An ecto-cyclic AMP-independent protein kinase in goat spermatozoa and its change of activity during forward motility

Goat cauda-epididymal intact spermatozoa have been shown to possess an ecto-cyclic AMP-independent protein kinase activity on the external surface that causes phosphorylation of the serine and threonine residues of exogenous phosvitin. The enzyme is neither a tyrosine kinase nor a catalytic subunit of the cyclic AMP-dependent protein kinase. It is not activated by Ca2+, calmodulin and phosphatidylserine. The intact-cell enzyme is capable of phosphorylating a variety of proteins including sperm plasma membrane-bound phosphoprotein(s). The enzymic activity of the intact spermatozoa was not due to contamination of broken or "leaky" cells. The kinase activity of the whole cells was strongly inhibited by the non-penetrating surface probes: p-chloromercuriphenylsulphonic acid (10 microM) and proteases (125 micrograms/ml). The specific activity of the ecto-kinase increased nearly 100% during vigorous forward progression of spermatozoa.     

Phosphorylation of external cell-surface proteins by an endogenous ecto-protein kinase of goat epididymal intact spermatozoa

Intact spermatozoa from goat cauda epididymides possess an ecto-(cyclic AMP-independent protein kinase) activity that causes transfer of the terminal phosphate of exogenously added [gamma-32P]ATP to the serine and threonine residues of several endogenous plasma-membrane phosphoproteins located on the external cell surface. Cyclic AMP, cyclic GMP, calmodulin and muscle cyclic AMP-dependent protein kinases I and II had no appreciable effect on the rate of phosphorylation of ecto-proteins by the intact cells. The ecto-enzyme is not derived from the catalytic subunit of a cyclic AMP-dependent kinase. Sperm ecto-kinase activity is not due to contamination of broken cells or any possible cell damage during incubation and isolation of spermatozoa. The phosphorylation reaction was linear for approx. 1 min and there was no detectable uptake of ATP by these cells. The activity of the ecto-kinase was strongly inhibited by proteinases and by the membrane-nonpenetrating surface probes. The products of the reaction were associated with the intact cells and the 32P of the labelled cells was largely lost when treated with Triton X-100 or proteinases: trypsin and pronase. These data are consistent with the view that the observed protein kinase and the phosphoproteins are located on the external surface of spermatozoa. Vigorously forward-motile whole spermatozoa showed a relatively high capacity to phosphorylate ecto-proteins that undergo rapid turnover. The results suggest the occurrence of a novel coupled-enzyme system (ecto-protein kinase and phosphoprotein phosphatase) on the sperm external surface that may modulate sperm physiology by determining the phosphorylated states of the ecto-proteins.     

Genetic evidence that a phorbol ester tumor promoter stimulates ornithine decarboxylase activity by a pathway that is independent of cyclic AMP-dependent protein kinases in CHO cells

Ornithine decarboxylase (ODC) inductions by cholera toxin and by the phorbol ester tumor promoter, TPA, were compared in wild-type Chinese hamster ovary (CHO) cells and in mutant cells having altered cyclic AMP-dependent protein kinase activity. The aim of these studies was to determine whether cyclic AMP-dependent protein kinase is involved in these inductions. The time course and the magnitude of ODC inductions by either 100 ng/ml cholera toxin or 100 ng/ml TPA were similar in wild-type cells with a maximum at 3-4 hours after treatment and a return to unstimulated levels by 8 hours. Induction of ODC by cholera toxin was suppressed more than 80% in the four protein kinase mutants studied (10215, 10248, 10260, and 10265), strongly implicating a cyclic AMP-dependent kinase step in the mechanism of induction. Similar results were found with the cyclic AMP analog 8-Br-cyclic AMP and the phosphodiesterase inhibitor, methyl-isobutylxanthine. The induction of ODC by TPA, on the other hand, was only partially inhibited (approximately 50%) in three of four mutants. Lower ODC activity in two mutants stimulated by cholera toxin or TPA whose kinetics were studied in more detail could not be ascribed to a reduced affinity (Km) of ornithine for the enzyme, but appeared to be due to reduced catalytic activity (Vmax) in the extracts. These results suggest that the induction of ODC by TPA proceeds by a mechanism which is only partially dependent on an intact cyclic AMP-dependent protein kinase activity.     

