Membrane-associated protein kinases in phorbol ester-activated human polymorphonuclear leukocytes

Membrane-associated protein kinases in human polymorphonuclear leukocytes were studied. In unstimulated polymorphonuclear leukocytes the protein kinase C was predominantly present in the cytosol but in phorbol 12-myristate 13-acetate- (PMA-) activated cells a time and dose-dependent translocation of the kinase to the particulate fraction occurred. Two new protein kinase activities also appeared in the particulate fraction upon PMA activation. The one had a Mr of 40,000 and its activity was independent of phospholipids. The other (Mr 90,000) as partially activated by phospholipids, but separated from protein kinase C on DEAE-cellulose chromatography.     

Protein kinase C in cytosol and cell membranes of concanavalin A-stimulated rat thymocytes

In concanavalin A-treated thymus cells and phytohemagglutinin-treated spleen cells, the distribution of protein kinase C is changed. Shortly after addition of plant lectins, the activity of protein kinase C increased in the cytosol and decreased in the particulate fraction. 2 h later, the activity of protein kinase C decreased in the cytosol and increased in the particulate fraction. Membrane binding of protein kinase C and tion of DNA synthesis showed the same dependency on concanavalin A concentration. A long-term membrane binding of protein kinase C seems to be essential for lymphocyte stimulation.     

Modulation of octopamine-mediated production of cyclic AMP by phorbol-ester-sensitive protein kinase C in an insect cell line

The presence of protein kinase C (EC 2.7.1.37) in an insect cell line has been demonstrated. Phorbol 12-myristate 13-acetate (PMA), in micromolar concentrations, activated protein kinase C with a translocation of the enzyme from the cytosol to the particulate fraction. Cyclic AMP production in the presence of PMA, octopamine and a combination of both increased in a dose-dependent and time-dependent fashion. The biologically inactive 4 alpha-phorbol 12,13-didecanoate had no effect on protein kinase C activity or on octopamine-mediated cyclic AMP production. Pretreatment of the cells with pertussis toxin had no effect on the response of cells to octopamine or PMA. However, pretreatment with cholera toxin resulted in increased cyclic AMP production which was further enhanced when both cholera toxin and PMA were used in combination. Our data indicate that the octopamine-mediated cyclic AMP production is modulated by protein kinase C.     

Contraction-associated translocation of protein kinase C in rat skeletal muscle

Electrical stimulation of the sciatic nerve of the anaesthetized rat in vivo led to a time-dependent translocation of protein kinase C from the muscle cytosol to the particulate fraction. Maximum activity of protein kinase C in the particulate fraction occurred after 2 min of intermittent short tetanic contractions of the gastrocnemius-plantaris-soleus muscle group and coincided with the loss of activity from the cytosol. Translocation of protein kinase C may imply a role for this kinase in contraction-initiated changes in muscle metabolism.     

Inhibition of T-lymphocyte activation by amiloride

The T-lymphocyte activation process involves a series of coordinately coupled biochemical events occurring in response to antigen or mitogen. These events have not been completely characterized. The present studies investigate the mechanism of protein synthesis during the initial phase of T-cell activation. Among the early biochemical changes, induction of protein synthesis was observed as early as 10 minutes after mitogen stimulation of T-lymphocytes. This early protein synthesis was inhibited by cycloheximide but was insensitive to actinomycin-D, indicating the presence of preformed mRNA in resting lymphocytes. Since early protein synthesis parallels the increase in protein kinase C activity in activated T-lymphocytes, these two biochemical events may be related. In the present report, amiloride, an inhibitor of Na+/H+ antiport and protein kinase C, significantly inhibited [3H]leucine and [3H]thymidine incorporation in a dose-dependent manner into phytohemagglutinin (PHA)-stimulated T-lymphocytes. Furthermore, when T-lymphocytes were stimulated by phorbol myristate acetate, a known activator of protein kinase C, a similar inhibition of protein and DNA synthesis by amiloride was observed. The partially purified cytosol fraction isolated from PHA-activated T-lymphocytes showed a 75% decrease in protein kinase C-mediated [32P] incorporation from ATP in the presence of 100 microM amiloride. These results suggest that the T-cell activation process following exposure to mitogens involves early protein synthesis, which may be mediated by protein kinase C.     

