Effect of phorbol esters on cytosolic protein kinase C content and activity in the human monoblastoid U937 cell

12-O-Tetradecanoylphorbol-13-acetate (TPA) stimulates the human monoblastoid U937 cell to differentiate into a mature monocyte/macrophage-like cell. Since TPA may produce cellular responses by activating protein kinase C, the effects of TPA on kinase activity in the U937 cell were investigated. Brief exposures (less than or equal to 60 min) to TPA dramatically diminished protein kinase C-dependent phosphorylation of histone and endogenous substrates. However, using a peptide substrate corresponding to residues 720-737 of protein kinase C-epsilon, Ca2(+)-, phospholipid-, and diacylglycerol-dependent kinase activity was reduced only modestly after exposure to TPA. This phospholipid-dependent kinase activity coeluted on DEAE chromatography with protein kinase C. Examination of cytosolic protein kinase C content by Western blot analysis demonstrated a moderate decline in kinase content after TPA treatment. The decline was due primarily to loss of an 80-kDa species with preservation of a 76-kDa protein. The immunoreactive 76-kDa protein observed after TPA treatment comigrated on DEAE chromatography with the kinase activity phosphorylating the protein kinase C-epsilon peptide and had an elution profile similar to protein kinase C derived from untreated cells. Using antisera recognizing the catalytic and regulatory domains of the kinase, no evidence for proteolytic degradation of protein kinase C was observed. Although incubation of extracts from vehicle and TPA-treated cells inhibited the activity of partially purified protein kinase C, the degree of inhibition was similar in the two extracts. These findings suggest that TPA markedly diminishes protein kinase C-dependent phosphorylation of histone and endogenous substrates in part by altering kinase substrate specificity. These observations provide evidence for a novel post-translational process that can modulate protein kinase C-dependent phosphorylation.     

Effect of phorbol esters on protein kinase C-zeta.

Protein kinase C-zeta (PKC-zeta) is a member of the protein kinase C gene family which using in vitro preparations has been described as being resistant to activation by phorbol esters. PKC-zeta was found to be expressed in several cell types as an 80-kDa protein. In vitro translation of a full-length PKC-zeta construct also yielded as a primary translation product an 80-kDa protein. In the U937 cell, PKC-zeta was slightly more abundant in the cytosol than in the particulate fraction. Acute exposure of U937 cells to tetradecanoyl-phorbol-13-acetate (TPA), phorbol dibutyrate, mezerin, or diacylglycerol derivatives did not induce translocation of this isoform to the particulate fraction. Chronic exposure to 1 microM TPA failed to translocate or down-regulate PKC-zeta in U937, HL-60, COS, or HeLa-fibroblast fusion cells. To examine whether PKC-zeta was activated by TPA, PKC activity was evaluated in COS cells transiently over-expressing this isoform. In non-transfected cells, two peaks of phospholipid- and TPA-dependent kinase activity were observed. Eluting at a lower salt concentration was a peak of activity associated with PKC-alpha. PKC-zeta eluted with the second peak of activity and at a higher salt concentration. In transfected cells which expressed PKC-zeta at 4-10-fold over endogenous levels, there was only a slight increase in activity associated with the second peak. The activity and quantity of PKC-zeta did not strictly correlate. Treatment with TPA under conditions that did not alter PKC-zeta content abolished detection of the second peak of PKC activity eluting from the Mono Q column. Thus, PKC-zeta does not translocate or down-regulate in response to phorbol esters or diacylglycerol derivatives. However, for reasons discussed these studies do not resolve the issue of whether this isoform is activated by TPA.     

Differences in the effects of phorbol esters and diacylglycerols on protein kinase C

The binding of protein kinase C (PKC) to membranes and appearance of kinase activity are separable events. Binding is a two-step process consisting of a reversible calcium-dependent interaction followed by an irreversible interaction that can only be dissociated by detergents. The irreversibly bound PKC is constitutively active, and the second step of binding may be a major mechanism of PKC activation [Bazzi & Nelsestuen (1988) Biochemistry 27, 7589]. This study examined the activity of other forms of membrane-bound PKC and compared the effects of phorbol esters and diacylglycerols. Like the membrane-binding event, activation of PKC was a two-stage process. Diacylglycerols (DAG) participated in forming an active PKC which was reversibly bound to the membrane. In this case, both activity and membrane binding were terminated by addition of calcium chelators. DAG functioned poorly in generating the constitutively active, irreversible PKC-membrane complex. These properties differed markedly from phorbol esters which activated PKC in a reversible complex but also promoted constitutive PKC activation by forming the irreversible PKC-membrane complex. The concentration of phorbol esters needed to generate the irreversible PKC-membrane complex was slightly higher than the concentration needed to activate PKC. In addition, high concentrations of phorbol esters (greater than or equal to 50 nM) activated PKC and induced irreversible PKC-membrane binding in the absence of calcium. Despite these striking differences, DAG prevented binding of phorbol esters to high-affinity sites on the PKC-membrane complex.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of phorbol esters on contraction and protein kinase C activation in rabbit aorta