佛波醇酯诱导HL-60细胞巨噬细胞样分化时蛋白激酶C活力及分布的改变

本文对佛波醇酶诱导人早幼粒白血病细胞系HL-60细胞分化为巨噬细胞样细胞对蛋白激酶c活力及其在亚细胞分布的变化进行了研究。蛋白激酶c活力在TPA处理1小时即明显降低,此低水平的酶活力持续整个实验时期。酶的亚细胞分布研究提示TPA处理细胞胞质组分酶活力剧烈降低,而颗粒组分存在一高盐浓度洗脱的酶活力峰。蛋白激酶c抑制剂三氟过(口了)嗪单独处理HL-60细胞导致胞质和颗粒组分酶活力升高,但并不诱导细胞分化;若与TPA合并处理细胞,酶活力又降低,此时细胞又分化为巨噬细胞样细胞。对上述结果的可能机理进行了讨论。     

Effects of phorbol esters, diglyceride, and cholinergic agonists on the subcellular distribution of protein kinase C in intact or digitonin-permeabilized adrenal chromaffin cells

Phorbol esters which activate protein kinase C increased the percentage of membrane-bound protein kinase C activity in bovine adrenal chromaffin cells from less than 10 to 20-50% within 30 min. Permeabilization of chromaffin cells with digitonin in the absence of Ca2+ and phorbol esters caused virtually 100% of the protein kinase C activity to leave the cells within 1 h, which is consistent with protein kinase C being soluble and cytosolic. However, if cells were incubated for 15-30 min with 12-O-tetradecanoylphorbol-13-acetate (TPA) prior to permeabilization, 50-60% of the protein kinase C activity exited from the cells within 1 h of permeabilization. In cells not incubated with phorbol ester, permeabilization in the presence of 1-10 microM Ca2+ also decreased the rate at which protein kinase C exited from the cells. The slower release of protein kinase C caused by prior incubation of the cells with TPA or because of the presence of micromolar Ca2+ in permeabilized cells was associated with increased membrane-bound protein kinase C. The effects of TPA and permeabilization in the presence of micromolar Ca2+ were approximately additive. Active phorbol esters had different abilities to cause retention of protein kinase C in digitonin-treated cells. Dioctanoylglycerol, which activates protein kinase C in vitro and enhanced Ca2+-dependent secretion from permeabilized chromaffin cells similarly to TPA, also increased membrane-bound protein kinase C in intact cells, but had no effect on the retention of protein kinase C in permeabilized cells in the presence or absence of Ca2+. The different abilities of protein kinase C activators to cause retention of protein kinase C in subsequently permeabilized cells suggest differences in the reversibility of the binding. The mixed nicotinic-muscarinic agonist carbachol and the nicotinic agonist 1,1-dimethyl-4-phenylpiperazinium, but not the muscarinic agonist muscarine, caused 3-10% of the total protein kinase C activity to become membrane-bound within 3 min in intact chromaffin cells. Thus, nicotinic stimulation of chromaffin cells may rapidly activate protein kinase C.     

Phosphorylation and dephosphorylation of human promyelocytic leukemia cell (HL-60) proteins by tumor promoter

Human promyelocytic leukemia cell line (HL-60) has been shown to be induced to the terminal differentiation into macrophage-like cells by a tumor promoter, 12-O-tetradecanoyl-phorbol-13-acetate (TPA). The present studies describe the effects of TPA on the phosphorylation of HL-60 cell proteins. A rapid decrease in the phosphorylation of a 75 kD protein was observed within a few minutes after treatment with TPA. On the other hand, TPA treatment of HL-60 cells caused rapid increase in the phosphorylation of a 67 kD protein and other minor proteins. Phorbol and 4α-phorbol-12,13-dodecanoate, both of which are biologically inactive derivatives of TPA, failed to cause any changes in protein phosphorylation in HL-60 cells. These results suggest that changes in protein phosphorylation are involved in mechanisms of the differentiation in HL-60 cells induced by TPA. Cell fractionation experiments revealed that 67K protein was located in cytosol. Though 75K protein also seemed to be located in cytosol, the phosphate moiety of 75K protein was almost lost during cell fractionation, suggesting that the phosphorylation of 75K protein was specifically regulated in HL-60 cells. Dimethyl sulfoxide (DMSO), retinoic acid (RA) and 1,25-dihydroxy-vitamin D₃, all of which induce the differentiation in HL-60 cells, did not cause any changes in protein phosphorylation. These results suggest that the changes in protein phosphorylation are specific for TPA. The possible mechanisms of changes in protein phosphorylation by TPA were discussed.     

