Phorbol ester-induced translocation of diacylglycerol kinase from the cytosol to the membrane in Swiss 3T3 fibroblasts

The tumor-promoting phorbol ester, 12-O-tetradecanoyl-phorbol-13-acetate, causes a rapid, partial redistribution of 1,2-sn-diacylglycerol kinase from the cytosol to the particulate fraction of quiescent, starved Swiss 3T3 fibroblasts. We utilized exogenous dioleoylglycerol as substrate for the kinase. The inactive alpha form of the phorbol ester does not cause any change in diacylglycerol kinase localization, and depletion of protein kinase C (Ca2+/phospholipid-dependent enzyme) by chronic administration of phorbol ester blocks the redistribution. Phorbol ester has no direct effect on Swiss 3T3 membrane-bound diacylglycerol kinase nor does it directly effect cytosolic diacylglycerol kinase. When phorbol ester is added to Swiss 3T3 membranes in the presence of ATP, magnesium, and calcium, there is no activation of membrane-bound kinase, indicating that phorbol ester does not activate membrane-bound kinase through phosphorylation by protein kinase C. Reconstitution studies show that the soluble rat brain diacylglycerol kinase binds to diacylglycerol-enriched membranes, produced by treatment of red cell ghosts with phospholipase C or calcium, suggesting that cytosolic diacylglycerol kinase may be capable of translocation to the membrane in response to elevated substrate concentration in the intact cell. Stimulation of the cells with phorbol ester increases the total mass of diacylglycerol. In protein kinase C-depleted cells, addition of a cell-permeable synthetic diacylglycerol, dioctanoylglycerol, results in a partial redistribution of cytosolic diacylglycerol kinase to the membrane, by 5 min, also suggesting that the translocation of diacylglycerol kinase activity is regulated primarily by substrate concentration.     

Translocation of diacylglycerol kinase from the cytosol to the membrane in phorbol ester-treated Swiss 3T3 fibroblasts

The tumor-promoting phorbol ester, 12-O-tetradecanoyl-phorbol-13-acetate, causes a rapid, partial redistribution of 1,2-diacylglycerol kinase from the cytosol to the particulate fraction of quiescent Swiss 3T3 fibroblasts. The inactive alpha form of the phorbol ester does not cause any change in diacylglycerol kinase localization, and depletion of protein kinase C by chronic administration of phorbol ester blocks the redistribution. Phorbol ester has no direct effect on membrane-bound diacylglycerol kinase in 3T3 cells. When phorbol ester is added to 3T3 membranes in the presence of ATP, Mg2+, and Ca2+, there is no activation of membrane-bound kinase, indicating that phorbol ester does not activate membrane-bound kinase through phosphorylation by protein kinase C. Stimulation of the cells with phorbol ester increases the total mass of diacylglycerol. In protein kinase C-depleted cells, addition of a cell-permeable synthetic diacylglycerol, dioctanoylglycerol, results in a partial redistribution of cytosolic diacylglycerol kinase to the membrane, also suggesting that the translocation of DAG kinase is regulated primarily by substrate concentration.     

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.     

Characterization and partial purification of rat submandibular gland protein kinase C

Membrane-bound protein kinase C of rat submandibular gland was characterized and the cytosolic kinase C of the tissue was partially purified. The membrane-bound kinase could be activated by Triton X-100 but not EGTA in the presence of both Ca2+ and phosphatidylserine (PS). The Km values for Ca2+ and PS were 150 microM and 5 micrograms, respectively. Addition of 10(-6) M diacylglycerol resulted in an increased affinity of the kinase for Ca2+ (Km = 10 microM). Phorbol 12,13-dibutyrate activated the kinase in the absence of exogenous Ca2+ and PS, suggesting that adequate amounts of each activator are present in the membrane itself. Polymyxin B inhibited the stimulated kinase C activity in a concentration-dependent manner. This inhibition could be overcome by addition of PS. The cytosolic kinase was partially purified 133-fold by chromatography on columns of DEAE-Sephacel and S-300 Sephacryl. The total kinase activity increased with respect to the kinase activity measured in the starting material with column chromatography, suggesting that an inhibitor is present in the cytosolic fraction of the tissue.     

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.     

High-Pressure Extraction of Membrane-Associated Protein Kinase C from Rat Brain

Extraction of rat brain membrane-associated protein kinase C with high specific activity was obtained by applying benzyl alcohol (a membrane fluidizer), EDTA, and high hydrostatic pressures. Approximately 50% of total brain-associated activity was extracted from membranes. The pressure-extracted activity had an eightfold enrichment in the lipid/protein ratio when compared with the cytosolic fraction. This may explain the inability of exogenous diacylglycerol to stimulate endogenous phosphorylation in pressure-extracted activity. The enzyme is extracted at greater than 1,300 atm, a result indicating it most likely has a portion inserted into the hydrophobic portion of the membrane bilayer. Perturbation of the native membrane induces a change in the membrane-associated protein kinase C-lipid interaction that permits extraction under conditions used for the cytosolic species. This is the first report of conversion of the endogenous membrane species to a cytosolic one and may be important in determining the role of protein kinase C in neuronal regulation.     

