Activation of Protein Kinase C by Trimethyltin: Relevance to Neurotoxicity

Abstract: The differentiated PC12 cell neuronal model was used to determine the effect of trimethyltin (TMT) on protein kinase C (PKC). Cells treated with 5–20 µ M TMT showed a partial and sustained PKC translocation within 30 min and persisted over a 24-h period. TMT treatment was accompanied by a low level of PKC down-regulation over 24 h, which was small compared with that produced by phorbol esters. Confocal imaging of differentiated PC12 cells showed that PKC translocates to the plasma membrane and the translocation is blocked by the PKC inhibitor chelerythrine (1 µ M ). Phorbol myristate-induced PKC down-regulation or inhibition with chelerythrine provided protection against TMT-induced cytotoxicity. It was concluded that TMT-induced PKC translocation and activation contribute to the cytotoxicity of TMT in differentiated PC12 cells.     

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.     

Proliferation of human breast cancer cells and anti-cancer action of doxorubicin and vinblastine are independent of PKC-alpha

Protein kinase C (PKC) has been considered for a potential target of anticancer chemotherapy. PKC-alpha has been associated with growth and metastasis of some cancer cells. However, the role of PKC-alpha in human breast cancer cell proliferation and anticancer chemotherapy remains unclear. In this study, we examined whether alterations of PKC-alpha by phorbol esters and PKC inhibitors could affect proliferation of human breast cancer MCF-7 cells and the cytotoxic effect of chemotherapeutic agents. Exposure for 24 h to doxorubicin (DOX) and vinblastine (VIN) caused a concentration-dependent reduction in proliferation of MCF-7 cells. However, these two anticancer drugs altered cellular morphology and growth pattern in distinct manners. Phorbol 12,13-dibutyrate (PDBu, 100 nM), which enhanced activities of PKC-alpha, increased cancer cell proliferation and attenuated VIN (1 microM)-induced cytotoxicity. These effects were not affected in the presence of 10 nM staurosporine. Phorbol myristate acetate (PMA, 100 nM) that completely depleted PKC-alpha also enhanced cancer cell proliferation and attenuated VIN-induced cytotoxicity. Three potent PKC inhibitors, staurosporine (10 nM), chelerythrine (5 microM) and bisindolylmaleimide-I (100 nM), had no significant effect on MCF-7 cell proliferation; staurosporine and chelerythrine, but not bisindolylmaleimide-I, attenuated VIN-induced cytotoxicity. Moreover, neither phorbol esters nor PKC inhibitors had an effect on cytotoxic effects of DOX (1 microM) on MCF-7 cell proliferation. Thus, these data suggest that MCF-7 cell proliferation or the anti-cancer action of DOX and VIN on breast cancer cells is independent of PKC-alpha.     

Enhanced generation of Alzheimer's amyloid-β following chronic exposure to phorbol ester correlates with differential effects on alpha and epsilon isozymes of protein kinase C

Alzheimer's amyloid precursor protein (APP) sorting and processing are modulated through signal transduction mechanisms regulated by protein phosphorylation. Notably, protein kinase C (PKC) appears to be an important component in signaling pathways that control APP metabolism. PKCs exist in at least 11 conventional and unconventional isoforms, and PKCα and PKCε isoforms have been specifically implicated in controlling the generation of soluble APP and amyloid-β (Aβ) fragments of APP, although identification of the PKC substrate phospho-state-sensitive effector proteins remains challenging. In the current study, we present evidence that chronic application of phorbol esters to cultured cells in serum-free medium is associated with several phenomena, namely: (i) PKCα down-regulation; (ii) PKCε up-regulation; (iii) accumulation of APP and/or APP carboxyl-terminal fragments in the trans Golgi network; (iv) disappearance of fluorescence from cytoplasmic vesicles bearing a green fluorescent protein tagged form of APP; (v) insensitivity of soluble APP release following acute additional phorbol application; and (vi) elevated cellular APP mRNA levels and holoprotein, and secreted Aβ. These data indicate that, unlike acute phorbol ester application, which is accompanied by lowered Aβ generation, chronic phorbol ester treatment causes differential regulation of PKC isozymes and increased Aβ generation. These data have implications for the design of amyloid-lowering strategies based on modulating PKC activity.     

