Activation of alpha-protein kinase C leads to association with detergent-insoluble components of GH4C1 cells

TRH and phorbol dibutyrate (PDBu) stimulate PRL secretion and synthesis from GH4C1 rat pituitary cells through activation of protein kinase C (PKC). TRH responses are mediated by increases in cellular levels of two PKC activators, Ca2+ and diacylglycerol (DAG), whereas PDBu acts as a DAG analog. We conducted experiments to compare the effects of Ca2+ and PDBu/DAG on alpha-PKC redistribution and to determine to what components of the particulate fraction activated alpha-PKC associates. Subcellular fractionation experiments demonstrated that TRH and PDBu both caused chelator-stable association of alpha-PKC with the particulate fraction. In contrast, Ca2+-mediated association with the particulate fraction was not chelator stable. Immunocytofluorescence experiments also demonstrated that TRH, PDBu, and increased cytosolic Ca2+ (due to ionomycin or K+ depolarization) caused redistribution. The effect of TRH was rapid and transient, similar to TRH stimulation of phospholipase C. The translocated alpha-PKC in the particulate fraction from TRH- or PDBu-treated cultures was not solubilized with Triton X-100. In comparable studies using an immunofluorescence assay, alpha-PKC immunofluorescence remained in detergent-insoluble preparations from TRH- and PDBu-stimulated, but not resting cells. The association of activated alpha-PKC with chelator- and detergent-insoluble material suggested that activated alpha-PKC may be associated with membrane and cytoskeletal components.     

Activation of protein kinase C inhibits human keratinocyte migration

The involvement of protein kinase C (PKC) in epidermal growth factor (EGF)-induced human keratinocyte migration was studied with the phagokinetic assay. It was concluded that PKC activation does not mediate, but rather inhibits, EGF-induced keratinocyte migration. The following experimental observations support these conclusions: 1) The PKC inhibitor H-7 did not inhibit EGF-induced migration but instead led to a modest enhancement. 2) PKC activators such as phorbol-12-myristate-13-acetate (PMA), phorbol-12,13-dibutyrate (PDBu), and 1,2-dioctanoly-sn-glycerol inhibited migration, but biologically inactive 4α-PMA had no effect. 3) PMA did not inhibit keratinocyte attachment and spreading but blocked migration almost immediately after addition. 4) Migration of PKC-depleted cells, which were produced by prolonged treatment with PDBu, was enhanced similarly to normal cells by EGF. 5) PKC-depleted cells were not susceptible to the inhibitory effects of phorbol esters on migration. Additional experiments, in which cells were preactivated with EGF, suggested that PKC inhibits the EGF effect at a post-receptor level. The inhibitory effect of PKC on keratinocyte migration was not restricted to EGF-induced migration; PKC activation also inhibited keratinocyte migration induced by bovine pituitary extract, insulin, insulin-like growth factor-1, and keratinocyte growth factor. © 1993 Wiley-Liss, Inc.     

Protein kinase C activity and phorbol ester binding to rat myogenic cells during growth and differentiation

Phorbol esters have been reported to induce opposite responses in fetal myoblasts and in satellite cells isolated from adult skeletal muscles. We examined the possibility that different levels of protein kinase C (PKC) activity and different phorbol ester binding characteristics account for these responses. For this purpose, the subcellular distributions of PKC were compared in primary cultures of myogenic cells from fetal and adult rat muscles and in the L6 cell line. Cells were used at the proliferative stage or after differentiation into myotubes. Binding of phorbol dibutyrate (PDBu) was assayed. In all three cell types, the levels of PKC specific activity were comparable at the proliferating and the differentiated stages, and partial translocation of PKC activity from the membrane to the cytosolic compartment was observed after differentiation into myotubes. PDBu binding, which had a Kd of 6 to 13 nM in proliferative cells, rose to between 30 and 52 nM in myotubes. Simultaneously, a small increase was observed in the total number of PDBu binding sites. These results suggest that the role of PKC might change with the stage of differentiation. They also imply that the difference described by others between the sensitivity to phorbol esters of fetal myoblasts and satellite cells is not connected with the phorbol ester receptor (i.e., PKC), but might be caused by events subsequent to PKC activation.     

Protein kinase C-alpha produces reciprocal effects on the phorbol ester stimulated tyrosine phosphorylation of a 50 kDa kinase in Jurkat cells.

