Persistent, membrane-associated protein kinase C: from model membranes to synaptic long-term potentiation.
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Arachidonic Acid↗
Biomedical subjects
D S Lester
Publications and source records attributed to D S Lester.
Cell specificity of molecular changes during memory storage.
The aeolid nudibranch, Hermissenda crassicornis, exhibits Pavlovian conditioning to paired light and rotational stimuli and it has been suggested that protein kinase C(PKC) may play a critical role in the cellular mechanism for this conditioned behavioral response in the B-cell photoreceptor. The present study was designed to further examine learning-specific PKC involvement in identified cellular areas, particularly those in the visual-vestibular network, of the Hermissenda nervous system after Pavlovian conditioning. As used in previous vertebrate studies, the highly specific PKC radioligand, [3H]phorbol-12,13-dibutyrate ([3H]-PDBU), was used to determine the binding characteristics of the molluscan protein receptor considered to be PKC. The binding was specific, saturable, and could be displaced by a soluble diacylglycerol analogue. The binding activity was distributed evenly between the cytosol and the membrane. All of these analyses suggest that [3H]PDBU binds primarily to PKC in Hermissenda as it does in many other systems. Computerized grain image analysis was then used to determine the cellular localization of PKC as a function of Pavlovian conditioning. The medial and intermediate B photoreceptor and the optic ganglion showed significantly increased [3H]PDBU binding in conditioned animals. The present results provide the first report of an associative learning change of a key signal transduction component in identified neurons.
Animals↗
Lipid vesicles which can bind to protein kinase C and activate the enzyme in the presence of EGTA.
Maximal protein kinase C activity with vesicles of phosphatidic acid and 1,2-dioleoyl-sn-glycerol is observed in the absence of added Ca2+. Addition of phosphatidylcholine to these vesicles restores some calcium dependence of enzyme activity. 1,2-Dioleoyl-sn-glycerol eliminates the Ca(2+)-dependence of protein kinase C activity found with phosphatidic acid alone. Phorbol esters do not mimic the action of 1,2-dioleoyl-sn-glycerol in this respect. This suggests that the 1,2-dioleoyl-sn-glycerol effect is a result of changes it causes in the physical properties of the membrane rather than to specific binding to the enzyme. The effect of 1,2-dioleoyl-sn-glycerol on the phosphatidic-acid-stimulated protein kinase C activity is dependent on the molar fraction of 1,2-dioleoyl-sn-glycerol used and results in a gradual shift from Ca2+ stimulation at low 1,2-dioleoyl-sn-glycerol concentrations to calcium inhibition at higher concentrations of 1,2-dioleoyl-sn-glycerol. Phosphatidylserine-stimulated activity is also shown to be largely independent of the calcium concentration at higher molar fractions of 1,2-dioleoyl-sn-glycerol. Thus, with certain lipid compositions, protein kinase C activity becomes independent of the calcium concentration or requires only very low, stoichiometric binding of Ca2+ to high affinity sites on the enzyme. Protein kinase C can bind to phosphatidic acid vesicles more readily than it can bind to phosphatidylserine vesicles in the absence of calcium. Addition of 1,2-dioleoyl-sn-glycerol to phosphatidylserine vesicles promotes the partitioning of protein kinase C into the membrane in the absence of added Ca2+. There is no isozyme specificity in this binding. These results suggest that a less-tightly packed headgroup region of the bilayer causes increased insertion of protein kinase C into the membrane. This is a necessary but not sufficient condition for activation of the enzyme in the presence of EGTA.
Animals↗
The mechanism of activation of protein kinase C: a biophysical perspective.
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Calcium↗
Identification of two distinct populations of protein kinase C in rat brain membranes.
The regulatory enzyme protein kinase C (PKC) is proposed to be activated on its translocation from the cytosol to the membrane. However, a portion of the native activity is always associated with the membrane fraction. Using a noninvasive procedure to extract this endogenous activity from rat brain membranes, it has been possible to characterize the activity in a partially purified reconstituted system bearing resemblance to the in vivo system. Two subpopulations of membrane-associated PKC were identified and characterized at the level of activation, inhibition, and isozyme immunologic characteristics and chromatographic properties. One peak had properties similar to those of cytosolic PKC, whereas the second population, extracted as protein-lipid complexes, had considerable constitutive activity that could be stimulated further on addition of PKC activators. This latter activity was relatively resistant to staurosporine inhibition and phorbol ester treatment, but it phosphorylated the exogenous PKC substrates, histone 1 and the epidermal growth factor receptor peptide KTRLRR. The constitutive activity was totally dependent on its endogenous associated lipids coextracted by the solubilization procedure. The ratio between these two populations was ontogenetically regulated and modulated by phorbol ester treatment, suggesting that different PKC populations may serve unique functions in the rat brain regulated by the lipid environment. Analyses of the phospholipids extracted in these protein-lipid complexes showed differences in the major classes correlating to age. However, apart from a markedly lower cholesterol content in these complexes, no direct relationship between a specific lipid composition and the amount of constitutive PKC activity was evident.
