Activation by phorbol esters of protein kinase C in MCF-7 human breast cancer cells

Exposure of MCF-7 human breast cancer cells to the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) leads to the inhibition of cell proliferation. We investigate here the short-term effects of TPA on subcellular distribution of protein kinase C, and on protein phosphorylation in cultured MCF-7 cells. We report a rapid and dramatic decrease in cytosolic protein kinase C activity after TPA treatment. Only 30% of the enzymatic activity lost in the cytosol was recovered in the particulate fraction. These data suggest that subcellular translocation of protein kinase C is accompanied by a rapid down-regulation of the enzyme (70%). Furthermore, TPA and other protein kinase C activators rapidly induce the phosphorylation of a 28 kDa protein in intact MCF-7 cells. Phorbol esters devoid of tumor-promoting activity are ineffective both for inducing these early biochemical events and for inhibiting cell proliferation.     

Regulation of phospholipase D by protein kinase C

In nearly all mammalian cells and tissues examined, protein kinase C (PKC) has been shown to serve as a major regulator of a phosphatidylcholine-specific phospholipase D (PLD) activity, At least 12 distinct isoforms of PKC have been described so far; of these enzymes only the α- and β-isoform were found to regulate PLD activity, While the mechanism of this regulation has remained unknown, available evidence suggests that both phosphorylating and non-phosphorylating mechanisms may be involved. A phosphatidylcholine-specific PLD activity was recently purified from pig lung, but its possible regulation by PKC has not been reported yet. Several cell types and tissues appear to express additional forms of PLD which can hydrolyze either phosphatidylethanolamine or phosphatidylinositol. It has also been reported that at least one form of PLD can be activated by oncogenes, but not by PKC activators, Similar to activated PKC, some of the primary and secondary products of PLD-mediated phospholipid hydrolysis, including phosphatidic acid, 1,2-diacylglycerol, choline phosphate and ethanolamine, also exhibit mitogenic/co-mitogenic effects in cultured cells. Furthermore, both the PLD and PKC systems have been implicated in the regulation of vesicle transport and exocytosis. Recently the PLD enzyme has been cloned and the tools of molecular biology to study its biological roles will soon be available. Using specific inhibitors of growth regulating signals and vesicle transport, so far no convincing evidence has been reported to support the role of PLD in the mediation of any of the above cellular effects of activated PKC.     

Conventional protein kinase C isoforms mediate neuroprotection induced by phorbol ester and estrogen

Rapid signal transduction pathways play a prominent role in mediating neuroprotective actions of estrogen in the CNS. We have previously shown that estrogen-induced neuroprotection of primary cerebrocortical neurons from beta-amyloid peptide (Abeta) toxicity depends on activation of protein kinase C (PKC). PKC activation with phorbol-12-myristate-13-acetate (PMA) also provides neuroprotection in this paradigm. Because the PKC family includes several isoforms that have opposing roles in regulating cell survival, we sought to identify which PKC isoforms contribute to neuroprotection induced by PMA and estrogen. We detected protein expression of multiple PKC isoforms in primary neuron cultures, including conventional (alpha, betaI, betaII), novel (delta, epsilon, theta) and atypical (zeta, iota/lambda) PKC. Using a panel of isoform-specific peptide inhibitors and activators, we find that novel and atypical PKC isoforms do not participate in the mechanism of either PMA or estrogen neuroprotection. In contrast, a selective peptide activator of conventional PKC isoforms provides dose-dependent neuroprotection against Abeta toxicity. In addition, peptide inhibitors of conventional, betaI, or betaII PKC isoforms significantly reduce protection afforded by PMA or 17beta-estradiol. Taken together, these data provide evidence that conventional PKC isoforms mediate phorbol ester and estrogen neuroprotection of cultured neurons challenged by Abeta toxicity.     

A phorbol ester and phospholipid-activated, calcium-unresponsive protein kinase in mouse epidermis: characterization and separation from protein kinase C

The phosphorylation of an Mr 82,000 protein (p82) in the Triton X-100 extract of the particulate fraction of mouse epidermis is dependent on the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) or diacylglycerol and phospholipid and, contrary to protein kinase C (PKC)-catalyzed phosphorylation, cannot be activated by calcium plus phospholipid. The novel p82 kinase differs also from PKC in many other respects, such as substrate specificity, turnover rate, and sensitivity to inhibitors. The p82 kinase can be separated from PKC by chromatography on phenyl sepharose and does not react with a polyclonal PKC antiserum. Like PKC, the novel kinase phosphorylates its substrate on threonine and serine, but not on tyrosine. Similar to PKC, the epidermal p82-kinase system is down-modulated after TPA treatment of mouse skin, with a half-life of around 5 h. Down-modulation is also accomplished by the phorbol ester RPA, but not by the Ca2+ ionophore A23187, and it is inhibited by the immunosuppressive agent cyclosporin A. In addition to down-modulation, TPA treatment of the animals activates a phosphatase that dephosphorylates phosphorylated p82 in the extract of the particulate fraction.     

