Structural requirements of diacylglycerols for binding and activating phospholipid-dependent, Ca++-sensitive protein kinase

Phospholipid-dependent, Ca++-sensitive protein kinase (protein kinase C) is activated by phorbol esters and diacylglycerols. A series of diacylglycerols was synthesized with different substituents at positions 1 and 2 in order to expand known structure-activity relationships for these compounds with respect to binding and activating purified protein kinase C. Compounds were synthesized with saturated and unsaturated long chain fatty acyl groups at position 1 and acetyl, butyryl, or hexanoyl groups at position 2. Binding to protein kinase C correlated well with in-vitro activation of the enzyme. These diacylglycerols activated protein kinase C in an intact cellular system causing the phosphorylation of pp60c-src. This indicates that the length of the fatty acyl group at C2 is critical and that the existence of unsaturation in the fatty acyl group at C1 is not essential.     

Tumor promoter chloroform is a potent protein kinase C activator

The major interaction site for tumor-promoting phorbol esters is the calcium-activated, phospholipid-dependent protein kinase (protein kinase C), a key-element in signal transduction. Binding of phorbol esters results in enzyme activation which mediates, at least in part, the action of these agents. We have investigated the effects of tumor promoter chloroform on protein kinase C activity. Like thrombin and 12-O-tetradecanoylphorbol-13-acetate (TPA), chloroform was able to activate protein kinase C in intact rabbit platelets. In addition, chloroform stimulated enzyme activity as well as TPA binding capacity in cell-free system. Scatchard analysis of the data has shown that chloroform increased the number of phorbol ester binding sites. Structurally related compounds, carbon tetrachloride and methylene chloride, activated the enzyme similarly.     

The glucocorticoid dexamethasone and the tumor-promoting artificial sweetener saccharin stimulate protein kinase C from T51B rat liver cells

Diacylglycerols, such as 1,2-diolein, and tumor-promoting phorbol compounds, such as TPA (12-0-tetradecanoyl phorbol-13-acetate), stimulate the Ca2+/phospholipid-dependent protein kinase C from T51B rat liver cells, probably by sensitizing it to activation by Ca2+, and they reduce the liver cells' content of EDTA-extractable (i.e., soluble) protein kinase C activity. Evidence is presented that indicates that the glucocorticoid, dexamethasone, and the tumor-promoting artificial sweetener, saccharin, also trigger a Ca2+-dependent increase in the activity of the protein kinase C from T51B liver cells and reduce the cells' content of EDTA-extractable protein kinase C activity. However, these novel stimulators do not activate the enzyme by binding to the same site as diacylglycerols and TPA, although they do alter this site as indicated by an increase in the binding of the TPA analogue PDBu (phorbol 12,13-dibutyrate).     

Osmotic activation of the Na+/H+ antiport in protein kinase C-depleted lymphocytes

The Na+/H+ antiport of rat thymic lymphocytes is activated when protein kinase C is stimulated by phorbol esters. A similar activation of the antiport is obtained when the cells are treated with hypertonic solutions. We tested the possibility that protein kinase C also mediates the osmotic activation of Na+/H+ exchange. Protein kinase C was depleted by preincubation of thymocytes for 24 hr in the presence of high concentrations of phorbol ester. Disappearance of the enzyme was assessed by direct measurement of phosphotransferase activity, and by the loss of biological responses to phorbol esters. The Na+/H+ antiport in protein kinase C-depleted cells was not stimulated by addition of phorbol ester, but responded normally to hypertonic treatment. The results indicate that the osmotic activation of countertransport does not require stimulation of protein kinase C.     

Control of glycogen phosphorylase interconversion by phorbol esters, diacylglycerols, Ca2+ and hormones in isolated rat hepatocytes.

