Role of specific interactions between protein kinase C and triphenylethylenes in inhibition of the enzyme.

The tumor promoter receptor protein kinase C (PKC) has been implicated as a key enzyme in cellular growth regulation. It is, therefore, believed that specific PKC inhibitors may include effective antiproliferative agents. Previously, we have shown that the antiestrogen tamoxifen and related triphenylethylenes are potent inhibitors of PKC. Although the mechanism of inhibition of PKC by triphenylethylenes clearly involves nonspecific interactions between the antiestrogens and the lipid cofactor of PKC, we recently demonstrated that PKC itself has specific triphenylethylene-binding sites, suggesting that the inhibitory mechanism also involves specific drug-protein interactions. In this report, we characterize the direct interactions between PKC and triphenylethylenes and demonstrate their relevance to the inhibitory action of triphenylethylenes against PKC. We show (a) that the triphenylethylene-binding sites of PKC are located in the catalytic domain of the enzyme, (b) that MgATP (i.e., 10 mM MgCl2 plus 1 mM ATP) competes with the triphenylethylenes for binding sites on PKC, and (c) that triphenylethylenes are competitive inhibitors of PKC with respect to MgATP. Taken together, these data provide strong evidence that triphenylethylenes can inhibit PKC by binding directly to the ATP-binding region of the active site of the enzyme. The specific interactions between triphenylethylenes and PKC characterized here may provide a rationale for developing more specific PKC inhibitors.

Triphenylethylenes: a new class of protein kinase C inhibitors.

The Ca2+- and phospholipid-dependent phosphotransferase activity of protein kinase C was inhibited by the triphenylethylene compounds clomiphene [drug concentration causing 50% inhibition (IC50) = 25 microM], 4-hydroxytamoxifen (IC50 = 25 microM), and N-desmethyltamoxifen (IC50 = 8 microM). The Ca2+- and phospholipid-independent phosphorylation of protamine sulfate, which is catalyzed by protein kinase C, was not inhibited by the triphenylethylenes, suggesting that they do not interact directly with the active site of protein kinase C. The inhibitory potency of each triphenylethylene was reduced when the phospholipid concentration was increased, providing evidence that these drugs inhibited protein kinase C by interacting with phospholipids. The potencies of the effects of the triphenylethylenes on protein kinase C in the lipid environment of intact cells were evaluated by determining their efficacies in the inhibition of [3H]phorbol 12,13-dibutyrate (PDBu) binding to mouse embryo C3H/10T1/2 cells. Micromolar concentrations of each drug inhibited [3H]PDBu binding in these cells. N-Desmethyltamoxifen, 4-hydroxytamoxifen, and tamoxifen inhibited protein kinase C with the same order of potency as that which has been reported for their inhibition of MCF-7 cell growth by Reddel et al. (1983). N-Desmethyltamoxifen and 4-hydroxytamoxifen were also more potent than tamoxifen in the inhibition of the growth of mouse embryo fibroblast C3H/10T1/2 cells. These correlations suggest that the mechanism of growth inhibition by tamoxifen and its metabolites includes interactions with protein kinase C.

Specific and direct binding of protein kinase C to an immobilized tamoxifen analogue.

We have previously demonstrated that tamoxifen and related triphenylethylene compounds are potent inhibitors of protein kinase C (PKC). The present study demonstrates that PKC binds specifically and reversibly to the antiestrogen N-didesmethyltamoxifen when the drug is coupled to CNBr-activated agarose through its primary amine, in the absence of lipid and other cofactors of the enzyme. PKC did not bind to 4-hydroxytamoxifen, which had been immobilized on epoxy-activated Sepharose through its hydroxyl moiety. This shows that the binding of PKC to immobilized N-didesmethyltamoxifen was not merely due to hydrophobic interactions, since N-didesmethyltamoxifen and 4-hydroxytamoxifen have nearly identical hydrophobicities. These results demonstrate that PKC has specific triphenylethylene-binding sites, which may mediate the inhibition of PKC activity by these antiestrogens.

