Interaction of protein kinase C with phosphatidylserine. 1. Cooperativity in lipid binding.

The basis for the apparent cooperativity in the activation of protein kinase C by phosphatidylserine has been addressed using proteolytic sensitivity, resonance energy transfer, and enzymatic activity. We show that binding of protein kinase C to detergent-lipid mixed micelles and model membranes is cooperatively regulated by phosphatidylserine. The sigmoidal dependence on phosphatidylserine for binding is indistinguishable from that observed for the activation of the kinase by this lipid [Newton & Koshland (1989) J. Biol. Chem. 264, 14909-14915]. Thus, protein kinase C activity is linearly related to the amount of phosphatidylserine bound. Furthermore, under conditions where protein kinase C is bound to micelles at all lipid concentrations, activation of the enzyme continues to display a sigmoidal dependence on the phosphatidylserine content of the micelle. This indicates that the apparent cooperativity in binding does not arise because protein kinase C senses a higher concentration of phosphatidylserine once recruited to the micelle. Our results reveal that the affinity of protein kinase C for phosphatidylserine increases as more of this lipid binds, supporting the hypothesis that a domain of phosphatidylserine is cooperatively sequestered around the enzyme.     

Interaction of protein kinase C with phosphoinositides.

Calcium/phosphatidylserine-dependent protein kinase C (PKC) is activated by phosphatidylinositol 4,5-bisphosphate (PIP2), as well as by diacylglycerol (DG) and phorbol esters. Here we report that PIP2, like DG, increases the affinity of PKC for Ca2+, and causes Ca(2+)-dependent translocation of the enzyme from the soluble to a particulate fraction (liposomes). Phosphatidylinositol 4-phosphate (PIP) also displaces phorbol ester from PKC and causes Ca(2+)-dependent translocation of the enzyme to liposomes, but is much less efficient than PIP2, and a much weaker activator, with a histone phosphorylation v(PIP)/v(PIP2) of approximately 0.15. Scatchard analysis indicates competitive inhibition between PIP and phorbol ester with Ki(PIP) = 0.26 mol% as compared with Ki(PIP2) = 0.043 mol%. No effect of phosphatidylinositol (PI) on phorbol ester binding to PKC, translocation of PKC, or activation of PKC was observed. These results suggest that both PIP and PIP2 can complex with PKC, but full activation of the enzyme takes place only when PIP is converted to PIP2. We suggest that an inositide interconversion shuttle has a role in the regulation of protein phosphorylation.     

Interaction of phosphoglycerate kinase with phosphatidylserine liposomes

The interaction of 3-phosphoglycerate kinase from bovine heart with natural phosphatidylserine (I) and synthetic dipalmitoyl phosphatidylserine (II) in form of liposomes was investigated by measuring fluorescence and activity of the enzyme. The addition of increasing amounts of I resulted in progressive quenching of protein fluorescence with no shift in the emission maximum. In contrast, II did not cause any change in the fluorescence. In the presence of low amounts of I and II (lipid/protein molar ratio 10-40) full enzymatic activity of 3-phosphoglycerate kinase was observed even after 80 min of incubation, whereas without phospholipids the activity considerably decreased. At higher lipid concentrations I strongly inactivated the enzyme and the inactivation by II was only insignificant. It was concluded that the phospholipid membrane protects the enzyme against thermal denaturation, whereas the inactivation is mainly due to phospholipid impurities.     

Specificity of protein phosphatases in the dephosphorylation of protein kinase C.

Protein kinase C can autophosphorylate in vitro and has also been shown to be phosphorylated in vivo. In order to investigate the factors that may determine the phosphorylation state of protein kinase C in vivo, we determined the ability of the ATP + Mg2+-dependent phosphatase and the polycation-stimulated (PCS) phosphatases to dephosphorylate protein kinase C in vitro. These studies show that all the oligomeric forms of the PCS phosphatases (PCSH1, PCSH2, PCSM and PCSL phosphatases) are effective in the dephosphorylation of protein kinase C, showing 34-82% of the activity displayed with phosphorylase a as substrate. In contrast both the catalytic subunit of the PCS phosphatase and that of the ATP+Mg2+-dependent phosphatase showed only weak activity with protein kinase C as substrate. All these phosphatases, however, were activated by protamine (Ka 14-16 micrograms/ml) through what appears to be a substrate-directed effect. The relative role of these phosphatases in the control of protein kinase C is discussed.     

