Insulin increases membrane and cytosolic protein kinase C activity in BC3H-1 myocytes

Insulin treatment stimulated the activity of the Ca2+- and phospholipid-dependent protein kinase (protein kinase C) in both cytosolic and membrane fractions of BC3H-1 myocytes. Within 60 s of insulin treatment, membrane protein kinase C activity increased 2-fold, diminished toward control levels transiently, and then increased 2-fold again after 15 min. Cytosolic protein kinase C activity increased more gradually and steadily up to 80% over a 20-min period. Increases in protein kinase C activity were dose-dependent and were not simply a result of translocation of cytosolic enzyme (although this may have occurred), as total activity was also increased. The increase in protein kinase C activity was not inhibited by cycloheximide (which also increased protein kinase C activity and 2-deoxyglucose transport) and was still evident following anion exchange chromatography. The insulin effect was decidedly different from those of 12-O-tetradecanoylphorbol-13-acetate and phenylephrine using histone III-S as substrate. Phenylephrine decreased cytosolic protein kinase C activity while increasing membrane activity; 12-O-tetradecanoylphorbol-13-acetate only decreased cytosolic protein kinase C activity. The early insulin-induced increases in membrane protein kinase C activity may be related to increased diacylglycerol generation from de novo phosphatidic acid synthesis, as there were rapid increases in [3H]glycerol incorporation into diacylglycerol, and transient increases in phospholipid hydrolysis, as there were transient rapid increases in [3H]diacylglycerol in cells prelabeled with [3H]arachidonate. Later, sustained increases in membrane and cytosolic protein kinase C activity may reflect the continuous activation of de novo phospholipid synthesis, as there were associated increases in [3H]glycerol incorporation into diacylglycerol at later, as well as very early time points.     

Protein kinase C in mouse kidney: subcellular distribution and endogenous substrates

The subcellular distribution, kinetic properties, and endogenous substrates of calcium-activated, phospholipid-dependent protein kinase (protein kinase C) were examined in mouse kidney cortex. Protein kinase C associated with the particulate, mitochondrial, and brush border membrane fractions was assayed after solubilization in 0.2% Triton X-100 under conditions shown to be noninhibitory to catalytic activity. Of recovered activity, 52% was associated with the cytosolic fraction; mitochondrial and brush border membrane associated protein kinase C constituted 12 and 3%, respectively, of the activity recovered in the particulate fraction. Protein kinase C associated with brush border membranes exhibited a high affinity for ATP (apparent Km = 62 +/- 10 microM) and the highest apparent maximal velocity (1146 +/- 116 pmol P/(mg protein.min] of the renal fractions examined. Maximal stimulation of protein kinase C by diacylglycerol (in the presence of phosphatidylserine) was achieved at both 25 and 300 microM calcium in all renal fractions. These results are consistent with previous reports demonstrating that diacylglycerol increases the apparent affinity of protein kinase C for calcium. Phorbol 12-myristate 13-acetate, but not 4 alpha-phorbol, was able to substitute for diacylglycerol and stimulate cytosolic and particulate renal protein kinase C. 1-(5-Isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride, a specific inhibitor of protein kinase C, led to significant inhibition of catalytic activity in all renal subcellular fractions. Endogenous substrates for protein kinase C were demonstrated in renal cytosolic (26, 45, 63, and 105 kilodaltons (kDa], particulate (26, 33, 68, and 105 kDa), mitochondrial (43 kDa), and brush border membrane (26, 41, 52, 88, and 105 kDa) fractions. The possible physiological significance of protein kinase C in mammalian kidney is discussed.     