Enhancement of murine tumor cell sensitivity to adriamycin by presentation of the drug in phosphatidylcholine-phosphatidylserine liposomes.

In vitro incubation of mouse UV-2237M fibrosarcoma cells with liposomes containing Adriamycin (ADR) produced significant cytotoxicity in drug-sensitive cells and in multidrug-resistant variants of this tumor. ADR was encapsulated in the aqueous space of multilamellar liposomes composed of phosphatidylcholine and phosphatidylserine. The preparation was stable in medium at 37 degrees C for up to 7 days. Free unencapsulated ADR produced cytostasis in parental ADR-sensitive cells but not in variant lines selected for resistance to the drug. In contrast, ADR encapsulated in multilamellar liposomes (MLV) produced high levels of cytostasis in both ADR-sensitive and ADR-resistant cells. The phospholipid composition of the MLV influenced the outcome of ADR-mediated cytostasis. ADR encapsulated in MLV consisting of only phosphatidylcholine did not produce cytostasis. Increasing the proportion of phosphatidylserine in the MLV increased the level of ADR-mediated cytotoxicity in cells resistant to free ADR. This effect was not due to simple modification of tumor cell surface by liposomes since ADR added to resistant cells together with liposomes containing buffer produced less cytostasis. The cytostasis of resistant cells by ADR in liposomes was not due to appreciable changes in the intracellular ADR concentration or localization within the cells because ADR-induced DNA cleavage was not found in ADR-resistant cells treated with cytostatic amounts of liposomal ADR. Whether the enhanced sensitivity of tumor cells to ADR was due to localized damage to the plasma membrane through a phosphatidylserine-mediated release of the drug to the cell surface is now under active investigation.

Inhibition of protein kinase C by tamoxifen.

The antiestrogen drug tamoxifen inhibits rat brain protein kinase C in vitro, whether the enzyme is activated by Ca2+ and phospholipid (50% inhibitory dose, 100 microM), 12-O-tetradecanoylphorbol-13-acetate and phospholipid (50% inhibitory dose, 40 microM), or teleocidin and phospholipid. Tamoxifen does not inhibit the Ca2+- and phospholipid-independent phosphorylation of protamine sulfate by protein kinase C, indicating that the drug does not interact with the active site of the enzyme. The binding of [3H]phorbol dibutyrate to high-affinity membrane receptors of cultured mouse fibroblast cells is inhibited by tamoxifen (50% inhibitory dose, 5 microM). Our findings suggest that the growth-inhibitory and cytotoxic effects of tamoxifen, which have been observed at microM concentrations of the drug, may be in part due to its effects on protein kinase C.

Altered levels of protein kinase C and Ca2+-dependent protein kinases in human colon carcinomas.

Protein kinase C (PKC) is a Ca2+- and phospholipid-dependent protein kinase which is implicated in tumor promotion, since it has been demonstrated to be a high affinity receptor for tumor promoters such as 12-O-tetradecanoylphorbol-13-acetate. Colon carcinogenesis appears to proceed through distinct stages of initiation and promotion. The present studies show that PKC and calcium-dependent protein kinase specific activities are reduced in human colon carcinomas when compared to their normal adjacent colon mucosa. There were significantly higher Ca2+-dependent protein kinase and PKC specific activities observed in both the cytosolic and particulate fractions of the normal mucosa relative to the corresponding values obtained with the carcinoma fractions. The average specific activity ratios were 5.1 (normal cytosolic/carcinoma cytosolic) and 3.7 (normal particulate/carcinoma particulate) for PKC. PKC activity was reduced in the carcinoma tissues with respect to both protein and tissue weight. The percentage of Ca2+-dependent protein kinase and PKC activities that were present in the particulate fraction of each of the samples varied considerably among tissues, and in general there was no systematic difference between the carcinoma and normal mucosa samples. However, in the carcinoma samples that contained an extensive admixture of benign adenomatous tissue, the particulate fractions consistently contained greater than 60% of the total Ca2+-dependent protein kinase and PKC activities. The present studies indicate that colon carcinogenesis is associated with alterations in cellular levels of protein kinase activities.

