Hormone- and phorbol ester-activated protein kinase C isozymes mediate a reorganization of the actin cytoskeleton associated with prolactin secretion in GH4C1 cells.

TRH regulates PRL secretion and synthesis in GH4C1 rat pituitary cells. TRH responses are associated with activation of protein kinase C (PKC) isozymes and elevation of cytosolic calcium. To determine which PKC isozymes are involved in TRH-directed responses, we evaluated the effect of TRH on GH cell alpha-, beta-, delta-, and epsilon-PKC isozymes. Immunoblot analysis demonstrated that TRH caused rapid redistribution of all isozymes to a Triton X-100-insoluble (i.e. cytoskeletal) fraction. Corollary immunocytofluorescence studies demonstrated that redistributed PKCs accumulate in cell peripheries. Exocytosis involves reorganization of the cytoskeleton, therefore, each of the GH cell PKCs is appropriately located to phosphorylate proteins important for cytoskeleton organization. To determine the relative contributions of calcium and PKC signal transduction pathways in mediating TRH responses, the effects of potassium depolarization (which increases cytosolic calcium) and phorbol dibutyrate (which activates all PKC isozymes without increasing calcium) were compared. The data indicate that TRH-mediated reorganization of vinculin proceeds via a calcium-mediated pathway, whereas fragmentation of actin filaments proceeds via a PKC-dependent pathway. Selective down-modulation of epsilon-PKC with prolonged TRH-treatment was used to demonstrate that epsilon-PKC is not necessary for certain TRH-stimulated biological responses.     

Activation of alpha-protein kinase C leads to association with detergent-insoluble components of GH4C1 cells

TRH and phorbol dibutyrate (PDBu) stimulate PRL secretion and synthesis from GH4C1 rat pituitary cells through activation of protein kinase C (PKC). TRH responses are mediated by increases in cellular levels of two PKC activators, Ca2+ and diacylglycerol (DAG), whereas PDBu acts as a DAG analog. We conducted experiments to compare the effects of Ca2+ and PDBu/DAG on alpha-PKC redistribution and to determine to what components of the particulate fraction activated alpha-PKC associates. Subcellular fractionation experiments demonstrated that TRH and PDBu both caused chelator-stable association of alpha-PKC with the particulate fraction. In contrast, Ca2+-mediated association with the particulate fraction was not chelator stable. Immunocytofluorescence experiments also demonstrated that TRH, PDBu, and increased cytosolic Ca2+ (due to ionomycin or K+ depolarization) caused redistribution. The effect of TRH was rapid and transient, similar to TRH stimulation of phospholipase C. The translocated alpha-PKC in the particulate fraction from TRH- or PDBu-treated cultures was not solubilized with Triton X-100. In comparable studies using an immunofluorescence assay, alpha-PKC immunofluorescence remained in detergent-insoluble preparations from TRH- and PDBu-stimulated, but not resting cells. The association of activated alpha-PKC with chelator- and detergent-insoluble material suggested that activated alpha-PKC may be associated with membrane and cytoskeletal components.     

Ca2(+)-independent secretory mechanism of thyrotropin-releasing hormone (TRH) involves protein kinase C in rat pituitary cells

Thyrotropin-releasing hormone (TRH) stimulates biphasic prolactin (PRL) secretion from rat pituitary GH3 cells. The pretreatment of cells with EGTA (100 microM) plus arachidonic acid (15 microM), a condition which decreased TRH-responsive intracellular Ca2+ pools, eliminated the activity of TRH on burst PRL secretion (2 min) but did not alter that on sustained PRL secretion (30 min). However, the treatment of cells with EGTA, arachidonic acid and H-7 (300 microM), a potent inhibitor of protein kinase C (PKC), almost completely suppressed the activity of TRH for sustained PRL secretion. In cells down-modulated for PKC, TRH abolished this Ca2(+)-independent sustained PRL secretion. These results suggest that TRH acts through a separate, Ca2(+)-independent secretory mechanism, besides by modulating the Ca2(+)-dependent mechanism and that PKC is involved in this Ca2(+)-independent secretory pathway.     