This study investigates the relationship between the contractile efficacy of phorbol esters and their ability to activate protein kinase C in intact rabbit aorta. Phorbol dibutyrate (PDB) induced a maximal contraction approximately 3.5-fold greater than that to phorbol myristate acetate (PMA). The magnitude of maximal PDB- and PMA-induced contraction correlated with the magnitude of protein kinase C activation, as assessed by the decrease in cytosolic protein kinase C activity. KCl (60mM) did not potentiate the PMA-induced decrease in cytosolic protein kinase C activity. These results suggest that the lack of efficacy of PMA is due to its inability to activate protein kinase C in the intact rabbit aorta. It is speculated that the different contractile efficacies of phorbol esters result from selective activation of protein kinase C isoforms, and that the amounts of these isoforms varies amongst vascular tissues.     

Rapid effects of phorbol esters on isolated rat adipocytes. Relationship to the action of protein kinase C

Treatment of isolated rat adipocytes with tumor-promoting phorbol esters, caused a fivefold stimulation of glucose oxidation, determined as 14CO2 production from [1-14C]glucose and a fivefold increase in the rate of lipid synthesis from [14C]glucose. Treatment of the cells with 12-O-tetradecanoylphorbol 13-acetate increased the rate of 86Rb+ uptake into the cells. Also phospholipase C was able to stimulate the rate of glucose oxidation; phospholipase C and 12-O-tetradecanoylphorbol 13-acetate stimulated glucose oxidation in a non-synergistic fashion, indicating a common mechanism for their action. Active phorbol esters and, in part, also phospholipase C, caused a translocation of protein kinase C activity from the soluble to the particulate fraction of the adipocytes. This process was rapid, being complete 30 s after the addition of phorbol ester, and resulted in the appearance of the kinase mainly in the mitochondrial and plasma membrane fractions. A comparison between the binding characteristics of adipocyte protein kinase C and the metabolic effects of the phorbol esters on the adipocytes revealed that the dose-response relationship did not correlate with binding of the phorbol esters, but, rather, a correlation was observed between the dose of phorbol esters required for translocation of protein kinase C and the intracellular effects. The results indicate that the intracellular translocation of protein kinase C might be a trigger for the effects of phorbol esters on the adipocyte and that binding of the esters to protein kinase C is not a sufficient event to cause this effect. Furthermore, it is suggested that activation of protein kinase C might be partly the action of hormones, such as insulin, on the fat cells.     

Expression of four protein kinase C isoforms in rat fibroblasts. Distinct subcellular distribution and regulation by calcium and phorbol esters.

Protein kinase C (PKC), the major receptor for tumor-promoting phorbol esters, consists of a family of at least eight distinct lipid-regulated enzymes. How the various PKC isozymes are regulated in vivo and how they couple to particular cellular responses is largely unknown. We have examined the expression and regulation of PKC isoforms in R6 rat embryo fibroblasts. Northern and Western blot analyses indicate that these cells express four PKC isoforms, cPKC alpha, nPKC epsilon, nPKC delta, and nPKC zeta; of which nPKC epsilon and nPKC delta are the most abundant. In agreement with the simultaneous presence of cPKC and nPKC isozymes, both Ca(2+)-dependent and -independent PKC activities were detected in extracts of these cells. cPKC alpha and nPKC zeta were predominantly localized in the cytosol when subcellular fractionation was carried out in the presence of [ethylenebis(oxyethylenenitrilo)]tetraacetic acid. When cell lysis was carried out in the presence of Ca2+, greater than 50% of cPKC alpha redistributed to the particulate fraction, whereas nPKC zeta remained in the cytosol. In contrast to cPKC alpha and nPKC zeta, 60-80% of nPKC epsilon and nPKC delta were located in a Ca(2+)-insensitive, membrane-bound form. Treatment of R6 cells with 12-O-tetradecanoyl phorbol 13-acetate (TPA), resulted in the translocation of all four PKC isozymes to the membrane fraction, and the subsequent down-regulation of cPKC alpha, nPKC zeta, and nPKC delta, nPKC epsilon, however, was only partially down-regulated in response to long-term TPA exposure. Overproduction of exogenous cPKC beta I in R6 cells conferred partial resistance of nPKC delta to TPA-induced down-regulation and potentiated the resistance of nPKC epsilon to down-regulation. These results demonstrate that the multiple isoforms of PKC which coexist within a single cell type are differentially regulated by extra- and intracellular stimuli and may thereby influence growth control and transformation via distinct mechanisms.     