Differential mechanisms of translocation of protein kinase C to plasma membranes in activated human neutrophils

Three classes of activators of human neutrophils that induce the intracellular translocation of protein kinase C from the cytosol to the particulate fraction were compared for their effects on the properties of the particulate (membrane-bound) enzyme. In cells stimulated with 10 ng/ml of phorbol-12-myristate-13-acetate (PMA) the particulate enzyme is almost fully active in the absence of added Ca2+ or phospholipids and this activity is not released by the Ca2+-chelator EDTA. In contrast, binding of protein kinase C to the particulate fraction in cells treated with the chemotactic factor f-Met-Leu-Phe (fMLF) or with the ionophore A-23187 plus Ca2+ is observed only when the cells are lysed in the presence of 1 mM Ca2+. With these stimuli the particulate enzyme retains a nearly absolute requirement for Ca2+ and phospholipids. Thus only the full intercalation of protein kinase C caused by PMA, which is resistant to removal by chelators stabilizes an active form of protein kinase C in the neutrophil membrane. In confirmation of this conclusion, in isolated plasma membranes loaded with partially purified protein kinase C by incubation with 5 microM Ca2+ further incubation with PMA, but not with fMLF, caused a significant fraction of the bound PKC to become resistant to removal by chelators, and to be nearly fully active in the absence of added activators.     

Proteolytic degradation of protein kinase C in the phorbol ester-induced interleukin-2 secreting thymoma cells

Effects of phorbol 12-myristate 13-acetate (PMA) on the fate of protein kinase C in two mouse thymoma cell lines, which are either responsive (EL4) or unresponsive (IEL4) to PMA-induced interleukin-2 (IL-2) production, were investigated with polyclonal antibodies raised against rat brain enzyme. These antibodies immunoprecipitated completely the protein kinase C from both cell lines and detected mainly an 82-kDa protein by immunoblot analysis of the crude homogenates as well as the partially purified kinase preparations. PMA elicited a time- and dose-dependent redistribution of protein kinase C from cytosol to the particulate fraction and proteolytic degradation of the kinase from both cell lines. The dose of PMA required for half-maximum protein kinase C translocation and degradation was at least five times lower for EL4 than for IEL4. In the presence of 16 nM PMA the rates of protein kinase C translocation and degradation were faster in EL4 than in IEL4, and the half-lives of protein kinase C in EL4 and IEL4 were less than 5 min and greater than 2 h, respectively. Analysis of the tryptic fragments of the immunoprecipitated enzyme, previously phosphorylated in the presence of [gamma-32P]ATP, revealed minor structural differences between the protein kinase C from these two cell lines. In neither cell line did the PMA-induced degradation of protein kinase C result in an accumulation of the Ca2+/phospholipid-independent kinase (catalytic unit) as analyzed by immunoblotting and gel filtration chromatography. Thus, activation of protein kinase C through the proteolytic conversion to the effector-independent catalytic unit plays little role in IL-2 production. The role of protein kinase C translocation and degradation in the PMA-induced responses in EL4 cells is unknown. However, IL-2 production in EL4 cells was reduced when PMA-induced degradation of protein kinase C was retarded by exogenously added protease inhibitors.     

Protein kinase C activity is altered in HL60 cells exposed to 60 Hz AC electric fields

We examined the effects of electric fields (EFs) on the activity and subcellular distribution of protein kinase C (PKC) of living HL60 cells. Sixty Hertz AC sinusoidal EFs (1.5–1.000 mV/cm p-p) were applied for 1 h to cells (10⁷/ml) in Teflon chambers at 37 °C in the presence or absence of 2 μM phorbol 12-myristate 13-acetate (PMA). PMA stimulation alone evoked intracellular translocation of PKC from the cytosolic to particulate fractions. In cells that were exposed to EFs (100–1,000 mV/cm) without PMA, a loss of PKC activity from the cytosol, but no concomitant rise in particulate PKC activity, was observed. In the presence of PMA. EFs (33–330 mV/cm) also accentuated the expected loss of PKC activity from the cytosol and augmented the rise in PKC activity in the particulate fraction. These data show that EFs alone or combined with PMA promote down-regulation of cytosolic PKC activity similar to that evoked by mitogens and tumor promoters but that it does not elicit the concomitant rise in particulate activity seen with those agents. © 1996 Wiley-Liss, Inc.     