Identification of naturally processed human ovarian peptides recognized by tumor-associated CD8+ cytotoxic T lymphocytes.

Identification of naturally processed peptides recognized by tumorspecific CTLs may lead to epitope-specific tumor vaccines. Because these epitopes may be expressed differently on epithelial tumors and may differ in their ability to induce CTL in vivo, we have isolated the HLA-A2-peptide complexes by immunoaffinity from an established ovarian tumor line transfected with and expressing HLA-A2 gene. High-performance liquid chromatography-fractionated peptides were used to reconstitute epitopes recognized on HLA-A2 by three HLA-A2+ CD8+ CTL lines. These lines recognized at least three of the same groups of fractions (designated SKOV3.A, -B, and -C) but showed differences in the pattern of recognition of other fractions. To gain insight in the epitope distribution by freshly isolated ovarian tumors, we compared the recognition of peaks SKOV3.B and -C with the corresponding peaks from an ovarian tumor (OVA-6) that expressed similar levels of HLA-A2, using one of these lines (CTL-OVA-5) as indicator. CTL-OVA-5 recognized a large number of epitopes from peaks B and C rechromatographed on more resolving high-performance liquid chromatography gradient. Although a number of peaks appeared to be coincident on both SKOV3 and OVA-6, an even higher number appeared either not to overlap or to overlap only partially. These findings, which represent the first analysis of the epitopes presented by a patient tumor, suggest that the use of tumor line-derived peptides for vaccination may require selection of the epitopes corresponding to the ones presented by freshly isolated human tumors.

Site-dependent differences in response of the UV-2237 murine fibrosarcoma to systemic therapy with adriamycin.

Murine fibrosarcoma UV-2237MM cells were implanted into different organs of syngeneic C3H/HeN mice. The resultant tumors were treated by i.v. administration of Adriamycin (ADR). Despite the high sensitivity of the fibrosarcoma cells to ADR in vitro, the established tumors growing in vivo exhibited marked differences in their responses to ADR. Tumors growing in the subcutis and the spleen were ADR-sensitive, whereas lung metastases were not. The resistance of lung metastases to ADR was not due to selection of a drug-resistant population since tumor cells isolated from lung metastases were highly sensitive to ADR under in vitro conditions. The responsiveness of skin and spleen tumors to ADR was due neither to increased blood supply nor to preferential accumulation of ADR, since both parameters were higher in lung metastases. Protein kinase C activity levels correlated with ADR resistance in the closely related murine fibrosarcoma cell line UV-2237 and its ADR-selected multidrug-resistant variants. However, nearly identical levels of protein kinase C activity were found in UV-2237MM tumors growing in the lung, spleen, and subcutis, indicating that protein kinase C activity levels did not account for the different responses to ADR. The present studies suggest that the organ environment influences the response of UV-2237MM to ADR administered systemically. This finding may have implications for the design of animal models for therapy of disseminated cancer.

Induction of transformation progression in type 5 adenovirus-transformed rat embryo cells by a cloned protein kinase C beta 1 gene and reversal of progression by 5-azacytidine.

Protein kinase C (PKC) is a key component in signal transduction in eucaryotic cells and when specific PKC isoforms are over-expressed in immortal mammalian cells they can induce transformation-associated properties. In the present study we demonstrate that a cloned PKC beta 1 gene can induce an enhanced expression of the transformed phenotype in type 5 adenovirus (Ad5)-transformed rat embryo (RE) cells (clone E11), a process termed transformation progression. E11 cells expressing the PKC beta 1 gene, clone B1/PKC, produce PKC beta 1 mRNA and display enhanced PKC enzymatic activity and binding of [3H]-phorbol-12,13-dibutyrate (PDBu) to cell surface phorbol ester receptors. B1/PKC cells grow with increased efficiency in agar in comparison with parental E11 cells and anchorage-independence is further enhanced in both cell types by addition of the tumor promoting agent 12-0-tetradecanoyl-phorbol-13-acetate (TPA). A single-exposure of B1/PKC cells to 5-azacytidine (AZA), followed by growth in the absence of this demethylating agent, results in B1/PKC-AZA clones which display a stable reversion of the progression phenotype to that of the unprogressed parental E11 clone. Loss of the progression phenotype corresponds with a reduction in PKC beta 1-induced biochemical and cellular changes. In contrast, progression-suppression does not involve an alteration in expression of the Ad5 transforming genes, E1A and E1B, or the endogenous PKC epsilon gene. TPA cannot induce the progression phenotype in B1/PKC-AZA cells, but it can reversibly induce an increase in the transcriptional rate and steady-state mRNA levels of PKC beta 1 and c-jun and it increases AP-1 DNA-binding. These results indicate that the PKC beta 1 gene can serve as a transformation progression-inducing gene in rat embryo cells previously transformed by Ad5 and progression may be mediated by the inactivation by methylation of an AZA-sensitive 'progression suppressor gene(s)'. The suppression process in B1/PKC cells is independent of expression of the Ad5-transforming genes but correlates directly with the reduced expression of the transfected PKC beta 1 gene in AZA-treated B1/PKC cells.