Involvement of protein kinase C in the regulation of ornithine decarboxylase mRNA by phorbol esters in rat hepatoma cells

The tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) stimulates a rapid increase in ornithine decarboxylase (EC 4.1.1.17; ODC) activity in target cells. Here we demonstrate that this process involves a rapid accumulation of ODC mRNA, which is maximal 3 h after treatment (three- to eightfold greater than control cells) and decays to control levels within 18 h. Stimulation of ODC mRNA by TPA is blocked by phorbol dibutyrate down-regulation of protein kinase C (PKC). ODC mRNA was also induced by the PKC activators, phospholipase C and 1-oleoyl-2-acetyl-rac-glycerol, and blocked by kinase inhibitors (trifluoroperazine, H7, and palmitoyl-L-carnitine), consistent with a requirement for PKC activation in the induction mechanism. However, the non-PKC-specific protein kinase inhibitor HA1004 also suppressed expression of ODC mRNA in response to TPA, under conditions where it did not inhibit PKC, suggesting that additional kinases may be involved in the intracellular signalling process. The stability of the ODC mRNA (control value = 6.2 +/- 1.6 h) is not significantly changed by either TPA (5.7 +/- 0.8 h) or by cycloheximide (6.0 h). These results are inconsistent with any contribution from altered mRNA half-life towards the accumulation of ODC mRNA following treatment with phorbol ester tumor promoters.     

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.     

Phorbol esters and calcium-mobilizing hormones increase membrane-associated protein kinase C activity in rat hepatocytes

Vasopressin, angiotensin II, epinephrine (alpha 1-adrenergic action) and phorbol 12-myristate 13-acetate (PMA) induce increases in membrane-associated protein kinase C activity concomitant with decreases in the cytosolic activity. The data indicate that the calcium-mobilizing hormones and the active phorbol ester induce translocation from the cytosol to the plasma membrane of this protein kinase. The protein kinase C inhibitor, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine, blocked the translocation to the membrane of this protein kinase induced by PMA and vasopressin.     

Effects of acetylcholine and nitroprusside on cGMP-dependent protein kinase in the perfused rat heart

The effects of acetylcholine and sodium nitroprusside on the activity of cGMP-dependent protein kinase were studied in the perfused rat heart. Acetylcholine produced a dose-dependent increase in cGMP levels and cGMP-dependent protein kinase activity, and reduced the force of contraction. Both acetylcholine and sodium nitroprusside produced rapid increases in cardiac cGMP, with nitroprusside being the more potent agent. Only acetylcholine, however, raised the activity ratio of the cGMP-dependent protein kinase and decreased the force of contraction. Whereas acetylcholine and nitroprusside were slightly additive in their effects on total cGMP levels, the increase in the activity ratio of the cGMP-dependent protein kinase and the decrease in the force of contraction produced by acetylcholine were unchanged by nitroprusside. The results suggest that the cGMP produced by acetylcholine, but not nitroprusside, was coupled to protein kinase activation in this tissue.     

Rapid assay of binding of tumor-promoting phorbol esters to protein kinase C1

Protein kinase C is generally accepted to be a receptor protein of tumor-promoting phorbol esters. The binding of [3H]phorbol-12,13-dibutyrate to protein kinase C can be assayed by a rapid filtration procedure using a glass-fiber filter that has been treated with a cationic polymer, polyethylenimine. The phorbol ester specifically binds to the protein kinase only in the presence of phosphatidylserine and calcium. Non-specific binding is less than 10%, at most, of the total binding. The binding is linear with respect to the concentration of protein kinase C, is dependent on the concentrations of phorbol ester and phosphatidylserine in a saturative manner, and is inhibited by diacylglycerol (an endogenous activator of the protein kinase).     