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.     

Characterization and distribution of protein kinase C in ovarian tissue

Although protein kinase C, an enzyme dependent on calcium, phospholipid and diacylglycerol, has been found in high levels in ovarian tissues, its biologic function is yet unknown. In initial studies on the role of this enzyme in regulating ovarian functions, we compared protein kinase C activity in subcellular fractions of porcine corpora lutea and medium follicles. Highest protein kinase C-specific activities were found in the cytosol, followed by microsomes and mitochondria for both follicles and luteal tissues. Solubilization of all membrane-containing fractions by 0.2% Triton X-100 was required for full expression (a 4-fold average increase) of protein kinase activity. Extraction of membrane fractions with 0.5 M NaCl or sonication in a hypotonic medium revealed that 90% of the total mitochondrial protein kinase C activity and 50% of the microsomal activity was tightly membrane-bound. Characterization of both cytosolic and Triton X-100 extracted membrane preparations of luteal tissue by diethylaminoethyl (DEAE)-cellulose chromatography revealed a single peak of protein kinase C activity eluting at 80 mM NaCl. Cytosolic fractions of corpora lutea contained 3 times more protein kinase C-specific activity than did cytosolic fractions of follicles. In contrast, mitochondria from medium follicles contained 30% more specific protein kinase C activity than did luteal mitochondria. These higher cytosolic levels of protein kinase C-specific activity in corpora lutea suggest that the enzyme may play an important role in the process of luteinization or in the regulation of luteal function.     

In Vivo Translocation and Down-Regulation of Protein Kinase C Following Intraventricular Administration of Tetanus Toxin

A single intraventricular injection into adult rats of 100 mouse lethal doses of tetanus toxin (TeTox) produces a marked intracellular redistribution of Ca2+/phosphatidylserine (PtdSer)-dependent protein kinase C (PKC) activity. Changes are particularly pronounced in hypothalamus, hippocampus, and spinal cord structures. Translocation of PKC from the inactive cytosolic compartment to a membrane-bound active form is followed by a time-dependent reduction in both total activity and enzyme protein. The down-regulation of PKC activity in the hypothalamus is accompanied by a marked increase in a Ca2+/PtdSer-independent kinase activity, predominantly in the cytosolic fraction. Our data identify PKC as a possible indirect target for TeTox and suggest that down-regulation of the enzyme may provide a clue for tetanus neurotoxicity.     

Correction of abnormal small intestinal cytosolic protein kinase C activity in streptozotocin-induced diabetes by insulin therapy.

Diabetes was induced in rats by administration of a single intraperitoneal injection of streptozotocin (50 mg/kg body wt). After 7 days, one group of diabetic animals was treated with insulin for an additional 5 days. Control, diabetic and diabetic + insulin rats were then killed, their distal small intestines were removed and the epithelial cells were examined and compared with respect to polyphosphoinositide turnover, total protein kinase C activity and cellular distribution, and 1,2-diacylglycerol mass and production. The results of these experiments demonstrated that, compared with their control counterparts, the intestines from diabetic rats had a decreased turnover of polyphosphoinositides, but an increase in 1,2-diacylglycerol mass which was a result, at least in part, of an increase in the synthesis of this lipid de novo. Total protein kinase C activity was decreased in the diabetic rats due to a decrease in cytosolic activity, with no significant change in particulate activity. Moreover, insulin administration for 5 days to diabetic animals did not affect their lowered intestinal polyphosphoinositide turnover, but did further accentuate their increased 1,2-diacylglycerol mass and synthesis de novo; this treatment also corrected total protein kinase C activity by increasing the cytosolic activity of this enzyme. These results indicate that signalling mechanisms involving polyphosphoinositides, 1,2-diacylglycerol and protein kinase C are abnormal in the intestines of diabetic rats and that some of these biochemical parameters can be modulated by insulin administration in vivo.     

Phosphatidylinositol kinase activities in normal and Rous sarcoma virus-transformed cells.

