Subcellular distribution of protein kinase C/phorbol ester receptors in differentiating mouse keratinocytes

The activation of protein kinase C (PKC) by diacylglycerol or tumor promoters plays a pivotal role in signal transduction and subsequent activation of cellular processes. Since the activity of this enzyme is dependent on its immediate lipid domain, its relative distribution within the cell may be an important regulatory mechanism. We report here a relative decrease in PKC/phorbol ester receptor associated with the particulate fraction of mouse keratinocytes induced to differentiate by two separate systems. First, proliferating keratinocytes maintained in low Ca2+ (0.09 mM) serum-free medium were induced to differentiate rapidly by the addition of Ca2+ (1.8 mM). A 1.4-fold decrease in the percent of total phorbol receptor binding activity present in the particulate fraction and concomitant increase in binding in the cytosol fraction was evident 20 min after the Ca2+ addition. Second, in keratinocytes that differentiate over a 6 day cultivation period in serum-containing medium with Ca2+ concentration of 1.8 mM, a significant decrease in the percent of the phorbol receptor binding activity present in the particulate fraction was observed as the culture begins to differentiate on days 3 and 4. Maximal phorbol ester binding in the particulate fraction corresponded to the proliferative phase of the culture (day 2), while lower levels of PKC/phorbol ester binding to particulate fractions were noted during the early differentiative phase (days 3 and 4). Addition of the synthetic diacylglycerols 1-oleoyl-2-acetylglycerol or L-alpha-1,2 dioctanyl glycerol at 30 micrograms/ml to proliferating keratinocyte cultures induced a modest increase in two markers of terminal differentiation: cornified envelope formation and transglutaminase levels. These findings, taken together, support the hypothesis that PKC activation plays a role in the initial signalling events for keratinocyte differentiation.     

Phorbol ester induces intracellular translocation of phospholipid/Ca2+-dependent protein kinase and stimulates amylase secretion in isolated pancreatic acini

Treatment of intact rat pancreatic acini with phorbol ester (12-O-tetradecanoyl-phorbol-13-acetate, TPA) resulted in a time- and concentration-dependent translocation of phospholipid/Ca2+-dependent protein kinase (PL/Ca-PK) from the soluble fraction. Redistribution of PL/Ca-PK was concurrent with stimulation of amylase secretion by TPA-treated acini. Polymyxin B, a potent and selective inhibitor of PL/Ca-PK completely inhibited TPA-induced amylase secretion. These findings are consistent with a role for PL/Ca-PK in the regulation of pancreatic exocrine secretion.     

New insights into the regulation of protein kinase C and novel phorbol ester receptors.

Protein kinase C (PKC), a family of related serine-threonine kinases, is a key player in the cellular responses mediated by the second messenger diacylglycerol (DAG) and the phorbol ester tumor promoters. The traditional view of PKCs as DAG/phospholipid-regulated proteins has expanded in the last few years by three seminal discoveries. First, PKC activity and maturation is controlled by autophosphorylation and transphosphorylation mechanisms, which includes phosphorylation of PKC isozymes by phosphoinositide-dependent protein kinases (PDKs) and tyrosine kinases. Second, PKC activity and localization are regulated by direct interaction with different types of interacting proteins. Protein-protein interactions are now recognized as important mechanisms that target individual PKCs to different intracellular compartments and confer selectivity by associating individual isozymes with specific substrates. Last, the discovery of novel phorbol ester receptors lacking kinase activity allows us to speculate that some of the biological responses elicited by phorbol esters or by activation of receptors coupled to elevation in DAG levels could be mediated by PKC-independent pathways.     