c-Src-dependent tyrosine phosphorylation of IKKbeta is involved in tumor necrosis factor-alpha-induced intercellular adhesion molecule-1 expression

The signaling pathway involved in tumor necrosis factor-alpha (TNF-alpha)-induced intercellular adhesion molecule-1 (ICAM-1) expression was further studied in human A549 epithelial cells. TNF-alpha- or 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced ICAM-1 promoter activity was inhibited by a protein kinase C (PKC) inhibitor (staurosporine), tyrosine kinase inhibitors (genistein and herbimycin A), or an Src-specific tyrosine kinase inhibitor (PP2). TNF-alpha- or TPA-induced IkappaBalpha kinase (IKK) activation was also blocked by these inhibitors, which slightly reversed TNF-alpha-induced but completely reversed TPA-induced IkappaBalpha degradation. c-Src and Lyn, two members of the Src kinase family, were abundantly expressed in A549 cells, and their activation by TNF-alpha or TPA was inhibited by the same inhibitors. Furthermore, the dominant-negative c-Src (KM) mutant inhibited induction of ICAM-1 promoter activity by TNF-alpha or TPA. Overexpression of the constitutively active PKC or wild-type c-Src plasmids induced ICAM-1 promoter activity, this effect being inhibited by the dominant-negative c-Src (KM) or IKKbeta (KM) mutant but not by the nuclear factor-kappaB-inducing kinase (NIK) (KA) mutant. The c-Src (KM) mutant failed to block induction of ICAM-1 promoter activity caused by overexpression of wild-type NIK. In co-immunoprecipitation and immunoblot experiments, IKK was found to be associated with c-Src and to be phosphorylated on tyrosine residues after TNF-alpha or TPA treatment. Two tyrosine residues, Tyr188 and Tyr199, near the activation loop of IKKbeta, were identified as being important for NF-kappaB activation. Substitution of these residues with phenylalanines abolished ICAM-1 promoter activity and c-Src-dependent phosphorylation of IKKbeta induced by TNF-alpha or TPA. These data suggest that, in addition to activating NIK, TNF-alpha also activates PKC-dependent c-Src. These two pathways converge at IKKbeta and go on to activate NF-kappaB, via serine phosphorylation and degradation of IkappaB-alpha, and, finally, to initiate ICAM-1 expression.     

Induction of plasminogen activator activity by phorbol ester in transformed fetal bovine aortic endothelial cells. Possible independence from protein kinase C

12-O-tetradecanoyl phorbol 13-acetate (TPA) and 1,2-dioctanoyl-sn-glycerol (diC8) activate protein kinase C (PKC) in transformed fetal bovine aortic endothelial GM 7373 cells. Both molecules cause a similar increase in membrane-associated PKC activity and in the phosphorylation of a PKC-specific endogenous 87-kDa substrate in intact cells. Even though both TPA and diC8 exert a mitogenic activity in GM 7373 cells, only TPA induces also an increase in cell-associated plasminogen activator (PA) activity. Down-regulation of PKC which follows TPA-pretreatment completely abolishes the mitogenic activity of diC8 and the mitogenic and PA-inducing activity of TPA. However, both the PKC inhibitor H-7 and the down-regulation of PKC which follows a prolonged stimulation with diC8 do not abolish the PA-inducing activity of TPA. The PA-inducing activity of TPA is instead inhibited in cultures incubated in the presence of 1 mM EGTA or in a calcium-free medium. The data indicate that TPA and diC8 induce a different pattern of cellular activation in GM 7373 cells and that the PA-inducing activity of TPA might not be mediated by PKC.     