In isolated rat hepatocytes: phosphorylase activation by the ionophore A23187 was enhanced in the presence of tumour-promoting phorbol esters and 1,2- (but not 1,3-) diacylglycerols (dioleoyl- and oleoylacetyl-glycerol), with a similar dose-dependency; the activation of phosphorylase by phenylephrine (1 microM) (but not by vasopressin or glucagon) was inhibited both by tumour-promoting phorbol esters and diacylglycerols, but with a different dose-dependency: complete inhibition was achieved with concentrations of phorbol esters two orders of magnitude lower than those of diacylglycerol; binding of the alpha 1-adrenergic antagonist [3H]prazosin and its displacement by unlabelled prazosin was not significantly affected in the presence of the phorbol esters. The possible involvement of protein kinase C in the control of phosphorylase interconversion is discussed.     

Structural analogies between protein kinase C activators

Phorbol esters and diacylglycerols activate protein kinase C but specific structural parameters appear to be required for the enzyme activation. We have analyzed the conformation of potent and not potent diacylglycerols and phorbol esters. The orientation of the CH20H group at C3 of 1,2 diolein is remarkably similar to that of the same group at C-20 of 4 beta phorbol didecanoate and crucial for potency in activating the enzyme. Our data suggest that the new conformational approach here described could be used to rationally design specific inhibitors preventing the effects of tumor promoters and to predict the structure of potential tumor promoters.     

Growth factor-stimulated protein phosphorylation in 3T3-L1 cells. Evidence for protein kinase C-dependent and -independent pathways

Exposure of serum-deprived 3T3-L1 fibroblasts to phorbol 12-myristate 13-acetate (PMA), synthetic diacylglycerols, platelet-derived growth factor (PDGF), or pituitary fibroblast growth factor (FGF) resulted in stimulated phosphorylation of an acidic, multicomponent, soluble protein of Mr 80,000. Phosphorylation of this protein was promoted to a lesser extent by epidermal growth factor; however, neither insulin nor dibutyryl cAMP was effective. Phosphoamino acid analysis and peptide mapping of the Mr 80,000 32P-protein after exposure of fibroblasts to PDGF revealed identical patterns to those obtained with PMA or diacylglycerols. In contrast to the Mr 80,000 protein, proteins of Mr 22,000 (and pI 4.4) and Mr 31,000 were also phosphorylated in response to insulin as well as to PMA, diacylglycerols, epidermal growth factor, PDGF, and FGF in these cells. Similar findings were noted in fully differentiated 3T3-L1 adipocytes. Preincubation of the cells with high concentrations of active phorbol esters abolished specific [3H]phorbol 12,13-dibutyrate binding, protein kinase C activity, and immunoreactivity and also prevented stimulated phosphorylation of the Mr 80,000 protein by PMA, diacylglycerols, PDGF, or FGF, supporting the contention that this effect was mediated through protein kinase C. The stimulated phosphorylation of the Mr 22,000 and 31,000 proteins in response to PMA was also abolished by such pretreatment. In contrast, the ability of insulin, PDGF, and FGF to promote phosphorylation of the Mr 22,000 and 31,000 proteins was unaffected in the protein kinase C-deficient cells. We conclude that PDGF and FGF may exert some of their effects on these cells through at least two distinct pathways of protein phosphorylation, phorbol ester-like (P) activation of protein kinase C, and an insulin-like (I) pathway exemplified by phosphorylation of the Mr 22,000 and 31,000 proteins.     

Differences in the effects of phorbol esters and diacylglycerols on protein kinase C

The binding of protein kinase C (PKC) to membranes and appearance of kinase activity are separable events. Binding is a two-step process consisting of a reversible calcium-dependent interaction followed by an irreversible interaction that can only be dissociated by detergents. The irreversibly bound PKC is constitutively active, and the second step of binding may be a major mechanism of PKC activation [Bazzi & Nelsestuen (1988) Biochemistry 27, 7589]. This study examined the activity of other forms of membrane-bound PKC and compared the effects of phorbol esters and diacylglycerols. Like the membrane-binding event, activation of PKC was a two-stage process. Diacylglycerols (DAG) participated in forming an active PKC which was reversibly bound to the membrane. In this case, both activity and membrane binding were terminated by addition of calcium chelators. DAG functioned poorly in generating the constitutively active, irreversible PKC-membrane complex. These properties differed markedly from phorbol esters which activated PKC in a reversible complex but also promoted constitutive PKC activation by forming the irreversible PKC-membrane complex. The concentration of phorbol esters needed to generate the irreversible PKC-membrane complex was slightly higher than the concentration needed to activate PKC. In addition, high concentrations of phorbol esters (greater than or equal to 50 nM) activated PKC and induced irreversible PKC-membrane binding in the absence of calcium. Despite these striking differences, DAG prevented binding of phorbol esters to high-affinity sites on the PKC-membrane complex.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phosphorylation of glycogen synthase by the Ca2+- and phospholipid-activated protein kinase (protein kinase C)