Altered catalytic properties of protein kinase C in phorbol ester treated cells

Exposure of various cell types (rat-1 fibroblasts, bovine adrenocortical cells, human lymphoid cells) to nanomolar concentrations of TPA, resulted in a rapid, apparent loss of cellular protein kinase C content, when the enzyme was assayed by its phospholipid and Ca2+-dependent histone (H1)-kinase activity, following solubilization and DEAE-cellulose chromatography isolation. By contrast, no loss of protein kinase C was detected when the enzyme was probed by its high affinity PDBu binding capacity nor when the kinase activity was assayed with protein substrates other than histones, such as vinculin and a cytochrome P-450. It is concluded that, in addition to the previously reported enzyme subcellular redistribution, following TPA treatment, the phorbol ester induces striking alterations of the cellular protein kinase C catalytic activities. The molecular mechanisms of these changes and their implication in the tumor promotion process remain to be clarified.     

Dissimilar effects of the protein kinase C inhibitors, staurosporine and H-7, on cholecystokinin-induced enzyme secretion from rabbit pancreatic acini.

The effects of two putative inhibitors of protein kinase C activity, staurosporine and H-7, on partially purified protein kinase C and amylase secretion from isolated rabbit pancreatic acini were investigated. Staurosporine dose-dependently inhibited amylase release stimulated by an optimal concentration of cholecystokinin C-terminal octapeptide. At a concentration of 100 nM, the drug inhibited the secretory response to the secretagogue by approximately 50%. At the same concentration, staurosporine inhibited 12-O-tetradecanoylphorbol 13-acetate-stimulated enzyme secretion by 90%. Moreover, the potentiating effect of this phorbol ester on cholecystokinin-induced amylase release was completely abolished in the presence of staurosporine. Interestingly, amylase release was decreased to the level observed with the combination of cholecystokinin and staurosporine. In contrast, H-7, potentiated rather than inhibited cholecystokinin-stimulated enzyme secretion, whereas the secretory response to 12-O-tetradecanoylphorbol 13-acetate was not affected by the drug. Both staurosporine and H-7, however, inhibited protein kinase C purified from exocrine pancreatic tissue. Kinetic analysis revealed that both compounds inhibited protein kinase C competitively with respect to ATP. The Ki value for staurosporine was 0.55 nM and for H-7 13.5 microM. Our results obtained with staurosporine are in line with a stimulatory role of protein kinase C in cholecystokinin-induced enzyme secretion from the exocrine pancreas. The results obtained with H-7 emphasize that care has to be taken in interpreting the biological effects of this drug.     

Participation of protein kinase C in the activation of human neutrophils by phorbol ester

The calmodulin antagonist N(6-aminohexyl)-5-chloro-1-naphthalene-sulfonamide (W-7) has been examined as an inhibitor of superoxide anion production and granule exocytosis in phorbol ester (PMA)-activated neutrophils. Inhibition of the respiratory burst was observed at a concentration of W-7 identical to that required for inhibition of native protein kinase C (PKC), whereas the concentration required to inhibit the secretory response was found to correspond to that required for inhibition of the proteolytically converted fully active PKC. The IC50 of W-7 was in both cases 5 and 12 fold higher than that required for inhibition of calmodulin dependent kinases. The results confirm the essential role for the membrane-bound PKC in the production of O2- radicals and provide a clear evidence of the direct participation of the proteolytically activated cytosolic PKC to the secretory response of PMA activated neutrophils.     

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

In unstimulated rat islets (2 mM glucose), most of the ion-exchange purified protein kinase C (PKC) activity was associated with the cytosolic fraction. Both carbachol and phorbol myristate acetate caused a significant translocation of PKC activity from cytosolic to membrane fractions, but under the same conditions, glucose (20 mM) did not cause such a redistribution of PKC activity. PMA-induced translocation of PKC to the membrane fraction was also observed in electrically permeabilised islets, in which recovery of the enzyme activity was enhanced by buffering the intracellular Ca2+ concentration to 50 nM and supplying the permeabilised islets with protease inhibitors.     