Animals↗
Chemical modification of arginine residues of protein kinase C.
Modification of basic residues of protein kinase C by phenylglyoxal results in a reversible, dose-dependent inhibition of autophosphorylation and substrate phosphorylation. The inhibition is not due to specific modification of the Ca2+ or ATP binding sites. Modified PKC bound more [3H] phorbol ester than unmodified providing further evidence that the binding of lipids and the catalytic phosphotransferase activity have different regulatory sequences. Additionally, the effects of these modifying reagents were different on enzyme stimulated by phorbol esters or by the endogenous activator, 1,2-diacylglycerol. These studies suggest that there is at least 1 arginine residue that is unique in the binding site of these different activators.
Adenosine Triphosphate↗
Arachidonic acid and diacylglycerol act synergistically to activate protein kinase C in vitro and in vivo.
Using a well-defined model membrane bilayer system, incorporation of both lipid second messengers, 1,2-diacylglycerol and arachidonic acid, at submaximal activating concentrations, resulted in a synergistic activation of protein kinase C in a Ca2+/phosphatidylserine-dependent manner as measured by monitoring phosphorylation of phosphoprotein substrates. The arachidonic acid appears to modulate membrane properties both at the hydrocarbon core and the membrane surface increasing the availability of the diacylglycerol which can bind to and subsequently activate the enzyme. Co-application of these two lipid activators to the Hermissenda photoreceptor reduced K+ channel conductance in a synergistic manner via a PKC-dependent pathway. Thus, these in vivo and in vitro studies suggest that the membrane bilayer properties of these PKC lipid activators interact to specifically regulate the cellular lipid microenvironment resulting in PKC activation.
Animals↗
Action of organic solvents on protein kinase C.
The action of organic solvents on the biochemical and biophysical properties of protein kinase C (PKC) was measured in a defined lipid vesicle system. Chloroform, benzyl alcohol and ethanol all partially activated PKC. They had no effect on the Ca(2+)- or anionic phospholipid-, phosphatidylserine-dependence. Their ability to activate PKC correlated with their lipid perturbing effect as measured by the decrease in fluorescence anisotropy. Chloroform was the most potent activator stimulating PKC phosphotransferase activity up to a level 40% of that obtained by the endogenous activator, diacylglycerol. The three fluidizers induced a more hydrophobic environment for at least one PKC tryptophan residue. This may have been due to insertion of an additional or different region of protein kinase C into the lipid bilayer as demonstrated by a blue shift in tryptophan fluorescence, providing an explanation for their inability to act as competitors of PKC binding of phorbol esters. Higher concentrations of the organic solvents resulted in a lipid bilayer that was too fluid to support membrane activity. This study demonstrates that these anesthetics and alcohols modulate lipid structure which subsequently induces PKC to undergo a different protein-lipid interaction in comparison to the endogenous activator, diacylglycerol. This supports the proposal that the biophysical state of the membrane plays a significant role in modulating PKC activity.
Animals↗
Divalent cation-induced changes in conformation of protein kinase C.
Using physical techniques, circular dichroism and intrinsic and extrinsic fluorescence, the binding of divalent cations to soluble protein kinase C and their effects on protein conformation were analyzed. The enzyme copurifies with a significant concentration of endogenous Ca2+ as measured by atomic absorption spectrophotometry, however, this Ca2+ was insufficient to support enzyme activity. Intrinsic tryptophan fluorescence quenching occurred upon addition to the soluble enzyme of the divalent cations, Zn2+, Mg2+, Ca2+ or Mn2+, which was irreversible and unaffected by monovalent cations (0.5 M NaCl). Far ultraviolet (200-250 nm) circular dichroism spectra provided estimations of secondary structure and demonstrated that the purified enzyme is rich in alpha-helices (42%) suggesting a rather rigid structure. At Ca2+ or Mg2+ concentrations similar to those used for fluorescence quenching, the enzyme undergoes a conformational transition (42-24% alpha-helix, 31-54% random structures) with no significant change in beta-sheet structures (22-26%). Maximal effects on 1 microM enzyme were obtained at 200 microM Ca2+ or 100 microM Mg2+, the divalent cation binding having a higher affinity for Mg2+ than for Ca2+. The Ca2(+)-induced transition was time-dependent, while Mg2+ effects were immediate. In addition, there was no observed energy transfer for protein kinase C with the fluorescent Ca2(+)-binding site probe, terbium(III). This study suggests that divalent cation-induced changes in soluble protein kinase C structure may be an important step in in vitro analyses that has not yet been detected by standard biochemical enzymatic assays.