The Ca2+- and phospholipid-dependent protein kinase (protein kinase C) has been found to phosphorylate and inactivate glycogen synthase. With muscle glycogen synthase as a substrate, the reaction was stimulated by Ca2+ and by phosphatidylserine. The tumor-promoting phorbol esters 12-O-tetradecanoyl phorbol 13-acetate was also a positive effector, half-maximal activation occurring at 6 nM. Phosphorylation of glycogen synthase, but not histone, was partially inhibited by glycogen, half-maximally at 0.05 mg/ml, probably via a substrate-directed mechanism. The rate of glycogen synthase phosphorylation was approximately half that for histone; the apparent Km for glycogen synthase was 0.25 mg/ml. Protein kinase C also phosphorylated casein, the preferred substrate among the individual caseins being alpha s1-casein. Glycogen synthase was phosphorylated to greater than 1 phosphate/subunit with an accompanying reduction in the -glucose-6-P/+glucose-6-P activity ratio from 0.9 to 0.5. Phosphate was introduced into serine residues in both the NH2-terminal and COOH-terminal CNBr fragments of the enzyme subunit. The two main tryptic phosphopeptides mapped in correspondence with the peptides that contain site 1a and site 2. Lesser phosphorylation in an unidentified peptide was also observed. Rabbit liver and muscle glycogen synthases were phosphorylated at similar rates by protein kinase C. The above results are compatible with a role for protein kinase C in the regulation of glycogen synthase as was suggested by a recent study of intact hepatocytes.     

The antioxidant butylated hydroxytoluene stimulates platelet protein kinase C and inhibits subsequent protein phosphorylation induced by thrombin

The phenolic antioxidant 2,6-bis(1,1-dimethyl ethyl)-4-methylphenol (BHT) evokes a transient phosphorylation of two platelet proteins of Mr 20,000 and 47,000 that are well-known substrates of protein kinase C (PKC) and, similarly to phorbol esters, a slight but persistent phosphorylation of a protein of Mr 26,000. These effects are observed both in the presence and in the absence of extracellular calcium, but are abolished in the presence of the protein kinase C inhibitor staurosporine. The phosphorylation of the 47 kDa protein takes place mostly at the serine and, to a lesser extent, at threonine residues. BHT induces an increased binding of tritiated phorbol dibutyrate to platelets indicating a PKC translocation from cytosol to plasma membrane. Addition of BHT (20 microM) a few min prior to thrombin causes inhibition of both agonist-evoked protein phosphorylation and increase in the Ca2+ concentration, the latter inhibition being counteracted by staurosporine. The inhibitory effect lasts for several minutes even after removal of BHT from the cellular suspending medium. Similar results are obtained with nordihydroguaiaretic acid, whereas 2- and 3-tert-butyl-4-methoxyphenol (BHA) produce only slight effects. BHT activates the protein kinase C purified from pig brain in a concentration-dependent manner (up to 200 microM), whereas it does not affect the activity of other purified protein kinases such as type 1 and 2 casein kinases, type II A, II B and III tyrosine protein kinases from rat spleen and the catalytic subunit of cyclic AMP-dependent protein kinase. It is concluded that, similarly to diacylglycerols and phorbol esters, these phenolic antioxidants activate the protein kinase C, which in turn desensitizes platelets towards subsequent phospholipase C activation.     

Direct effects of phorbol esters and diacylglycerols on the T-tubule Mg(2+)-ATPase.