The relationship between AMP-activated protein kinase activity and AMP concentration in the isolated perfused rat heart.

The objective of this study was to define the relationship among AMP-activated protein kinase (AMPK) activity, AMP concentration ([AMP]), and [ATP] in perfused rat hearts. Bromo-octanoate, an inhibitor of beta-oxidation, and amino-oxyacetate, an inhibitor of the malate-aspartate shuttle, were used to modify substrate flux and thus increase cytosolic [AMP]. Cytosolic [AMP] was calculated using metabolites measured by (31)P NMR spectroscopy. Rat hearts were perfused with Krebs-Henseleit solution containing glucose and either no inhibitor, the inhibitors, or the inhibitors plus butyrate, a substrate that bypasses the metabolic blocks. In this way, [AMP] changed from 0.2 to 27.9 microm, and [ATP] varied between 11.7 and 6.8 mm. AMPK activity ranged from 7 to 60 pmol.min(-1).microg of protein(-1). The half-maximal AMPK activation (A(0.5)) was 1.8 +/- 0.3 microm AMP. Measurements in vitro have reported similar AMPK A(0.5) at 0.2 mm ATP, but found that A(0.5) increased 10-20-fold at 4 mm ATP. The low A(0.5) of this study despite a high [ATP] suggests that in vivo the ATP antagonism of AMPK activation is reduced, and/or other factors besides AMP activate AMPK in the heart.     

The protein kinase inhibitor staurosporine, like phorbol esters, induces the association of protein kinase C with membranes

Staurosporine induced the association of purified protein kinase C (PKC) with inside-out vesicles from erythrocyte membranes. This effect was Ca2+ and concentration dependent, and maximum PKC translocation was observed at 50 nM staurosporine and 0.5 microM Ca2+, or higher. A significant effect of staurosporine was already obtained at free Ca2+ concentrations in the range found in resting cells. Under these conditions, the PKC activator 4-phorbol 12,13-dibutyrate was by itself inactive, but enhanced translocation by staurosporine. Protein phosphorylation by staurosporine-translocated PKC was inhibited in the presence or absence of phorbol esters. Translocation and inhibition of PKC occurred in the same staurosporine concentration range.     

Cellular distribution of gastric chief cell protein kinase C activity: differential effects of diacylglycerol, phorbol esters, carbachol, and cholecystokinin.

Stimulation of chief cells with carbachol or cholecystokinin (CCK) results in the production of inositol trisphosphate (IP3) and diacylglycerol (DAG). Although IP3 increases cell calcium concentration, thereby stimulating pepsinogen secretion, the role of DAG and its target, protein kinase C (PKC), is less clear. To examine the relation between the cellular distribution of PKC activity and pepsinogen secretion, we determined PKC activity in cytosolic and membrane fractions from dispersed chief cells from guinea pig stomach. To validate our assay, we studied the actions of the phorbol ester PMA. PMA caused a rapid, dose-dependent, 6-fold increase in pepsinogen secretion and membrane-associated PKC activity. Similarly, dose-response curves for pepsinogen secretion and the increase in membrane-associated PKC activity induced by a membrane-permeant DAG (1-oleoyl-2-acetylglycerol) were superimposable. In contrast, CCK (0.1 nM to 1.0 microM) and carbachol (0.1 microM to 1.0 mM) caused a 4-fold increase in pepsinogen secretion, but did not alter the distribution of PKC activity. These results indicate that in gastric chief cells, PMA- and DAG-induced pepsinogen secretion is accompanied by increased membrane-associated PKC activity. However, the cellular distribution of PKC activity is not altered by CCK or carbachol.     