Possible role of Ca2(+)-independent protein kinase C isozyme, nPKC epsilon, in thyrotropin-releasing hormone-stimulated signal transduction: differential down-regulation of nPKC epsilon in GH4C1 cells

Protein kinase C (PKC) molecular species of GH4C1 cells were analyzed after separation by hydroxyapatite column chromatography. A novel Ca2(+)-independent PKC, nPKC epsilon, was identified together with two conventional Ca2(+)-dependent PKCs, PKC alpha and beta II by analysis of kinase and phorbol ester-binding activities, immunoblotting using isozyme-specific antibodies, and Northern blotting. These PKCs are down-regulated differently when cells are stimulated by outer stimuli; phorbol esters deplete PKC beta II and nPKC epsilon from the cells more rapidly than PKC alpha, whereas thyrotropin-releasing hormone (TRH) at 200 nM depletes nPKC epsilon exclusively with a time course similar to that induced by phorbol esters. However, translocation of PKC alpha and beta II to the membranes is elicited by both TRH and phorbol esters. These results suggest that TRH and phorbol ester activate PKC alpha and beta II differently but that nPKC epsilon is stimulated similarly by both stimuli. Thus, in GH4C1 cells, Ca2(+)-independent nPKC epsilon may play a crucial role distinct from that mediated by Ca2(+)-dependent PKC alpha and beta II in a cellular response elicited by both TRH and phorbol esters.     

Activation of multifunctional Ca2+/calmodulin-dependent protein kinase in GH3 cells.

We report that the rat pituitary cell line GH3 contains a Ca2(+)- and calmodulin-dependent protein kinase with properties characteristic of multifunctional Ca2+/calmodulin-dependent protein kinase (CaM kinase) from rat brain. The GH3 kinase exhibits the hallmark of authentic CaM kinase: conversion from Ca2(+)-dependent to Ca2(+)-independent activity following a brief initial phosphorylation in vitro. This phosphorylation occurs at a site which is similar or identical to that of the "autonomy" site of the rat brain enzyme and thus may be an autophosphorylation event. GH3 CaM kinase is phosphorylated and becomes Ca2(+)-independent in situ. Depolarization of intact cells with K+ opens calcium channels and leads to the phosphorylation of CaM kinase at the autonomy site, and the kinase becomes significantly and persistently Ca2(+)-independent. Treatment of cells with thyrotropin-releasing hormone (TRH), which activates the phosphatidylinositol signaling pathway, also generates a Ca2(+)-independent CaM kinase in situ. The primary effect of TRH on CaM kinase activity is transient and correlates with the spike of Ca2+ released from intracellular stores and the rapid phase of prolactin release from GH3 cells. This study demonstrates that CaM kinase is able to detect and respond to both calcium that enters the cell through voltage-sensitive Ca2+ channels and calcium released from internal stores via the phosphatidylinositol pathway. We find that TRH, a hormone that causes release of prolactin and was previously believed to activate primarily protein kinase C, also significantly activates CaM kinase in intact cells.     

Effects of extracellular calcium on the subcellular translocation of bovine parathyroid PKC isozymes

The release of parathyroid hormone is regulated by the extracellular concentration of Ca2+ through a sensor(s) on the surface of the parathyroid cells, but few details are known on the further relay of the signal inside the cell. Activation of protein kinase C (PKC) isozymes is associated with their translocation from the cell soluble fraction to the particulate fraction of the cell. Therefore, identification of a subcellular localization of a PKC isozyme in parathyroid cells as a response to changes in extracellular Ca2+ should be an indication for its putative role in signal transduction coupled to the Ca2+ sensor. We have determined the subcellular localization of six PKC isozymes (alpha, betaI, betaII, epsilon, zeta, and iota) in nonstimulated parathyroid cells and in those treated with low (0.5 mM) and high (3.0 mM) extracellular Ca2+ by confocal microscopy. At the physiological concentration of serum Ca2+, all PKC isozymes studied were localized mainly to the cytosol, although to different extents. Low extracellular Ca2+ caused a redistribution of PKCalpha to the periphery of the cells. In contrast, PKCbetaI, -epsilon, -zeta, and -iota were translocated to the periphery of the cells at high extracellular Ca2+. These results indicate that PKCalpha, -betaI, -epsilon, -zeta, and -iota are involved in the response of parathyroid cells to changes in extracellular Ca2+.     