Tyrosine protein phosphorylation is required for protein kinase C-mediated proliferation in T cells.

We have studied the role of tyrosine kinase in PMA-stimulated T cells. Protein kinase C (PKC)-mediated D10A cell proliferation is inhibited by the specific inhibitor of tyrosine kinase, tyrphostin. This inhibitor selectively blocks the mRNA expression of the proto-oncogene c-myc in response to the phorbol ester, PMA. On the other hand, the same doses of this inhibitor do not affect the mRNA expression of the proto-oncogene c-fos in PMA-stimulated D10A cells. Phorbol esters induce in this T cell line the tyrosine phosphorylation of a unique protein of 42 kDa and the enzyme PKC is required for this activity.     

Endothelin, vasopressin, and angiotensin II enhance tyrosine phosphorylation by protein kinase C-dependent and -independent pathways in glomerular mesangial cells.

Protein tyrosine phosphorylation has not been considered to be important for cellular activation by phospholipase C-linked vasoactive peptides. We found that endothelin, angiotensin II, and vasopressin (AVP), peptides that signal via phospholipase C activation, rapidly enhanced tyrosine phosphorylation of proteins of approximate molecular mass 225, 190, 135, 120, and 70 kDa in rat renal mesangial cells. The phosphorylated proteins were cytosolic or membrane-associated, and none were integral to the membrane, suggesting that the peptide receptors are not phosphorylated on tyrosine. Epidermal growth factor (EGF), which does not activate phospholipase C in these cells, induced the tyrosine phosphorylation of its own 175-kDa receptor, in addition to five proteins of identical molecular mass to those phosphorylated in response to endothelin, AVP, and angiotensin II. This suggests that in mesangial cells there is a common signaling pathway for phospholipase C-coupled agonists and agonists classically assumed to signal via receptor tyrosine kinase pathways, such as EGF. The phorbol ester, phorbol 12-myristate 13-acetate, and the synthetic diacylglycerol, oleoyl acetylglycerol, stimulated the tyrosine phosphorylation of proteins identical to those phosphorylated by the phospholipase C-linked peptides, suggesting that protein kinase C (PKC) activation is sufficient to active tyrosine phosphorylation. However, the PKC inhibitor, staurosporine, and down-regulation of PKC activity by prolonged exposure to phorbol esters completely inhibited tyrosine phosphorylation in response to PMA but not to endothelin, AVP, or EGF. In conclusion, endothelin, angiotensin II, and AVP enhances protein tyrosine phosphorylation via at least two pathways, PKC-dependent and PKC-independent. Although activation of PKC may be sufficient to enhance protein tyrosine phosphorylation, PKC is not necessary and may not be the primary route by which these agents act. At least one of these pathways is shared with the growth factor EGF, suggesting not only common intermediates in the signaling pathways for growth factors and vasoactive peptides but also perhaps common cellular tyrosine kinases which phosphorylate these intermediates.     

Interaction of insulin and phorbol esters on the regulation of DNA synthesis in rat hepatoma cells

Insulin and phorbol esters stimulated DNA synthesis in rat H4 hepatoma cells. Insulin and phorbol ester induction of thymidine incorporation was dose-dependent, with a maximal 4.2- and 3.0-fold increases at concentrations of 1 x 10(-9)M and 1 microM, respectively. Phorbol esters in combination with increasing concentrations of insulin resulted in additive effects, but only at submaximal insulin concentrations. The combination failed to increase thymidine incorporation above the maximal effects produced by insulin alone. When cells were pretreated with phorbol esters for 24h to produce protein kinase-C (PKC) deficiency, basal DNA synthesis was depressed. Pretreatment with phorbol esters abolished the effects of phorbol esters to induce DNA synthesis but did not impair the magnitude of insulin-induced DNA synthesis. Thus, although phorbol ester-activatable PKC-activity was necessary for basal DNA synthesis, it was not necessary for insulin-induction of DNA synthesis in H4 cells.     

