Purified Protein Kinase C Activity Research Articles

Anti-inflammatory activity of Chios mastic gum is associated with inhibition of TNF-alpha induced oxidative stress

BackgroundGum of Chios mastic (Pistacia lentiscus var. chia) is a natural antimicrobial agent that has found extensive use in pharmaceutical products and as a nutritional supplement. The molecular mechanisms of its anti-inflammatory activity, however, are not clear. In this work, the potential role of antioxidant activity of Chios mastic gum has been evaluated.MethodsScavenging of superoxide radical was investigated by electron spin resonance and spin trapping technique using EMPO spin trap in xanthine oxidase system. Superoxide production in endothelial and smooth muscle cells stimulated with TNF-α or angiotensin II and treated with vehicle (DMSO) or mastic gum (0.1-10 μg/ml) was measured by DHE and HPLC. Cellular H2O2 was measured by Amplex Red. Inhibition of protein kinase C (PKC) with mastic gum was determined by the decrease of purified PKC activity, by inhibition of PKC activity in cellular homogenate and by attenuation of superoxide production in cells treated with PKC activator phorbol 12-myristate 13-acetate (PMA).ResultsSpin trapping study did not show significant scavenging of superoxide by mastic gum itself. However, mastic gum inhibited cellular production of superoxide and H2O2 in dose dependent manner in TNF-α treated rat aortic smooth muscle cells but did not affect unstimulated cells. TNF-α significantly increased the cellular superoxide production by NADPH oxidase, while mastic gum completely abolished this stimulation. Mastic gum inhibited the activity of purified PKC, decreased PKC activity in cell homogenate, and attenuated superoxide production in cells stimulated with PKC activator PMA and PKC-dependent angiotensin II in endothelial cells.ConclusionWe suggest that mastic gum inhibits PKC which attenuates production of superoxide and H2O2 by NADPH oxidases. This antioxidant property may have direct implication to the anti-inflammatory activity of the Chios mastic gum.

A Direct Redox Regulation of Protein Kinase C Isoenzymes Mediates Oxidant-induced Neuritogenesis in PC12 Cells

In this study, we have used the PC12 cell model to elucidate the mechanisms by which sublethal doses of oxidants induce neuritogenesis. The xanthine/xanthine oxidase (X/XO) system was used for the steady state generation of superoxide, and CoCl(2) was used as a representative transition metal redox catalyst. Upon treatment of purified protein kinase C (PKC) with these oxidants, there was an increase in its cofactor-independent activation. Redox-active cobalt competed with the redoxinert zinc present in the zinc-thiolates of the PKC regulatory domain and induced the oxidation of these cysteine-rich regions. Both CoCl(2) and X/XO induced neurite outgrowth in PC12 cells, as determined by an overexpression of neuronal marker genes. Furthermore, these oxidants induced a translocation of PKC from cytosol to membrane and subsequent conversion of PKC to a cofactor-independent form. Isoenzyme-specific PKC inhibitors demonstrated that PKCepsilon plays a crucial role in neuritogenesis. Moreover, oxidant-induced neurite outgrowth was increased with a conditional overexpression of PKCepsilon and decreased with its knock-out by small interfering RNA. Parallel with PKC activation, an increase in phosphorylation of the growth-associated neuronal protein GAP-43 at Ser(41) was observed. Additionally, there was a sustained activation of extracellular signal-regulated kinases 1 and 2, which was correlated with activating phosphorylation (Ser(133)) of cAMP-responsive element-binding protein. All of these signaling events that are causally linked to neuritogenesis were blocked by antioxidant N-acetylcysteine (both L and D-forms) and by a variety of PKC-specific inhibitors. Taken together, these results strongly suggest that sublethal doses of oxidants induce neuritogenesis via a direct redox activation of PKCepsilon.