Mechanism of inhibition by cyclic AMP of protein kinase C-triggered respiratory burst in Ehrlich ascites tumour cells

The superoxide anion generation in Ehrlicg ascites tumour (EAT) cells increased more than two-fold in the presence of the tumour promoter, tetradecanoyl phorbol myristate acetate (TPA). Epinephrine and dibutryl cAMP (Bt₂ cAMP) inhibited in a dose-dependent manner, both basal and TPA-triggered superoxide generation in EAT cells. The kinetics of inhibition of superoxide generation showed a maximum inhibition between 30 and 40 min of preincubation with epinephrine or Bt₂ cAMP of EAT cells and coincided with an increase in activity of a phosphoprotein phosphatase. In TPA-treated EAT cells, epinephrine or Bt₂ cAMP increased the phosphatase activity in a dose-dependent manner. In vitro EGTA, EDTA and sodium fluoride inhibited phosphatase activity. Superoxide generation in response to TPA in Triton-permeabilized EAT cells was inhibited by inclusion of the phosphatase in the assay. Taken together, these results clearly suggest that the phosphatase activity in EAT cells develops as a result of protein kinase A (PKA) and protein kinase C (PKC)-mediated phosphorylation of the phosphatase which then mediates dephosphorylation of the PKC-triggered phosphorylation of proteins to inhibit respiratory burst. A cross-talk between PKA and PKC pathways negatively modulates superoxide generation in EAT cells.     

Dimethylsulfoxide, retinoic acid and 12-O-tetradecanoylphorbol-13-acetate induce a selective decrease in the phosphorylation of P150, a surface membrane phosphoprotein of HL60 cells resistant to adriamycin

Studies have been carried out to analyze protein phosphorylation in membranes isolated from adriamycin resistant HL60 cells which have been grown for various time periods in the presence of dimethylsulfoxide (DMSO), retinoic acid (RA) or 12-O-tetradecanoylphorbol-13-acetate (TPA). The results show that membranes isolated from cells treated with these agents are defective in the phosphorylation of P150, a membrane phosphoprotein associated with drug resistance in HL60 cells. This response is highly selective since only a few membrane proteins show decreased phosphorylation levels under these conditions. Magnesium dependent protein kinase activity in membranes from cells treated with DMSO, RA or TPA is not altered relative to untreated membranes under conditions where there is a major decrease in P150 phosphorylation. Additional studies also show that treatment of resistant cells with TPA results in a major decrease in the in vivo phosphorylation of P150. These results thus demonstrate that agents capable of inducing differentiation in HL60 cells can selectively modulate the phosphorylation of P150. This system should be of value in clarifying mechanisms involved in the phosphorylation of this protein.     

Phorbol ester causes desensitization of gonadotropin-responsive adenylate cyclase in a murine Leydig tumor cell line

The murine Leydig tumor cell line, MLTC-1, contains gonadotropin receptors and a gonadotropin-responsive adenylate cyclase system that became refractory (desensitized) when exposed to human chorionic gonadotropin (hCG). MLTC-1 cells also contain phorbol ester receptors with a Kd of 53 nM for [3H]phorbol dibutyrate. Exposing cells to 12-O-tetradecanoyl phorbol 13-acetate (TPA) also causes desensitization of the hCG response. TPA-induced desensitization was similar to hCG-induced desensitization by every criteria tested. Both TPA- and hCG-induced desensitization caused approximately 50% loss of the hormone response within 30 min. Neither TPA or hCG altered receptor affinity for hCG. The dose response of adenylate cyclase to hCG or GTP in isolated membranes was not affected by either hCG- or TPA-induced desensitization. Similarly the dose response to hCG of cAMP accumulation in intact cells was not altered by desensitization with hCG or TPA. It was determined that MLTC-1 cells have Ca2+/phospholipid-dependent protein kinase activity that displayed a dose-dependent response to TPA. The concentration of TPA required to activate the protein kinase was similar to that required for desensitization. Phorbol esters that were unable to activate protein kinase C were also unable to desensitize MLTC-1 cells. The protein kinase from MLTC-1 cells was also activated by diacylglycerol. In addition, diacylglycerols caused desensitization of the hCG response. TPA- and diacylglycerol-induced desensitization is probably mediated by protein kinase C, and the similarities between hCG- and TPA-induced refractoriness suggests a convergence of mechanisms at some point of MLTC-1 cell desensitization.     