Down-regulation of protein kinase C in Swiss 3T3 fibroblasts is independent of its phosphorylating activity

The phorbol ester TPA induces down-regulation of protein kinase C (PKC) in Swiss-3T3 fibroblasts, as determined by the use of an alpha, beta, gamma PKC-specific antiserum. PKC is almost completely degraded 10 hours after TPA treatment of the cells and recovers within 72 hours. The staurosporine derivative K252a, known to inhibit PKC activity, causes strong suppression of TPA-induced (PKC-catalyzed) protein phosphorylation in Swiss-3T3 cells. Inhibition of protein phosphorylation by K252a is still effective when the process of down-regulation is completed. However, K252a does not influence TPA-induced down-regulation of PKC at all. Thus, down-regulation of PKC is not dependent on the enzyme's phosphorylating activity and, therefore, most likely not on its autophosphorylation as has been suggested by Ohno et al. [J. Biol. Chem. 265, 6296-6300 (1990)].     

Targeting protein kinase C and "non-kinase" phorbol ester receptors: emerging concepts and therapeutic implications

Phorbol esters, natural compounds that mimic the action of the lipid second messenger diacylglycerol (DAG), are known to exert their biological actions through the activation of classical and novel protein kinase C (PKC) isozymes. Phorbol esters, via binding to the PKC C1 domains, cause major effects on mitogenesis by controlling the activity of cyclin-cdk complexes and the expression of cdk inhibitors. In the last years it became clear that phorbol esters activate other molecules having a C1 domain in addition to PKCs. One of the most interesting families of "non-kinase" phorbol ester receptors is represented by the chimaerins, lipid-regulated Rac-GAPs that modulate actin cytoskeleton reorganization, migration, and proliferation. The discovery of the chimaerins and other "non-kinase" phorbol ester receptors has major implications in the design of agents for cancer therapy.     

Active site inhibitors protect protein kinase C from dephosphorylation and stabilize its mature form

Conformational changes acutely control protein kinase C (PKC). We have previously shown that the autoinhibitory pseudosubstrate must be removed from the active site in order for 1) PKC to be phosphorylated by its upstream kinase phosphoinositide-dependent kinase 1 (PDK-1), 2) the mature enzyme to bind and phosphorylate substrates, and 3) the mature enzyme to be dephosphorylated by phosphatases. Here we show an additional level of conformational control; binding of active site inhibitors locks PKC in a conformation in which the priming phosphorylation sites are resistant to dephosphorylation. Using homogeneously pure PKC, we show that the active site inhibitor Gö 6983 prevents the dephosphorylation by pure protein phosphatase 1 (PP1) or the hydrophobic motif phosphatase, pleckstrin homology domain leucine-rich repeat protein phosphatase (PHLPP). Consistent with results using pure proteins, treatment of cells with the competitive inhibitors Gö 6983 or bisindolylmaleimide I, but not the uncompetitive inhibitor bisindolylmaleimide IV, prevents the dephosphorylation and down-regulation of PKC induced by phorbol esters. Pulse-chase analyses reveal that active site inhibitors do not affect the net rate of priming phosphorylations of PKC; rather, they inhibit the dephosphorylation triggered by phorbol esters. These data provide a molecular explanation for the recent studies showing that active site inhibitors stabilize the phosphorylation state of protein kinases B/Akt and C.     

Inhibition and stimulation of growth of Entamoeba histolytica in culture: association with PKC activity and protein phosphorylation

We studied the role of protein kinase C (PKC) and protein threonine phosphorylation in the inhibition and stimulation of growth of the protozoan parasite Entamoeba histolytica. PKC was activated after serum deprivation in E. histolytica and during this period proteins became threonine phosphorylated. Conversely, on serum stimulation of serum-deprived cells, PKC activation was rapidly reversed and the threonine phosphorylation of proteins quickly declined. Growth of E. histolytica was not affected by either PKC inhibitors H-7 and GF109203X or by down-regulation of PKC by Phorbol 12-Myristate 13-Acetate (PMA). Interestingly, very low doses of PMA which caused activation of PKC and were unable to down-regulate PKC after 48 h of culture, negatively influenced the growth of E. histolytica. Serine/threonine phosphatase inhibitors Okadaic acid and Calyculin A drastically inhibited growth of E. histolytica. In conclusion, the growth of E. histolytica is not adversely affected by PKC down-regulation. On the contrary, growth inhibition of E. histolytica is associated with activation of Ca(2+), Diacylglycerol (DAG)-dependent PKC, and threo nine phosphorylation of proteins.     