A detergent extract isolated from the enriched fraction of integral membrane proteins of Jurkat cells showed an enhanced tyrosine phosphate level when phosphorylated in the presence of phorbol 12-myristate 13-acetate (TPA) and phorbol 12,13-dibutyrate (PDBu). The enhanced tyrosine phosphorylation was observed when the reaction time exceeded 6 min; at shorter incubation times, however, TPA inhibited tyrosine phosphorylation. When the reaction proceeded for a constant time period longer than 6 min and phorbol esters were added at different times after the start of the reaction, two phases of an enhanced tyrosine phosphorylation of a 50 kDa protein were observed. An increased phosphorylation of the 50 kDa protein was correlated with an enhanced phosphorylation of poly(Glu4,Tyr1). The two phases of enhanced phosphorylation differed in their TPA and PDBu requirement and in the proteins that were tyrosine phosphorylated. Studies with protein kinase C (PKC) inhibitors showed a negatively correlated effect on the enhanced tyrosine phosphorylation in phase I; tyrosine phosphorylation was further augmented. In phase II the regulation of tyrosine phosphorylation correlated with the efficiency of the PKC inhibitors on the alpha-isoform of PKC which was found in the cell extract. Separation of the proteins present in the investigated cell extract by gel filtration revealed a co-migration of the alpha-PKC and the 50 kDa protein. The metabolic labeling of intact Jurkat cells with 32Pi indicated that phorbol esters are also able to induce tyrosine phosphorylation of the 50 kDa protein underin vivo conditions. These data suggest an activation of two different tyrosine phosphorylation pathways by phorbol esters involving tyrosine phosphorylation/autophosphorylation of a 50 kDa kinase, as confirmed by 5'-p-fluorosulfonylbenzoyladenosine (FSBA) labeling, that are accurately regulated by alpha-PKC.     

Biphasic effect of protein kinase C on rat renal cortical Na+, K+-ATPase.

We examined the dependence of rat renal Na+, K+-ATPase activity on protein kinase C (PKC) stimulation. Infusion of either phorbol 12, 13-dibutyrate (PDBu) or phorbol 12-myristate 13-acetate (PMA) into rat abdominal aorta resulted in dose-dependent changes of renal cortical Na+, K+-ATPase activity. Low doses of these esters (3 x 10(-11) mol/kg/min) increased activity of Na+, K+-ATPase whereas high doses (3 x 10(-9) mol/kg/min) decreased it. The changes in Na+, K+-ATPase activity induced by PDBu and PMA were prevented by staurosporine, a PKC inhibitor. 4Alpha phorbol didecanoate (4alpha PDD), phorbol ester which does not activate PKC had no effect on cortical Na+, K+-ATPase. PDBu and PMA did not change Na+, K+-ATPase activity in the renal medulla. The stimulatory effect of PDBu (3 x 10(-11) mol/kg/min) was neither mimicked by amphotericin B, a sodium ionophore nor blocked by amiloride, an inhibitor of Na+/H+-exchanger. The inhibitory effect of 3 x 10(-9) mol/kg/min PDBu was not mimicked by amiloride indicating that the observed effects of PKC stimulation are not secondary to alterations in intracellular sodium concentration. The inhibitory effect of PDBu was prevented by infusion of ethoxyresorufin, an inhibitor of cytochrome P450-dependent arachidonate metabolism. These results suggest that the inhibitory effect of PKC on renal cortical Na+, K+-ATPase is mediated by cytochrome P450-dependent arachidonate metabolites.     

Regulation of Na+/K+/Cl- cotransport and [3H]bumetanide binding site density by phorbol esters in HT29 cells