The phosphatidylinositol (PI), phosphatidylinositol 4-phosphate (PIP), and diacylglycerol kinase activities in the plasma membrane-rich fraction of chicken embryo fibroblasts infected with a temperature-sensitive mutant of Rous sarcoma virus increased when the cells were shifted from the nonpermissive temperature, 41 degrees C, to the permissive temperature, 35 degrees C. Temperature shift from 35 to 41 degrees C decreased the lipid kinase activities in the membrane vesicles. These changes accompanied the changes observed in pp60v-src protein kinase activity. Thermal inactivation at 41 degrees C did not appreciably reduce PI and PIP kinase activities in membrane vesicles prepared from uninfected or Rous sarcoma virus-transformed cells, whereas pp60v-src protein kinase activity in the membrane vesicles was rapidly inactivated under the same conditions. These data suggest that pp60v-src may indirectly enhance PI and PIP phosphorylation but not directly contribute to this pathway.     

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.     

Substance P stimulates translocation of protein kinase C in brain microvessels

The action of substance P on protein kinase C in cerebral microvessels, isolated from bovine, was investigated. We found that in untreated microvessels 85% of the total activity was localized to the cytosolic fraction. Substance P caused a shift of activity to the membrane fraction, accompanied by a loss of activity in the cytosolic fraction. This effect resulted in dose-dependence and it was evident after 5 min treatment. These data suggest that substance P may be involved in the regulation of processes underlying protein phosphorylation in the blood-brain barrier.     

Participation of protein kinase C in hormonal regulation of pepsin secretion in the rat stomach]

It was shown that pentagastrin (0.5 micrograms/100 g of body mass) increases the activity of Ca2+ and phospholipid-dependent protein kinase C in the membrane fraction of rat gastric mucosa cells. This effect of pentagastrin is accompanied by a decrease of the protein kinase C activity in the cytosolic fraction. Chromatography of the membrane fraction revealed an additional peak of the enzyme activity. Analysis of isolated gastric mucosa cells demonstrated that pentagastrin (10(-8)-10(-6) M) (but not 10(-4) M histamine) added to the incubation mixture increased the protein kinase C concentration in the membranes. The pentagastrin effect was directly correlated with the amount of pepsin-producing chief cells in the cellular pools. Carbacholine, another well-known pepsin secretion stimulator, was able to activate, similar to pentagastrin, the protein kinase C activity. It is concluded that protein kinase C plays a prominent role in hormonal regulation of the chief gastric cell function.     

Phytohemagglutinin treatment of T lymphocytes stimulates rapid increases in activity of both particulate and cytosolic protein kinase C

We have measured the activity of protein kinase C in particulate and cytosolic fractions prepared from lymphocytes following stimulation with phytohemagglutinin. Activity in the particulate fraction increased approximately three-fold within 5 min, and declined to nearly zero between 20 and 60 min. Cytosolic activity increased in a biphasic manner, with an initial increase at 5 min, a decline at 10 min, and a further increase by 20 min, which was sustained for at least 60 min. By contrast, 12-O-tetradecanoylphorbol-13-acetate caused a rapid translocation of protein kinase C from cytosol to the particulate fraction which was sustained for at least 1 h. The results suggest that agents, such as phytohemagglutinin, which both generate diacylglycerol and mobilize intracellular Ca2+ stores, result in changes in subcellular distribution and activity of protein kinase C which are different from those elicited by 12-O-tetradecanoylphorbol-13-acetate.     

Role of the aggregation factor in the regulation of phosphoinositide metabolism in sponges. Possible consequences on calcium efflux and on mitogenesis

The aggregation factor (AF) of the marine sponge Geodia cydonium recognizes the aggregation receptor (AR) which is inserted in the plasma membrane, under formation of species-specific aggregates. The specific cell-binding fragment of the AF was used to investigate for the first time the phosphoinositide metabolism in a lower avertebrate system. We found that after binding of the cell-binding fragment to the aggregation receptor a strong and rapid stimulation of the phosphate incorporation into phosphatidylinositol occurs followed by an increased turnover of phosphoinositides in the Geodia cells. The consequences of an increased degradation of phosphatidylinositol 4,5-bisphosphate into the two second messengers inositol-1,4,5-trisphosphate and diacylglycerol are 2-fold. First, after the addition of the extracellular stimulus the cytosolic Ca2+ concentration rises, resulting in a rapid increased Ca2+ efflux rate. The functional consequence of the increase of the extracellular Ca2+ level is an initiation of the aggregate formation that is mediated by the collagen assembly factor (= primary aggregation factor). Second, some experimental evidences are presented, showing that the other second messenger formed, diacylglycerol, causes a translocation of protein kinase C within the cell. Incubation of Geodia cells with the cell-binding fragment of the AF, or with the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate, resulted within 5 min after treatment in a 70% decrease in protein kinase C activity in the cytosolic fraction and in a 700% increase in enzyme activity in the membrane fraction. It is proposed that by membrane association protein kinase C becomes activated. As a result of this event a series of cellular proteins are phosphorylated, a process which ultimately leads to an unusually strong induction of DNA polymerase alpha activity. From these data we conclude that inositol trisphosphate and protein kinase C also play a fundamental role in cellular signal transduction in lower eukaryotes.     