Activation of conventional and novel protein kinase C isozymes by different diacylglycerol molecular species

A variety of diacylglycerol (DG) molecular species are produced in stimulated cells. Conventional (α, βII and γ) and novel (δ, ε, η and θ) protein kinase C (PKC) isoforms are known to be activated by DG. However, a comprehensive analysis has not been performed. In this study, we analyzed activation of the PKC isozymes in the presence of 2–2000 mmol% 16:0/16:0-, 16:0/18:1-, 18:1/18:1-, 18:0/20:4- or 18:0/22:6-DG species. PKCα activity was strongly increased by DG and exhibited less of a preference for 18:0/22:6-DG at 2 mmol%. PKCβII activity was moderately increased by DG and did not have significant preference for DG species. PKCγ activity was moderately increased by DG and exhibited a moderate preference for 18:0/22:6-DG at 2 mmol%. PKCδ activity was moderately increased by DG and exhibited a preference for 18:0/22:6-DG at 20 and 200 mmol%. PKCε activity moderately increased by DG and showed a moderate preference for 18:0/22:6-DG at 2000 mmol%. PKCη was not markedly activated by DG. PKCθ activity was the most strongly increased by DG and exhibited a preference for 18:0/22:6-DG at 2 and 20 mmol% DG. These results indicate that conventional and novel PKCs have different sensitivities and dependences on DG and a distinct preference for shorter and saturated fatty acid-containing and longer and polyunsaturated fatty acid-containing DG species, respectively. This differential regulation would be important for their physiological functions.     

Protein kinase C as a possible receptor protein of tumor-promoting phorbol esters

A tumor-promoting phorbol ester, [3H]phorbol-12,13-dibutyrate, may bind to a homogeneous preparation of Ca2+-activated, phospholipid-dependent protein kinase (protein kinase C) in the simultaneous presence of Ca2+ and phospholipid. This tumor promoter does not bind simply to phospholipid nor to the enzyme per se irrespective of the presence and absence of Ca2+. All four components mentioned above appear to be bound together, and the quaternary complex thus produced is enzymatically fully active for protein phosphorylation. Phosphatidylserine is most effective. Various other phorbol derivatives which are active in tumor promotion compete with [3H]phorbol-12,13-dibutyrate for the binding, and an apparent dissociation binding constant of the tumor promoter is 8 nM. This value is identical with the activation constant for protein kinase C and remarkably similar to the dissociation binding constant that is described for intact cell surface receptors. The binding of the phorbol ester is prevented specifically by the addition of diacylglycerol, which serves as activator of protein kinase C under physiological conditions. Scatchard analysis suggests that one molecule of the tumor promoter may bind to every molecule of protein kinase C in the presence of Ca2+ and excess phospholipid. It is suggestive that protein kinase C is a phorbol ester-receptive protein, and the results presented seem to provide clues for clarifying the mechanism of tumor promotion.     

Activation of protein kinase C in neutrophil cytoplasts: Localization of protein substrates and possible relationship with stimulus-response coupling

Treatment of enucleated, granule-free neutrophil cytoplasts with the protein kinase C activator phorbol 12O-myristate-13-acetate (PMA) causes an increased -³²P-incorporation into a variety of polypeptides. Permeabilization of PMA-stimulated, ³²P-labeled cytoplasts by 0.01% digitonin fully releases the majority of these phosphorylated proteins. A statistically significant correlation is found between the extent of PMA-induced activation of generation of Superoxide anion (O⁻₂) and the phosphorylation of a cytosolic polypeptide with an apparent M_r, of 46000, whose -³²P-labeling is also enhanced by the treatment of cytoplasts with 1-oleyl-2-acetylglycerol, the Ca²⁺ ionophore ionomycin or latex beads. Furthermore, treatment of cytoplasts with the protein kinase C inhibitor trifluoperazine markedly inhibits the ³²P-labeling of proteins in the 40000 M_r range, including the 46 kDa polypeptide, and almost totally abolishes the activation of O⁻₂ production by PMA.     