T-tubule membrane vesicles isolated from skeletal muscle contain a very active Mg(2+)-ATPase (EC 3.6.1.34) which is modulated by lectins and is located in the junctional region near the sarcoplasmic reticulum membranes (1). The effects of several prominent lipophilic agents upon the ATPase have led us to evaluate the action of diacylglycerols and phorbol esters upon the enzyme. The ATPase is inhibited by submicromolar levels of the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), and the diacylglycerol, 1-oleoyl-2-acetyl-sn-glycerol (sn-OAG), with K0.5s of 0.2 and 0.5 microM, respectively. Significantly, 4-alpha-phorbol 12,13-didecanoate (4-alpha-phorbol) the TPA analogue shown to be inactive toward protein kinase C (PKC), inhibited the ATPase with a K0.5 of 0.3 microM, and 1-stearoyl-2-arachidonyl-sn-glycerol, the preferred endogenous activator of PKC, was not inhibitory toward the ATPase. 1-(5-Isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (a membrane permeant PKC inhibitor) and peptide 19-36 (the highly specific PKC pseudosubstrate inhibitor) were both without effect upon the ATPase and did not affect TPA inhibition. ATPase activity was not altered under phosphorylating conditions in experiments using exogenous rat brain PKC. ConA protected ATPase activity against inhibition by TPA, 4-alpha-phorbol, and sn-OAG. Additionally, phorbol-12,13-dibutyrate binding studies demonstrated that the ATPase was capable of significant phorbol binding with ConA protection. The data are consistent with a direct and specific effect of phorbol esters and diacylglycerols upon the ATPase, without any participation of PKC. We conclude that the transverse tubule (T-tubule) ATPase is an alternate receptor for diacylglycerol and TPA in skeletal muscle and that the mode of action of these agents upon the ATPase (inhibition) is opposite to their mode of action on PKC (activation). The data demonstrate that substantial care must be taken in ascribing either cellular or subcellular effects of phorbol esters and diacylglycerols exclusively to the activation of PKC and that alternate receptors may exist. Criteria are recommended for the demonstration of PKC-independent modulation by phorbols and diacylglycerols.     

Role of protein kinase C in transmembrane signaling

Many extracellular signals elicit Ca2+ mobilization and diacylglycerol formation in their target cells. Diacylglycerol is derived from the receptor-linked phosphoinositide turnover and serves as a second messenger for the activation of protein kinase C in the presence of Ca2+ and phosphatidylserine. Unique diacylglycerols such as 1-oleoyl-2-acetyl-glycerol, which activate intracellular protein kinase C when added to intact cells, have been synthesized. Tumor-promoting phorbol esters substitute for such diacylglycerols and directly activate protein kinase C in both intact cell and cell-free systems. Under appropriate conditions, the synthetic diacylglycerols and phorbol esters induce protein kinase C activation without Ca2+ mobilization, whereas Ca2+ ionophore A23187 induces Ca2+ mobilization without protein kinase C activation. Using these substances, we have obtained evidence that both protein C and Ca2+ are involved in and play a synergistic role in exocytosis, cell division, and other cellular functions. In this article, the role of protein kinase C in transmembrane signaling is discussed.     

Interplay of C1 and C2 domains of protein kinase C-alpha in its membrane binding and activation.

The regulatory domain of conventional protein kinase C (PKC) contains two membrane-targeting modules, the C2 domain that is responsible for Ca2+-dependent membrane binding of protein, and the C1 domain composed of two cysteine-rich zinc fingers (C1a and C1b) that bind diacylglycerols and phorbol esters. To understand the individual roles and the interplay of the C1 and C2 domains in the membrane binding and activation of PKC, we functionally expressed isolated C1 and C2 domains of PKC-alpha and measured their vesicle binding and monolayer penetration. Results indicate that the C2 domain of PKC-alpha is responsible for the initial Ca2+- and phosphatidylserine-dependent electrostatic membrane binding of PKC-alpha, whereas the C1 domain is involved in subsequent membrane penetration and diacylglycerol binding, which eventually lead to enzyme activation. To determine the roles of individual zinc fingers in the C1 domain, we also mutated hydrophobic residues in the C1a (Trp58 and Phe60) and C1b (Tyr123 and Leu125) domains of the native PKC-alpha molecule and measured the effects of mutations on vesicle binding, enzyme activity and monolayer penetration. Results show that the hydrophobic residues in the C1a domain are essential for the membrane penetration and activation of PKC-alpha, whereas those in the C1b domain are not directly involved in these processes. Based on these results in conjunction with our previous structure-function studies of the C2 domain (Medkova, M., and Cho, W. (1998) J. Biol. Chem. 273, 17544-17552), we propose a mechanism for the in vitro membrane binding and activation of conventional PKC that accounts for the temporal and spatial sequences of PKC activation.     