Evidence for increased synthesis as well as increased degradation of protein kinase C after treatment of human osteosarcoma cells with phorbol ester

Phorbol 12-myristate 13-acetate (PMA) induces time-dependent changes in protein kinase C subcellular distribution and enzymatic activity in the human osteosarcoma cell line SaOS-2. Short (less than 60 min) incubations with PMA caused decreased cytosolic enzyme activity and a concomitant increase in particulate protein kinase; after 3 h, particulate protein kinase C activity also declined to reach less than 10% of basal activity by 24 h (Krug, E., and Tashjian, Jr., A. H., (1987) Cancer Res. 47, 2243-2246). In order to determine whether the loss in enzyme activity was due to decreased enzyme protein, Western blot analyses were performed using a polyclonal antibody against protein kinase C raised in rabbits. This approach confirmed the previously reported time-related changes: 80-kDa immunoreactive protein kinase C initially translocated from the cytosol to the particulate cell fraction and later disappeared completely from the particulate fraction. Loss of protein kinase C enzymatic activity thus results from actual loss of the 80-kDa protein; we found no evidence for generation of a calcium/phospholipid-independent protein kinase C-like form of the enzyme. Membrane association was confirmed by immunoprecipitation experiments using [35S]methionine-labeled cells. Brief exposure to PMA caused a marked loss in the [35S]methionine-labeled cytosolic protein kinase C band and an increase in the labeled particulate band. Protein kinase C immunoprecipitated from cells treated with PMA for 14 h displayed an increase in [35S]methionine label despite a greater than 80% loss of enzyme activity. The high specific radioactivity of the remaining 80-kDa protein leads us to conclude that long term treatment with PMA causes an increase in the rate of protein kinase C synthesis accompanied by a still greater increase in the rate of enzyme degradation in SaOS-2 cells.     

Influence of calcium on the subcellular distribution of protein kinase C in human neutrophils. Extraction conditions determine partitioning of histone-phosphorylating activity and immunoreactivity between cytosol and particulate fractions

Activation of the neutrophil respiratory burst is thought to involve a translocation and activation of protein kinase C. We report that the presence of Ca2+ during the disruption of unstimulated human neutrophils and cytoplasts resulted in an increase in protein kinase C activity (histone phosphorylation) and immunoreactive protein kinase C species in the particulate (membrane) fraction and a reduction in such activities in the cytosol. This Ca2+-induced translocation of activity was concentration-dependent and occurred at physiologically relevant concentrations of Ca2+ (30-500 nM). The Ca2+-induced membrane association of protein kinase C could be reversed by removal of Ca2+. These findings indicate that the Ca2+ concentration of the extraction buffer can determine the subcellular distribution of protein kinase C in disrupted cells and suggest that the observed location of this enzyme activity in cell fractions may not necessarily reflect the localization in intact cells. These results also raise the possibility that the distribution of protein kinase C between cytosol and membrane is a dynamic equilibrium controlled by levels of free Ca2+. Thus, Ca2+ might regulate distribution as well as activation of protein kinase C.     

Early effects of TPA on protein kinase activity in murine thymocytes. Reduction of protein kinase C activity in the cytosol and increase of Ca2+ and phospholipid-independent kinase activity in the particulate fractions

Brief treatment of intact thymocytes with TPA and other tumor promoters causes a reduction in protein kinase C activity from the cytosol and an increase in kinase activity in the particulate fraction. In contrast to the activity in the cytosol, which is absolutely dependent on the addition of Ca2+, phosphatidylserine and diolein, the activity in the particulate fraction is independent of these agents. Analysis of target specificity of the particulate kinase activity using exogenous and endogenous substrates suggests that the increased phosphorylation in the particulate fraction is catalysed by protein kinase C with altered catalytic properties. Although interleukin-1 and TPA are both co-mitogens for murine thymocytes, interleukin-1 does not share with TPA its property to alter protein kinase activity in the cytosolic and particulate fractions.     

Regulation of membrane-bound PKC in adult cardiac ventricular myocytes

Activation of protein kinase C (PKC) is thought to involve translocation to the particulate fraction. The present study demonstrates a membrane-associated, inactive pool of PKC in adult rat ventricular myocytes. Membranes were isolated from stimulated (phorbol 12-myristate 13-acetate (PMA), endothelin-1 (ET-1)) or control myocytes and PKC activity determined in the absence (active PKC) or presence (total PKC) of PMA. An inactive, PMA-responsive, pool of PKC was detected. In intact myocytes, PMA or ET-1 induced a translocation of PKC epsilon from the cytosol into the particulate fraction. In contrast, ET-1 decreased both total and active PKC in the membranes: this decrease was associated with a loss of PKC epsilon immunoreactivity. PMA increased the amount of membrane-associated, inactive PKC. Our results demonstrate the presence of a membrane-associated pool of PKC in cardiac myocytes that is differentially modulated by ET-1 or PMA.     