Heregulin-induced apoptosis is mediated by down-regulation of Bcl-2 and activation of caspase-7 and is potentiated by impairment of protein kinase C alpha activity.

Heregulins are a group of growth factors that play diverse and critical roles in the signaling network of the human epidermal growth factor receptor (HER or EGFR) superfamily. Our earlier studies have shown that recombinant heregulinbeta1 (HRG) induces apoptosis in SKBr3 breast cancer cells that overexpress HER2. Here we report molecular mechanisms of HRG-induced apoptosis. HRG treatment of SKBr3 cells for 72 h decreased the level of Bcl-2 protein. HRG treatment led to degradation of poly (ADP-ribose) polymerase (PARP) and activated both caspase-9 and caspase-7. No significant activation of caspase-3, -6, or -8 was detected. Expression of exogenous caspase-7 by adenovirus-caspase-7 (Ad-casp-7) in SKBr3 cells resulted in apoptosis, which mimicked the effect of HRG treatment. Expression of exogenous caspase-7 had no impact on Bcl-2 expression, but promoted PARP degradation. Two highly selective inhibitors of protein kinase C (PKC), GF109203X (GF) and Ro318425 (Ro), significantly enhanced HRG-induced apoptosis as determined by flow cytometric analysis and DNA fragmentation assay. Accordingly, the PKC inhibitor GF further decreased the level of Bcl-2 protein and further degraded PARP in HRG-treated cells. Assay of PKC activity indicated that HRG activated PKC in SKBr3 cells, predominantly affecting the PKCalpha isoform. To confirm which PKC isoform(s) mediated potentiation of HRG-induced apoptosis, the profile of PKC isoforms was measured in SKBr3 cells. Five PKC isoforms, PKCalpha, PKCiota, PKCzeta, PKClambda, and PKCdelta as well as their receptors (RACK1) were expressed in this cell line. Treatment with PKC inhibitors GF and Ro decreased protein levels of both PKCalpha and PKCdelta at 24 h. PKCalpha levels were still depressed at 72 h. GF and Ro had little effect on the expression of other PKC isoforms. An inhibitor of classical PKC isoforms (Go6976) enhanced HRG-induced apoptosis, whereas the PKCdelta selective inhibitor rottlerin did not. As PKCalpha was the only classical isoform expressed in SKBr3 cells, the effect of Go6976 on HRG-induced apoptosis largely related to inhibition of PKCalpha. Constitutive expression of wild-type PKCalpha attenuated the apoptosis produced by HRG and GF. Consequently, HRG-induced apoptosis in SKBr3 cells appeared to involve down-regulation of Bcl-2 protein, activation of caspase-9 and caspase-7, and degradation of PARP. Inhibition of PKC function enhanced HRG-induced apoptosis, leading to synergistic down-regulation of Bcl-2 expression. Impairment of the PKCalpha isoform alone was sufficient to potentiate HRG-induced apoptosis.

Activation of tumoricidal properties in macrophages by lipopolysaccharide requires protein-tyrosine kinase activity.