Calcium and phosphatidylserine stimulate the self-association of conventional protein kinase C isoforms.

Conflicting evidence exists as to whether "conventional" protein kinase C isoforms (cPKCs) function as monomers or oligomers. In this report, we demonstrate that purified cPKC isoforms can be rapidly cross-linked by the sulfhydryl-selective cross-linker bis(maleimido)hexane, but only in the presence of both Ca(2+) and phosphatidylserine; cross-linking was minimal in the presence of either of these activators alone. In addition, cross-linking of these cPKCs did not require Mg(2+) or ATP. Among the various phospholipids tested, phosphatidylserine was found to be the most effective in the promotion of cPKC self-association and for the stimulation of protein kinase activity toward the exogenous substrate histone. Phosphatidic acid and phosphatidylinositol were less effective in this regard, whereas phosphatidylcholine exhibited little ability to induce cPKC self-association or to stimulate kinase activity. An examination of the mechanism by which the cPKC isoforms self-associate in the presence of phospholipid/Ca(2+) revealed that this process occurred independently of phospholipid aggregation. Moreover, self-association was not inhibited by saturating the enzyme active site with a peptide substrate, suggesting that self-association is distinct from an enzyme-substrate interaction. Isoform-specific antibodies revealed that all cPKC isoforms (alpha, beta, and gamma) self-associate and that, in a mixture of cPKC isoforms, PKC-alpha forms primarily alpha-alpha homodimers. Besides cPKC interactions detected with purified enzyme, PKC-alpha also appeared capable of self-association in murine B82L fibroblasts that were treated with calcium ionophore, phorbol ester, or epidermal growth factor but not in untreated cells. Collectively, these data indicate that self-association occurs in parallel with cPKC activation, that self-association is not mediated by the substrate binding site, and, at least in the case of PKC-alpha, that the formation of isoform homodimers predominates.     

Interaction of protein kinase C isozymes with phosphatidylinositol 4,5-bisphosphate.

Interaction of protein kinase C (PKC) isozymes with phosphatidylinositol 4,5-bisphosphate (PIP2) was investigated by monitoring the changes in the intrinsic fluorescence of the enzyme, the kinase activity, and phorbol ester binding. Incubation of PKC I, II, and III with PIP2 resulted in different rates of quenching of PKC fluorescence and different degrees of inactivation of these enzymes. Other inositol-containing phospholipids such as phosphatidylinositol and phosphatidylinositol 4-phosphate also caused differential rates of quenching of the intrinsic fluorescence of these enzymes. These latter two phospholipids were, however, less potent in the inactivation of PKCs than PIP2. The IC50 of PIP2 were 2, 4, and 11 microM for PKC I, II, and III, respectively. Inactivation of PKCs by PIP2 cannot be reversed by extensive dilution of PIP2 with Nonidet P-40 nor by digestion of PIP2 with phospholipase C. Interaction of PIP2 with the various PKC isozymes was greatly facilitated in the presence of Mg2+ or Ca2+ as evidenced by the accelerated quenching of the PKC fluorescence, however, these divalent metal ions protected PKC from the PIP2-induced inactivation. Binding of PIP2 to PKC in the absence of divalent metal ion also caused a reduction of [3H]phorbol 12,13-dibutyrate binding as a result of reducing the affinity of the enzyme for phorbol ester. Based on gel filtration chromatography, it was estimated that one molecule of PKC interacted with one PIP2 micelle with an aggregation number of 80-90. The PIP2-bound PKC could further interact with phosphatidylserine in the presence of Ca2+ to form a larger complex. Binding of PKC to both PIP2 and phosphatidylserine in the presence of Ca2+ was also evident by changes in the intrinsic fluorescence of PKC. As the interaction of PKC with PIP2, but not with phosphatidylserine, could be enhanced by millimolar concentrations of Mg2+, we propose that PIP2 may be a component of the membrane anchor for PKC under basal physiological conditions when [Ca2+]i is low and Mg2+ is plentiful. Under the in vitro assay conditions, PIP2 could stimulate PKC activity to a level approximately 10-20% of that by diacylglycerol. The stimulatory effect of PIP2 on PKC apparently is not due to binding to the same site recognized by diacylglycerol or phorbol ester, because PIP2 cannot effectively compete with phorbol 12,13-dibutyrate in the binding assay.     