Early effects of TPA on protein kinase activity in murine thymocytes : Reduction of protein kinase C activity in the cytosol and increase of Ca and phospholipid-independent kinase activity in the particulate fractions

Brief treatment of intact thymocytes with TPA and other tumor promoters causes a reduction in protein kinase C activity from the cytosol and an increase in kinase activity in the pariculate fraction. In contrast to the activity in the cytosol, which is absolutely dependent on the addition of Ca²⁺, phosphatidylserine and diolein, the activity in the particulate fraction is independent of these agents. Analysis of target specificity of the particulate kinase activity using exogenous and endogenous substrates suggests that the increased phosphorylation in the particulate fraction is catalysed by protein kinase C with altered catalytic properties. Although interleukin-1 and TPA are both co-mitogens for murine thymocytes, interleukin-1 does not share with TPA its property to alter protein kinase activity in the cytosolic and particulate fractions.     

Effect of phorbol esters on the distribution and total activity of protein kinase C in the perfused rat heart

1. The perfused rat heart was treated with the tumour-promoter and protein kinase C activator, phorbol 12-myristate 13-acetate and the distribution of protein kinase C activity between cytosolic and particulate fractions determined. 2. Phorbol ester treatment led to a rapid loss of protein kinase C activity from the cytosol (t0.5 = 2 min) with a corresponding translocation into the particulate fraction. Translocated protein kinase C activity was tightly bound to the particulate fraction, could only be extracted with buffers containing 2% Triton X-100 and could therefore be misinterpreted as being down-regulated. 3. Claims of rapid down-regulation of protein kinase C activity by phorbol esters need to be supported by rigorous procedures for extraction of the particulate material.     

Tumor-promoting phorbol esters stimulate the proliferation of interleukin-3 dependent cells

To determine whether activation of protein kinase C is involved in the proliferation of interleukin-3 (IL-3) -dependent cells, we examined the effect of tumor-promoting phorbol esters on the in vitro proliferation of the IL-3-dependent cell lines FD and DA-1. The viability of FD and DA-1 cells cultured for 24 hours in 100 nM phorbol myristate acetate (PMA) and 10% FCS was similar to that of cells cultured in 25% WEHI-3 conditioned medium as a source of IL-3, and 10% FCS. FD cells failed to proliferate in concentrations of FCS of up to 50%, while DA-1 cell proliferation was not markedly influenced by FCS. By contrast, PMA promoted the proliferation of FD and DA-1 cells in the absence of FCS and enhanced their proliferation in the presence of 10% FCS, 60- and 20-fold, respectively. Stimulation of proliferation was achieved with as little as 10 nM PMA and was maximal at 100 nM PMA. Low concentrations (0.05-0.1%) of WEHI-3 CM promoted the proliferative response of FD and DA-1 cells to PMA, but at concentrations of WEHI-3 CM greater than 0.8%, no further increment in proliferation was obtained with PMA. As little as 1/2 hour of exposure to phorbol esters was sufficient to cause translocation of protein kinase C from the cytosol to the membranes of DA-1 cells, and 1 hour of exposure to phorbol esters was sufficient to stimulate DNA synthesis. A protein kinase C inhibitor, H-7, at a concentration of 10 microM inhibited phorbol ester-induced stimulation of DA-1 cell proliferation. When DA-1 cells were exposed to the calcium ionophore A23187 in addition to both a phorbol ester and IL-3, their proliferation was enhanced over that stimulated by only the phorbol ester and IL-3. The data indicate that stimulation of proliferation of IL-3-dependent cells involves the activation of protein kinase C.     

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.     

The involvement of calpain in the activation of protein kinase C in neutrophils stimulated by phorbol myristic acid

The Ca2+/phospholipid-dependent protein kinase (protein kinase C) of human neutrophils is converted to a proteolytically modified Ca2+/phospholipid-independent form (Inoue, M., Kishimoto, A., Takai, Y.U., and Nishizuka, Y. (1977) J. Biol. Chem. 252, 7610-7616) on incubation with neutrophil membranes in the presence of micromolar concentrations of Ca2+ and an endogenous Ca2+-requiring proteinase (Melloni, E., Pontremoli, S., Michetti, M., Sacco, O., Sparatore, B., Salamino, F., and Horecker, B. L. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 6435-6439). We have now demonstrated the appearance of a similar Ca2+/phospholipid-independent kinase in intact human neutrophils stimulated by phorbol 12-myristate 13-acetate (PMA). The following evidence supports the conclusion that the Ca2+/phospholipid-independent protein kinase recovered from the PMA-treated cells is a proteolytically modified form of the "native" protein kinase C. 1) In cells exposed to PMA, the rate of disappearance of Ca2+/phospholipid-dependent protein kinase C activity is correlated with the rate of appearance of the Ca2+/phospholipid-independent kinase. 2) The chromatographic behavior of the new protein kinase and its molecular size (approximately 65 kDa) are identical to those previously reported for the proteolytically modified form of protein kinase C. 3) The modified protein kinase no longer binds to the cell membrane and is recovered almost entirely in the cytosol fraction. 4) In neutrophils preloaded with inhibitors of the Ca2+-requiring proteinase, stimulation with PMA results in translocation of protein kinase C from the cytosol fraction to the particulate fraction, but the appearance of the soluble, Ca2+/phospholipid-dependent form is prevented. We conclude that binding of protein kinase C to the plasma membrane and its proteolytic conversion are related, but independent, processes both elicited by exposure of neutrophils to the phorbol ester. Proteolytic cleavage of the membrane-bound protein kinase C provides an alternative mechanism for its activation and may account for certain of the cellular responses observed in PMA-stimulated neutrophils.     

