Differential localization of protein kinase C delta by phorbol esters and related compounds using a fusion protein with green fluorescent protein

Enzyme localization often plays a controlling role in determining its activity and specificity. Protein kinase C (PKC) has long been known to translocate in response to physiological stimuli as well as to exogenous ligands such as the phorbol esters. We report here that different phorbol derivatives and related ligands, selected for differences in chemical structure and profile of biological activity, induce distinct patterns of redistribution of PKC delta. Localization of a PKC delta-green fluorescent protein (GFP) fusion construct was monitored in living Chinese hamster ovary cells as a function of ligand, concentration, and time using confocal laser scanning microscopy. delta-PKC-GFP was expressed predominantly in the cytoplasm, with some in the nucleus and perinuclear region. Phorbol 12-myristate 13-acetate (PMA) induced plasma membrane translocation followed by slower nuclear membrane translocation. As the concentration of PMA increased, the proportion of nuclear to plasma membrane localization increased markedly. In contrast to PMA, bryostatin 1, a unique activator of PKC that induces a subset of PMA-mediated responses while antagonizing others, at all doses induced almost exclusively nuclear membrane translocation. Like PMA, the complete tumor promoter 12-deoxyphorbol 13-tetradecanoate induced plasma membrane and slower nuclear membrane translocation, whereas the inhibitor of tumor promotion 12-deoxyphorbol 13-phenylacetate, which differs only in its side chain, induced a distinctive distribution of PKC delta-GFP. Finally, the novel constrained diacylglycerol derivative B8-DL-B8 induced a slow Golgi localization. We speculate that differential control of PKC delta localization may provide an interesting strategy for producing ligands with differential biological consequences.     

Inhibition of stimulated cell responses by phorbol esters and other activators of protein kinase C: sites of action

The addition of the protein kinase C activator phorbol esters to cell suspension a few minutes prior to stimulation inhibits the agonists-induced biochemical changes and cell responses. This inhibition is prevented by protein kinase C inhibitors. Activation of protein kinase C down regulates the stimulated responses by affecting one or more of the steps in the exitation-response coupling. This includes the receptors, the quanine-nucleotide-binding protein, the activity or distribution of phospholipase C, and other steps.     

Association of protein kinase C with phospholipid vesicles

The Ca2+- and phospholipid-dependent protein kinase, protein kinase C (PKC), was purified from bovine brain by a modified procedure that provided sufficient quantities of stable protein for analysis of physical properties of protein-membrane binding. The binding of PKC to phospholipid vesicles of various compositions was investigated by light-scattering and fluorescence energy transfer measurements. The binding properties for membranes of low phosphatidylserine (PS) content were consistent with a peripheral membrane association; PKC showed Ca2+ -dependent binding to phospholipid vesicles containing phosphatidylserine, phosphatidylinositol, or phosphatidylglycerol. Membranes containing 0-20% PS (the remainder of the phospholipid was phosphatidylcholine) bound less protein than membranes containing greater than 20% PS; the factor limiting protein binding to membranes containing low PS appeared to be the availability of acidic phospholipids. Increasing the PS content above 20% did not increase the amount of membrane-bound protein at saturation, and the limiting factor was probably steric packing of protein on the membrane surface. The membranes bound about 1 g of protein/g of phospholipid at steric saturation. Binding was of relatively high affinity (Kd less than 5 nM), and the association rate was rapid on the time scale of the experiments. Addition of ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid to phospholipid-bound PKC caused dissociation of the complex, and the properties of this dissociation indicated an equilibrium binding of protein to membrane. However, only partial dissociation of PKC was achieved when the PS content of the vesicles exceeded 20%. A number of comparisons revealed that binding of protein to the membrane, even in the presence of phorbol esters, was insufficient for development of enzyme activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phosphorylation of purified rat brain Na+ channel reconstituted into phospholipid vesicles by protein kinase C.