Platelet-activating factor affects cytosolic free calcium concentration and prolactin secretion in GH4C1 rat pituitary cells.

Platelet-activating factor (PAF) is a naturally occurring pleiotropic mediator which acts via specific membrane receptors. In certain target cells, PAF causes elevations in cytosolic free Ca2+ concentration ([Ca2+]i); however, little is known of the effects of PAF on endocrine cells. Therefore, we have investigated the actions of PAF on [Ca2+]i in prolactin-secreting GH4C1 cells and have compared the effects with the well documented actions on these cells of thyrotropin-releasing hormone (TRH). GH4C1 cells were loaded with quin2/AM and fluorescence was measured in suspended populations. PAF induced a dose-dependent (10-100 microM) rise in [Ca2+]i which was slower in onset than that caused by TRH, peaking (200 to 400% above basal [Ca2+]i) at about 12 sec, and decaying over about 3 min to basal [Ca2+]i. Unlike TRH, PAF did not cause a secondary plateau phase of rise in [Ca2+]i. The terpene PAF receptor antagonist BN52021 inhibited the action of PAF on [Ca2+]i. Voltage-dependent Ca2+ channel blocker, verapamil (200 microM), antagonized the action of PAF on [Ca2+]i as did chelation of extracellular Ca2+. PAF also stimulated the secretion of prolactin in a dose-dependent manner (10 to 50 microM). The concentrations of PAF required to evoke responses in GH4C1 cells were considerably higher than those required in several other known PAF target cell types. The high concentration requirement in GH4C1 cells may be due to rapid degradation of PAF or the presence of low affinity receptors. We conclude that PAF can act, via cell surface receptors, on pituitary GH4C1 cells to alter [Ca2+]i by a pathway that enhances influx of extracellular Ca2+ through voltage-gated channels and then to enhance the secretion of prolactin.     

Isoform-specific protein kinase C activity at variable Ca2+ entry during coronary artery contraction by vasoactive eicosanoids.

Vasoactive eicosanoids have been implicated in the pathogenesis of coronary vasospasms. The signaling mechanisms of eicosanoid-induced coronary vasoconstriction are unclear, and a role for protein kinase C (PKC) has been suggested. Activated PKC undergoes translocation to the surface membrane in the vicinity of Ca2+ channels; however, the effect of Ca2+ entry on the activity of the specific PKC isoforms in coronary smooth muscle is unknown. In the present study, 45Ca2+ influx and isometric contraction were measured in porcine coronary artery strips incubated at increasing extracellular calcium concentrations ([Ca2+]e) and stimulated with prostaglandin F2alpha (PGF2alpha) or the stable thromboxane A2 analog U46619, while in parallel, the cytosolic (C) and particulate (P) fractions were examined for PKC activity and reactivity with anti-PKC antibodies using Western blot analysis. At 0-300 microM [Ca2+]e, both PGF2alpha and U46619 (10(-5) M) significantly increased PKC activity and contraction in the absence of a significant increase in 45Ca2+ influx. At 600 microM [Ca2+]e, PGF2alpha and U46619 increased P/C PKC activity ratio to a peak of 9.52 and 14.58, respectively, with a significant increase in 45Ca2+ influx and contraction. The 45Ca2+ influx--PKC activity--contraction relationship showed a 45Ca2+-influx threshold of approximately 7 micromol x kg(-1) x min(-1) for maximal PKC activation by PGF2alpha and U46619. 45Ca2+ influx > 10 micromol x kg(-1) x min(-1) was associated with further increases in contraction despite a significant decrease in PKC activity. Western blotting analysis revealed alpha-, delta-, epsilon-, and zeta-PKC in porcine coronary artery. In unstimulated tissues, alpha- and epsilon-PKC were mostly distributed in the cytosolic fraction. Significant eicosanoid-induced translocation of epsilon-PKC from the cytosolic to the particulate fraction was observed at 0 [Ca2+]e, while translocation of alpha-PKC was observed at 600 microM [Ca2+]e. Thus, a significant component of eicosanoid-induced coronary contraction is associated with significant PKC activity in the absence of significant increase in Ca2+ entry and may involve activation and translocation of the Ca2+-independent epsilon-PKC. An additional Ca2+-dependent component of eicosanoid-induced coronary contraction is associated with a peak PKC activity at submaximal Ca2+ entry and may involve activation and translocation of the Ca2+-dependent alpha-PKC. The results also suggest that a smaller PKC activity at supramaximal Ca2+ entry may be sufficient during eicosanoid-induced contraction of coronary smooth muscle.     