Redistribution of protein kinase C isoforms in rat pancreatic acini during lactation and weaning

Freshly enzymatically isolated pancreatic acini from lactating and weaning Wistar rats were used to investigate the role of protein kinase C (PKC) isoforms during these physiologically relevant pancreatic secretory and growth processes. The combination of immunoblot and immunohistochemical analysis shows that the PKC isoforms alpha, delta, and epsilon are present in pancreatic acini from control, lactating and weaning rats. A vesicular distribution of PKC-alpha, -delta, and -epsilon was detected by immunohistochemical analysis in the pancreatic acini from all the experimental groups. PKC-delta showed the strongest PKC immunoreactivity (PKC-IR). In this vesicular distribution, PKC-IR was located at the apical region of the acinar cells. No differences were observed between control, lactating and weaning rats. However, the immunoblot analysis of pancreatic PKC isoforms during lactation and weaning showed a significant translocation of PKC-delta from the cytosol to the membrane fraction when compared with control animals. Translocation of PKC isoforms (alpha, delta and epsilon) in response to 12-O-tetradecanoyl phorbol 13-acetate (TPA) 1 microM (15 min, 37 degrees C) was comparable in pancreatic acini from control, lactating and weaning rats. In the control group, a significant translocation of all the isoforms (alpha, delta and epsilon) from the cytosol to the membrane was observed. The PKC isoform most translocated by TPA was PKC-delta. In contrast, no statistically significant increase in PKC-delta translocation was detected in pancreatic acini isolated from lactating or weaning rats. These results suggest that the PKC isoforms are already translocated to the surface of the acinar cells from lactating or weaning rats. In addition, they suggest that isoform specific spatial PKC distribution and translocation occur in association with the growth response previously described in the rat exocrine pancreas during lactation and weaning.     

Inhibition of phorbol ester binding and protein kinase C activity by heavy metals

Other laboratories have reported biphasic effects of heavy metals on protein kinase C activity: stimulation followed by inhibition at higher concentrations. We demonstrate that these earlier findings most likely resulted from a combination of the effect of the heavy metals to liberate Ca2+ from Ca2+-EGTA buffer systems and the direct inhibitory effects of the metals on protein kinase C. Simulations of such interactions substantiate this conclusion. When soluble protein kinase C is prepared without the addition of Ca2+ or chelator, heavy metals (Cd2+, Cu2+, Hg2+, Zn2+, in the 10 microM range) inhibit the activity of, and the binding of regulatory ligands to, protein kinase C. Heavy metals inhibit the extent of [3H]phorbol dibutyrate binding without affecting the affinity of the interaction, an inhibition that is not surmounted by excess phospholipid. Heavy metals also inhibit the phospholipid-dependent catalytic activity of protein kinase C in a manner that excess phosphatidylserine can overcome. The inhibition of enzyme activity by heavy metals cannot be surmounted by excess Ca2+ or Mg2+. The inhibitory effects of heavy metals are not confined to protein kinase C. Heavy metals also inhibit cyclic AMP binding to cyclic AMP-dependent protein kinase and the catalytic activity of that kinase, but in a distinctly different pattern.     

Synergy between zinc and phorbol ester in translocation of protein kinase C to cytoskeleton

Protein kinase C was measured in the cytoskeletal fraction of lymphocytes, platelets and HL60 cells, by specific binding of [3H]phorbol dibutyrate and by immunoblotting with antibody to a consensus sequence in the regulatory domain of alpha-, beta- and gamma-isozymes of protein kinase C. Treatment of cells for 40 min with a combination of zinc (2-50 microM), zinc ionophore pyrithione and unlabelled phorbol dibutyrate (200 nM) caused up to a ten-fold increase in cytoskeletal protein kinase C and a corresponding decrease in other cellular compartments. Omission of any of the reagents resulted in much less or no translocation. These effects were inhibited by 1,10-phenanthroline, which chelates zinc, and were not seen with calcium. Increase in cytoskeletal protein kinase C persisted for several hours and appeared to involve attachment of the enzyme to actin microfilaments. We propose that zinc, like calcium, regulates the distribution of PKC in cells. However, unlike calcium which controls the binding of PKC to the lipid component on cell membranes, zinc controls the distribution of PKC to membrane cytoskeleton, possibly actin.     