Phosphorylation of voltage-sensitive Na+ channels in neurons by protein kinase C slows Na+ channel inactivation and reduces peak Na+ currents. Na+ channels purified from rat brain and reconstituted into phospholipid vesicles under conditions that restore Na+ channel function were rapidly phosphorylated by protein kinase C on their 260-kDa alpha subunit. The phosphorylation reaction required Ca2+, diolein, and phosphatidylserine for activation of protein kinase C, and the rate of phosphorylation of reconstituted Na+ channels was 3- to 4-fold faster than for Na+ channels in detergent solution. Phosphorylation was on serine residues in three distinct tryptic phosphopeptides designated A, B, and C. Up to 2.5 mol of phosphate were incorporated per mol of Na+ channel. Following maximum phosphorylation by protein kinase C, cAMP-dependent protein kinase was able to incorporate more than 2.25 mol of phosphate per mol of Na+ channel indicating that these two kinases phosphorylate distinct sites. However, prior phosphorylation by cAMP-dependent protein kinase prevented phosphorylation of phosphopeptide B indicating that both kinases phosphorylate the site in this peptide. Phosphopeptide B shown here to be phosphorylated by protein kinase C and phosphopeptide 7 previously shown to be phosphorylated by cAMP-dependent protein kinase co-migrate on two-dimensional phosphopeptide maps and evidently are identical. The reduction in peak Na+ currents caused by both protein kinase C and cAMP-dependent protein kinase may result from phosphorylation of this single common site.     

The insertion of D-beta-hydroxybutyrate apodehydrogenase into phospholipid monolayers and phospholipid vesicles

The strong interaction of D-beta-hydroxybutyrate dehydrogenase with phospholipid monomolecular films is demonstrated by the surface pressure increase of a film compressed up to 33 mN/m. Although the D-beta-hydroxybutyrate apodehydrogenase was able to penetrate many phospholipid monolayers, it interacted preferentially with negatively charged monolayers such as those made from diphosphatidylglycerol. The weakest interaction was found with phosphatidylcholine, which is the reactivating phospholipid for the enzyme. These interactions were dependent on the phospholipid chain length, ionic strength, and pH. At basic pH the apoenzyme lost its specificity for negatively charged phospholipids, suggesting the deprotonation of a cationic amino acid residue of the enzyme polypeptide chain. The charge effects are in agreement with results obtained using phospholipid vesicles. Beside the electrostatic interactions, the influence of phospholipid chain length and the ionic strength indicate that D-beta-hydroxybutyrate apodehydrogenase penetrates into the hydrophobic part of the lipid interface.     

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.     

The nature of protein kinase C activation by physically defined phospholipid vesicles and diacylglycerols

Protein kinase C is activated by a 1,2-sn-diacylglycerol and phospholipid at low calcium concentrations. Of the various phospholipids studied, phosphatidylserine has been shown to be the most effective one and is usually used in assaying the enzyme (Kaibuchi, K., Takai, Y., and Nishizuka, Y. (1981) J. Biol. Chem. 256, 7146-7149). It is shown here that under the conditions of the enzymatic assay, phosphatidylserine does not form typical fluid bilayer structures as seen by electron microscopy and fluorescence polarization. On the other hand, 1:4 phosphatidylserine/phosphatidylcholine bilayer vesicles can be formed which support protein kinase C activation. They have the advantage in that they are characterizable, form physiologically relevant bilayer structures, and are readily and reproducibly formed. In addition, they do not support protein kinase C activation in the absence of added diacylglycerol, a property that makes them invaluable in studying the role of diacylglycerol structure in protein kinase C activation. It is further demonstrated that the rat brain enzyme is activated by 1,2-sn-diolein but not by 2,3-sn-diolein nor 1,3-diolein, demonstrating the high specificity of the kinase toward the glycerol backbone. 1,2-rac-Dielaidin, 1,2-rac-distearin, and 1,2-sn-dipalmitin are all active, which is consistent with the idea that the specificity of protein kinase C is not directed toward the fatty acid side chain of the diacylglycerols.     

Association of protein kinase C with phospholipid monolayers: two-stage irreversible binding

The association of protein kinase C (PKC) with phospholipid (PL) monolayers spread at the air-water interface was examined. PKC-PL binding induced surface pressure changes that were dependent on the amount of PKC, the phospholipid composition of the monolayers, the presence of Ca2+, and the initial surface pressure of the monolayer (pi 0). Examination of surface pressure increases induced by PKC as a function of phospholipid surface pressure, pi 0, revealed that PKC-phosphatidylserine (PS) association had a critical pressure of 43 dyn/cm. Above this surface pressure, PKC cannot cause further surface pressure changes. This high critical pressure indicated that PKC should be able to penetrate many biological membranes which appear to have surface pressures of about 30 dyn/cm. PKC-induced surface pressure changes were Ca2+ dependent only for PL monolayers spread at a pi 0 greater than 26 dyn/cm. PKC alone (in the absence of PL) formed a film at the air-water interface with a surface pressure of about 26 dyn/cm. Calcium-dependent binding was studied at the higher surface pressures which effectively excluded PKC from the air-water interface. Subphase depletion measurements suggested that association of PKC with PS monolayers consisted of two stages: a rapid Ca2+-dependent interaction followed by a slower process that resulted in irreversible binding of PKC to the monolayer. The second stage appeared to involve penetration of PKC into the hydrocarbon region of the phospholipid. The commonly used in vitro substrates for PKC, histone and protamine sulfate, also associated with and penetrated PS monolayers with critical pressures of 50 and 60 dyn/cm, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