Gonadotropin release and redistribution of calcium-activated, phospholipid-dependent protein kinase in phorbol-stimulated rat pituitary cells

The effect of phorbol esters on calcium-activated, phospholipid-dependent kinase (protein kinase C) and luteinizing hormone (LH) secretion was examined in cultured rat anterior pituitary cells. The potent tumor promoter 12-O-tetra-decanoylphorbol-13-acetate (TPA) stimulated LH secretion and activated pituitary protein kinase C in the presence of calcium and phosphatidylserine. The enzyme activity present in cytosol and particulate fractions was eluted at about 0.05 M NaCl during DE52-cellulose chromatography. Preincubation of pituitary cells with TPA markedly decreased cytosolic protein kinase C activity and increased enzyme activity in the particulate fraction. The maximal TPA-induced change in enzyme activity, with a 76% decrease in cytosol and a 4.3-fold increase in the particulate fraction, occurred within 10 min. The dose-dependent changes in protein kinase C redistribution in TPA-treated cells were correlated with the stimulation of LH release by the phorbol ester. These results suggest that activation of protein kinase C by TPA is associated with intracellular redistribution of the enzyme and is related to the process of secretory granule release from gonadotrophs.     

Redistribution of protein kinase C activity in human monocytes: correlation with activation of the respiratory burst

Protein kinase C (PKC) was found to be present in purified human monocytes and lymphocytes isolated by countercurrent centrifugal elutriation. In unstimulated monocytes and lymphocytes, approximately 90% of the PKC activity was cytosolic when the cells were disrupted in the presence of EGTA. The role of this kinase in the stimulation of the respiratory burst in monocytes was investigated. Phorbol esters capable of triggering the release of O2- caused a loss of PKC activity from the cytosol and the appearance of the kinase activity in the particulate cell fraction. Kinase activity was partially extractable from the particulate fraction by 0.1% Triton X-100, whereupon it demonstrated calcium and lipid dependence. The EC50 for the phorbols in initiating the respiratory burst correlated well with their EC50 for stimulating the appearance of PKC activity in the particulate fraction (R = 0.998). Redistribution of PKC activity in monocytes by phorbol myristate acetate (PMA) was rapid and appeared to precede the release of O2-. PMA also shifted PKC activity from the cytosol to the extractable particulate fraction of lymphocytes. We conclude that redistribution of PKC activity by active phorbols or other cell stimulants could provide substrate specificity for phosphorylation reactions. By shifting PKC activity to the monocyte particulate fraction, active phorbols may initiate the phosphorylation of a substrate required for stimulation of the respiratory burst.     

Translocation of PKC isoforms in bovine aortic smooth muscle cells exposed to strain

Our laboratory has previously reported that the exposure of smooth muscle cells (SMC) to the cyclic strain results in significant stimulation of protein kinase C (PKC) activity by translocating the enzyme from the cytosol to the particulate fraction. We now sought to examine the strain-induced translocation of individual PKC isoforms in SMC. Confluent bovine aortic SMC grown on collagen type I-coated plates were exposed to cyclic strain for up to 100 s at average 10% strain with 60 cycles/min. Immunoblotting analysis demonstrates that SMC express PKC-alpha, -beta and -zeta in both cytosolic and particulate fractions. Especially, PKC-alpha and -zeta were predominantly expressed in the cytosolic fraction. However, cyclic strain significantly (P < 0.05) increased PKC-alpha and -zeta in the particulate fraction and decreased in the cytosolic fraction. Thus, the cyclic strain-mediated stimulation of PKC activity in SMC may be due to the translocation of PKC-alpha and -zeta from the cytosolic to the particulate fraction. These results demonstrate that mechanical deformation causes rapid translocation of PKC isoforms, which may initiate a cascade of proliferation responses of SMC since NF-kappaB, which is involved in the cellular proliferation has been known to be activated by these PKC isoforms.