Animals↗
Activation of protein kinase C phosphorylation pathways: a role for storage of associative memory.
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Animals↗
Extracellular dephosphorylation in the parasite, Leishmania major.
Leishmania major promastigotes were analyzed for the presence of protein phosphatase activity in intact cells and membrane-enriched fractions. Parasite phosphoproteins, phosphorylated in live cells with [gamma-32P]adenosine 5'-triphosphate (ATP) and an endogenous leishmanial ectokinase, were dephosphorylated by endogenous protein phosphatase-like activity in intact cells and a membrane-rich fractions. An alkaline phosphatase-like activity was also identified using the artificial substrate, p-nitrophenyl phosphate (pNPP). This activity was localized on the extracellular membrane of intact parasites, as well as in the particulate fraction of lysed cells. The phosphatase activity measure using pNPP had inhibition properties and a pH profile between protein phosphatase and general alkaline phosphatases. This study supports the observation that there is extracellular protein phosphorylation/dephosphorylation in L. major which may play a significant role in host cell-parasite recognition and infection.
Alkaline Phosphatase↗
Application of high-pressure to subfractionate membrane protein-lipid complexes: a case study of protein kinase C.
Current procedures for solubilization of membrane proteins involve the use of detergents. A procedure using high hydrostatic pressures without detergent has been applied in this study to subfractionate membrane proteins and their endogenously associated lipids. Rat brain membrane preparations were suspended in hypotonic buffer containing the membrane fluidizer benzyl alcohol in a sealed pressure cell and subjected to hydrostatic pressures of up to 1500 atmospheres (approx 22,000 psi) in a French press. Under these conditions, specific membrane proteins including protein kinase C, phospholipase A2, calmodulin-binding proteins, G-proteins, and microtubule-associated proteins all coextracted and were associated to lipid particles, suggesting inherent physical contact. Two populations of membrane-associated protein kinase C were identified according to molecular weight estimations. The first coeluted with the lipid particles composed predominantly of phospholipids, while the second contained much less lipid and was similar to the soluble monomer, i.e., cytosolic protein kinase C. This procedure provides an important technique for selective subfractionation of membrane proteins in their native lipid environment which could be used for structure-function studies.
Animals↗
In vitro linoleic acid activation of protein kinase C.
The importance of membrane fluidity in the activation of protein kinase C (PKC) was examined using the membrane fluidizer, linoleic acid, in a well-defined model membrane system. Biochemical and biophysical properties of the system were monitored. Linoleic acid activated PKC to a level of 50% of that observed for diacylglycerol. In contrast, linoleic acid did not directly interact with the phorbol ester binding site as did diacylglycerol. This was determined by the lack of involvement of the ionizable group of the fatty acid with activity and the enhancement of phorbol ester binding by linoleic acid and its ester analogs. The membrane fluidity of this model membrane system in the presence of linoleic acid was increased as determined by fluorescence polarization. This increased the availability of phospholipids, thus, explaining the linoleic acid-induced enhancement of phorbol ester binding. The PKC conformation as determined from intrinsic tryptophan fluorescence spectra was different for lipid mixtures containing linoleic acid or diacylglycerol correlating with the difference in biochemical activation properties. This study provides evidence that membrane fluidization is not the predominant function of the lipid activator in PKC activation, but may play a role in obtaining the preferred membrane state for maximal activation.
Animals↗
Lipid dependence of surface conformations of protein kinase C.