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.

     

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)     

Induction of Epstein-Barr virus lytic cycle by tumor-promoting and non-tumor-promoting phorbol esters requires active protein kinase C.

Exposure to the tiglian 12-O-tetradecanoylphorbol-13-acetate (TPA) represents one of the most efficient and widely used protocols for inducing Epstein-Barr virus (EBV)-infected cells from latent into lytic cycle. Since TPA is both a potent tumor promoter and a potent activator of the cellular protein kinase C (PKC), we sought to determine whether either of these activities was closely linked to EBV lytic cycle induction. A panel of TPA structural analogs, encompassing tiglians with different spectra of biological activities, was assayed on a number of EBV-positive B-lymphoid cell lines. Lytic cycle induction correlated with the capacity to activate PKC, not with tumor promoter status; some nonpromoting tiglians were as efficient as TPA in inducing lytic cycle antigen expression. We then sought more direct evidence for an involvement of PKC in the induction process. In initial experiments, 1-(5-isoquinolinyl sulphonyl)-2-methylpiperazine (H-7), the best available pharmacological inhibitor of PKC, completely blocked the induction of the lytic cycle by TPA and its active analogs. This is consistent with, but does not prove, a requirement for active PKC in the induction process, since H-7 targets PKC preferentially but also has some effects on other kinases. We therefore turned to the synthetic pseudosubstrate peptide PKC(19-36) as a means of specific PKC inhibition and to the closely related but inactive peptide PKC(19-Ser-25-36) as a control. Using the technique of scrape loading to deliver the peptides into cells of an adherent EBV-positive target line, we found that the pseudosubstrate peptide PKC(19-36) completely and specifically blocked tiglian-induced entry of the cells into the lytic cycle. The evidence both from TPA analogs and from enzyme inhibition studies therefore indicates that the pathway linking TPA treatment to lytic cycle induction involves active PKC. Interestingly, inhibition of PKC had no effect upon the spontaneous entry into lytic cycle which occurs in naturally productive cell lines, suggesting that spontaneous entry is signalled by another route.     

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.     

Inhibition of human skin fibroblast proliferation by histamine and phorbol esters is mediated by protein kinase C

The proliferation of human skin fibroblasts in culture was examined using a [3H]thymidine incorporation assay. Histamine inhibited thymidine incorporation with an IC50 of about 0.2 microM. This effect was blocked by the H1 receptor antagonist mepyramine but not by the H2 receptor antagonist cimetidine. Protein kinase C activators, including several phorbol esters and mezerine, also inhibited thymidine incorporation. The IC50 for beta-phorbol 12,13-didecanoate was less than 0.1 nM. The alpha-isomer of this compound was inactive. Long-term treatment of cells with the beta-isomer eliminated the ability of both histamine and phorbol ester to inhibit thymidine incorporation, presumably due to downregulation of protein kinase C. Our results suggest that histamine H1 receptors are linked to activation of protein kinase C and that activation of this enzyme leads to an inhibition of cell proliferation.     

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.     

Effect of spermine on association of protein kinase C with phospholipid vesicles

The in vitro mechanism by which polyamines affect protein kinase C (PK C) activation process was investigated in a reconstituted system consisting of purified enzyme and phospholipid vesicles of various phosphatidylserine content. It was found that the addition of spermine greatly interferes with the association of PK C to liposomes. This tetramine, at micromolar concentrations, was most potently effective while other polyamines such as spermidine and putrescine were almost ineffective; therefore the modulatory action appeared to be structure specific. The spermine effect is dramatically influenced by the density of the phosphatidylserine present on the liposome, suggesting the complex formation with the acidic component on phospholipid vesicles to be the mechanism by which this polyamine exerts its modulatory action.     

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.     

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).     

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.