Complexities of protein kinase C regulation

A report from the Keystone Protein kinase C: structure, regulation and cellular function meeting, New Mexico, February 5-10, 2000.     

Interaction of protein kinase C isozymes with Rho GTPases

Evidence is provided for direct protein-protein interactions between protein kinase C (PKC) alpha, betaI, betaII, gamma, delta, epsilon, and zeta and members of the Rho family of small GTPases. Previous investigations, based on the immunoprecipitation approach, have provided evidence consistent with a direct interaction, but this remained to be proven. In the study presented here, an in vitro assay, consisting only of purified proteins and the requisite PKC activators and cofactors, was used to determine the effects of Rho GTPases on the activities of the different PKC isoforms. It was found that the activity of PKCalpha was potently enhanced by RhoA and Cdc42 and to a lesser extent by Rac1, whereas the effects on the activities of PKCbetaI, -betaII, -gamma, -delta, -epsilon, and -zeta were much reduced. These results indicate a direct interaction between PKCalpha and each of the Rho GTPases. However, the Rho GTPase concentration dependencies for the potentiating effects on PKCalpha activity differed for each Rho GTPase and were in the following order: RhoA > Cdc42 > Rac1. PKCalpha was activated in a phorbol ester- and Ca(2+)-dependent manner. This was reflected by a substantial decrease in the phorbol ester concentration requirements for activity in the presence of Ca(2+), which for each Rho GTPase was induced within a low nanomolar phorbol ester concentration range. The activity of PKCalpha also was found to be dependent on the nature of the GTP- or GDP-bound state of the Rho GTPases, suggesting that the interaction may be regulated by conformational changes in both PKCalpha and Rho GTPases. Such an interaction could result in significant cross-talk between the distinct pathways regulated by these two signaling elements.     

Reversible exposure of the pseudosubstrate domain of protein kinase C by phosphatidylserine and diacylglycerol.

The lipid activators of protein kinase C, phosphatidylserine and diacylglycerol, induce a reversible conformational change that exposes the auto-inhibitory pseudosubstrate domain of the enzyme. The pseudosubstrate domain of beta-II protein kinase C is cleaved after the first residue, arginine 19, by the endoproteinase Arg-C only when the kinase is bound to the activating lipid phosphatidylserine. Exposure of this residue is markedly enhanced by diacylglycerol. In contrast, the pseudosubstrate domain is not cleaved in the absence of lipids, when protein kinase C is bound to non-activating acidic lipids, when the kinase has autophosphorylated on the amino terminus, or after dilution of the activating lipids. This work reveals specificity in the interaction of protein kinase C with phosphatidylserine since only this phospholipid causes the specific conformational change detected in the regulatory domain of the enzyme, and demonstrates that allosteric regulators expose the intramolecular auto-inhibitory domain of a kinase.     