Thyrotropin-releasing hormone and phorbol esters induce phosphatidylcholine synthesis in GH3 pituitary cells. Evidence for stimulation via protein kinase C

Phorbol esters have been shown to stimulate phosphatidylcholine synthesis via the CDP-choline pathway. The present study compares the effects of phorbol esters and thyrotropin-releasing hormone (TRH) on phosphatidylcholine metabolism in GH3 pituitary cells. In a previous study (Kolesnick, R.N., and Paley, A.E. (1987) J. Biol. Chem. 262, 9204-9210), the potent phorbol ester, 12-O-tetradecanoylphorbol 13-acetate (TPA) induced time- and concentration-dependent incorporation of 32Pi and [3H]choline into phosphatidylcholine in short-term labeling experiments. In this study, TPA is shown to activate choline-phosphate cytidylyltransferase (EC 2.7.7.15), the regulatory enzyme of the CDP-choline pathway, by stimulating redistribution of the inactive cytosolic form of the enzyme to the membrane. Redistribution was quantitative. TPA reduced cytosolic activity from 3.5 +/- 0.4 to 1.5 +/- 0.3 nmol . min-1 x 10(7) cells-1 and enhanced particulate activity from 2.5 +/- 0.4 to 4.9 +/- 0.6 nmol . min-1 x 10(7) cells-1. TRH also stimulated time- and concentration-dependent 32Pi and [3H]choline incorporation into phosphatidylcholine. An increase was detectable after 5 min; and after 30 min, the levels were 164 +/- 9 and 150 +/- 11% of control, respectively; EC50 congruent to 2 X 10(-10) M TRH. These events correlated directly with TRH-induced 32Pi incorporation into phosphatidylcholine. TRH also stimulated redistribution of cytidylyl-transferase specific activity. TRH reduced cytosolic activity 45% and enhanced particulate activity 51%. Neither TRH nor TPA stimulated phosphatidylcholine degradation. In cells down-modulated for protein kinase C (Ca2+/phospholipid-dependent protein kinase), the effects of TPA and TRH on 32Pi incorporation into phosphatidylcholine were abolished. However, TRH-induced incorporation into phosphatidylinositol still occurred. These studies provide evidence that hormones may regulate phosphatidylcholine metabolism via the protein kinase C pathway.     