Effects of a phorbol ester and clomiphene on protein phosphorylation and insulin secretion in rat pancreatic islets.

The potentiation of glucose-stimulated insulin release induced by 100 nM-12-O-tetradecanoylphorbol 13-acetate (TPA) was inhibited by clomiphene, an inhibitor of protein kinase C (PK C), in a dose-dependent manner. Clomiphene at concentrations up to 50 microM had a modest inhibitory action (27%) on insulin release stimulated by 10 mM-glucose alone, but had no effect on the potentiation of insulin release induced by forskolin. Islet PK C activity, associated with a particulate fraction, was stimulated maximally by 100 nM-TPA. This stimulation was blocked by clomiphene in a dose-dependent manner, with 50% inhibition at 30 microM. Incubation of intact islets with TPA after preincubation with [32P]Pi and 10 mM-glucose to label intracellular ATP resulted primarily in enhanced phosphorylation of a 37 kDa protein (mean value, +/- S.E.M., 36,700 +/- 600 Da; n = 7). This increased phosphorylation was blocked by the simultaneous inclusion of clomiphene. Subcellular fractionation revealed the presence of the 37 kDa phosphoprotein in a 24,000 g particulate fraction of islet homogenates. Neither clomiphene nor TPA affected the rate of glucose oxidation by islets. These results show that the phosphorylation state of a 37 kDa membrane protein parallels the modulation of insulin release induced by TPA and clomiphene and support a role for PK C in the insulin-secretory mechanism.     

Concanavalin A and phorbol ester cause opposite subcellular redistribution of protein kinase C

Concanavalin A and phorbol ester induce human blood monocytes to produce superoxide. We tested whether activation of human monocytes by these agents is accompanied by a subcellular redistribution of protein kinase C. Phorbol ester predictably caused a profound shift of the enzyme from the cytosol to the particulate fraction. In contrast concanavalin A induced a shift of the enzyme from the particulate fraction to the cytosol. The opposite effect of these agents on kinase C translocation was observed also by analysis of the phosphorylation of cytosolic proteins. Kinase C is either not involved in monocyte activation or does so by distinct pathways determined by the activating agent.     

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.     

Phosphatidylinositol 4,5-bisphosphate competitively inhibits phorbol ester binding to protein kinase C

Calcium phospholipid dependent protein kinase C (PKC) is activated by diacylglycerol (DG) and by phorbol esters and is recognized to be the phorbol ester receptor of cells; DG displaces phorbol ester competitively from PKC. A phospholipid, phosphatidylinositol 4,5-bisphosphate (PIP2), can also activate PKC in the presence of phosphatidylserine (PS) and Ca2+ with a KPIP2 of 0.04 mol %. Preliminary experiments have suggested a common binding site for PIP2 and DG on PKC. Here, we investigate the effect of PIP2 on phorbol ester binding to PKC in a mixed micellar assay. In the presence of 20 mol % PS, PIP2 inhibited specific binding of [3H]phorbol 12,13-dibutyrate (PDBu) in a dose-dependent fashion up to 85% at 1 mol %. Inhibition of binding was more pronounced with PIP2 than with DG. Scatchard analysis indicated that the decrease in binding of PDBu in the presence of PIP2 is the result of an altered affinity for the phorbol ester rather than of a change in maximal binding. The plot of apparent dissociation constants (Kd') against PIP2 concentration was linear over a range of 0.01-1 mol % with a Ki of 0.043 mol % and confirmed the competitive nature of inhibition between PDBu and PIP2. Competition between PIP2 and phorbol ester could be demonstrated in a liposomal assay system also. These results indicate that PIP2, DG, and phorbol ester all compete for the same activator-receiving region on the regulatory moiety of protein kinase C, and they lend support to the suggestion that PIP2 is a primary activator of the enzyme.     

Bimodal effect of phorbol ester on B cell activation. Implication for the role of protein kinase C.