The purpose of these studies was to determine whether triggering murine peritoneal macrophages to a tumoricidal state by lipopolysaccharide (LPS) requires protein-tyrosine phosphorylation. The LPS-triggered activation of mouse macrophages to lyse syngeneic B16 melanoma cells was significantly inhibited in a dose-dependent manner by the protein-tyrosine kinase (PTK) inhibitors genistein, herbimycin A, and tyrphostin. Genistein was effective only when added to macrophages prior to or simultaneously with LPS. Genistein potently inhibited the productive interaction of macrophages with LPS but had only a minor effect on the action of interferon-gamma. The effects of genistein on LPS-triggered macrophage activation were not due to nonspecific changes in macrophage metabolism or toxicity because genistein did not prevent lysis of tumor cells by activated macrophages, nor did it reduce the capacity of macrophages to phagocytose antibody-opsonized sheep erythrocytes. Western blot analysis with antiphosphotyrosine monoclonal antibody revealed that incubation of macrophages with LPS produced a rapid increase in tyrosine phosphorylation of several proteins and that the induced phosphorylation could be inhibited by effective concentrations of genistein, herbimycin A, or tyrphostin. Taken together, these data indicate that protein-tyrosine phosphorylation plays an important role in LPS-induced tumoricidal activation of macrophages.

The activation of protein kinase C by the polyphosphoinositides phosphatidylinositol 4,5-diphosphate and phosphatidylinositol 4-monophosphate.

Protein kinase C(PKC) is a Ca2+- and phospholipid-dependent protein kinase which can be activated by diacylglycerol, a product of polyphosphoinositide hydrolysis. In this report, we show that the polyphosphoinositides L-alpha-phosphatidylinositol 4-monophosphate (PI 4P) and L-alpha-phosphatidylinositol 4,5-diphosphate (PI 4.5DP) can serve as phospholipid cofactors of isolated rat brain PKC. The order of potency of the phosphoinositides in the activation of PKC, PI greater than PI 4P greater than PI 4,5DP, shows a negative correlation with the degree of acidity of the phospholipid head group, whether 1 mM Ca2+ or 200 nM TPA is present in the reaction assay mixture. Although the polyphosphoinositides are by themselves weaker activators of PKC than PI, small amounts of PI 4,5DP cause a two-fold enhancement of PKC in the presence of Ca2+ and PI. While the endogenous phospholipid cofactors of PKC remain to be identified, these results suggest that the small amounts of polyphosphoinositides which are present in cell membranes may play a direct role in the activation of PKC in vivo, by serving as phospholipid cofactors of the enzyme.

N-alpha-Tosyl-L-lysine chloromethyl ketone and N-alpha-tosyl-L-phenylalanine chloromethyl ketone inhibit protein kinase C.

TLCK (N-alpha-tosyl-L-lysine chloromethyl ketone) inhibits protein kinase C whether or not the enzyme is under the regulation of Ca2+ and phospholipid. TLCK (IC50 = 1 mM) is a much more potent inhibitor of protein kinase C than TPCK (N-alpha-tosyl-L-phenylalanine chloromethyl ketone) (IC50 = 8 mM), suggesting that the lysyl moiety of TLCK may be specifically recognized by the active site of protein kinase C. These results extend the evidence that the active site of protein kinase C recognizes basic amino acids, and suggest that the active sites of protein kinase C and the cAMP-dependent protein kinase, which is also inhibited by TLCK and TPCK, are structurally related.

Level of protein kinase C activity correlates directly with resistance to adriamycin in murine fibrosarcoma cells.

In this report, we demonstrate a direct correlation between protein kinase C (PKC) activity and adriamycin (ADR) resistance in mouse fibrosarcoma cells. PKC activity was measured in four murine UV-2237M fibrosarcoma cell lines that differed in the degrees to which they expressed resistance to ADR, which is an inhibitor of PKC. A comparison of the four cell lines revealed a positive correlation between the level of PKC activity and resistance to ADR. Incubation of the cells with the PKC inhibitor H-7 produced a partial reversal of ADR resistance. Taken together, these results suggest a role for PKC in the mechanism of ADR resistance.