Evidence for TRH-induced influx of extracellular Ca2+ in pituitary GH4C1 cells

The aim of the study was to investigate the relationship between thyrotropin-releasing hormone (TRH)-induced changes in intracellular free Ca2+ ([Ca2+]i), and influx of extracellular Ca2+ in Fura 2 loaded pituitary GH4C1 cells. Stimulating the cells with TRH in a Ca(2+)-containing buffer induced a biphasic change in [Ca2+]i. First, a transient increase in [Ca2+]i, followed by a sustained phase. In cells stimulated with TRH in a Ca(2+)-free buffer, the transient increase in [Ca(2+)]i was decreased (p less than 0.05), and the sustained phase was totally abolished. Addition of Ni2+ prior to TRH blunted the component of the TRH-induced transient increase in [Ca2+]i dependent on influx of Ca2+. In the presence of extracellular Mn2+, TRH stimulated quenching of Fura 2 fluorescence. This quenching was blocked by Ni2+. The results indicate that both the TRH-induced transient increase in [Ca2+]i as well as the sustained phase in [Ca2+]i in GH4C1 cells is dependent on influx of extracellular Ca2+.     

Granulocyte-macrophage colony-stimulating factor can stimulate macrophage proliferation via persistent activation of Na+/H+ antiport. Evidence for two distinct roles for Na+/H+ antiport activation.

Thapsigargin stimulates an increase of cytosolic free Ca2+ concentration [( Ca2+]c) in, and 45Ca2+ efflux from, a clone of GH4C1 pituitary cells. This increase in [Ca2+]c was followed by a lower sustained elevation of [Ca2+]c, which required the presence of extracellular Ca2+, and was not inhibited by a Ca2(+)-channel blocker, nimodipine. Thapsigargin had no effect on inositol phosphate generation. We used thyrotropin-releasing hormone (TRH) to mobilize Ca2+ from an InsP3-sensitive store. Pretreatment with thapsigargin blocked the ability of TRH to cause a transient increase in both [Ca2+]c and 45Ca2+ efflux. The block of TRH-induced Ca2+ mobilization was not caused by a block at the receptor level, because TRH stimulation of InsP3 was not affected by thapsigargin. Rundown of the TRH-releasable store by Ca2(+)-induced Ca2+ release does not appear to account for the action of thapsigargin on the TRH-induced spike in [Ca2+]c, because BAY K 8644, which causes a sustained rise in [Ca2+]c, did not block Ca2+ release caused by TRH. In addition, caffeine, which releases Ca2+ from intracellular stores in other cell types, caused an increase in [Ca2+]c in GH4C1 cells, but had no effect on a subsequent spike in [Ca2+]c induced by TRH or thapsigargin. TRH caused a substantial decrease in the amount of intracellular Ca2+ released by thapsigargin. We conclude that in GH4C1 cells thapsigargin actively discharges an InsP3-releasable pool of Ca2+ and that this mechanism alone causes the block of the TRH-induced increase in [Ca2+]c.     

Expression of four protein kinase C isoforms in rat fibroblasts. Distinct subcellular distribution and regulation by calcium and phorbol esters.

Protein kinase C (PKC), the major receptor for tumor-promoting phorbol esters, consists of a family of at least eight distinct lipid-regulated enzymes. How the various PKC isozymes are regulated in vivo and how they couple to particular cellular responses is largely unknown. We have examined the expression and regulation of PKC isoforms in R6 rat embryo fibroblasts. Northern and Western blot analyses indicate that these cells express four PKC isoforms, cPKC alpha, nPKC epsilon, nPKC delta, and nPKC zeta; of which nPKC epsilon and nPKC delta are the most abundant. In agreement with the simultaneous presence of cPKC and nPKC isozymes, both Ca(2+)-dependent and -independent PKC activities were detected in extracts of these cells. cPKC alpha and nPKC zeta were predominantly localized in the cytosol when subcellular fractionation was carried out in the presence of [ethylenebis(oxyethylenenitrilo)]tetraacetic acid. When cell lysis was carried out in the presence of Ca2+, greater than 50% of cPKC alpha redistributed to the particulate fraction, whereas nPKC zeta remained in the cytosol. In contrast to cPKC alpha and nPKC zeta, 60-80% of nPKC epsilon and nPKC delta were located in a Ca(2+)-insensitive, membrane-bound form. Treatment of R6 cells with 12-O-tetradecanoyl phorbol 13-acetate (TPA), resulted in the translocation of all four PKC isozymes to the membrane fraction, and the subsequent down-regulation of cPKC alpha, nPKC zeta, and nPKC delta, nPKC epsilon, however, was only partially down-regulated in response to long-term TPA exposure. Overproduction of exogenous cPKC beta I in R6 cells conferred partial resistance of nPKC delta to TPA-induced down-regulation and potentiated the resistance of nPKC epsilon to down-regulation. These results demonstrate that the multiple isoforms of PKC which coexist within a single cell type are differentially regulated by extra- and intracellular stimuli and may thereby influence growth control and transformation via distinct mechanisms.     