Phosphorylation of liver gap junction protein by protein kinase C

The 27 kDa protein, a major component of rat liver gap junctions, was shown to be phosphorylated in vitro by protein kinase C. The stoichiometry of the phosphorylation indicated that approx. 0.33 mol phosphate was incorporated per mol 27 kDa protein. Phosphorylation was entirely dependent on the presence of calcium and was virtually specific for serine residues. For comparison, the gap junction protein was also examined for its phosphorylation by cAMP-dependent protein kinase, the extent of phosphorylation being one-tenth that exerted by protein kinase C.     

Protein kinase C isoforms in pituitary cells displaying differential sensitivity to phorbol ester

Investigations with protein kinase C (PKC) isoform-specific antisera, revealed distinct profiles of PKC isoform content amongst pituitary tissues. Western analysis revealed the and isoforms of PKC are present in rat anterior and posterior pituitary tissue as well as in the GH₃ somatomammotrophic cell line. AtT-20/D16-V corticotrophic and T3-1 gonadotrophic murine cell lines contained no PKC-. The or isoforms were undetected in any pituitary tissue. PKC activity measurements revealed Ca²⁺-independent PKCs in T3-1 and GH₃ cells which were more sensitive to activation by phorbol-dibutyrate (PDBu) than the corresponding PKC activity found in COS cells. However, Ca²⁺-dependent PKC activities were of similar sensitivity to PDBu in GH₃, T3-1 and COS cells, indicating that functional differences observed in PDBu-sensitivity in these cells may be due to differential activation of Ca²⁺-independent PKC isoforms. Moreover, substrate-specificity of these PKCs were also compared indicating that the amount of Ca²⁺-dependency of the observed PKC activity from the same pituitary tissue is dependent upon the substrate utilized by the PKC isotypes present. These findings explain differential sensitivities of PKC-mediated actions that have previously been observed in a range of pituitary cells. (Mol Cell Biochem 000-000, 1999)     

Mutual regulation of conventional protein kinase C and a ubiquitin ligase complex

Several isoforms of protein kinase C (PKC) are degraded by the ubiquitin-proteasome pathway after phorbol ester-mediated activation. However, little is known about the ubiquitin ligase (E3) that targets activated PKCs. We recently showed that an E3 complex composed of HOIL-1L and HOIP (LUBAC) generates linear polyubiquitin chains and induces the proteasomal degradation of a model substrate. HOIL-1L has also been characterized as a PKC-binding protein. Here we show that LUBAC preferentially binds activated conventional PKCs and their constitutively active mutants. LUBAC efficiently ubiquitinated activated PKC in vitro, and degradation of activated PKCalpha was delayed in HOIL-1L-deficient cells. Conversely, PKC activation induced cleavage of HOIL-1L and led to downregulation of the ligase activity of LUBAC. These results indicate that LUBAC is an E3 for activated conventional PKC, and that PKC and LUBAC regulate each other for proper PKC signaling.     

Ceramide does not inhibit protein kinase C β-dependent phospholipase D activity stimulated by anti-Fas monoclonal antibody in A20 cells

We have investigated the roles of ceramide in Fas signalling leading to phospholipase D (PLD) activation in A20 cells. Upon stimulation of Fas signalling by anti-Fas monoclonal antibody, sphingomyelin hydrolysis and activation of PLD were induced. Also, the translocation of protein kinase C (PKC) βI and βII and the elevation of diacylglycerol (DAG) content were induced by Fas cross-linking. When phosphatidylcholine-specific phospholipase C (PC-PLC) was inhibited by D609, the Fas-induced changes in PLD activity, DAG content, and PKC translocation were inhibited. In contrast, D609 had no effect on Fas-induced alterations in sphingolipid metabolism, suggesting that changes in ceramide content do not account for Fas-induced PLD activation. Furthermore, C6-ceramide had no effect on Fas-induced PLD activation and PKC translocation. Taken together, these data might suggest that ceramide generated by Fas cross-linking does not affect PKC β-dependent PLD activity stimulated by anti-Fas monoclonal antibody in A20 cells.     