Modulation of transglutaminase activity in mononuclear phagocytes and macrophage-like tumor cell lines by differentiation agents

The effect of glucocorticosteroids, retinoids, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) and the tumor promoter phorbol myristate acetate (TPA) on the expression of transglutaminase activity in vitro differentiating bone marrow-derived mouse and rat mononuclear phagocytes (BMDMP) and mouse and human myeloid leukemia cell lines was assessed. Dexamethasone was found to induce an increase of about 100% in transglutaminase activity in mouse and rat BMDMP. The effect was time- and dose-dependent, and specific for steroids with glucocorticoid activity. Retinoic acid (RA) suppressed transglutaminase activity in mouse BMDMP (approximately 50%) and enhanced it in rat BMDMP (100-200%). Other retinoids were less effective. 1,25(OH)2D3 had little effect on transglutaminase expression in mouse BMDMP and suppressed it in rat BMDMP (approximately 60%). TPA exerted a suppressive effect (approximately 50%) on transglutaminase activity of both rat and mouse BMDMP. In murine (P388D1 and J774.2) and human (ML3, HL-60, KG-1, HEL, U937) myeloid leukemia cell lines, dexamethasone enhanced transglutaminase activity to a varying degree (100-1,000%), RA suppressed it in P388D1 cells (approximately 70%) and enhanced it in the other cell lines (100-1,500%), 1,25(OH)2D3 induced a rather small augmentation of enzyme expression, whereas TPA suppressed enzyme expression (70-100%). The species-specific differences previously observed by us for the effect of RA, dexamethasone and 1,25(OH)2D3 on the formation of BMDMP from mouse and rat bone marrow progenitor cells are now shown to extend also to effects on expression of transglutaminase activity. From a mechanistic point of view it is of interest that dexamethasone uniformly enhanced transglutaminase activity, whereas TPA suppressed it. RA and 1,25(OH)2D3 induced either suppression or enhancement in the various cell types, with no correlation between the direction of the effect of the two agents. The data suggest that modulation of transglutaminase activity by the four agents occurs via disparate mechanisms.     

Interferon gamma bound to endothelial cells is phosphorylated by ecto-protein kinases

The presence of protein kinase activity and its phosphorylated products has been demonstrated on the outer surface of the plasma membrane of endothelial cells. Extracellular phosphorylation was detected by incubation of primary endothelial cells (HUVEC's) and endothelial cell line EA.hy 926 with [gamma-32P]ATP. The reaction products were subjected to SDS/PAGE, autoradiography and scanning densitometry. Under the experimental conditions, five proteins with apparent molecular masses of 19, 23, 55, 88, and 110 kDa were prominently phosphorylated in both types of cells. Phosphorylation of the 19 kDa protein was the most rapid reaching maximum after 60 s and then the protein became dephosphorylated. Ecto-protein kinases responsible for the surface labeling of membrane proteins were characterized by using (a) protein kinase C inhibitors: K-252b, chelerythrine chloride, and [Ala113] myelin basic protein (104-118), (b) protein kinase A inhibitor Kemptide 8334, and (c) casein kinase II inhibitor 5,6-dichloro-1-beta-D-ribofuranosyl benzimidazole (DRB). Stimulation of endothelial cells with tumor necrosis factor alpha (TNF alpha) and interferon gamma (IFN gamma) is associated with 20-80% reduction of extracellular phosphorylation of all membrane proteins. IFN gamma bound to membrane receptors becomes rapidly phosphorylated. Only in the case of IFN gamma it was associated with the appearance of a strongly phosphorylated band of 17 kDa corresponding to IFN gamma itself. Phosphorylation of this 17 kDa exogenous substrate was prevented by an ecto-kinase inhibitor K-252b. The existence of ecto-phosphoprotein phosphatase activity in endothelial cells was evidenced by testing the effect of microcystin LR--a membrane impermeable reagent that inhibits both PP-1 and PP-2a phosphoprotein phosphatases. The extent of phosphorylation of 19 kDa and 110 kDa phosphoproteins significantly increased in the presence of microcystin. Our results suggest the presence of at least two ecto-kinase activities on endothelial cells that may play a significant role(s) in the regulation of cytokines function.     