The role of protein kinase C PKC in B cell activation is controversial. These studies were undertaken to determine whether protein kinase C has a stimulatory or inhibitory role in B cell activation. We found that treatment of B cells for a short period of time (30 min) with the PKC activator phorbol 12,13-dibutyrate (PDBU) primed the cells for enhanced proliferative responses to anti-immunoglobulin (anti-Ig) antibody whereas treatment for a longer period of time (3 h or more) resulted in suppression of proliferation. The enhanced proliferative response to treatment of B cells with PDBU for short periods of time was associated with inhibition of anti-Ig-stimulated increases in phosphatidyl 4,5-bisphosphate (PIP2) hydrolysis and inhibition of increases in [Ca2+]i, indicating that activation of PKC per se might be sufficient for enhancing B cell activation. The time-dependent effect of phorbol esters on the inhibition of B cell proliferation was found to be closely correlated with the kinetics of disappearance of PKC as measured by Western blot and by enzymatic activity but not with inhibition of [Ca2+]i and PIP2. These data demonstrate a bimodal time-dependent effect of PDBU on B cell activation and suggest that (a) the inhibitory effect of phorbol ester on anti-Ig-induced proliferation may be due to the disappearance of PKC rather than to the inhibition of PIP2 and Ca2+; and (b) the early activation of PKC is a stimulatory rather than an inhibitory signal in the induction of B lymphocyte proliferation by anti-Ig.     

Calcium concentration and amylase secretion in guinea pig pancreatic acini: interactions between carbachol, cholecystokinin octapeptide and the phorbol ester, 12-O-tetradecanoylphorbol 13-acetate

The effects of the phorbol ester, 12-O-tetradecanoylphorbol 13-acetate (TPA) on amylase secretion and cytoplasmic free calcium concentration ([Ca2+]i) were investigated in dispersed guinea pig pancreatic acini. Carbachol evoked dose-dependent increases in amylase secretion and [Ca2+]i with half-maximal responses at 2.5 and 5 microM, respectively. Carbachol-induced calcium transients could be blocked by atropine. In the presence of a maximal effective dose of carbachol, cholecystokinin octapeptide caused no further increase in [Ca2+]i, suggesting that both agonists act on the same pool of trigger calcium. TPA (10(-9)-10(-6) M) stimulated amylase secretion with no change in [Ca2+]i. Maximum amylase secretion occurred at 0.5 microM TPA. Preincubation of acini in the presence of TPA resulted in a time- and dose-dependent inhibition (IC50 = 30 nM) of the carbachol-induced rise in [Ca2+]i, the maximal effect being observed within 3 min. The inactive phorbol ester, 4 alpha-phorbol 12,13-didecanoate was ineffective in inhibiting the carbachol-stimulated rise in [Ca2+]i. These findings suggest that, in addition to stimulating amylase secretion, probably through protein kinase C, TPA may also exert a negative feedback control over secretagogue-induced calcium transients.     

Differentiative action of K252a on protein kinase C and a calcium-unresponsive, phorbol ester/phospholipid-activated protein kinase

The Triton X-100 extract of the particulate fraction of porcine spleen contains a protein kinase which can be activated by phospholipid and the phorbol ester TPA but does not respond to phospholipid and calcium. The partially purified kinase has a molecular weight of 76 kDa (p76-kinase) and hence is somewhat smaller than the similarly behaving p82-kinase from mouse epidermis and spleen. The p76-kinase shows strong autophosphorylation. The protein kinase inhibitor K252a clearly differentiates between the Ca2+-unresponsive p76-kinase and Ca2+-responsive PKC. At concentrations of up to 5 x 10(-7)M it fails to suppress p76-kinase-catalyzed autophosphorylation and histone phosphorylation, but it inhibits PKC-catalyzed phosphorylation up to 50%. The IC50 values of K252a regarding PKC and the p76-kinase differ by two orders of magnitude. At low concentrations, K252a appears to slightly activate further TPA-activated p76-kinase. It is not able, however, to replace TPA and to stimulate the p76-kinase in the presence of phospholipid alone.     