Synergistic activation of protein kinase Calpha, -betaI, and -gamma isoforms induced by diacylglycerol and phorbol ester: roles of membrane association and activating conformational changes

Protein kinase Calpha (PKCalpha) has been shown to contain two discrete activator sites with differing binding affinities for phorbol esters and diacylglycerols. The interaction of diacylglycerol with a low-affinity phorbol ester binding site leads to enhanced high-affinity phorbol ester binding and to a potentiated level of activity [Slater, S. J., Ho, C., Kelly, M. B., Larkin, J. D. , Taddeo, F. J., Yeager, M. D., and Stubbs, C. D. (1996) J. Biol. Chem. 271, 4627-4631]. In this study, the mechanism of this enhancement of activity was examined with respect to the Ca2+ dependences of membrane association and accompanying conformational changes that lead to activation. The association of PKCalpha with membranes containing 12-O-tetradecanoylphorbol 13-acetate (TPA) or 1, 2-dioleoylglycerol (DAG), determined from tryptophan to dansyl-PE resonance energy transfer (RET) measurements, was found to occur at relatively low Ca2+ levels (相似文献   

Downregulation of protein kinase C-γ is independent of a functional kinase domain

Prolonged activation of protein kinase C (PKC) types and β by tumor-promoting phorbol esters leads to desensitization of the phorbol ester response, downregulation of protein kinase C activity and depletion of the protein kinase C polypeptide. When the γ isoenzyme of PKC is transiently expressed in COS-1 cells and exposed to phorbol esters, PKC-γ is downregulated in COS cells although these cells do not normally express this subtype. A point mutation in the purative ATP-binding site (Lys-380→Met-380) of the protein kinase C γ isoenzyme which results in a kinase-deficient enzyme does not interfere with this downregulation. Our results suggest that autophosphorylation or constitutive signalling through the protein kinase C-γ kinase domain is not a prerequisite for downregulation of PKC activity.     

Calcium transients in human platelets monitored by aequorin, fura-2 and quin-2: effects of protein kinase C activation and inhibition

Tumour-promoting phorbol esters and 1,2-dioctanoyl-sn-glycerol both induce calcium transients in platelets. However, these can only be detected in platelets loaded with aequorin, but not in those loaded with the fluorescent probes quin-2 and fura-2 presumably because of intracellular calcium buffering. Several effects induced by phorbol esters and diacylglycerols, including the rise in (Ca2+)i, the stimulation of Na+/H+ transporter and the inhibition of the effects of thrombin alone on (Ca2+)i are potently antagonised by staurosporine, a compound known to inhibit protein kinase C. Higher concentrations of staurosporine themselves inhibit the thrombin-induced calcium transient. Staurosporine inhibits the effects of phorbol esters and dioctanoyl glycerol with equal potency although the latter does not cause enzyme translocation of cytosolic protein kinase C to membranes. These results therefore suggest that some, if not all, the effects of protein kinase C activation can occur without translocation of the enzyme.     

Inhibition of protein kinase C by calphostin C is light-dependent.

Calphostin C, a secondary metabolite of the fungus Cladosporium cladosporioides, inhibits protein kinase C by competing at the binding site for diacylglycerol and phorbol esters. Calphostin C is a polycyclic hydrocarbon with strong absorbance in the visible and ultraviolet ranges. In characterizing the activity of this compound, we unexpectedly found that the inhibition of [3H]phorbol dibutyrate binding was dependent on exposure to light. Ordinary fluorescent light was sufficient for full activation. The inhibition of protein kinase C activity in cell-free systems and intact cells also required light. Light-dependent cytotoxicity was seen at concentrations about 5-fold higher than those inhibiting protein kinase C.     