Distinct patterns of phorbol ester-induced downregulation of protein kinase C activity in adriamycin-selected multidrug resistant and parental murine fibrosarcoma cells.

Specific activators of protein kinase C (PKC), including the phorbol-ester tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA), can reduce the chemosensitivities of a variety of mammalian tumor cell lines and their cytotoxic drug-selected multidrug resistant (MDR) variants to MDR-linked drugs, thus implicating PKC in the MDR phenotype. Previously, we reported that the adriamycin-selected MDR murine fibrosarcoma cell line UV-2237M-ADRR has approximately twice as much PKC activity as the parental UV-2237M line. In this report, we show that the level of [3H]phorbol-12,13-dibutyrate specific binding activity was elevated 3.5-fold in the MDR cells, thus establishing that phorbol-ester responsive PKC is overexpressed in the MDR line. Phorbol esters mediate downregulation of PKC by stimulating proteolysis of the enzyme, without altering the rate of PKC synthesis. We report that the kinetics of TPA-induced downregulation of PKC activity differ markedly in parental and MDR UV-2237M cells, providing evidence that the overexpression of phorbol-ester responsive PKC in adriamycin-selected MDR UV-2237M-ADRR cells results, at least in part, from a reduced rate of PKC degradation in the cells.

Inhibition of protein kinase C by a synthetic peptide corresponding to cytoplasmic domain residues 828-848 of the human immunodeficiency virus type 1 envelope glycoprotein.

This report describes the inhibition of protein kinase C (PKC) by a synthetic peptide corresponding to a viral sequence expressed in mammalian cells. The peptide corresponds to cytoplasmic domain residues 828-848 of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (gp41), and it inhibits Ca(2+)- and phosphatidylserine (PS)-dependent phosphorylation of synthetic peptide substrates and histone by purified PKC with IC50 values ranging from 9 to 32 microM. Although previously described pKC-inhibitory synthetic peptides corresponding to sequences expressed in mammalian cells are also effective against the phosphorylation of synthetic peptide substrates, they fail to affect PKC-catalysed phosphorylation of potent protein substrates such as histone. This may limit their usefulness as inhibitors of PKC-catalysed protein phosphorylation in cellular systems. PKC activation is a major contributing factor in multidrug resistance (MDR) in cancer. Our observation that the synthetic peptide gp41(828-848) inhibits pKC-catalysed phosphorylation of a protein substrate suggests the potential value of expressing the viral sequence gp41(828-848) in cancer cells as a novel in vitro model system of MDR reversal.

Inhibition of protein kinase C by the 12-O-tetradecanoylphorbol-13-acetate antagonist glycyrrhetic acid.

Glycyrrhetic acid is an anti-inflammatory agent isolated from licorice root that inhibits 12-O-tetradecanoylphorbol-13-acetate (TPA)-mediated tumor promotion in mouse skin. Although it has been established that glycyrrhetic acid inhibits a number of events induced by the phorbol ester tumor promoter TPA in cultured cells, its mechanisms of action has remained obscure. In this report, we demonstrate that glycyrrhetic acid inhibits the Ca2+-and phospholipid-dependent phosphotransferase activity of protein kinase C (PKC), the phorbol ester tumor promoter receptor. Therefore, inhibition of PKC may play a role in the anti-promoting activity of glycyrrhetic acid.

In vitro inhibition of rat brain protein kinase C by rhodamine 6G. Profound effects of the lipid cofactor on the inhibition of the enzyme.