Angoline and Chelerythrine, Benzophenanthridine Alkaloids That Do Not Inhibit Protein Kinase C

Starting with an extract derived from the stem of Macleaya cordata (Papaveraceae) that was active in the process of inhibiting phorbol 12,13-dibutyrate binding to partially purified protein kinase C (PKC), the benzophenanthridine alkaloid angoline was isolated and identified. This discovery appeared in context, as a related benzophenanthridine alkaloid, chelerythrine, has been reported to mediate a variety of biological activities, including potent and selective inhibition of protein kinase C (PKC). However, in our studies, angoline was not observed to function as a potent inhibitor of PKC. Moreover, we were unable to confirm the reported inhibitory activity of chelerythrine. In a comprehensive series of studies performed with various PKC isozymes derived from a variety of mammalian species, neither chelerythrine nor angoline inhibited activity with high potency. To the contrary, chelerythrine stimulated PKC activity in the cytosolic fractions of rat and mouse brain in concentrations up to 100 microM. In addition, chelerythrine and angoline did not inhibit [3H]phorbol 12,13-dibutyrate binding to the regulatory domain of PKC at concentrations up to 40 microg/ml, and no significant alteration of PKC-alpha, -beta, or -gamma translocation was observed with human leukemia (HL-60) cells in culture. Further, chelerythrine did not inhibit 12-O-tetradecanoylphorbol 13-acetate-induced ornithine decarboxylase activity with cultured mouse 308 cells, but angoline was active in this capacity with an IC50 value of 1.0 microg/ml. A relatively large number of biological responses have been reported in studies conducted with chelerythrine, and alteration of PKC activity has been considered as a potential mechanism of action. In light of the current report, mechanisms independent of PKC inhibition should be considered as responsible for these effects.

Identification of a protein kinase C (PKC) activator, daphnoretin, that suppresses hepatitis B virus gene expression in human hepatoma cells

We examined the antiviral activity of a crude extract prepared from a Chinese medicinal herb Wikstroemia indica C.A. Mey. One active component, daphnoretin (7-hydroxyl-6-methoxy-3,7′-dicoumarylether), was identified, which showed strong suppressive effects on the expression of the hepatitis B surface antigen (HBsAg) in human hepatoma Hep3B cells. To examine the signaling pathway of daphnoretin on the Hep3B cells, we pretreated Hep3B cells with 12- O-tetradecanoylphorbol-13- O-acetate (200 nM) for 24 hr to down-regulate intracellular protein kinase C (PKC) levels and found that the PKC-down-regulated Hep3B cells did not respond at all to daphnoretin. Furthermore, daphnoretin induced translocation of PKC from the cytosol to the membrane and down-regulated intracellular PKC levels in the Hep3B cells, indicating that it may directly activate PKC. This hypothesis was supported by the observation that daphnoretin directly competed with [ 3H]phorbol dibutyrate for the binding of PKC in the whole cell and activated purified PKC activity in vitro. Our results demonstrated that daphnoretin, with a structure distinct from phorbol ester, is a PKC activator and has suppressive effects on HBsAg gene expression in human hepatoma cells.

Phosphatidic acid activation of protein kinase C in LA-N-1 neuroblastoma cells

Phosphatidic acid (PA), a hydrolytic product of phospholipase D activity, stimulated cytosolic protein kinase C (PKC) activity when LA-N-1 neuroblastoma cells in culture were treated with PA, without translocating the enzyme to the membrane. Treatment of cells with 12- O-tetradecanoylphorbol-13-acetate (TPA) translocated and activated PKC in a dogmatic manner. Partially purified PKC activity derived from LA-N-1 neuroblastoma cells was stimulated by PA alone or in the presence of phosphatidylserine or TPA, without affecting [ 3H]phorbol dibutyrate binding, indicating that the site of action of PA was different from the phorbol ester or diacylglycerol binding site. These results suggest an unorthodox pattern of PKC stimulation mediated by PA which appears to be yet another mode of PA signal transduction.