Interaction of palmitoylcarnitine with protein kinase C in neuroblastoma NB-2a cells

As reported previously [Acta Neurobiol. Exp. 57 (1997) 263], palmitoylcarnitine was observed to promote differentiation of neuroblastoma NB-2a cells with a concomitant inhibition of proliferation and of the phorbol ester stimulated activity of the protein kinase C (PKC). In the present study, palmitoylcarnitine was observed to inhibit phosphorylation of the PKC peptide substrate and to completely diminish binding of phorbol 12-myristate-13-acetate (PMA), although the effect was found to be uncompetitive. The exposure of NB-2a cells to palmitoylcarnitine in the presence of PMA resulted in a dramatic decrease in phosphorylation of the conventional and novel isozymes of PKC, mainly on serine. This effect was observed to be dose dependent. Inhibitors of serine/threonine phosphatases were not influencing the effect of palmitoylcarnitine what can point to an interaction between PKC and palmitoylcarnitine, affecting the process of autophosphorylation. These findings suggest that pamitoylcarnitine could be a natural modulator of PKC activity, thus regulating the process of cell differentiation.     

Ca(2+) bridges the C2 membrane-binding domain of protein kinase Calpha directly to phosphatidylserine

The C2 domain acts as a membrane-targeting module in a diverse group of proteins including classical protein kinase Cs (PKCs), where it plays an essential role in activation via calcium-dependent interactions with phosphatidylserine. The three-dimensional structures of the Ca(2+)-bound forms of the PKCalpha-C2 domain both in the absence and presence of 1, 2-dicaproyl-sn-phosphatidyl-L-serine have now been determined by X-ray crystallography at 2.4 and 2.6 A resolution, respectively. In the structure of the C2 ternary complex, the glycerophosphoserine moiety of the phospholipid adopts a quasi-cyclic conformation, with the phosphoryl group directly coordinated to one of the Ca(2+) ions. Specific recognition of the phosphatidylserine is reinforced by additional hydrogen bonds and hydrophobic interactions with protein residues in the vicinity of the Ca(2+) binding region. The central feature of the PKCalpha-C2 domain structure is an eight-stranded, anti-parallel beta-barrel with a molecular topology and organization of the Ca(2+) binding region closely related to that found in PKCbeta-C2, although only two Ca(2+) ions have been located bound to the PKCalpha-C2 domain. The structural information provided by these results suggests a membrane binding mechanism of the PKCalpha-C2 domain in which calcium ions directly mediate the phosphatidylserine recognition while the calcium binding region 3 might penetrate into the phospholipid bilayer.     

Interaction of the C2 domain from protein kinase C(epsilon) with model membranes

The C2 domain from protein kinase Cepsilon (PKCepsilon) binds to membranes but does not require Ca2+ to do so. This work examines the mode in which the conformation and organization of the phospholipids present in model membranes are altered by the presence of the C2 domain from PKCepsilon (C2-PKCepsilon). It is concluded from the results of differential scanning calorimetry that the protein shifted the temperature of the gel to the fluid phase transition of pure 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphate (POPA), widening the transition and increasing it to a higher temperature. When POPA was mixed with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), the changes in the transition were smaller and no phase separation was observed. Experiments performed using magic angle spinning NMR showed that this C2 domain specifically affected POPA when the phospholipid was mixed with POPC, as indicated by the downfield shift in the isotropic resonance of POPA, the widening of the resonance peak, the decrease in T2, and the decrease in T1 observed at all temperatures. All these effects were quite marked compared with the very small effect observed with POPC, indicating the specificity of the effect. The presence of the C2-PKCepsilon protein changed the conformation of the polar head group of POPA, as shown by infrared spectroscopy. All these results clearly illustrate the electrostatic interaction that takes place between this C2 domain and membranes which contain POPA in the absence of Ca2+.     

The mechanism of protein kinase C regulation

Protein kinase C (PKC) is a family of serine/threonine protein kinases that plays a central role in transducing extracellular signals into a variety of intracellular responses ranging from cell proliferation to apoptosis. Nine PKC genes have been identified in the human genome, which encode 10 proteins. Each member of this protein kinase family displays distinct biochemical characteristics and is enriched in different cellular and subcellular locations. Activation of PKC has been implicated in the regulation of cell growth and differentiation. This review summarizes works of the past years in the field of PKC biochemistry that covers regulation and activation mechanism of different PKC isoforms.     

