the cysteine-rich sequences of about 50 residues in the C1 domains of these receptors. Fifty-one-residue RasGRP C1 peptides except for RasGRP2 showed significant phorbol 12,13-dibutyrate (PDBu) binding, but the K d values of the RasGRP1 and RasGRP3 C1 peptides were about 10-fold larger than those for the corresponding whole enzymes. Addition of the C-terminal basic amino acid cluster decreased their K d values about 10-fold, suggesting that the positive charges of these C1 peptides play an important role in the PDBu binding in the presence of negatively-charged phosphatidylserine. The 51-mer chimaerin C1 peptides showed potent PDBu binding, while the Unc13 and Munc13-1 C1 peptides without sufficient positive charges hardly bound PDBu. By the rapid screening system using this C1 peptide library, 5-prenyl-indolactam-V was identified as a promising lead for the novel protein kinase C isozyme specific ligands.

Ligand structure-activity requirements and phospholipid dependence for the binding of phorbol esters to protein kinase D.

Although protein kinase D (PKD), like protein kinase C (PKC), possesses a C1 domain that binds phorbol esters and diacylglycerol, the structural differences from PKC within this and other domains of PKD imply differential regulation by lipids and ligands. We characterized the phorbol ester and phospholipid binding properties of a glutathione S-transferase-tagged full-length PKD and compared them with those of PKC-alpha and -delta. We found that PKD is a high-affinity phorbol ester receptor for a range of structurally and functionally divergent phorbol esters and analogs and showed both similarities and differences in structure-activity relations compared with the PKCs examined. In particular, PKD had lower affinity than PKC for certain diacylglycerol analogs, which might be caused by a lysine residue at the 22 position of the PKD-C1b domain in place of the tryptophan residue at this position conserved in the PKCs. The membrane-targeting domains in PKD are largely different from those in PKC; among these differences, PKD contains a pleckstrin homology (PH) domain that is absent in PKC. However, phosphatidylinositol-4,5-bisphosphate PIP2, a lipid ligand for some PH domains, reconstitutes phorbol 12,13-dibutyrate (PDBu) binding to PKD similarly as it does to PKC-alpha and -delta, implying that the PH domain in PKD may not preferentially interact with PIP2. Overall, the requirement of anionic phospholipids for the reconstitution of [3H]PDBu binding to PKD was intermediate between those of PKC-alpha and -delta. We conclude that PKD is a high-affinity phorbol ester receptor; its lipid requirements for ligand binding are approximately comparable with those of PKC but may be differentially regulated in cells through the binding of diacylglycerol to the C1 domain.

Synergistic effects of adenosine A1 and P2Y receptor stimulation on calcium mobilization and PKC translocation in DDT1 MF-2 cells.

1. The effect of adenosine analogues and of nucleotides, alone or in combination, on intracellular calcium, accumulation of inositol (1,4,5) trisphosphate (InsP3), and on activation of protein kinase C (PKC) was studied in DDT1 MF2 cells derived from a Syrian hamster myosarcoma. These cells were found to express mRNA for A1 and some as yet unidentified P2Y receptor(s). 2. Activation of either receptor type stimulated the production of InsP3 and raised intracellular calcium in DDT1 MF2 cells. Similarly, the A1 selective agonist N6-cyclopentyladenosine (CPA) increased PKC-dependent phosphorylation of the substrate MBP(4-14) and induced a PKC translocation to the plasma membrane as determined using [3H]-phorbol dibutyrate (PDBu) binding in DDT1 MF-2 cells. However, neither adenosine nor CPA induced a significant translocation of transiently transfected gamma-PKC-GFP from the cytosol to the cell membrane. In contrast to adenosine analogues, ATP and UTP also caused a rapid but transient translocation of gamma-PKC-GFP and activation of PKC. 3. Doses of the A1 agonist CPA and of ATP or UTP per se caused barely detectable increases in intracellular Ca2+ but when combined, they caused an almost maximal stimulation. Similarly, adenosine (0.6 microM) and UTP (or ATP, 2.5 microM), which per se caused no detectable translocation of either gamma- or epsilon-PKC-GFP, caused when combined a very clear-cut translocation of both PKC subforms, albeit with different time courses. These results show that simultaneous activation of P2Y and adenosine A1 receptors synergistically increases Ca2+ transients and translocation of PKC in DDT1 MF-2 cells. Since adenosine is rapidly formed by breakdown of extracellular ATP, such interactions may be biologically important.