Evidence for protein kinase C independent activation of phospholipase D by phorbol esters in lymphocytes

Recently it was reported that tumor-promoting phorbol esters stimulate the production of phosphatidylethanol (PEt) in lymphocytes through the activation of phospholipase D (PLD). However, it remains unclear whether this activation is mediated through protein kinase (PKC). The study reported here shows that tumor promoters 12-0-tetradecanoylphorbol-13-acetate (TPA), phorbol dibutyrate (PDBU), 12-deoxyphorbol-13-phenylacetate (DOPP), 12-deoxyphorbol-13-phenylacetate-20-acetate (DOPPA) and mezerin activated PLD, as measured by the formation of PEt, whereas Concanavalin A (ConA) had no effect. Inhibitors of PKC, sphingosine (2 x 10(-6) M - 5 x 10(-6) M), H-7, HA1004 (5 x 10(-7) - 5 x 10(-6) M) and K252a (1 x 10(-7) - 1 x 10(-6) M) failed to block the PEt synthesis induced by TPA. In fact, sphingosine increased it. Other PKC activators, 1-oleoyl-2-acetylglycerol (OAG) and dioctanoylglycerol (DiC8) had no effect on lymphocyte PLD activity. Analysis of the phospholipid contents after stimulation by TPA showed that only phosphatidylcholine (PC) was significantly decreased. Interestingly, TPA activated PLD in intact cells but not in lysates or subcellular fractions. These observations suggest that stimulation of PLD-catalyzed PEt synthesis by TPA is not solely mediated through PKC activation.     

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

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

Activation of protein kinase C by phorbol esters induces DNA synthesis and protein phosphorylations in glomerular mesangial cells

The tumor-promoting phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) is shown to be mitogenic for quiescent glomerular mesangial cells cultured in serum-free conditions. TPA induces DNA synthesis measured by [3H]thymidine incorporation in a dose-dependent manner with an ED50 of 7 ng/ml and an optimal response for 50 ng/ml. The phorbol ester action is potentiated by insulin with an increase of the maximal effect from 232 +/- 15% for TPA alone to 393 +/- 96% for TPA plus insulin. Down-regulation of protein kinase C by prolonged exposure to TPA completely abolishes the mitogenic effect of the phorbol ester. Using a highly resolutive 2D electrophoresis, we have shown that TPA is able to stimulate the phosphorylation of 2 major proteins of Mr 80,000, pl 4.5 (termed 80K) and Mr 28,000, pI 5.7-5.9 (termed 28K). The 80K protein phosphorylation is time- and dose-dependent with an ED50 of 8 ng/ml TPA. Exposure of mesangial cells to heat-shock induces synthesis of a 28K protein among a set of other proteins suggesting that the 28K protein kinase C substrate belongs to the family of low molecular mass stress proteins. Mitogenic concentrations of TPA and phorbol 12,13-dibutyrate inhibit [125 I]epidermal growth factor binding and stimulate the 80K protein phosphorylation with the same order of potency. The inactive tumor-promoter 4 alpha-phorbol was found to be ineffective both on these 2 parameters and on DNA synthesis. These results suggest a positive role for protein kinase C on mesangial cell proliferation and indicate the existence in this cell line of 2 major protein kinase C substrates.     

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.     

Differential translocation of protein kinase C isozymes by phorbol esters, EGF, and ANG II in rat liver WB cells

The protein kinaseC (PKC) family represents an important group of enzymes whoseactivation is associated with their translocation from the cytosol todifferent cellular membranes. In this study, the spatial distributionof PKC-, - and - in rat liver epithelial (WB) cells has beenexamined by Western blot analysis after subcellular fractionation.Cytosolic, membrane, nuclear, and cytoskeletal fractions were obtainedfrom cells stimulated with phorbol 12-myristate 13-acetate (PMA),angiotensin II (ANG II), or epidermal growth factor (EGF). PMA causedmost of the PKC-, - and - initially present in the cytosol tobe transported to the membrane and nuclear fractions. In contrast, bothANG II and EGF induced only a minor translocation of PKC- to themembrane fraction but caused a statistically significantmembrane-directed movement of PKC- and -. Translocation ofPKC- and - to the nucleus induced by ANG II and EGF was transient and quantitatively smaller than that induced by PMA. PKC- and -were present in the cytoskeleton of resting cells, but although PMA,ANG II, and EGF caused some changes in their content, these werevariable, suggesting that the cytoskeleton fraction was heterogeneous. PKC depletion inhibited ANG II-induced mitogenesis and the sustained activation of Raf-1 and extracellular regulated protein kinase (ERK).However, although PKC depletion inhibited EGF-induced mitogenesis, themaximum EGF-induced activation of the ERK pathway was only slightlyretarded. We hypothesize that PKC- and - are involved inmitogenesis via both ERK-dependent and ERK-independent mechanisms. These results support the notion that specific PKC isozymes exert spatially defined effects by virtue of their directed translocation todistinct intracellular sites.