Protein kinase C activity can desensitize the gonadotropin-responsive adenylate cyclase in Leydig tumor cells. But hCG-induced desensitization does not involve protein kinase C activation

The murine Leydig tumor cell line, MLTC-1, contains a gonadotropin receptor-coupled adenylate cyclase. Although the binding of human choriogonadotropin (hCG) initially causes cells to accumulate cAMP, in time, the response to hCG is attenuated by desensitization. Treating intact cells with the tumor promoter, 12-O-tetradecanoyl-phorbol-13-acetate (TPA), or with diacylglycerol also causes desensitization of the hCG response. These compounds are activators of calcium/phospholipid-dependent protein kinase (PKC). Treating MLTC-1 cells with TPA or dioctanoylglycerol increased the portion of PKC in the cell membrane fraction. This phenomenon is associated with activation of PKC. Treating isolated membranes with purified PKC desensitize the hCG response. Thus, desensitization caused by TPA or dioctanoylglycerol is probably mediated by PKC. PKC is normally activated when phosphoinositides are metabolized to diacylglycerol and inositol phosphates. There was no significant accumulation of inositol phosphates when cells were treated with hCG. hCG did not increase the portion of PKC in the cell membrane fraction. However, hCG could desensitize isolated membranes, but TPA could not. We conclude that although protein kinase C activity can desensitize the gonadotropin response, hCG does not cause desensitization by activating PKC. The implications of this observation are discussed.     

Diacylglycerol modulates binding and phosphorylation of the epidermal growth factor receptor

Tumor promoters cause a variety of effects in cultured cells, at least some of which are thought to result from activation of the Ca2+-phospholipid-stimulated protein kinase C. One action of tumor promoters is the modulation of the binding and phosphorylation of the epidermal growth factor (EGF) receptor in A431 cells. To determine if these compounds act on the EGF receptor by substituting for the endogenous activator of C kinase, diacylglycerol, we compared the effects of the potent tumor promoter 12-O-tetradecanoyl phorbol 13-acetate (TPA) with those of the synthetic diacylglycerol analog 1-oleyl 2-acetyl diglycerol (OADG). When A431 cells were treated with TPA, the subcellular distribution of C kinase activity shifted from a predominantly cytosolic location to a membrane-associated state; OADG also caused the disappearance of cytosolic C kinase activity. The shift in the subcellular distribution of C kinase, caused by TPA or OADG, correlated with changes in binding and phosphorylation of the EGF receptor. OADG, like TPA, caused loss of binding to an apparent high affinity class of receptors, blocked EGF-induced tyrosine phosphorylation of the EGF receptor, and stimulated phosphorylation of the EGF receptor at both serine and threonine residues. No difference between the phosphopeptide maps of receptors from cells treated with OADG or TPA was observed. Thus, it appears that tumor promoters can exert their effects on the EGF receptors by substituting for diacylglycerol, presumably by activating protein kinase C. Further, these results suggest that endogenously produced diacylglycerol may have a role in normal growth regulatory pathways.     