Effects of angiotensin II and of phorbol ester on protein kinase C activity and on prostacyclin production in cultured rat aortic smooth-muscle cells.

A procedure is described for isolation of the pterin molybdenum cofactor, in the active molybdenum-containing state, starting from purified milk xanthine oxidase. The method depends on the use of anaerobic-glove-cabinet techniques and on working in aqueous solution, in the presence of 1 mM-Na2S2O4. SDS was used to denature the protein, followed by ion-exchange chromatography and gel filtration. The cofactor, obtained at concentrations up to 0.5-1.0 mM, was fully active in the nit-1 assay [Hawkes & Bray (1984) Biochem. J. 214, 481-493], with a specific activity of 22 nmol of NO2-/min per pg-atom of Mo (with 15% molybdate-dependence). The Mr, determined by gel filtration, was about 610, consistent with the structure proposed by Kramer, Johnson, Ribeiro, Millington & Rajagopalan [(1987) J. Biol. Chem. 262, 16357-16363]. At pH 5.9, under anaerobic conditions, the cofactor was stable for at least 300 h at 20-25 degrees C.     

Regulation by the protein kinase C activator phorbol ester of calcium channels in polymorphonuclear leukocytes

The Quin fluorescence in gamma-hexachlorocyclohexane-stimulated polymorphonuclear leukocytes is rapidly increased, which points to the increase in Ca2+in concentration during leukotriene B4 synthesis in leukocytes. An addition of EGTA and calcium antagonists (nifedipine, verapamil, diltiazem) to cell suspensions does not affect the basal level of internal Ca2+ but results in the inhibition of the gamma-hexachlorocyclohexane-induced Ca2+ increase. Two mechanisms of calcium homeostasis regulation in neutrophils are proposed. One of them, cAMP regulation, is coupled with a potent inhibiting effect of prostacyclin, an adenylate cyclase activator, on Ca2+in increase in stimulated neutrophils. The other one is the activation of protein kinase C catalyzed by 4 beta-phorbol-12 beta-myristate-13 alpha-acetate. The experimental results suggest that such an activation blocks Ca2+ influx into the cells via the closure of Ca2+ channels. The synergism of action of the above mechanisms in the regulation of calcium homeostasis in neutrophils is demonstrated.     

Ca2+ and phorbol ester activation of protein kinase C at intracellular Ca2+ concentrations and the effect of TMB-8

A calcium-activated, phospholipid-dependent protein kinase (protein kinase C) was purified to near homogeneity from human polymorphonuclear leukocytes and shown to be identical to bovine protein kinase C. The Ca2+ activation of the enzyme was studied and the Ca2+ concentrations required to activate the enzyme were compared to free cytosolic Ca2+ concentrations in resting and activated polymorphonuclear leukocytes. The free calcium concentrations in the cytosol and in the enzyme assay mixture were determined using the calcium indicator quin 2. The enzyme activity was almost totally dependent upon phosphatidylserine and could be strongly activated by Ca2+ concentrations in the micromolar range, but was not activated by phosphatidylserine at Ca2+ concentrations corresponding to the intracellular free Ca2+ concentration under resting conditions. However, at similar Ca2+ concentrations (less than 2.5 X 10(-7) M) the enzyme was highly activated by phorbol 12-myristate 13-acetate (PMA) or diolein in the presence of phosphatidylserine. It was demonstrated that PMA stimulation of human polymorphonuclear leukocytes did not induce any increase in the level of the intracellular free calcium concentration. It was concluded that PMA activation of protein kinase C occurred independently of a rise in the intracellular Ca2+ concentration. K0.5 (half-maximal activation) for the PMA activation of purified protein kinase C was shown to be equivalent to the K0.5 for PMA stimulation of superoxide (O-2) production in human polymorphonuclear leukocytes, suggesting that protein kinase C is involved in activation of the NADPH oxidase. The presumed intracellular Ca2+ antagonist TMB-8 inhibited the PMA-induced superoxide production, but neither by an intracellular Ca2+ antagonism nor by a direct inhibition of protein kinase C activity.