AKAP79 selectively enhances protein kinase C regulation of GluR1 at a Ca2+-calmodulin-dependent protein kinase II/protein kinase C site

Enhancement of AMPA receptor activity in response to synaptic plasticity inducing stimuli may arise, in part, through phosphorylation of the GluR1 AMPA receptor subunit at Ser-831. This site is a substrate for both Ca(2+)-calmodulin-dependent protein kinase II (CaMKII) and protein kinase C (PKC). However, neuronal protein levels of CaMKII may exceed those of PKC by an order of magnitude. Thus, it is unclear how PKC could effectively regulate this common target site. The multivalent neuronal scaffold A-kinase-anchoring protein 79 (AKAP79) is known to bind PKC and is linked to GluR1 by synapse-associated protein 97 (SAP97). Here, biochemical studies demonstrate that AKAP79 localizes PKC activity near the receptor, thus accelerating Ser-831 phosphorylation. Complementary electrophysiological studies indicate that AKAP79 selectively shifts the dose-dependence for PKC modulation of GluR1 receptor currents approximately 20-fold, such that low concentrations of PKC are as effective as much higher CaMKII concentrations. By boosting PKC activity near a target substrate, AKAP79 provides a mechanism to overcome limitations in kinase abundance thereby ensuring faithful signal propagation and efficient modification of AMPA receptor-mediated responses.     

Endothelial cell sodium-potassium-chloride cotransport. Evidence of regulation by Ca2+ and protein kinase C

Previous studies have shown that vascular endothelial cells exhibit a highly active Na-K-Cl cotransport system that is regulated by a variety of vasoactive hormones and neurotransmitters, suggesting that the cotransporter may play an important role in endothelial cell function. In this study, the regulation of endothelial cell Na-K-Cl cotransport was further investigated by probing the stimulus-transfer pathway by which vasoactive agents stimulate the cotransporter. Specifically, three peptides previously shown to stimulate cotransport activity (angiotensin II, vasopressin, and bradykinin) were evaluated. Na-K-Cl cotransport was assessed in cultured bovine aortic endothelial cells as bumetanide-sensitive K+ influx. Stimulation of Na-K-Cl cotransport by angiotensin II, vasopressin, or bradykinin was found to be reduced either by removal of extracellular Ca2+ or by treatment of the cells with 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate or 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid. In addition, the calmodulin antagonist W-7 was found to prevent stimulation of endothelial cell Na-K-Cl cotransport by the three peptides. These findings suggest that regulation of endothelial cell cotransport by these vasoactive peptides may be both Ca(2+)- and calmodulin-dependent. Angiotensin II, vasopressin, and bradykinin were also found to elevate phosphatidylinositol hydrolysis in the cultured endothelial cells. Thus, the possibility that regulation of endothelial Na-K-Cl cotransport by these vasoactive peptides also involves diacylglycerol activation of protein kinase C was investigated. A 10-min exposure of the endothelial cells to low doses of phorbol 12-myristate 13-acetate was found to reduce Na-K-Cl cotransport whether in the presence or absence of angiotensin II, vasopressin, or bradykinin. However, down-regulation of protein kinase C by a 40-h exposure to higher doses of the phorbol ester was found to elevate Na-K-Cl cotransport activity under both control and agonist-stimulated conditions, indicating that activation of protein kinase C results in inhibition of endothelial cell Na-K-Cl cotransport. Thus, protein kinase C activation may serve as negative feedback in the stimulus-transfer pathway by which these agonists regulate endothelial cell Na-K-Cl cotransport.     