Lack of translocation of protein kinase C from the cytosol to the membranes in vasopressin-stimulated hepatocytes.

The ability of Ca2(+)-mobilizing hormones to promote changes in the subcellular distribution of protein kinase C (PKC) was studied in isolated hepatocytes. In recently isolated cells the distribution of PKC between the soluble and particulate fractions was 47 and 53% respectively. Exposure of the hepatocytes to 100 nM-vasopressin produced an increased phosphoinositide turnover, as reflected by the changes in the concentrations of inositol trisphosphate and Ca2+, and in glycogen phosphorylase a activity. However, the distribution of both PKC activity and [3H]phorbol dibutyrate binding between the cytosol and the membranes remained unchanged under these conditions. To determine the threshold values of the concentrations of Ca2+ and diacylglycerol required to produce a redistribution of PKC, the hepatocytes were treated with the Ca2+ ionophore ionomycin, and with permeant diacylglycerol derivatives. Hepatocytes incubated in the presence of 100 nM-vasopressin required concentrations of Ca2+ 2.5 times those produced physiologically by the hormone to produce translocation of PKC from the cytosol to the membranes. These studies suggest that, at least in hepatocytes, activation of PKC in response to Ca2(+)-mobilizing hormones involves only the pre-existent membrane-bound enzyme without affecting the soluble enzyme.     

Thyrotropin-releasing hormone induces redistribution of protein kinase C in GH4C1 rat pituitary cells

Thyrotropin-releasing hormone (TRH) affects hormone secretion and synthesis in GH4C1 cells, a clonal strain of rat pituitary cells. Recent evidence suggests that the intracellular mediators, inositol 1,4,5-trisphosphate and 1,2-diacylglycerol, which are generated as a result of TRH-induced hydrolysis of the polyphosphatidylinositols, may be responsible for some of the physiological events regulated by TRH. Because diacylglycerol is an activator of protein kinase C, we have examined a role for this enzyme in TRH action. The subcellular distribution of protein kinase C in control and TRH-treated cells was determined by measuring both enzyme activity and 12,13-[3H]phorbol dibutyrate binding in the cytosol and by measuring enzyme activity in the particulate fraction. Acute exposure of GH4C1 cells to TRH resulted in a decrease of cytosolic protein kinase C, and an increase in the level of the enzyme associated with the particulate fraction. The redistribution of protein kinase C induced by TRH was dose- and time-dependent, with maximal effects occurring within the first minute of TRH treatment. Analogs of TRH which do not bind to the TRH receptor did not induce redistribution of protein kinase C, while the active analog, methyl-TRH, did promote redistribution. Treatment of GH4C1 cells with phorbol myristate acetate also resulted in a shift in protein kinase C distribution, although the response was slower than that produced by TRH. TRH-induced redistribution of protein kinase C implies translocation of the enzyme from a soluble to a membrane-associated form. Because protein kinase C requires a lipid environment for activity, association with the membrane fraction of the cell suggests activation of the enzyme; thus, protein kinase C may play a role in some of the actions of TRH on GH4C1 cells.     