Extracellular phosphorylation in the parasite, Leishmania major

Intact promastigotes or cell-free extracts of the parasite Leishmania major were labelled with adenosine 5'[gamma-32P]-triphosphate (ATP). This resulted in the identification of eleven phosphoproteins. [gamma-32P]ATP incorporation into endogenous and exogenous substrates was insensitive to most of the commonly used protein kinase inhibitors and activators indicating that the leishmanial enzyme(s) may represent a new class of kinase(s). In addition, exogenous substrate specificity was inconsistent with the preferences of second messenger-dependent protein kinases. Cyclic AMP had differential effects on phosphorylation in intact cells and lysates. The majority of kinase activity could be attributed to an externally oriented membrane-associated protein kinase(s), as no specific cytosolic phosphoproteins were found and intact cells phosphorylated exogenous substrates. Labelled ATP did not cross the membrane and [alpha-32P]ATP was an unsuitable substrate for the phosphorylation activity. The ectokinase activity on live Leishmania exhibited a different substrate preference when compared to the protein kinase activity in the particulate fraction, suggesting that more than one protein kinase may be present in L. major. Three serine-labelled phosphoproteins were specifically released into the medium. The presence of an ecto-kinase and these released phosphoproteins may play a significant role in host-parasite interactions.     

Interferon-gamma-induced epithelial ICAM-1 expression and monocyte adhesion. Involvement of protein kinase C-dependent c-Src tyrosine kinase activation pathway.

Interferon-gamma (IFN-gamma) induced intercellular adhesion molecule-1 (ICAM-1) expression in human NCI-H292 epithelial cells, as shown by enzyme-linked immunosorbent assay and immunofluorescence staining. The enhanced ICAM-1 expression resulted in increased adhesion of U937 cells to NCI-H292 cells. Tyrosine kinase inhibitors (genistein or herbimycin), Src family inhibitor (PP2), or a phosphatidylinositol-phospholipase C inhibitor (U73122) attenuated the IFN-gamma-induced ICAM-1 expression. Protein kinase C (PKC) inhibitors (staurosporine or Ro 31-8220) also inhibited IFN-gamma-induced response. 12-O-Tetradecanoylphorbol-13-acetate (TPA), a PKC activator, stimulated ICAM-1 expression; this effect was inhibited by tyrosine kinase or Src inhibitor. ICAM-1 promoter activity was enhanced by IFN-gamma and TPA in cells transfected with pIC339-Luc, containing the downstream NF-kappaB and gamma-activated site (GAS) sites, but not in cells transfected with GAS-deletion mutant, pIC135 (DeltaAP2). Electrophoretic gel mobility shift assay demonstrated that GAS-binding complexes in IFN-gamma-stimulated cells contained STAT1alpha. The IFN-gamma-induced ICAM-1 promoter activity was inhibited by tyrosine kinase inhibitors, a phosphatidylinositol-phospholipase C inhibitor, or PKC inhibitors, and the TPA-induced ICAM-1 promoter activity was also inhibited by tyrosine kinase inhibitors. Cotransfection with a PLC-gamma2 mutant inhibited IFN-gamma- but not TPA-induced ICAM-1 promoter activity. However, cotransfection with dominant negative mutants of PKCalpha or c-Src inhibited both IFN-gamma- and TPA-induced ICAM-1 promoter activity. The ICAM-1 promoter activity was stimulated by cotransfection with wild type PLC-gamma2, PKCalpha, c-Src, JAK1, or STAT1. An immunocomplex kinase assay showed that both IFN-gamma and TPA activated c-Src and Lyn activities and that these effects were inhibited by staurosporine and herbimycin. Thus, in NCI-H292 epithelial cells, IFN-gamma activates PLC-gamma2 via an upstream tyrosine kinase to induce activation of PKC-alpha and c-Src or Lyn, resulting in activation of STAT1alpha, and GAS in the ICAM-1 promoter, followed by initiation of ICAM-1 expression and monocyte adhesion.     