Rhodamine 6G inhibited protein kinase C (PKC) when the enzyme was activated by Ca2+ plus phosphatidylserine, 12-O-tetradecanoyl-phorbol-13-acetate (TPA) or mezerein plus phosphatidylserine, Ca2+ plus arachidonic acid, or arachidonic acid alone. Rhodamine 6G did not affect protein kinase C activity in the absence of lipid cofactor and, thus, does not appear to inhibit the enzyme through direct interactions with the active site. The inhibitory potency of the drug was affected dramatically by the nature of the lipid cofactor. Thus, 50 microM rhodamine 6G inhibited the Ca2+ plus arachidonic acid dependent protein kinase activity approximately 50%, whereas 800 microM rhodamine 6G was required to cause 50% inhibition of the Ca2+ plus phosphatidylserine dependent protein kinase activity. These results, along with studies demonstrating a reversal of inhibition by high lipid concentrations, provide evidence that rhodamine 6G exerts its inhibitory effect on PKC through drug-lipid interactions. The dramatic effect of the lipid cofactor on the potency of rhodamine 6G as a PKC inhibitor suggests that the lipid environment of the cell may profoundly affect the abilities of rhodamine 6G and related cationic lipophilic drugs to inhibit PKC in vivo.

Binding of protein kinase C to N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide through its ATP binding site.

Protein kinase C (PKC) is a Ca2+- and phospholipid-dependent protein kinase which has been implicated as a key enzyme in the regulation of cellular growth. The naphthalenesulfonamide W7 [N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide] is representative of a number of cationic amphiphilic inhibitors of PKC which appear to inhibit PKC by interacting with the acidic phospholipid cofactor of the enzyme, according to kinetic studies. In a previous report, we demonstrated that PKC binds directly to W7 when the naphthalenesulfonamide is immobilized on agarose. In the present report, we have defined the mechanism of the binding of PKC to W7-agarose, and its relevance to the inhibitory mechanism of the naphthalenesulfonamide. We demonstrate that PKC bound W7-agarose through the catalytic domain of the enzyme. An active catalytic fragment of PKC was generated by limited proteolysis, and we found that this fragment bound W7-agarose and coeluted with intact PKC upon the addition of Triton X-100. W7 inhibited PKC activity by two different mechanisms. As previously reported, W7 inhibited PKC by interacting with the phospholipid cofactor of the enzyme (IC50 = 260 microM). However, at higher concentrations of W7, we found that this naphthalenesulfonamide inhibited PKC by serving as a competitive inhibitor with respect to the substrate ATP, according to a kinetic analysis of the inhibition of the active catalytic fragment of PKC by W7. W7 inhibited the active catalytic fragment of PKC as well as PKC-catalyzed phosphorylation of protamine sulfate, a reaction which is independent of Ca2+ and phospholipid, with similar potencies. Consistent with the kinetic evidence that W7 serves as a competitive inhibitor of PKC with respect to ATP, we found that, in the presence of 10 mM MgCl2, 1 mM ATP was sufficient to elute PKC from W7-agarose. Thus, naphthalenesulfonamide PKC inhibitors may include both agents which primarily function by interacting with the phospholipid cofactor of the enzyme and agents which primarily serve as active site inhibitors of PKC.

Effects of resveratrol on the autophosphorylation of phorbol ester-responsive protein kinases: inhibition of protein kinase D but not protein kinase C isozyme autophosphorylation.

The natural product resveratrol is a potent antagonist of phorbol ester-mediated tumor promotion and in vitro cellular responses to phorbol-ester tumor promoters, but it is only weakly inhibitory against the phosphorylation of conventional exogenous substrates by phorbol ester-responsive protein kinase C (PKC) isozymes. In this report, we compare the effects of resveratrol against the autophosphorylation reactions of PKC isozymes versus the novel phorbol ester-responsive kinase, protein kinase D (PKD). We found that resveratrol inhibits PKD autophosphorylation in a concentration-dependent manner, but has only negligible effects against the autophosphorylation reactions of representative members of each PKC isozyme subfamily (cPKC-alpha, -beta(1), and -gamma, nPKC-delta and -epsilon, and aPKC-zeta). Resveratrol was comparably effective against PKD autophosphorylation (IC(50) = 52 microM) and PKD phosphorylation of the exogenous substrate syntide-2 (IC(50) = 36 microM). The inhibitory potency of resveratrol against PKD is in line with the potency of resveratrol observed in cellular systems and with its potency against other purified enzymes and binding proteins that are implicated in the cancer chemopreventive activity of the polyphenol. Thus, PKD inhibition may contribute to the cancer chemopreventive action of resveratrol.