Memory impairment induced by chronic intracerebroventricular infusion of beta-amyloid (1–40) involves downregulation of protein kinase C

Signaling pathways underlying the cognitive deficit of the Alzheimer’s disease (AD) are not completely understood. Protein kinase C (PKC), a major neuronal protein plays a critical role in cellular signal transduction and it is known to be subjected to modulation in AD. We showed previously that, chronic infusion of β-amyloid (1–40) into rat cerebroventricle leads to deficit in spatial and non-spatial memory formation. As an attempt to identify the cellular correlates of the memory deficit, in the present study we investigated the PKC activation in different brain areas. Chronic infusion of β-amyloid (1–40) for 14 days into the rat cerebroventricle decreased the activity of soluble protein kinase C (PKC) in the hippocampus. Subcellular translocation of PKC to membrane fraction in hippocampal slices of rats treated with β-amyloid (1–40) was completely abolished under acute stimulation with 0.5 μM phorbol-dibutyrate (PDBu). We also reported a decreased affinity ( k D) for PDBu binding in the hippocampus, cerebral cortex and striatum. The total number of binding sites for PDBu ( B max) was increased, in the three brain areas analyzed on the day 14, but the changes were not statistically significant. Our data indicate that chronic accumulation of β-amyloid (1–40) into the rat brain reduced activation of PKC, effect that would substantially contribute to the memory deficit found in these animals.

Diacylglycerol Kinase γ Is One of the Specific Receptors of Tumor-Promoting Phorbol Esters

Diacylglycerol kinase (DGK) and protein kinase C (PKC) are two different enzyme families that interact with diacylglycerol. Both enzymes contain cysteine-rich C1 domains with a zinc finger-like structure. Most of the C1 domains of PKCs show strong phorbol-12,13-dibutyrate (PDBu) binding with nanomolar dissociation constants (Kd's). However, there has been no experimental evidence that phorbol esters bind to the C1 domains of DGKs. We focused on DGKγ because its C1A domain has a high degree of sequence homology to those of PKCs, and because DGKγ translocates from the cytoplasm to the plasma membrane following 12-O-tetradecanoylphorbol-13-acetate treatment similar to PKCs. Two C1 domains of DGKγ (DGKγ-C1A and DGKγ-C1B) were synthesized and tested for their PDBu binding along with whole DGKγ (Flag-DGKγ) expressed in COS-7 cells. DGKγ-C1A and Flag-DGKγ showed strong PDBu binding affinity, while DGKγ-C1B was completely inactive. Scatchard analysis of DGKγ-C1A and Flag-DGKγ gave Kd's of 3.1 and 4.4 nM, respectively, indicating that the major PDBu binding site of DGKγ is C1A. This is the first evidence that DGKγ is a specific receptor of tumor-promoting phorbol esters.

Memory deficits and increased emotionality induced by beta-amyloid (25-35) are correlated with the reduced acetylcholine release and altered phorbol dibutyrate binding in the hippocampus.

In the present study we found that chronic infusion of beta-amyloid fragment (25-35) at nanomolar concentration into rat cerebral ventricle impairs learning and memory. At a concentration of 3 nmol/day but not 0.3 nmol/day, beta-amyloid significantly reduced the spontaneous alternation behavior and the memory performance in the water maze and multiple passive avoidance tests. A significant increase in anxiety was also found in the animals infused with 3 nmol/day beta-amyloid fragment. Memory deficits and the increased emotionality were correlated with a decreased nicotine-evoked acetylcholine release from the frontal cortex/hippocampus, as assessed by microdialysis, in freely moving rats. The amyloid fragment infused either at pico- or nanomolar concentrations reduced the affinity of [3H] phorbol dibutyrate binding, an index of activated protein kinase C (PKC), and increased the total number of binding sites in the hippocampal particulate fraction. Our results suggest that the amnesic and anxiogenic effects of chronic infusion of beta-amyloid (25-35) are related to the decreased acetylcholine release and reduced PKC activation.