Blocking with phorbol ester, a protein kinase C activator, of receptor-dependent platelet calcium channels

The regulation of receptor-operated calcium channels of human platelets by phospholipid-dependent, Ca2+- and diacylglycerol-activated protein kinase C was studied. In order to induce the activation of endogenous protein kinase C, a cell-penetrable structural diacylglycerol analog, 4 beta-phorbol 12 beta-myristate-13 alpha-acetate (FMA), was used. Using two independent approaches, i. e., the fluorescent probe for Ca2+, quin-2, and 45Ca2+ absorption technique, it was demonstrated that FMA (10(-10) - 10(-8) g/ml) blocks Ca2+ influx into the platelets induced by aggregation factors, e. g., ADP, vasopressin, platelet activating factor, thrombin and thromboxane A2 receptor agonist U46619. The half-maximum inhibition of the receptor-sensitive influx of Ca2+ was observed at (3-6) X 10(-10) g/ml of FMA. Under physiological conditions, protein kinase C is activated with an increase in Ca2+ concentration in the cytoplasm in the presence of diacylglycerol. Since the above-mentioned inducers besides Ca2+ influx stimulate diacylglycerol synthesis, it was assumed that the activation of protein kinase C triggers a negative feedback mechanism which blocks the receptor-operated calcium channels.     

Regulation of protein kinase C activity by gangliosides

The activity of protein kinase C (Ca2+/phospholipid-dependent enzyme) in the presence of phosphatidylserine and its physiological regulator, diacylglycerol, could be suppressed by a mixture of brain gangliosides. Half-maximal inhibition was observed at 30 microM and was nearly complete at 100 microM. Inhibition was observed at all concentrations of Ca2+ between 10(-8) and 10(-4) M. Inhibition of protein kinase C activity could not be reversed by increasing the concentration of diacylglycerol or the substrate, histone. Inhibition was also observed when myelin basic protein or a synthetic myelin basic protein peptide was used as substrate. Among the individual gangliosides, the rank order of potency was GT1b greater than GD1a = GD1b greater than GM3 = GM1. Our results suggest that gangliosides may regulate the responsiveness of protein kinase C to diacylglycerol.     

Stimulation of phosphatidylcholine hydrolysis, diacylglycerol release, and arachidonic acid production by oncogenic ras is a consequence of protein kinase C activation

Swiss-3T3 cells were scrape-loaded with oncogenically activated p21ras protein. 10-20 min after introducing Val12p21ras into the cell, diacylglycerol levels were increased, but levels of inositol phosphates were unaltered. However, cellular choline and phosphocholine levels were increased with a similar time course to that observed for diacylglycerol production, suggesting that ras increases phosphatidylcholine turnover but not phosphatidylinositol turnover. Down-regulation of protein kinase C (by prolonged exposure to phorbol esters prior to scrape loading) blocked the ability of ras protein to elevate the levels of diacylglycerol, choline, and phosphocholine. Oncogenic ras can, therefore, cause a substantial increase in diacylglycerol (which correlates with increased phosphatidylcholine breakdown) in a protein kinase C-dependent fashion. Val12p21ras also increased arachidonic acid release, which was also dependent on protein kinase C activation. Induction of DNA synthesis by oncogenic ras was unaffected by inhibitors of prostaglandin synthesis, indicating that conversion of the released arachidonic acid to various prostaglandins is not required for stimulation of DNA synthesis by ras. We suggest that ras rapidly activates protein kinase C, which in turn activates a number of cellular signalling systems, leading to a sustained increase in diacylglycerol levels. This elevation of diacylglycerol could sustain protein kinase C activation over the 12-15 h required for initiation of DNA synthesis.     

Alpha 1-adrenergic receptors promote phosphatidylcholine hydrolysis in MDCK-D1 cells. A mechanism for rapid activation of protein kinase C