Modulation of adenylate cyclase in human keratinocytes by protein kinase C

Adenylate cyclase (ATP-pyrophosphate lyase (cyclizing); EC 4.6.1.1) in the human keratinocyte cell line SCC 12F was potentiated by 12-O-tetradecanoyl-phorbol-13-acetate (TPA), phorbol-12,13-diacetate, and 1,2-dioctanoylglycerol. Keratinocytes exposed to TPA showed a 2-fold enhancement of adenylate cyclase activity when assayed in the presence of isoproterenol or GTP. The half-maximal effective concentration (EC50) for both isoproterenol and GTP were unaltered by TPA treatment of the cells. Basal adenylate cyclase activity in membranes from TPA-treated cultures was also increased 2-fold relative to activity in control membranes. Potentiation of adenylate cyclase activity was dependent on the concentration of TPA to which the keratinocytes were exposed (EC50 for TPA = 3 nM). TPA actions on adenylate cyclase were maximal after 15 min of incubation of the cells with the compound, correlating well with the time course of translocation of protein kinase C (Ca2+/phospholipid-dependent enzyme) from cytosol to membrane. The action of cholera toxin on adenylate cyclase was additive with TPA. In contrast, pertussis toxin actions on adenylate cyclase were not additive with TPA. Treatment of control cells with pertussis toxin activated adenylate cyclase 1.5-fold, whereas cells exposed to pertussis toxin for 6 h followed by TPA for 15 min showed the same 2-fold increase in adenylate cyclase activity as observed in membranes from cells exposed to TPA without prior exposure to pertussis toxin. Pertussis toxin catalyzed ADP-ribosylation was increased 2-fold in membranes from SCC 12F cells exposed to TPA, indicating an increase in the alpha beta gamma form of Gi. These data suggest that exposure of human keratinocytes to phorbol esters increases adenylate cyclase activity by a protein kinase C-mediated increase in the heterotrimeric alpha beta gamma form of Gi resulting in decreased inhibition of basal adenylate cyclase activity.     

Phorbol ester-induced alteration of protein kinase C catalytic properties occurs at the membrane level and is not reproduced by physiological stimuli

Potent tumor promoter TPA (1-100 nM) has previously been shown to induce a striking alteration of protein kinase C catalytic properties in target cells (C. Cochet et al., 1986, Biochem. Biophys. Res. Comm. 134, 1031-1037). This alteration contributes to the apparent loss of cellular protein kinase C, secondary to TPA treatment, when the enzyme is probed by its phospholipid-dependent histone kinase activity. This effect was observed as well when rat-1 cells were treated by other tumor promoters such as mezerein, teleocidin, aplysiatoxin and palytoxin, whereas inactive phorbol ester structures were ineffective. On the other hand, 1,2-dioctanoyl glycerol did not induce that effect. This protein kinase C alteration was shown to occur at the cellular membrane level. It is suggested that membrane translocation and activation of protein kinase C induced by potent tumor promoter structures are not functionally equivalent to that secondary to physiological stimuli. Although the mechanisms underlying this phenomenon remains to be understood at the molecular level, it may be of significance in the process of tumor promotion.     

Differential regulation of phosphatidylcholine biosynthesis by 12-O-tetradecanoylphorbol-13-acetate and diacylglycerol in NG108-15 neuroblastoma x glioma hybrid cells

12-O-Tetradecanoylphorbol-13-acetate (TPA), a tumor promoter and potent activator of protein kinase C, stimulates [3H]choline incorporation into phosphatidylcholine (PtdCho) in NG108-15 cells (Liscovitch, M., Freese, A., Blusztajn, J. K. and Wurtman, R. J. (1986) J. Neurochem. 47, 1936-1941). In the present study we demonstrate that two cell-permeant diacylglycerols, sn-1-oleoyl-2-acetylglycerol and sn-1,2-dioctanoylglycerol, also stimulate [3H]choline incorporation into PtdCho. However, the effect of diacylglycerol is additional to that produced by a maximally effective concentration of TPA (0.5 microM), suggesting that the two agents may not act via the same mechanism. In addition, the protein kinase inhibitor 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (at 200 microM) inhibits the action of TPA by 59% while not affecting that of diacylglycerol. Finally, preincubation of the cells with TPA (0.1 microM) for 24 h reduces protein kinase C activity in the cells and completely abolishes the effect of additional TPA on choline incorporation. In contrast, diacylglycerol-induced stimulation of PtdCho biosynthesis was not inhibited in the cells that were desensitized to TPA. These results suggest that the effect of the two cell-permeant diacylglycerols on PtdCho biosynthesis either is not mediated by protein kinase C activation, or, is mediated by a TPA-insensitive isoenzyme of protein kinase C.