The involvement of protein kinase C in the regulation of Na+/K+/Cl- cotransport was investigated in cultured HT29 human colonic adenocarcinoma cells. We have demonstrated previously the presence of a Na+/K+/Cl- cotransport pathway in HT29 cells (Kim, H.D., Tsai, Y-S., Franklin, C.C., and Turner, J.T. (1989) Biochim. Biophys. Acta 946, 397-404). Treatment of cells with the phorbol esters phorbol 12-myristate 13-acetate (PMA) and phorbol 12,13-dibutyrate (PDBu) caused an increase in membrane-associated protein kinase C activity that was accompanied by a concomitant decrease in cytosolic protein kinase C activity. PMA also produced a rapid transient increase in cotransport to 137% of control values by 5 min followed by a progressive decrease to 19% of control values by 2 h. To determine the underlying mechanism for the reduction in Na+/K+/Cl- cotransport, changes in cotransporter number and/or affinity were determined in radioligand binding studies using [3H]bumetanide. PMA and PDBu produced essentially identical time- and dose-dependent decreases in specific [3H]bumetanide binding that were similar to the observed decreases in cotransport. Analysis of saturation and competition binding data indicated that the decrease in binding was due to a lowered Bmax with no change in affinity. Both the decrease in binding and the changes in cotransport elicited by PMA were prevented by the protein kinase inhibitor H7. These findings suggest that phorbol esters cause a decrease in the number of cotransporters in HT29 cells, resulting in a reduction in Na+/K+/Cl- cotransport activity.     

Pregnancy-associated reduction in vascular protein kinase C activity rebounds during inhibition of NO synthesis

Vascular reactivity has been shown to be reduced during pregnancy and to be enhanced during chronic inhibition of nitric oxide (NO) synthesis in pregnant rats; however, the cellular mechanisms involved are unclear. The purpose of this study was to investigate whether the pregnancy-induced changes in vascular reactivity are associated with changes in the amount and/or activity of vascular protein kinase C (PKC). Active stress as well as the amount and activity of PKC was measured in deendothelialized thoracic aortic strips from virgin and pregnant rats untreated or treated with the NO synthase inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME). In virgin rats, the PKC activator phorbol 12,13-dibutyrate (PDBu, 10(-6) M) and the alpha-adrenergic agonist phenylephrine (Phe, 10(-5) M) caused significant increases in active stress and PKC activity that were inhibited by the PKC inhibitors staurosporine and calphostin C. Western blot analysis in aortic strips of virgin rats showed significant amount of the alpha-PKC isoform. Both PDBu and Phe caused significant translocation of alpha-PKC from the cytosolic to the particulate fraction. Compared with virgin rats, the PDBu- and Phe-stimulated active stress and PKC activity as well as the amount and the PDBu- and Phe-induced translocation of alpha-PKC were significantly reduced in late pregnant rats but significantly enhanced in pregnant rats treated with L-NAME. The PDBu- and Phe-induced changes in active stress and the amount, distribution, and activity of alpha-PKC in virgin rats treated with L-NAME were not significantly different from that in virgin rats, whereas the changes in pregnant rats treated with L-NAME + the NO synthase substrate L-arginine were not significantly different from that in pregnant rats. These results provide evidence that a PKC-mediated contractile pathway in vascular smooth muscle is reduced during pregnancy and significantly enhanced during chronic inhibition of NO synthesis. The results suggest that one possible mechanism of the pregnancy-associated changes in vascular reactivity may involve changes in the amount and activity of the alpha-PKC isoform.     

Properties of membrane-inserted protein kinase C

Protein kinase C (PKC) interacted with phospholipid vesicles in a calcium-dependent manner and produced two forms of membrane-associated PKC: a reversibly bound form and a membrane-inserted form. The two forms of PKC were isolated and compared with respect to enzyme stability, cofactor requirements, and phorbol ester binding ability. Membrane-inserted PKC was stable for several weeks in the presence of calcium chelators and could be rechromatographed on gel filtration columns in the presence of EGTA without dissociation of the enzyme from the membrane. The activity of membrane-inserted PKC was not significantly influenced by Ca2+, phospholipids, and/or PDBu. Partial dissociation of this PKC from phospholipid was achieved with Triton X-100, followed by dialysis to remove the detergent. The resulting free PKC appeared indistinguishable from original free PKC with respect to its cofactor requirements for activation (Ca2+, phospholipid, and phorbol esters), molecular weight, and phorbol 12,13-dibutyrate (PDBu) binding. The binding of PDBu to free and membrane-inserted PKC was measured under equilibrium conditions using gel filtration techniques. At 2.0 nM PDBu, free PKC bound PDBu with nearly 1:1 stoichiometry in the presence of Ca2+ and phospholipid. No PDBu binding to the free enzyme was observed in the absence of Ca2+. In contrast, membrane-inserted PKC bound PDBu in the presence or the absence of Ca2+; calcium did enhance the affinity of this interaction.(ABSTRACT TRUNCATED AT 250 WORDS)     

Biochemical and immunological studies of protein kinase C from Trypanosoma cruzi.