Hydrolysis of phosphoinositides can lead to mobilization of calcium and production of diacylglycerol, which together are proposed to activate protein kinase C. We have shown that phosphoinositide hydrolysis mediated by alpha 1-adrenergic receptors on Madin-Darby canine kidney cells (MDCK-D1) occurred with an early lag and increased over a prolonged time course (Slivka, S.R., and Insel, P.A. (1987) J. Biol. Chem. 262, 4200-4207). In this study we characterize another type of receptor-mediated phospholipid hydrolysis in MDCK-D1 cells, alpha 1-adrenergic receptor-mediated hydrolysis of phosphatidylcholine. The predicted products of this hydrolysis, phosphorylcholine and diacylglycerol, were detectable as early as 0.5 min after alpha 1-adrenergic receptor stimulation by epinephrine. This hydrolysis appears to be a primary event after receptor occupancy because it occurred in the presence of neomycin, an inhibitor of polyphosphoinositide hydrolysis, and the protein kinase C inhibitors, sphingosine and 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7). In addition, we demonstrate alpha 1-adrenergic receptor-mediated activation of protein kinase C in MDCK-D1 cells. This activation was measured as a rapid translocation (0.5 min) of protein kinase C activity from the cytosolic fraction to the membrane fraction. This translocation also was not inhibited by neomycin. The time course and agonist concentration dependence of both phosphatidylcholine hydrolysis and protein kinase C activation by alpha 1-adrenergic receptors were similar. Thus, we propose that agonists acting at alpha 1-adrenergic receptors promote hydrolysis of phosphatidylcholine which results in rapid generation of diacylglycerol for the activation of protein kinase C.     

Does protein kinase C activation mediate thrombin-induced arachidonate release in human platelets?

Thrombin stimulated rapid formation of diacylglycerol, inositol 1,4,5-trisphosphate (IP3) and thromboxane B2 (TXB2) in human platelets. Formation of diacylglycerol and IP3 appeared to precede that of TXB2. Activation of protein kinase C by diacylglycerol combining with Ca+2 mobilization by IP3 has been implicated in mediating arachidonate release. However, addition of the protein kinase C inhibitor 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7) to platelet suspension did not inhibit thrombin-stimulated arachidonate release and TXB2 synthesis, whereas addition of the Ca+2 antagonist, 3,4,5-trimethoxybenzoic acid 8-(diethylamino) octyl ester (TMB-8) or the calmodulin antagonist N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) abolished arachidonate release. The correlation of IP3 production with arachidonate release on increasing the concentrations of thrombin was further examined. IP3 production reached near maximum at 0.2 U/ml, whereas TXB2 synthesis continued to increase at 1 U/ml. These results suggest that protein kinase C activation may not mediate arachidonate release and that Ca+2 mobilization by IP3 may only partially account for arachidonate release in platelets stimulated with relatively high concentrations of thrombin.     

The interaction of protein kinase C and other specific cytoplasmic proteins with phospholipid bilayers

The role of lipid composition in the interaction of purified protein kinase C with large unilamellar vesicles was determined by the extent of photolabelling of the enzyme with 5-[125I]iodonaphthalene-I-azide. The protein kinase C was only slightly labelled when exposed to phosphatidylcholine (PC) liposomes. The addition of phorbol 12-myristate 13-acetate (PMA) or of diacylglycerol to the PC liposomes enhanced significantly the labelling of the protein kinase C at low calcium concentrations. A further enhancement in the photolabelling of the protein kinase C was observed in liposomes containing 2% phosphatidylserine (PS). At low calcium concentrations, the binding of the enzyme to these liposomes increased in the presence of added PMA or diacylglycerol. Raising the levels of PS beyond 2% in the liposomes did not enhance the binding of the protein kinase C. However, when the enzymatic activity of the protein kinase C was measured using basic histones as substrates, maximum phosphorylation was obtained in liposomes with a PC to PS ratio of 1. The fact that the translocation of the protein kinase C from solution to the surface of the liposomes could be monitored by its labelling with 5-iodonaphthalene 1-azide prompted us to determine whether other cytoplasmic proteins might share this property. The interaction of cytoplasmic proteins from HeLa cells with PC liposomes gave trace labelling irrespective of whether calcium was added. When the HeLa cell cytoplasmic proteins were allowed to interact with liposomes containing PS, selective 5-iodonaphthalene-1-azide photolabelling was observed in distinct proteins. Addition of calcium and of PMA or diacylglycerol modified the labelling of some but not all of these proteins. These results suggest that the methodology developed might serve to identify proteins that move to the membrane during stimulation of cells by phorbol esters or by growth factors which induce the generation of diacylglycerol. These results also suggest a role for the phospholipid composition of the plasma membrane (or any intracellular membrane) in the modulation of the activation processes of specific phospholipid-dependent proteins, in particular protein kinase C.     