Scanning mutagenesis studies reveal multiple distinct regions within the human protein kinase C alpha regulatory domain important for phorbol ester-dependent activation of the enzyme

While phorbol ester-binding sites within protein kinase C alpha (PKCalpha) have been identified and characterized utilizing fragments of the enzyme, it remains unclear whether additional regions within the enzyme may play an important role in its ability to be activated by phorbol ester. To examine this hypothesis, we generated 20 glutathione-S-transferase-tagged, V1-deficient, human PKCalpha holoenzyme constructs in which tandem six or 12 amino acid residue stretches along the full regulatory domain were changed to alanine residues. Each protein was assessed for its ability to bind phorbol ester and to induce growth repression when its catalytic activity was activated by phorbol ester upon expression in yeast cells. Mutagenesis of residues 99-158 potently reduced phorbol binding, consistent with previously published findings on the importance of the C1b region in phorbol binding. In addition, we identified a number of regions within the PKC regulatory domain that, when mutagenized, blocked the activation of PKC-mediated growth repression by phorbol ester while actually enhancing phorbol ester binding in vitro (residues 33-62, and 75-86). This study thus helps distinguish regions important for phorbol binding from regions important for the ability of phorbol ester to activate the enzyme. Our findings also suggest that multiple regions within C2 are necessary for full activation of the enzyme by phorbol ester, in particular residues 231-254. Finally, three regions, when mutagenized, completely, blocked catalytic domain activity in vivo (residues 33-62, 75-86, and 123-146), underscoring the important role of regulatory domain sequences in influencing catalytic domain function, even in the absence of the V1 region containing the pseudosubstrate sequence. This is the first tandem mutagenesis study for PKC that assesses the importance of regions for both phorbol binding and for phorbol-dependent activation in the context of the entire holoenzyme.     

Sphingosine reverses growth inhibition caused by activation of protein kinase C in vascular smooth muscle cells.

In certain cell systems, including neonatal vascular smooth muscle (VSM) cells, phorbol esters are growth inhibitory. Here we show that 1,2-dioctanoyl-sn-glycerol (DiC8), when added 2 h after alpha-thrombin, reverses by greater than 95% the induction of DNA synthesis in VSM cells by alpha-thrombin. Sphingosine, a naturally occurring lysosphingolipid inhibitor of protein kinase C, and its synthetic analogues N-acetylsphingosine and C11-sphingosine were used to investigate this phenomenon further. Neither phorbol 12-myristate 13-acetate (PMA;200 ng/ml) nor sphingosine (up to 10 microM) alone had any effect upon basal DNA synthesis in VSM cells. Like DiC8, PMA totally blocked the induction of DNA synthesis by alpha-thrombin. This inhibitory effect of PMA was reversed by sphingosine in a dose-dependent manner with complete reversal at 10 microM. Neither N-acetylsphingosine nor C11-sphingosine exhibited any effect on DNA synthesis in VSM cells. The effect of sphingosine and its analogues on the activity of protein kinase C extracted from VSM cells was measured by histone III-S phosphorylation. Protein kinase C activity was inhibited 50% by 300 microM sphingosine, but less than 15% by similar concentrations of N-acetylsphingosine and C11-sphingosine. To assess the effects of sphingosine and analogues on protein kinase C in intact cells, we examined the effect of the lipids on [3H]phorbol dibutyrate binding. Sphingosine (at greater than 5 microM), but not N-acetylsphingosine or C11-sphingosine, blocked [3H]phorbol dibutyrate binding in a dose- and time-dependent fashion. Thus the mechanism of growth inhibition by DiC8 and PMA in neonatal VSM cells appears to be through activation of protein kinase C by these compounds. Sphingosine reverses this growth inhibition through interference with the binding to protein kinase C of phorbol esters or other activators of this enzyme.