Phorbol ester binding was studied in protein kinase C-containing extracts obtained from Trypanosoma cruzi epimastigote forms. Specific 12-O-tetradecanoyl phorbol 13-acetate, [3H]PMA, or 12,13-O-dibutyryl phorbol, [3H]PDBu, binding activities, determined in T. cruzi epimastigote membranes, were dependent on ester concentration with a Kd of 9x10(-8) M and 11.3x10(-8) M, respectively. The soluble form of T. cruzi protein kinase C was purified through DEAE-cellulose chromatography. Both protein kinase C and phorbol ester binding activities co-eluted in a single peak. The DEAE-cellulose fraction was further purified into three subtypes by hydroxylapatite chromatography. These kinase activity peaks were dependent on Ca2+ and phospholipids and eluted at 40 mM (PKC I), 90 mM (PKC II) and 150 mM (PKC III) phosphate buffer, respectively. Western blot analysis of the DEAE-cellulose fractions, using antibodies against different isoforms of mammalian protein kinase C enzymes, revealed that the parasite expresses high levels of the alpha-PKC isoform. Immunoaffinity purified T. cruzi protein kinase C, isolated with an anti-protein kinase C antibody-sepharose column, were subjected to phosphorylation in the absence of exogenous phosphate acceptor. A phosphorylated 80 kDa band was observed in the presence of Ca2+, phosphatidylserine and diacylglycerol.     

Properties of the protein kinase C-phorbol ester interaction

The properties of the protein kinase C (PKC)-phorbol ester interaction were highly dependent on assay methods and conditions. Binding to cation-exchange materials or adsorption to gel matrices resulted in PKC that was capable of binding phorbol 12,13-dibutyrate (PDBu). The extraneous interactions were eliminated by measuring phorbol ester binding with a gel filtration chromatography assay in the presence of bovine serum albumin (BSA). In the absence of calcium, free PKC did not bind PDBu or phospholipids. Calcium caused structural changes in PKC which enhanced its interaction with surfaces such as the gel chromatography matrix. While BSA prevented this interaction, it did not interfere with PKC association with acidic phospholipids. Interaction of PKC with phospholipid resulted in two forms of membrane-associated PKC. The initial calcium-dependent and reversible form of membrane-associated PKC was capable of binding PDBu. Both PKC and PDBu were released from this complex by calcium chelation. Sustained interaction with phospholipid vesicles resulted in a PKC-membrane complex that could not be dissociated by calcium chelation and appeared to result from insertion of PKC into the hydrocarbon portion of the phospholipid bilayer. Membrane insertion was observed at calcium concentrations of 2-500 microM and with membrane compositions of 10-50% acidic phospholipid. However, the extent of insertion was dependent on the binding conditions and was promoted by high phospholipid to PKC ratios, high calcium, the presence of phorbol esters, high membrane charge, and long incubations.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phorbol esters can persistently replace interleukin-2 (IL-2) for the growth of a human IL-2-dependent T-cell line

A selected clone from an IL-2-dependent human T-cell line was persistently propagated in the presence of phorbol esters with the ability to activate protein kinase C (PKC), such as 12-O-tetradecanoylphorbol-13-acetate (TPA) or phorbol-12,13-dibutylate (PDBu). Thus, a TPA(PDBu)-dependent T-cell line, designated TPA-Mat, was established from IL-2-dependent T cells. The TPA-dependency of TPA-Mat was not lost during cultivation for more than a year in the presence of TPA, and TPA-Mat cells still showed IL-2-dependent growth. However, the TPA (PDBu)-dependent growth of TPA-Mat did not seem to be mediated by an autocrine mechanism of IL-2 or by any other growth factor production, because these factors were not detected in TPA-Mat cell supernatants. Therefore, the phorbol esters substituted for IL-2 and may be directly involved in transduction of growth signals in TPA-Mat cells. Although activity of PKC was down-regulated, messenger ribonucleic acid (mRNA) of the PKC beta-gene was detected in TPA-Mat cells cultured with PDBu. Furthermore, the growth of TPA-Mat cells was stimulated not only by phorbol esters but also by nonphorbol ester tumor promoters with the ability to activate PKC. These observations suggest that the sustained activation of PKC by the phorbol esters could induce continuous growth of the IL-2-dependent TPA-Mat cells.     