Rapid dephosphorylation of protein kinase C substrates by protein kinase A activators results from inhibition of diacylglycerol release

The biochemical events encompassing the dephosphorylation of protein kinase C substrates by protein kinase A activators have been investigated in a neurotumor cell line, NCB-20. Treatment of [32P]orthophosphate-labeled cells with protein kinase A activators (e.g. forskolin, dibutyryl cAMP, prostaglandin E1) resulted in an inhibition of protein kinase C activity due to a failure of the protein kinase C complex to translocate into the membrane. Phospholipase C activity, as measured by the synchronous release of diacylglycerol and inositol phosphates (inositol 1,4,5-trisphosphate, inositol 1,4-bisphosphate, and inositol 1-phosphate) in response to bradykinin, was inhibited up to 50% following exposure to protein kinase A activators. At the same time, phospholipase C-specific inositol phospholipid substrates (phosphatidylinositol, phosphatidylinositol 4-phosphate, and phosphatidylinositol 4,5-bisphosphate) were found to accumulate in NCB-20 cells following treatment with protein kinase A activators. This suggests that phospholipase C may be altered through protein kinase A-mediated protein phosphorylation. Second messenger generation (inositol phosphates, diacylglycerol, and Ca2+) is therefore inhibited through cyclic AMP-mediated shutdown of the inositol lipid cycle at the level of phospholipase C.     

Protein kinase C from small intestine epithelial cells

Protein kinase C activity has been identified in cytosolic and membrane fractions from rat and rabbit small intestine epithelial cells. The cytosolic fraction comprised about the 75% of total activity. Protein kinase C activity was resolved from other protein kinase activities by ion exchange chromatography. Phosphatidylserine or phosphatidylinositol were required for protein kinase C to be active. In addition, the activity was enhanced by the presence of a diacylglycerol. Diolein and dimyristin were the most effective (13-14 fold activation). In the presence of phosphatidylserine and diolein, the Ka for activation by Ca2+ was 10(-7)M. The phorbol ester TPA substituted for diacylglycerol in activating protein kinase C. Brush border and basolateral membranes contained protein kinase C activity, although the specific activity of the basal lateral membranes was four-fold higher than the specific activity of the brush border membranes. The presence of PKC in small intestine epithelial cells might have important implications in the Ca2+ mediated control of ionic transport in this tissue.     

Involvement of protein kinase C in Ca(2+)-independent contraction of rat uterine smooth muscle

The contribution of protein kinase C to the contraction by oxytocin of rat uterine longitudinal smooth muscle in Ca(2+)-free solution was investigated. Immunological analysis revealed that type II (beta) and III (alpha) protein kinase C subspecies were present in rat uterine smooth muscle. The pretreatment of a diacylglycerol kinase inhibitor R59022 to accumulate diacylglycerol potentiated the Ca(2+)-independent contraction. The contractile activity was diminished with the depletion of protein kinase C, when the contraction was evoked repeatedly by oxytocin during the prolonged exposure to a tumor-promoting phorbol ester 12-O-tetradecanoylphorbol 13-acetate. These results suggested the involvement of protein kinase C in oxytocin-induced contraction in Ca(2+)-free solution.     

Interaction of protein kinase C with phosphatidylserine. 2. Specificity and regulation.

The roles of specific and nonspecific interactions in the regulation of protein kinase C by lipid have been examined. Binding and activity measurements reveal two mechanisms by which protein kinase C interacts with membranes: (1) a specific binding to the activating lipid phosphatidylserine and (2) a nonspecific binding to nonactivating, acidic lipids. The specific interaction with phosphatidylserine is relatively insensitive to ionic strength, surface charge, and the presence of nonactivating lipids. The two second messengers of the kinase, diacylglycerol and Ca2+, increase markedly the affinity of the kinase for phosphatidylserine. In contrast, the nonspecific interaction is sensitive to ionic strength and surface charge, and is unaffected by diacylglycerol. These results suggest that electrostatic interactions promote the binding of protein kinase C to membranes but the cooperative and selective binding of phosphatidylserine is the dominant driving force in a productive protein-lipid interaction.     