Toward the identification of selective modulators of protein kinase C (PKC) isozymes: establishment of a binding assay for PKC isozymes using synthetic C1 peptide receptors and identification of the critical residues involved in the phorbol ester binding

Conventional and novel protein kinase C (PKC) isozymes contain two cysteine-rich C1 domains (C1A and C1B), both of which are candidate phorbol-12,13-dibutyrate (PDBu) binding sites. We previously synthesized C1 peptides (of approximately 50 residues) corresponding to all PKC isozymes and measured their PDBu binding affinity. While many of these peptide receptors exhibited PDBu affinities comparable to the respective complete isozyme, some of the C1A peptides could not be used because they undergo temperature dependent inactivation. This problem was however eliminated by 4 °C incubation or elongation of the 50-mer C1 peptides at both N- and C-termini to increase their folding efficiency and stability. These findings enabled us to determine the K d's of PDBu for all PKC C1 peptides (except for θ-C1A) and establish the value of these peptides as readily available, stable, and easily handled surrogates of the individual isozymes. The resultant C1 peptide receptor library can be used to screen for new ligands with PKC isozyme and importantly C1 domain selectivity. Most of the C1 peptide receptors showed strong PDBu binding affinities with K d's in the nanomolar range (0.45–7.4 nM). Two peptides (δ-C1A and θ-C1A) bound PDBu over 100-fold less tightly. To identify the residues that contribute to this affinity difference, several mutants of δ-C1A and θ-C1A were synthesized. Both the G9K mutant of δ-C1A and the P9K mutant of θ-C1A showed K d's of 2–3 nM. This approach provides a useful procedure to determine the role of each C1 domain of the PKC isozymes by point mutation.

Role of Hydrophobic Residues in the C1b Domain of Protein Kinase C δ on Ligand and Phospholipid Interactions

The C1 domains of conventional and novel protein kinase C (PKC) isoforms bind diacylglycerol and phorbol esters with high affinity. Highly conserved hydrophobic residues at or near the rim of the binding cleft in the second cysteine-rich domain of PKC-delta (PKC-deltaC1b) were mutated to probe their roles in ligand recognition and lipid interaction. [(3)H]Phorbol 12,13-dibutyrate (PDBu) binding was carried out both in the presence and absence of phospholipids to determine the contribution of lipid association to the ligand affinity. Lipid dependence was determined as a function of lipid concentration and composition. The binding properties of a high affinity branched diacylglycerol with lipophilicity similar to PDBu were compared with those of PDBu to identify residues important for ligand selectivity. As expected, Leu-20 and Leu-24 strongly influenced binding. Substitution of either by aspartic acid abolished binding in either the presence or absence of phosphatidylserine. Mutation of Leu-20 to Arg or of Leu-24 to Lys caused a dramatic (340- and 250-fold, respectively) reduction in PDBu binding in the presence of lipid but only a modest reduction in the weaker binding of PDBu observed in the absence of lipid, suggesting that the main effect was on C1 domain -phospholipid interactions. Mutation of Leu-20 to Lys or of Trp-22 to Lys had modest (3-fold) effects and mutation of Phe-13 to Tyr or Lys was without effect. Binding of the branched diacylglycerol was less dependent on phospholipid and was more sensitive to mutation of Trp-22 to Tyr or Lys, especially in the presence of phospholipid, than was PDBu. In terms of specific PKC isoforms, our results suggest that the presence of Arg-20 in PKC-zeta may contribute to its lack of phorbol ester binding activity. More generally, the results emphasize the interplay between the C1 domain, ligand, and phospholipid in the ternary binding complex.