Phosphatidylinositol 4,5-bisphosphate competitively inhibits phorbol ester binding to protein kinase C

Calcium phospholipid dependent protein kinase C (PKC) is activated by diacylglycerol (DG) and by phorbol esters and is recognized to be the phorbol ester receptor of cells; DG displaces phorbol ester competitively from PKC. A phospholipid, phosphatidylinositol 4,5-bisphosphate (PIP2), can also activate PKC in the presence of phosphatidylserine (PS) and Ca2+ with a KPIP2 of 0.04 mol %. Preliminary experiments have suggested a common binding site for PIP2 and DG on PKC. Here, we investigate the effect of PIP2 on phorbol ester binding to PKC in a mixed micellar assay. In the presence of 20 mol % PS, PIP2 inhibited specific binding of [3H]phorbol 12,13-dibutyrate (PDBu) in a dose-dependent fashion up to 85% at 1 mol %. Inhibition of binding was more pronounced with PIP2 than with DG. Scatchard analysis indicated that the decrease in binding of PDBu in the presence of PIP2 is the result of an altered affinity for the phorbol ester rather than of a change in maximal binding. The plot of apparent dissociation constants (Kd') against PIP2 concentration was linear over a range of 0.01-1 mol % with a Ki of 0.043 mol % and confirmed the competitive nature of inhibition between PDBu and PIP2. Competition between PIP2 and phorbol ester could be demonstrated in a liposomal assay system also. These results indicate that PIP2, DG, and phorbol ester all compete for the same activator-receiving region on the regulatory moiety of protein kinase C, and they lend support to the suggestion that PIP2 is a primary activator of the enzyme.     

Phorbol esters specifically inhibit induction of immunoglobulin secretion in a murine B cell leukemia

We have examined the effect of tumor-promoting phorbol esters such as phorbol myristate acetate (PMA) on the murine B cell leukemia BCL-1 and its in vitro adapted derivative CW.13.20. Phorbol esters, including PMA and phorbol dibutyrate (PDBu), were potent inhibitors of BCL-1 IgM secretion induced by either LPS or lymphokines; half-maximal inhibition was obtained with 0.1 nM PMA and 0.8 nm PDBu. The inhibitory action of PDBu on BCL-1 cells was reversible for over 1 hr, but after 5 hr 70% of the inhibition was irreversible. Irreversible inhibition could be blocked by cycloheximide, suggesting a requirement for protein synthesis. The specificity of PDBu inhibition was examined by comparing the patterns of protein synthesis in PDBu-treated and control BCL-1 cells. Total incorporation of [35S]methionine into protein by BCL-1 cells cultured in the presence of PDBu was similar to that of untreated cells. Analysis of radiolabeled proteins by SDS-PAGE and autoradiography revealed no consistent changes in the pattern of protein synthesis except at those positions corresponding to the heavy and light chains of IgM. Immunoprecipitation with an affinity-purified anti-IgM indicated that PDBu inhibited the increased synthesis of heavy and light chain that follows stimulation by lymphokine but did not diminish control IgM synthesis. Induced IgM secretion from CW.13.20 cells was also inhibited by phorbol esters, indicating a direct action on B cells. Delaying the addition of phorbol ester relative to lymphokine or LPS by 24 hr significantly reduced inhibition of induced IgM secretion from both BCL-1 and CW.13.20 cells. This suggests that phorbol esters specifically interfere with the signal for induction of IgM secretion by both lymphokine and LPS.     

Synthesis and phorbol ester binding of the cysteine-rich domains of diacylglycerol kinase (DGK) isozymes. DGKgamma and DGKbeta are new targets of tumor-promoting phorbol esters