The lipophosphoglycan of Leishmania and macrophage protein kinase C

Lipophosphoglycan (LPG), the major cellsurface glycoconjugote of Leishmania promastigotes, is on essential virulence determinant. One feature of LPG resides in its strong inhibitory effect on the activity of purified protein kinase C (PKC). In this article, Albert Descoteaux and Salvatore Turco briefly review the evidence that LPG effectively inhibits PKC activity in the macrophage, and discuss the implication of such inhibition on Leishmania intromocrophoge survival.

Inhibition of macrophage protein kinase C-mediated protein phosphorylation by Leishmania donovani lipophosphoglycan.

The cell surface lipophosphoglycan (LPG) from Leishmania donovani promastigotes is a potent inhibitor of purified protein kinase C (PKC) activity in vitro. In this study, we have investigated the effect of LPG on the activation process of PKC in murine bone marrow-derived macrophages. The extent and kinetics of calcium ionophore A23187-induced [3H] phorbol dibutyrate binding to macrophages were not affected by LPG pretreatment or infection with either wild-type or LPG-deficient promastigotes, indicating no effect on the association of calcium-dependent PKC with the plasma membrane. In contrast, LPG inhibited the phosphorylation of both the PKC-specific VRKRTRLLR substrate peptide and MARCKS, and endogenous PKC substrate, in 1-oleoyl-2-acetyl-glycerol-stimulated macrophages. These observations provide direct evidence that LPG effectively inhibits PKC activity in intact macrophages. Finally, depletion of PKC rendered macrophages more permissive for the proliferation of L. donovani, suggesting that inhibition of PKC-dependent events contributes to the survival of this parasite within its host cell.

Purification of PKC-I, an endogenous protein kinase C inhibitor, and types II and III protein kinase C isoenzymes from human neutrophils.

Human neutrophil protein kinase C (PKC) activity is inhibited by an endogenous protein found primarily in the pellet fraction from homogenized specific granules, which was both heat- and proteinase-sensitive [Balazovich, Smolen & Boxer (1986) J. Immunol. 137, 1665-1673]. We now report that two PKC isoenzymes and the endogenous PKC inhibitor, which we named PKC-I, were purified from human neutrophils. A neutrophil soluble fraction that was subjected to DEAE-Sephacel chromatography yielded highly enriched PKC because, by definition, enzymic activity was strictly dependent on Ca2+ and phosphatidylserine. Hydroxyapatite chromatography resolved two peaks of PKC activity. Type II and Type III PKC isoenzymes were each identified on Western blots by using isoenzyme-specific monoclonal antibodies. Unlike rat brain, from which PKC isoenzymes were also purified, Type I PKC was not detected in human neutrophils. Western blots indicated that both Type II and Type III PKC isoenzymes had molecular masses near 80 kDa. In agreement with other reports, PKC was autophosphorylated in vitro. PKC-I, an endogenous neutrophil inhibitor of PKC, was purified to apparent homogeneity by DEAE-Sephacel and S-400 Sephacel chromatography. PKC-I had a molecular mass of 41 kDa. PKC-I inhibited purified PKC activity stimulated by 1,2-diacylglycerols in a concentration-dependent manner, and inhibited PKC-dependent phosphorylation of proteins present in neutrophil cytosol.

Translocation of protein kinase C in rat islets of Langerhans Effects of a phorbol ester, carbachol and glucose

In unstimulated rat islets (2 mM glucose), most of the ion-exchange purified protein kinase C (PKC) activity was associated with the cytosolic fraction. Both carbachol and phorbol myristate acetate caused a significant translocation of PKC activity from cytosolic to membrane fractions, but under the same conditions, glucose (20 mM) did not cause such a redistribution of PKC activity. PMA-induced translocation of PKC to the membrane fraction was also observed in electrically permeabilised islets, in which recovery of the enzyme activity was enhanced by buffering the intracellular Ca 2+ concentration to 50 nM and supplying the permeabilised islets with protease inhibitors.