Phorbol ester-induced activation of protein kinase C leads to increased formation of diacylglycerol in human neutrophils

Human neutrophils stimulated with a phorbol ester (phorbol 12-myristrate 13-acetate or phorbol 12,13-dibutyrate) responded with an increase in diacylglycerol, considered the natural activator of protein kinase C. The amounts of diacylglycerol formed were considerable, reaching 700-900% of basal after 20 min. In contrast, 4-alpha-phorbol 12-myristate 13-acetate did not induce any detectable formation of diacylglycerol. Simultaneously, phorbol 12-myristate 13-acetate exposure caused increased breakdown of both phosphatidylcholine and phosphatidylinositol 4,5-bisphosphate. These results suggest that once activated, protein kinase C can positively modulate its own activity by inducing additional formation of diacylglycerol from at least two different sources.     

Effects of phorbol ester and cholecystokinin on the intracellular distribution of protein kinase C in rabbit pancreatic acini.

Treatment of rabbit pancreatic acini with the phorbol ester, 12-O-tetradecanoylphorbol 13-acetate (TPA), resulted in a time- and dose-dependent decrease of soluble protein kinase C activity coinciding with an increase of protein kinase C activity in the particulate fraction. After 5 min, soluble protein kinase C activity had decreased to almost 10% of the corresponding control. Total extractable protein kinase C activity, however, remained unchanged, indicating that the decrease of soluble protein kinase C activity was not due to TPA-induced inactivation of the enzyme. The biologically inactive phorbol ester, 4 alpha-phorbol 12,13-didecanoate, did not induce such a translocation of protein kinase C. The half-maximal concentration for TPA-induced translocation of protein kinase C was 40 nM, and was equal to that for TPA-induced amylase secretion from isolated acini. This suggests that translocation of protein kinase C to the particulate fraction is an important step in TPA-induced activation of protein kinase C and enzyme secretion. On the other hand, cholecystokinin, a secretagogue of the calcium-mobilizing type, whose secretory action is thought to be mediated, at least in part, by protein kinase C, did not change the subcellular distribution of protein kinase C. In the presence of R59022 6-(2-[(4-fluorophenyl)phenylmethylene]-1-piperidinyl ) ethyl-7-methyl-5H-thiazolo[3,2-a]pyrimidin-5-one, an inhibitor of diacylglycerol kinase activity, cholecystokinin produced a small but significant translocation of protein kinase C, suggesting that the inability of the hormone to induce translocation is not due to a rapid conversion of the diacylglycerol formed into phosphatidic acid.     

Delta protein kinase C interacts with the d subunit of the F1F0 ATPase in neonatal cardiac myocytes exposed to hypoxia or phorbol ester. Implications for F1F0 ATPase regulation

Mitochondrial protein kinase C isozymes have been reported to mediate both cardiac ischemic preconditioning and ischemia/reperfusion injury. In addition, cardiac preconditioning improves the recovery of ATP levels after ischemia/reperfusion injury. We have, therefore, evaluated protein kinase C modulation of the F(1)F(0) ATPase in neonatal cardiac myocytes. Exposure of cells to 3 or 100 nM 4beta-phorbol 12-myristate-13-acetate induced co-immunoprecipitation of delta protein kinase C (but not alpha, epsilon, or zeta protein kinase C) with the d subunit of the F(1)F(0) ATPase. This co-immunoprecipitation correlated with 40+/-3% and 72+/-9% inhibitions of oligomycin-sensitive F(1)F(0) ATPase activity, respectively. We observed prominent expression of delta protein kinase C in cardiac myocyte mitochondria, which was enhanced following a 4-h hypoxia exposure. In contrast, hypoxia decreased mitochondrial zetaPKC levels by 85+/-1%. Following 4 h of hypoxia, F(1)F(0) ATPase activity was inhibited by 75+/-9% and delta protein kinase C co-immunoprecipitated with the d subunit of F(1)F(0) ATPase. In vitro incubation of protein kinase C with F(1)F(0) ATPase enhanced F(1)F(0) activity in the absence of protein kinase C activators and inhibited it in the presence of activators. Recombinant delta protein kinase C also inhibited F(1)F(0) ATPase activity. Protein kinase C overlay assays revealed delta protein kinase C binding to the d subunit of F(1)F(0) ATPase, which was modulated by diacylglycerol, phosphatidylserine, and cardiolipin. Our results suggest a novel regulation of the F(1)F(0) ATPase by the delta protein kinase C isozyme.     