Diacylglycerol kinase (DGK) and protein kinase C (PKC) are two distinct enzyme families associated with diacylglycerol. Both enzymes have cysteine-rich C1 domains (C1A, C1B, and C1C) in the regulatory region. Although most PKC C1 domains strongly bind phorbol esters, there has been no direct evidence that DGK C1 domains bind phorbol esters. We synthesized 11 cysteine-rich sequences of DGK C1 domains with good sequence homology to those of the PKC C1 domains. Among them, only DGKgamma-C1A and DGKbeta-C1A exhibited significant binding to phorbol 12,13-dibutyrate (PDBu). Scatchard analysis of rat-DGKgamma-C1A, human-DGKgamma-C1A, and human-DGKbeta-C1A gave K(d) values of 3.6, 2.8, and 14.6 nm, respectively, suggesting that DGKgamma and DGKbeta are new targets of phorbol esters. An A12T mutation of human-DGKbeta-C1A enhanced the affinity to bind PDBu, indicating that the beta-hydroxyl group of Thr-12 significantly contributes to the binding. The K(d) value for PDBu of FLAG-tagged whole rat-DGKgamma (4.4 nm) was nearly equal to that of rat-DGKgamma-C1A (3.6 nm). Moreover, 12-O-tetradecanoylphorbol 13-acetate induced the irreversible translocation of whole rat-DGKgamma and its C1B deletion mutant, not the C1A deletion mutant, from the cytoplasm to the plasma membrane of CHO-K1 cells. These results indicate that 12-O-tetradecanoylphorbol 13-acetate binds to C1A of DGKgamma to cause its translocation.     

The C4 hydroxyl group of phorbol esters is not necessary for protein kinase C binding

To investigate the role of the hydroxyl group at position 4 of the phorbol esters in protein kinase C (PKC) binding and function, 4beta-deoxy-phorbol-12,13-dibutyrate (4beta-deoxy-PDBu, 5a) and 4beta-deoxy-phorbol-13-acetate (6a) were synthesized from phorbol (1). The binding affinities of these 4beta-deoxy compounds (5a, 6a) to the 13 PKC isozyme C1 domains were quite similar to those of the corresponding 4beta-hydroxy compounds (4a, 4b), suggesting that the C4 hydroxyl group of phorbol esters is not necessary for PKC binding. Moreover, functional assays showed that 4beta-deoxy-PDBu (5a) exhibited biological activities (Epstein-Barr virus induction and superoxide generation) equally potent to those of PDBu (4a). These solution phase results differ from expectations based on the previously reported solid-phase structure of the complex of PKCdelta-C1B and phorbol-13-acetate (4b).     

Protein kinase C contains two phorbol ester binding domains

A series of deletion and truncation mutants of protein kinase C (PKC) were expressed in the baculovirus-insect cell expression system in order to elucidate the ability of various domains of the enzyme to bind phorbol dibutyrate (PDBu). A PKC truncation mutant consisting of only the catalytic domain of the enzyme did not bind [3H]PDBu, whereas a PKC truncation mutant consisting of the regulatory domain (containing the tandem cysteine-rich putative zinc finger regions) bound [3H]PDBu. Deletion of the second conserved region (C2) of PKC did not abolish [3H]PDBu binding, whereas a deletion of the first conserved region (C1) of PKC, containing the two cysteine-rich sequences, completely abolished [3H]PDBu binding. Additional truncation and deletion mutants helped to localize the region necessary for [3H]PDBu binding; all PKC mutants that contained either one of the cysteine-rich zinc finger-like regions possessed phorbol ester binding activity. Scatchard analyses of these mutants indicated that each bound [3H]PDBu with equivalent affinity (21-41 nM); approximately 10-20-fold less than the native enzyme. In addition, a peptide of 146 amino acid residues from the first cysteine-rich region, as well as a peptide of only 86 amino acids residues from the second cysteine-rich region, both bound [3H]PDBu with high affinity (31 +/- 4 and 59 +/- 13 nM, respectively). These data establish that PKC contains two phorbol ester binding domains which may function in its regulation.     

The involvement of protein kinase C in the regulation of choline cotransport in Limulus.

The involvement of protein kinase C (PKC) in the regulation of [3H]choline cotransport was studied in Limulus brain hemi-slice preparations. The PKC activators, phorbol 12-myristate 13-acetate (PMA) or phorbol 12,13-dibutyrate (PDBu), significantly decreased [3H]choline cotransport. Conversely, the PKC inhibitors, staurosporine (STAURO) and polymyxin B (PMB), each increased [3H]choline cotransport. These PKC inhibitors prevented the phorbol ester-induced reduction of transport. Both the PMA induced decrease and the STAURO induced increase in [3H]choline cotransport were paralleled by respective and comparable changes in [3H]hemicholinium-3 (HC-3) specific binding. Pre-exposure of brain hemi-slices to elevated potassium chloride (120 mM KCl) resulted in a doubling of [3H]choline cotransport and [3H]HC-3 binding. The enhancement of [3H]choline cotransport by STAURO and antecedent 120 mM KCl treatment were additive. PMA did not significantly alter elevated potassium stimulated transport. Moreover, arachidonyltrifluoromethyl ketone (AACOCF3) and quinacrine (QUIN), both phospholipase A2 (PLA2) inhibitors, markedly decreased enhanced [3H]choline transport and [3H]HC-3 binding induced by antecedent exposure to depolarizing concentrations of potassium. These results suggest that PKC and PLA2 are involved in the regulation of [3H]choline cotransport but at different regulatory sites.     