1,2-Diacylglycerols, but not phorbol esters, activate a potential inhibitory pathway for protein kinase C in GH3 pituitary cells. Evidence for involvement of a sphingomyelinase

It has been suggested that sphingoid bases may serve as physiologic inhibitors of protein kinase C. Because 1,2-diacylglycerols, but not phorbol esters, enhance sphingomyelin degradation via a sphingomyelinase in GH3 pituitary cells (Kolesnick, R. N. (1987) J. Biol. Chem. 262, 16759-16762), the effects of phorbol esters, 1,2-diacylglycerols, and sphingomyelinase on protein kinase C activation were assessed. Under basal conditions, the inactive cytosolic form of protein kinase C predominated. 1,2-Diacylglycerols stimulated transient protein kinase C redistribution to the membrane. 1,2-Dioctanoylglycerol (200 micrograms/ml) reduced cytosolic protein kinase C activity to 67% of control from 72 to 48 pmol.min-1.10(6) cells-1 and enhanced membrane-bound activity to 430% of control from 6 to 25 pmol.min-1.10(6) cells-1 after 4 min of stimulation. Thereafter, protein kinase C activity returned to the cytosol. In contrast, the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), stimulated redistribution to the membrane without return to the cytosol. Exogenous sphingomyelinase reduced membrane-bound protein kinase C activity to 30% of control, yet did not alter cytosolic activity. Sphingomyelinase, added after phorbol ester-induced redistribution was completed, restored activity to the cytosol. In these studies, TPA (10(-8) M) reduced cytosolic activity to 62% of control and elevated membrane-bound protein kinase C activity to 650% of control. Sphingomyelinase restored cytosolic activity to 84% of control and reduced membrane-bound activity to 297% of control. Similarly, the free sphingoid bases, sphingosine, sphinganine, and phytosphingosine, reversed phorbol ester-induced protein kinase C redistribution. Since 1,2-diacylglycerols activate a sphingomyelinase and sphingomyelinase action can reverse protein kinase C activation, these studies suggest that a pathway involving a sphingomyelinase might comprise a physiologic negative effector system for protein kinase C. Further, the failure of phorbol esters to activate this system might account for some differences between these agents.     

Effect of membrane perturbation on protein kinase C activation: treatment with exogenous phospholipase C decreases translocation of enzyme to cellular membranes

Incubation of mouse epidermal HEL-37 cells with C. perfringens phospholipase C for 30 min caused a partial loss of protein kinase C activity after 30 min incubation. Essentially all the kinase activity was present in the cytosolic fraction of both control and treated cells, despite the continued hydrolysis of phospholipid by the exogenous phospholipase. At shorter incubation times with phospholipase C (5 and 10 min) an association of protein kinase with particulate material was observed, presumably reflecting the accumulation of diacylglycerol. It is proposed that further incubation with phospholipase C renders the plasma membrane unable to bind protein kinase C and that already bound enzyme is destroyed by proteolysis.     

Proteolytic activation of protein kinase C by membrane-bound protease in rat liver plasma membrane

Incubation of rat liver plasma membrane produced histone phosphorylating activity at 75 mM Mg2+ in the soluble fraction. The release of the kinase activity was inhibited by leupeptin and bovine pancreatic trypsin inhibitor, suggesting the involvement of membrane-bound protease. When partially purified protein kinase C from rat liver cytosol was treated with the trypsin-like protease purified from rat liver plasma membrane, histone phosphorylating kinase which was independent of Ca2+ and phospholipids, produced with a molecular weight of about 5 X 10(4). These results suggest that membrane-bound, trypsin-like protease activates protein kinase C in plasma membrane and the activated kinase is released from the membrane to the soluble fraction.     

Involvement of protein kinase C in the phosphorylation of an insulin-granule membrane protein.

The involvement of protein kinase C in the Ca2+-dependent phosphorylation of a 29 000-Mr insulin-granule membrane protein prepared from a rat insulinoma was investigated. Protein kinase C activity towards exogenous lysine-rich histone was detected in a cytosolic fraction prepared from an insulinoma homogenate in the presence of EGTA. This activity bound reversibly to insulin granules in a Ca2+-dependent manner. Phosphatidylserine liposomes removed both protein kinase C activity and the 29 000-Mr protein-phosphorylating activity from the cytosolic fraction in a Ca2+-dependent fashion. Protein kinase C activity and the enzymic activity responsible for the phosphorylation of the 29 000-Mr granule protein behaved identically on sucrose-density-gradient centrifugation, ion-exchange chromatography, (NH4)2SO4 fractionation and gel filtration of the cytosolic fraction. These results are consistent with protein kinase C being the enzyme responsible for the phosphorylation of the 29 000-Mr insulin-granule membrane protein.