Protein kinase C activation by cis-fatty acid in the absence of Ca2+ and phospholipids

Protein kinase C has been shown to be a phospholipid/Ca2+-dependent enzyme activated by diacylglycerol (Nishizuka, Y. (1984) Nature 308, 693-697; Nishizuka, Y. (1984) Science 225, 1365-1370). We have reported that unsaturated fatty acids (oleic acid and arachidonic acid) can activate protein kinase C independently of Ca2+ and phospholipid (Murakami, K., and Routtenberg, A. (1985) FEBS Lett. 192, 189-193). This study shows that other cis-fatty acids such as linoleic acid also fully activate protein kinase C in the same manner. None of the saturated fatty acids (C:4 to C:18) nor the detergents (sodium dodecyl sulfate and Triton X-100) tested here were as effective as oleic acid. Unlike oleic acid, these detergents strongly inhibited protein kinase C activity induced by Ca2+/phosphatidylserine (PS) and diacylglycerol. Lowering the critical micelle concentration of oleic acid by increasing ionic strength also strongly inhibited oleic acid activation of protein kinase C activity. Dioleoylphosphatidylserine activated protein kinase C effectively (Ka = 7.2 microM). On the other hand, dimyristoylphosphatidylserine, which contains saturated fatty acids at both acyl positions, failed to activate protein kinase C even in the presence of Ca2+. These observations suggest that: protein kinase C activation by free fatty acid is specific to the cis-form and is not due to their detergent-like action, cis-fatty acid activation is due to the direct interaction of protein kinase C with the monomeric form of cis-fatty acids and not with the micelles of fatty acids, and cis-fatty acids at acyl positions in PS are also important for Ca2+/PS activation of protein kinase C.     

A role of calcium-activated phospholipid-dependent protein kinase in human platelet activation. Comparison of thrombin and collagen actions

In human platelets stimulated by thrombin and collagen, diacylglycerol is rapidly produced from phosphatidylinositol. Concurrently, an endogenous protein having a molecular weight of about 40,000 (40K protein) is phosphorylated, and serotonin is released. These reactions are all inhibited by a prior treatment of platelets with prostaglandin E1, dibutyryl cyclic AMP, sodium nitroprusside, or with 8-bromo-cyclic GMP, which are known as potent inhibitors for platelet activation. Ca2+-activated phospholipid-dependent protein kinase (protein kinase C) preferentially phosphorylates 40K protein. As judged by fingerprint analysis, the sites in 40K protein that are phosphorylated during the platelet activation appear to be identical with those phosphorylated by protein kinase C in a purified cell-free system. 12-O-Tetradecanoylphorbol-13-acetate, which directly activates protein kinase C by substituting for diacylglycerol, stimulates 40K protein phosphorylation and release reaction without inducing diacylglycerol formation. Tetracaine, which inhibits protein kinase C by competing with phospholipid, blocks 40K protein phosphorylation and serotonin release without inhibiting the receptor-linked diacylglycerol formation. The results indicate that thrombin and collagen activate platelets in almost similar mechanisms and that protein kinase C may lie on a common pathway which leads to the release of serotonin. However, analysis with indomethacin indicates that the role of thromboxane A2 appears to be more predominant for the action of collagen, and it is suggestive that this arachidonate metabolite activates platelets in an analogous mechanism to thrombin.     