Phorbol esters and related analogs regulate the subcellular localization of beta 2-chimaerin, a non-protein kinase C phorbol ester receptor

The novel phorbol ester receptor beta2-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, beta2-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, beta2-chimaerin is translocated by phorbol esters from the cytosolic to particulate fraction. Phorbol esters also induce translocation of alpha1 (n)- and beta1-chimaerins, suggesting common regulatory mechanisms for all chimaerin isoforms. The subcellular redistribution of beta2-chimaerin by phorbol esters is entirely dependent on the C1 domain, as revealed by deletional analysis and site-directed mutagenesis. Interestingly, beta2-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 beta2-chimaerin and PKCalpha. Strikingly, the mezerein analog thymeleatoxin is not able to translocate beta2-chimaerin, although it very efficiently translocates PKCalpha. Phorbol esters also promote the association of beta2-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.     

Diacylglycerol kinase gamma 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 (K(d)'s). However, there has been no experimental evidence that phorbol esters bind to the C1 domains of DGKs. We focused on DGK gamma because its C1A domain has a high degree of sequence homology to those of PKCs, and because DGK gamma translocates from the cytoplasm to the plasma membrane following 12-O-tetradecanoylphorbol-13-acetate treatment similar to PKCs. Two C1 domains of DGK gamma (DGK gamma-C1A and DGK gamma-C1B) were synthesized and tested for their PDBu binding along with whole DGK gamma (Flag-DGK gamma) expressed in COS-7 cells. DGK gamma-C1A and Flag-DGK gamma showed strong PDBu binding affinity, while DGK gamma-C1B was completely inactive. Scatchard analysis of DGK gamma-C1A and Flag-DGK gamma gave K(d)'s of 3.1 and 4.4 nM, respectively, indicating that the major PDBu binding site of DGK gamma is C1A. This is the first evidence that DGK gamma is a specific receptor of tumor-promoting phorbol esters.     

Diacylglycerol (DAG)-lactones, a new class of protein kinase C (PKC) agonists, induce apoptosis in LNCaP prostate cancer cells by selective activation of PKCalpha

Phorbol esters, the archetypical (PKC) activators, induce apoptosis in androgen-sensitive LNCaP prostate cancer cells. In this study we evaluate the effect of a novel class of PKC ligands, the diacylglycerol (DAG)-lactones, as inducers of apoptosis in LNCaP cells. These unique ligands were designed using novel pharmacophore- and receptor-guided approaches to achieve highly potent DAG surrogates. Two of these compounds, HK434 and HK654, induced apoptosis in LNCaP cells with much higher potency than oleoyl-acetyl-glycerol or phorbol 12,13-dibutyrate. Moreover, different PKC isozymes were found to mediate the apoptotic effect of phorbol 12-myristate 13-acetate (PMA) and HK654 in LNCaP cells. Using PKC inhibitors and dominant negative PKC isoforms, we found that both PKCalpha and PKCdelta mediated the apoptotic effect of PMA, whereas only PKCalpha was involved in the effect of the DAG-lactone. The PKCalpha selectivity of HK654 in LNCaP cells contrasts with similar potencies in vitro for binding and activation of PKCalpha and PKCdelta. Consistent with the differences in isoform dependence in intact cells, PMA and HK654 show marked differences in their abilities to translocate PKC isozymes. Both PMA and HK654 induce a marked redistribution of PKCalpha to the plasma membrane. On the other hand, unlike PMA, HK654 translocates PKCdelta predominantly to the nuclear membrane. Thus, DAG-lactones have a unique profile of activation of PKC isozymes for inducing apoptosis in LNCaP cells and represent the first example of a selective activator of a classical PKC in cellular models. An attractive hypothesis is that selective activation of PKC isozymes by pharmacological agents in cells can be achieved by differential intracellular targeting of each PKC.