Sphingosine kinase-dependent directional migration of leukocytes in response to phorbol ester

Syndecan-4 participates in focal adhesion by non-G protein-dependent activation of protein kinase C. Ligation of syndecan-4 with antithrombin elicits pertussis toxin-sensitive chemotaxis of leukocytes. As activation of protein kinase C stimulates release of sphingosine-1-phosphate, a chemoattracting G protein-coupled receptor agonist, we studied directional migration of leukocytes in response to phorbol myristate acetate (PMA), a direct activator of protein kinase C. Human peripheral blood neutrophils, monocytes, and lymphocytes were purified and tested for chemotactic migration in micropore filter assays in response to PMA. Dose-dependent stimulation of migration was seen only when leukocytes were exposed to concentration gradients of PMA; in the absence of such a gradient, inhibition of random migration was induced. Dimethylsphingosine inhibited PMA-induced leukocyte chemotaxis, indicating that activation of sphingosine kinase for enhanced production of sphingosine-1-phosphate mediates the chemotactic response to PMA. Pertussis toxin abrogated the chemotactic response to PMA, suggesting involvement of G protein-coupled sphingosine-1-phosphate receptor. Dimethylsphingosine also inhibited leukocyte chemotaxis toward antithrombin, indicating that similar mechanisms may be involved upon syndecan-4 ligation. Data show that protein kinase C-dependent activation of sphingosine kinase may play a central role in leukocyte chemotaxis toward non-G protein-coupled receptor agonists.     

A point mutation at phenylalanine 663 abolishes protein kinase C alpha's ability to translocate to the perinuclear region and activate phospholipase D1

Previous research showed that protein kinase C alpha (PKC alpha) translocated to the perinuclear region and activated phospholipase D1, but the mechanism involved was not clear. Here, we provide evidence that Phe 663 (the 10th amino acid from C-terminus) of PKC alpha is essential for its translocation. A point mutation (F663D) completely blocked PKC alpha's binding to and activation of phospholipase D1. Further studies showed that deletion of the C-terminal nine amino acids of PKC alpha did not alter its translocation to the perinuclear region but deletion of the C-terminal 10 amino acids and the F663D mutation abolished this translocation. The F663D mutant was found to be resistant to dephosphorylation, which might account for its inability to translocate to the perinuclear region and activate PLD1, since dephosphorylation of PKC alpha is required for its relocation from plasma membrane to the perinuclear region.     

Dual regulation of phospholipase D1 by protein kinase C alpha in vivo

The regulation of phospholipase D1 (PLD1), which has been shown to be activated by protein kinase C (PKC) alpha, was investigated in the human melanoma cell lines. In G361 cell line, which lacks PKCalpha, 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced PLD activation was potentiated by introducing PKCalpha by the adenovirus vector. The kinase-negative PKCalpha elevated TPA-induced PLD activity less significantly than the wild type. A PKC specific inhibitor GF109203X lowered PLD activation in the cells expressing PKCalpha, but did not prevent PLD potentiation induced by the kinase-negative PKCalpha. Expression of PKCbetaII and the kinase-negative PKCbetaII enhanced TPA-stimulated PLD activity moderately in MeWo cell line, in which PKCbetaII is absent. Furthermore, the TPA treatment increased the association of PKCalpha, PKCbetaII, and their kinase-negative mutants with PLD1 in melanoma cells. These results indicate that PLD1 is dually regulated through phosphorylation as well as through the protein-protein interaction by PKCalpha, and probably by PKCbetaII, in vivo.     

Structure and function of the protein kinase C gene family

Protein kinase C is a serine/threonine protein kinase which is activated in the cell in response to production of diacylglycerol. Gene cloning has revealed the presence of a highly related family of enzymes, which can be sub-divided into groups on the basis of sequence conservation. Differences are seen in both isoform distribution and associated biochemical activity, for example in substrate specificity and activator requirements. Comparison of the protein sequences andin vitro activities of the protein kinase C isoforms has identified regions important for particular aspects of kinase function. Some of these regions are also found associated with other proteins, allowing confirmation of the assigned activity. Site-directed mutagenesis has confirmed the presence of an autoinhibitory sequence involved in protein kinase C regulation and generated constitutively activated proteins which can be used to study differential isoform function. These same sequences have been shown to play a role in substrate selection, perhaps by competition for binding to the active site. Protein kinase C is known to be a phosphoprotein and the identification of regulatory sites phosphorylated by a ‘PKC-kinase’ suggest a possible alternative route for regulation of protein kinase C activity.     