Interaction between protein kinase C and sphingomyelin/cholesterol

Physical characteristics of binding of protein kinase C with sphingomyelin/cholesterol lipid bilayers were analysed using three complementary approaches: acrylodan fluorescence, fluorescence energy transfer and quenching of tryptophan fluorescence. It was demonstrated that sphingomyelin/cholesterol lipid membranes were available for protein kinase C binding. The intensity of the binding was dependent on the sphingomyelin content. The results of quenching of intrinsic tryptophan fluorescence showed that the enzyme molecule penetrated the sphingomyelin/cholesterol lipid bilayer to the C-16 position of labeled fatty acid probes. Our results also showed sphingomyelin itself restrains protein kinase C activity. A possible explanation for our results is that caveolae function as signaling storage devices.     

The C1 and C2 domains of protein kinase C are independent membrane targeting modules, with specificity for phosphatidylserine conferred by the C1 domain

Protein kinase C is specifically activated by binding two membrane lipids: the second messenger, diacylglycerol, and the amino phospholipid, phosphatidylserine. This binding provides the energy to release an autoinhibitory pseudosubstrate from the active site. Interaction with these lipids recruits the enzyme to the membrane by engaging two membrane-targeting modules: the C1 domain (present as a tandem repeat in most protein kinase Cs) and the C2 domain. Here we dissect the contribution of each domain in recruiting protein kinase C betaII to membranes. Binding analyses of recombinant domains reveal that the C2 domain binds anionic lipids in a Ca(2+)-dependent, but diacylglycerol-independent, manner, with little selectivity for phospholipid headgroup beyond the requirement for negative charge. The C1B domain binds membranes in a diacylglycerol/phorbol ester-dependent, but Ca(2+)-independent manner. Like the C2 domain, the C1B domain preferentially binds anionic lipids. However, in striking contrast to the C2 domain, the C1B domain binds phosphatidylserine with an order of magnitude higher affinity than other anionic lipids. This preference for phosphatidylserine is, like that of the full-length protein, stereoselective for sn-1, 2-phosphatidyl-L-serine. Quantitative analysis of binding constants of individual domains and that of full-length protein reveals that the full-length protein binds membranes with lower affinity than expected based on the binding affinity of isolated domains. In addition to entropic and steric considerations, the difference in binding energy may reflect the energy required to expel the pseudosubstrate from the substrate binding cavity. This study establishes that each module is an independent membrane-targeting module with each, independently of the other, containing determinants for membrane recognition. The presence of each of these modules, separately, in a number of other signaling proteins epitomizes the use of these modules as discreet membrane targets.     

Role of protein kinase C in cellular regulation

Protein kinase C (PKC) consists of a family of closely related enzymes ubiquitously present in animal tissues. These enzymes respond to second messengers, Ca2+, diacylglycerol and arachidonic acid, to express their activities at membrane locations. Numerous hormones, neurotransmitters, growth factors and antigens are believed to transmit their signals by activation of a variety of phospholipases to generate these messengers. The various PKC isozymes, which exhibit distinct biochemical characteristics and unique cellular and subcellular localizations, may be differentially stimulated depending on the duration and strength of these messengers. Activation of PKC has been linked to the regulation of cell surface receptors, ion channels, secretion, gene expression, and neuronal plasticity and toxicity. The mechanisms of action of PKC in the regulation of these cellular functions are not entirely clear. Further study to identify the target substrates relevant to the various cellular functions is essential to define the functional diversity of this enzyme family.     

Specificity of activated human protein C.

Peptide p-nitroanilide substrates and peptidylchloromethane inhibitors were used to examine the specificity of activated human Protein C. Substrates with arginine in the P1 position had the highest activity. The best substrates and inhibitors, as judged by the second-order rate constant for their interaction with the enzyme, had an apolar residue in the P2 position. In contrast with thrombin [Kettner & Shaw (1981) Methods Enzymol. 80, 826-842], activated Protein C was able to accommodate large hydrophobic residues such as phenylalanine and leucine in the P2 position. In the P3 position, the enzyme preferred an apolar D-amino acid residue. The results of the present study have also indicated a suitable substrate and inhibitor to be used in the assay of functional protein C and of thrombomodulin.