Regulation of K+ transport in tomato roots by the TSS1 locus. Implications in salt tolerance

The tss1 tomato (Lycopersicon esculentum) mutant exhibited reduced growth in low K+ and hypersensitivity to Na+ and Li+. Increased Ca2+ in the culture medium suppressed the Na+ hypersensitivity and the growth defect on low K+ medium of tss1 seedlings. Interestingly, removing NH4+ from the growth medium suppressed all growth defects of tss1, suggesting a defective NH4(+)-insensitive component of K+ transport. We performed electrophysiological studies to understand the contribution of the NH4(+)-sensitive and -insensitive components of K+ transport in wild-type and tss1 roots. Although at 1 mm Ca2+ we found no differences in affinity for K+ uptake between wild type and tss1 in the absence of NH4+, the maximum depolarization value was about one-half in tss1, suggesting that a set of K+ transporters is inactive in the mutant. However, these transporters became active by raising the external Ca2+ concentration. In the presence of NH4+, a reduced affinity for K+ was observed in both types of seedlings, but tss1 at 1 mm Ca2+ exhibited a 2-fold higher Km than wild type did. This defect was again corrected by raising the external concentration of Ca2+. Therefore, membrane potential measurements in root cells indicated that tss1 is affected in both NH4(+)-sensitive and -insensitive components of K+ transport at low Ca2+ concentrations and that this defective transport is rescued by increasing the concentration of Ca2+. Our results suggest that the TSS1 gene product is part of a crucial pathway mediating the beneficial effects of Ca2+ involved in K+ nutrition and salt tolerance.     

Protein kinase C translocates from cytosol to membrane upon hormone activation: effects of thyrotropin-releasing hormone in GH3 cells

The subcellular distribution of protein kinase C (PK C) was examined in thyrotropin-releasing hormone (TRH)--responsive GH3 pituitary cells. TRH treatment, which is known to stimulate polyphosphoinositide turnover and diacylglycerol generation, resulted in a rapid (less than or equal to 15 sec) and transient redistribution of the enzyme from cytosol to membrane fraction. Other agents which either stimulate PK C directly (1-oleoyl-2-acetyl-sn-glycerol and 12-O-tetradecanoyl phorbol-13-acetate) or elevate cellular diglyceride levels (phospholipase C) also promoted a redistribution of the enzyme from cytosol to membrane. These results provide evidence for the concept that cell-surface receptor-mediated phosphoinositide breakdown activates PK C. It appears that translocation of PK C to the membrane is an early step in the cellular activation of this enzyme.     

Properties of membrane-inserted protein kinase C

Protein kinase C (PKC) interacted with phospholipid vesicles in a calcium-dependent manner and produced two forms of membrane-associated PKC: a reversibly bound form and a membrane-inserted form. The two forms of PKC were isolated and compared with respect to enzyme stability, cofactor requirements, and phorbol ester binding ability. Membrane-inserted PKC was stable for several weeks in the presence of calcium chelators and could be rechromatographed on gel filtration columns in the presence of EGTA without dissociation of the enzyme from the membrane. The activity of membrane-inserted PKC was not significantly influenced by Ca2+, phospholipids, and/or PDBu. Partial dissociation of this PKC from phospholipid was achieved with Triton X-100, followed by dialysis to remove the detergent. The resulting free PKC appeared indistinguishable from original free PKC with respect to its cofactor requirements for activation (Ca2+, phospholipid, and phorbol esters), molecular weight, and phorbol 12,13-dibutyrate (PDBu) binding. The binding of PDBu to free and membrane-inserted PKC was measured under equilibrium conditions using gel filtration techniques. At 2.0 nM PDBu, free PKC bound PDBu with nearly 1:1 stoichiometry in the presence of Ca2+ and phospholipid. No PDBu binding to the free enzyme was observed in the absence of Ca2+. In contrast, membrane-inserted PKC bound PDBu in the presence or the absence of Ca2+; calcium did enhance the affinity of this interaction.(ABSTRACT TRUNCATED AT 250 WORDS)