Peptidyl-prolyl isomerase Pin1 controls down-regulation of conventional protein kinase C isozymes

The down-regulation or cellular depletion of protein kinase C (PKC) attendant to prolonged activation by phorbol esters is a widely described property of this key family of signaling enzymes. However, neither the mechanism of down-regulation nor whether this mechanism occurs following stimulation by physiological agonists is known. Here we show that the peptidyl-prolyl isomerase Pin1 provides a timer for the lifetime of conventional PKC isozymes, converting the enzymes into a species that can be dephosphorylated and ubiquitinated following activation induced by either phorbol esters or natural agonists. The regulation by Pin1 requires both the catalytic activity of the isomerase and the presence of a Pro immediately following the phosphorylated Thr of the turn motif phosphorylation site, one of two C-terminal sites that is phosphorylated during the maturation of PKC isozymes. Furthermore, the second C-terminal phosphorylation site, the hydrophobic motif, docks Pin1 to PKC. Our data are consistent with a model in which Pin1 binds the hydrophobic motif of conventional PKC isozymes to catalyze the isomerization of the phospho-Thr-Pro peptide bond at the turn motif, thus converting these PKC isozymes into species that can be efficiently down-regulated following activation.     

Protein kinase C protects from DNA damage-induced necrotic cell death by inhibiting poly(ADP-ribose) polymerase-1

The goal of the current study, conducted in freshly isolated thymocytes was (1) to investigate the possibility that the activation of poly(ADP-ribose) polymerase-1 (PARP-1) in an intact cell can be regulated by protein kinase C (PKC) mediated phosphorylation and (2) to examine the consequence of this regulatory mechanism in the context of cell death induced by the genotoxic agent. In cells stimulated by the PKC activating phorbol esters, DNA breakage was unaffected, PARP-1 was phosphorylated, 1-methyl-3-nitro-1-nitrosoguanidine-induced PARP activation and cell necrosis were suppressed, with all these effects attenuated by the PKC inhibitors GF109203X or G?6976. Inhibition of cellular PARP activity by PKC-mediated phosphorylation may provide a plausible mechanism for the previously observed cytoprotective effects of PKC activators.     

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.     

Phorbol Ester- and Retinoic Acid-Induced Regulation of the Protein Kinase C Substrate MARCKS in Immortalized Hippocampal Cells

Abstract: The expression of MARCKS, a major protein kinase C (PKC) substrate, was examined in the immortalized hippocampal cell line HN33, following differentiation using phorbol esters or retinoic acid. In cells exposed to phorbol esters, MARCKS protein levels were reduced through an apparent PKC-dependent mechanism. Exposure to 1 µ M phorbol 12-myristate 13-acetate (PMA) for 10 min resulted in a rapid loss of PKC activity in the soluble fraction with a concurrent increase in membrane-associated PKC activity. PKC activity was reduced to <20% of control values in both soluble and membrane fractions following 1 h of PMA exposure. Significant reductions in MARCKS protein levels were initially observed in membrane and soluble fractions following PMA exposure for 4 and 8 h, respectively. The reduction in MARCKS protein levels was maximal following 24 h of PMA exposure. MARCKS protein expression was also down-regulated in a dose-dependent manner on exposure of HN33 cells to retinoic acid. In cells exposed to 10 µ M retinoic acid, the MARCKS protein level was reduced in the membrane fraction within 4 h. Reduction of MARCKS protein levels was maximal (>90%) by 12 h with no evidence for any alteration in PKC activity. Reduced levels of MARCKS protein were also observed in the soluble fraction of retinoic acid-exposed cells, but to a significantly lesser extent. Addition of the PKC inhibitor GF109203X blocked the down-regulation of MARCKS protein in PMA-treated cultures but not in retinoic acid-treated cells. These findings suggest that the down-regulation of MARCKS may play an important role in both phorbol ester- and retinoic acid-induced differentiation in cells of neuronal origin.