RasGRP3 mediates phorbol ester-induced, protein kinase C-independent exocytosis

Phorbol esters are involved in neurotransmitter release and hormone secretion via activation of protein kinase C (PKC). In addition, it has been recently reported to enhance neurotransmitter release in a PKC-independent manner. However, the exocytotic machinery is not fully clarified. Nowadays members of the RasGRP family are being identified as novel molecules binding to diacylglycerol and calcium, representing a new class of guanine nucleotide exchange factor that activates small GTPases including Ras and Rap1. In the present study, we demonstrated that RasGRP3 is expressed in endocrine tissues and mediates phorbol ester-induced exocytosis. Furthermore, the effects were partially blocked by PKC inhibitor but not mitogen-activated protein kinase kinase inhibitor, although both significantly suppressed the phorbol ester-induced phosphorylation of extracellular signal-regulated kinase 1/2. These results indicate that RasGRP3 is implicated in phorbol ester-induced, PKC-independent exocytosis.     

The membrane effects of 17beta-estradiol on chondrocyte phenotypic expression are mediated by activation of protein kinase C through phospholipase C and G-proteins

Growth plate chondrocytes from both male and female rats have nuclear receptors for 17β-estradiol (E₂); however, recent studies indicate that an alternative pathway involving a membrane receptor may also be involved in the female cell response. E₂ directly affects the fluidity of chondrocyte membranes derived from female, but not male, rats. In addition, E₂ activates PKC in a nongenomic manner in female cells, and chelerythrine, a specific inhibitor of PKC, inhibits E₂-dependent alkaline phosphatase activity in these cells, indicating PKC is involved in the signal transduction mechanism. The aims of this study were: (1) to examine if PKC mediates the effect of E₂ on chondrocyte proliferation, differentiation, and matrix synthesis; and (2) to determine the pathway that mediates the membrane effect of E₂ on PKC. Confluent, fourth passage resting zone (RC) and growth zone (GC) chondrocytes from female rat costochondral cartilage were treated with 10⁻¹⁰ to 10⁻⁷ M E₂ in the presence or absence of the PKC inhibitor chelerythrine, and changes in alkaline phosphatase specific activity, proteoglycan sulfation, and [³H]thymidine incorporation were measured. To examine the pathway of PKC activation, chondrocyte cultures were treated with E₂ in the presence or absence of genistein (an inhibitor of tyrosine kinases), U73122 or D609 (inhibitors of phospholipase C [PLC]), quinacrine (an inhibitor of phospholipase A₂ [PLA₂]), and melittin (an activator of PLA₂). Alkaline phosphatase specific activity and proteoglycan sulfation were increased and [³H]thymidine incorporation was decreased by E₂. The effects of E₂ on all parameters were blocked by chelerythrine. Treatment of the cultures with E₂ produced a significant dose-dependent increase in PKC. U73122 dose-dependently inhibited the activation of PKC in E₂-stimulated female chondrocyte cultures. However, the classical receptor antagonist ICI 182780 was unable to block the stimulatory effect of E₂ on PKC. Moreover, the classical receptor agonist diethylstilbestrol (DES) had no effect on PKC, nor did it alter the stimulatory effect of E₂. Inhibition of tyrosine kinase and PLA₂ had no effect on the activation of PKC by E₂. The PLA₂ activator also had no effect on PKC activation by E₂. E₂ stimulated PKC activity in membranes isolated from the chondrocytes, demonstrating a direct membrane effect for this steroid hormone. These data indicate that the rapid nongenomic effect of E₂ on PKC activity in chondrocytes from female rats is sex-specific and dependent upon a G-protein-coupled phospholipase C.     

Induction of apoptosis by protein kinase C pseudosubstrate lipopeptides in several human cells

Intracellular enzymes or receptors are interesting targets for thepharmacomodulation of cellular metabolism. We have previously shown thatmodification of relatively long peptides by a palmitoyl-lysine residue couldfacilitate their delivery into the cytoplasm of living cells. Severalpeptides containing pseudosubstrate sequences of protein kinase C (PKC) havebeen evaluated for their ability to modulate phosphorylation of modelsubstrate, neuronal morphology or tumor necrosis factor secretion. In thiswork we have evaluated the effect of palmitoyl-modified PKC-pseudosubstratepeptides on induction of apoptosis. We have established that these peptidesare able to induce apoptosis in different human cell types (primaryfibroblasts, T- and B-lymphocyte cell lines) as assessed by (terminal deoxynucleotidyl transferase dUTP nick-end labelling) and DNAfragmentation. In contrast, control peptides (non-lipidicPKC-pseudosubstrate peptides and irrelevant lipopeptides) had no or littleeffect on programmed cell death. This work highlights the pharmacologicalinterest of lipopeptides and argues in favor of the potential role of PKC(s)in the cell death machinery.