Constitutive activity of membrane-inserted protein kinase C

Incubation of purified protein kinase C (PKC) with phospholipid vesicles produced two populations of membrane-bound PKC: one population was dissociated by calcium chelation and the other was not. The second population appeared to be inserted into the membrane. The activity of membrane-inserted PKC was Ca2+-independent and was only modestly sensitive to phorbol esters. Insertion was caused by high calcium concentrations or by phorbol esters plus low calcium. These conditions correlated with those needed to activate PKC; insertion into the membrane may be a primary mechanism of PKC activation. PKC may be a long-term cell regulator which becomes inserted into the membrane upon appearance of the second messengers, calcium and diacylglycerol, and remains in an active membrane-bound state when the second messengers have been removed.

The lipophilic muramyltripeptide MTP-PE, a biological response modifier, is an activator of protein kinase C

The lipophilic immunomodulator MTP-PE is able to activate purified protein kinase C (PKC) by substituting phosphatidylserine (PS) or the synthetic diacylglycerol, DiC 8, in the assay system. In addition, MTP-PE inhibited [ 3H]-phorbol-12, 13-dibutyrate ([ 3H]-PDBu) binding to PKC in a reconstituted receptor system as well as on intact cells (MCF-7). Furthermore, MTP-PE was also able to reduce the epidermal growth factor binding of MCF-7 cells to an extent similar to that found with DiC 8 or PDBu. These data indicate that MTP-PE is able to compete for the phorbol ester binding site on PKC both in vivo and in vitro. The components of the MTP-PE molecule, MTP (muramyltripeptide) and PE (phosphatidylethanolamine) exerted only marginal effects on PKC activity, did not affect the phorbol ester binding of PKC and the EGF binding of intact MCF-7 cells. Our results suggest that only the complete molecule of the immunomodulator MTP-PE is able to interact with PKC.

Possible involvement of a lanthanide-sensitive protein kinase C substrate in lanthanide promotion of neoplastic transformation.

The rare earth elements lanthanum and terbium (0.1-1.0 mM), pharmacological analogs of calcium, induced neoplastic transformation of 12-O-tetradecanoylphorbol-13-acetate (TPA)-sensitive (P+) and to a lesser extent TPA-resistant (P-) preneoplastic mouse JB6 epidermal cells. A maximum of 2500 anchorage-independent colonies per 10(4) cells were induced in P+ lines, a response comparable to that induced by phorbol esters (1.6-16 nM). The maximum lanthanide-induced colony yield in P- lines was 20% of that in P+ lines (approximately 550 colonies), and was observed under conditions where TPA induced less than 30 colonies per 10(4) cells. Lanthanides and TPA produced a synergistic effect on colony size in P+ cells. Lanthanides are not promoting transformation merely by mimicking high calcium: adding exogenous extracellular calcium (up to 50.0 mM) or using calcium ionophore (up to toxic concentrations) to increase intracellular calcium does not promote transformation. Lanthanum will substitute for calcium in activating partially purified protein kinase C (PKC), the calcium-dependent phorbol ester receptor. However, lanthanides must be promoting transformation by a mechanism other than PKC activation because lanthanides failed to activate PKC in intact JB6 cells. Three independent experiments showed a lack of lanthanum effect on PKC-dependent events in intact cells. First, in contrast to TPA, lanthanum pretreatment of JB6 cells did not produce elevated phosphorylation of an 80-kd substrate. Second lanthanum pretreatment did not cause decreased PKC activity after prolonged exposure. Third, lanthanum and TPA affected epidermal growth factor binding with a different magnitude, time course and calcium dependency. We found, however, a PKC substrate in P+, P- and tumorigenic cell lines that is sensitive to lanthanum and increases its migration in sodium dodecylsulfate-polyacrylamide gels from 23 to 21 kd. The above data suggest that: (i) alterations in cation binding may be sufficient for inducing the transformed phenotype; and (ii) lanthanide promotion of neoplastic transformation may be linked to a lanthanide-sensitive PKC substrate, but is not due to a direct PKC activation.