Phospholipase A2 activation in chemotactic peptide-stimulated HL60 granulocytes: synergism between diacylglycerol and Ca2+ in a protein kinase C-independent mechanism

In dimethylsulfoxide-differentiated HL60 granulocytes, the chemotactic peptide N-formyl-Met-Leu-Phe (FMLP) augments arachidonic acid (AA) release via phospholipase A2 activity induced by the Ca2+-ionophore, A23187. Evidence indicates that this augmentation is mediated by diacylglycerols formed endogenously during FMLP receptor activation: The augmentation is mimicked by the synthetic diglyceride 1-oleoyl-2-acetyl-glycerol (OAG) and the tumor promoting phorbol ester 12-O-tetradecanoylphorbol-13-acetate; Pertussis toxin inhibits FMLP-induced augmentation but not OAG-induced augmentation: At suboptimal concentrations FMLP and OAG act cooperatively to augment ionophore A23187-induced AA release but not at optimal concentrations. These data indicate that phospholipase A2 activation in FMLP-stimulated HL60 granulocytes involves cooperative interactions between diacylglycerol formed endogenously and Ca2+. Interestingly, this effect of diacylglycerol appears not to be mediated by protein kinase C, since a specific protein kinase C inhibitor, 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H7) does not inhibit receptor-mediated release of AA by stimulated HL60 granulocytes.     

Activation of calcium and phospholipid-dependent protein kinase by epidermal growth factor (EGF) in A431 cells: attenuation by 12-0-tetradecanoylphorbol-13-acetate (TPA)

A calcium and phospholipid-dependent protein kinase (protein kinase C) was detected in the crude soluble extracts of A431 human epidermoid carcinoma cells. The enzyme required calcium, phosphatidylserine or phosphatidylinositol, and diacylglycerol (DG) for maximal activation. Protein kinase C phosphorylated both endogenous cytosolic proteins and various histones. Addition of epidermal growth factor (EGF) to A431 cultures resulted in a 2 to 3-fold stimulation of protein kinase activity. 12-0-tetradecanoylphorbol-13-acetate (TPA) in concert with EGF attenuated the EGF-induced enhanced phosphorylation of endogenous proteins. It is conceivable that DG, derived from phosphatidylinositol turnover, acts as a natural activator of protein kinase C activity.     

Human neutrophil protein kinase C: calcium-induced changes in the solubility of the enzyme do not always correlate with enzymatic activity

We hypothesized that calcium and 1,2-diacylglycerols stimulated human neutrophil (PMN) protein kinase C (EC 2.7.1.37) in a two-step mechanism. The proposed mechanism entails (1) increased insoluble protein kinase C activity and (2) endogenous protein phosphorylation, events which have not been biochemically dissociated. PMN which were treated with 100 nM ionomycin shifted protein kinase C activity from being mostly soluble to insoluble. Concentrations of ionomycin greater than 300 nM stimulated a doubling of total cellular (soluble + insoluble) protein kinase activity and stimulated increased endogenous phosphorylation of PMN proteins. Intracellular calcium (measured with fura-2) increased from 65 nM (basal) to 680 nM using 500 nM ionomycin; calcium increases were dose-dependent. The anti-inflammatory agents acetylsalicylic acid and sodium salicylate (but not ibuprophen, indomethacin or acetaminophen) inhibited ionomycin-induced protein kinase C activation and protein phosphorylation in a dose-dependent manner by inhibiting the production of diacylglycerols. 1-Oleoyl-2-acetylglycerol reversed the inhibitory effect of salicylates. In contrast to the effect of acetylsalicylates on protein kinase C functional activity the distribution of phorbol receptors was unaffected in acetylsalicylate-treated, ionomycin-stimulated PMN using a phorbol-binding assay. Our results show that ionomycin increased intracellular diacylglycerol levels 3.5-fold over those present in control PMN, while acetylsalicylate decreased diacylglycerol production in ionomycin-stimulated PMN below baseline values. These results support the hypothesis that increased intracellular calcium activated protein kinase C leading to protein phosphorylation in two distinct dissociable events: (1) increased intracellular calcium; and (2) increased 1,2-diacylglycerol levels.