Protein Kinases C Translocation Responses to Low Concentrations of Arachidonic Acid

Arachidonic acid (AA) directly activates protein kinases C (PKC) and may thereby serve as a regulatory signal during cell stimulation. The effect, however, requires a > or =20 microm concentration of the fatty acid. We find that human polymorphonuclear neutrophils (PMN) equilibrated with a ligand for the diacylglycerol receptor on PKC, [(3)H]phorbol dibutyrate (PDB), increased binding of [(3)H]PDB within 15 s of exposure to > or =10-30 nm AA. Other unsaturated fatty acids, but not a saturated fatty acid, likewise stimulated PDB binding. These responses, similar to those caused by chemotactic factors, resulted from a rise in the number of diacylglycerol receptors that were plasma membrane-associated and therefore accessible to PDB. Unlike chemotactic factors, however, AA was fully active on cells overloaded with Ca(2+) chelators. The major metabolites of AA made by PMN, leukotriene B(4) and 5-hydroxyicosatetraenoate, did not mimic AA, and an AA antimetabolite did not block responses to AA. AA also induced PMN to translocate cytosolic PKCalpha, beta(II), and delta to membranes. This response paralleled PDB binding with respect to dose requirements, time, Ca(2+)-independence, resistance to an AA antimetabolite, and induction by another unsaturated fatty acid but not by a saturated fatty acid. Finally, HEK 293 cells transfected with vectors encoding PKCbeta(I) or PKCdelta fused to the reporter enhanced green fluorescent protein (EGFP) were studied. AA caused EGFP-PKCbeta translocation from cytosol to plasma membrane at > or =0.5 microm, and EGFP-PKCdelta translocation from cytosol to nuclear and, to a lesser extent, plasma membrane at as little as 30 nm. We conclude that AA induces PKC translocations to specific membrane targets at concentrations 2-4 orders of magnitude below those activating the enzymes. These responses, at least as they occur in PMN, do not require changes in cell Ca(2+) or oxygenation of the fatty acid. AA seems more suited for signaling the movement than activation of PKC.

Altered brain protein kinase C in depression: a post-mortem study

[ 3H]Phorbol 12,13-dibutyrate (PDBu) binding was measured in soluble and particulate fractions of frontal cortex and hippocampus from suicides, with a firm retrospective diagnosis of depression, and individually matched controls. Suicides were divided into those who had been free of antidepressant drugs for at least 3 months and those in whom prescription of antidepressants was clearly documented. In frontal cortex, there was a significantly higher number (by 75%) of [ 3H]PDBu binding sites in the soluble fraction in antidepressant-free suicides compared to controls; significant differences were also seen in the proportion of sites in the soluble and particulate fractions. Higher numbers of [ 3H]PDBu binding sites in the particulate fraction of hippocampus in antidepressant-free suicides was restricted to those who died by violent means. No significant differences in the number of [ 3H]PDBu binding sites were found in antidepressant-treated suicides compared to controls. This study provides evidence for the involvement of protein kinase C in the pathophysiology of depression.

Regional and Temporal Profiles of Phorbol 12,13-Dibutyrate Binding After Myocardial Infarction in Rats: Effects of Captopril Treatment

Phosphoinositide turnover and protein kinase C (PKC) mediate the signaling of angiotensin II, which plays a pivotal role in ventricular remodeling after myocardial infarction (MI). To determine whether PKC is activated after MI, rat hearts after MI were subjected to in vitro quantitative autoradiography with [3H]phorbol 12,13-dibutyrate (PDBu), which is highly selective for PKC. [3H]PDBu binding in the infarcted area increased significantly compared with the non-infarcted region 7 and 21 days after MI, but not 1 and 3 days and 10 months after MI. [3H]PDBu binding in the noninfarcted area was similar to that in the sham-operated rats. Immunohistochemical analysis revealed that abundant macrophages (7 days after MI), fibroblasts, and myofibroblasts (7 and 21 days after MI) occupied the infarcted region. To investigate whether myocardial [3H]PDBu binding is affected by captopril, hearts were subjected to in vitro autoradiography with [3H]PDBu after 1- or 3-week captopril treatment or no treatment. Captopril treatment significantly suppressed [3H]PDBu binding in the infarcted area 3 weeks after MI, but not 1 week after MI nor in the noninfarcted areas. These results suggest that PKC is upregulated during the healing and fibrogenic process after MI and that captopril treatment suppresses the upregulation in the infarcted area.