The change of conformation of protein kinase C interacting with the surface of a mercury electrode directly from a solution or through a lipid monolayer was inferred from the number of cystine residues exposed and reduced on the electrode and from their reduction potentials. Soluble protein kinase C was estimated to have 5-6 disulfide bonds which could potentially react with the mercury electrode. Two major reduction peaks of cystine at different microenvironments within the protein molecule adsorbed to a mercury surface. They were observed in a.c. polarograms and cyclic voltamograms at two distinct potentials. The potential of these peaks became more negative as the pH of the solution increased, which was consistent with relaxation or decrease in alpha-helicity (ordered structure) of the protein as determined by circular dichroism (CD) estimations of secondary structure. The peak at the more positive potentials (-0.46 V relative to NAg/AgCl electrode at pH 7.4) tended to vanish upon cyclic reduction and reoxidation of the cystine, while the more negative peak (-0.62 V at pH 7.4) was enhanced. Addition of Mg2+ or Ca2+ had no significant effect on the potential but there was a reduction in their amplitude which appeared to affect the disappearance of these peaks upon pH adjustment. This suggests that the tertiary structure of the molecule is stabilized by Ca2+ and Mg2+, as substantiated by CD spectral analysis of secondary structures. Protein kinase C penetrated lipid monolayers to some extent. Addition of diacylglycerol or phorbol ester to the lipid monolayers facilitated this penetration. These compounds stabilized the protein surface conformation by destabilizing the monolayer at more positive potentials, resulting in an enhanced reduction peak at -0.42 V. This phenomenon was not significantly affected by Mg2+ or by Ca2+. The region of the protein kinase C (PKC) sequence which penetrated the monolayer contains cysteines and a primary amine(s), and may have homology to a region of phospholipase A2 which has been proposed as a phospholipid binding site for the two enzymes. Additionally, these polarographic studies suggest that PKC associates with and penetrates monolayers in a divalent cation-independent manner in agreement with our previous physical analyses of PKC interactions with lipid bilayers.
Adsorption↗
Extracellular phosphorylation in the parasite, Leishmania major.
Intact promastigotes or cell-free extracts of the parasite Leishmania major were labelled with adenosine 5'[gamma-32P]-triphosphate (ATP). This resulted in the identification of eleven phosphoproteins. [gamma-32P]ATP incorporation into endogenous and exogenous substrates was insensitive to most of the commonly used protein kinase inhibitors and activators indicating that the leishmanial enzyme(s) may represent a new class of kinase(s). In addition, exogenous substrate specificity was inconsistent with the preferences of second messenger-dependent protein kinases. Cyclic AMP had differential effects on phosphorylation in intact cells and lysates. The majority of kinase activity could be attributed to an externally oriented membrane-associated protein kinase(s), as no specific cytosolic phosphoproteins were found and intact cells phosphorylated exogenous substrates. Labelled ATP did not cross the membrane and [alpha-32P]ATP was an unsuitable substrate for the phosphorylation activity. The ectokinase activity on live Leishmania exhibited a different substrate preference when compared to the protein kinase activity in the particulate fraction, suggesting that more than one protein kinase may be present in L. major. Three serine-labelled phosphoproteins were specifically released into the medium. The presence of an ecto-kinase and these released phosphoproteins may play a significant role in host-parasite interactions.
Adenosine Triphosphate↗
Structural distinction between soluble and particulate protein kinase C species.
A number of peripheral membrane proteins functioning as regulatory enzymes are distributed between soluble and particulate fractions upon homogenization and subcellular fractionation. One such enzyme, the Ca2+/phospholipid-dependent protein kinase, protein kinase C, was analyzed in order to examine this characteristic of differential localization. The soluble and particulate forms of this enzyme were purified to relative homogeneity, and their biochemical and biophysical properties were analyzed and compared. Based on biochemical activities, the particulate form required lower phospholipid concentrations for maximal activation than for the soluble species. The particulate species had a more hydrophobic structure as demonstrated by a hydrophobic fluorescence probe, and had almost 50% more alpha-helical structures according to secondary structure estimation, determined from far ultra-violet-circular dichroism spectra (200-250 nm). Using Fourier transform infrared spectroscopy, specific lipid spectra were detected associated with the soluble protein kinase C species. Further analyses with a fluorescent neutral membrane probe suggested that there was more lipid associated with the purified particulate form, which was of a less mobile nature than those associated with the soluble species. These structural differences provide an explanation for the preferential localization of the enzyme and may prove to be the basis for distribution of other membrane-active peripheral membrane regulatory enzymes.
Chemical Phenomena↗
Protein kinase C penetration into lipid bilayers.
Physical characteristics of the association and subsequent penetration of protein kinase C into defined lipid bilayers were analyzed using four different fluorescence probes. The enzyme demonstrated strong hydrophobic and electrostatic interactions with the bilayer as suggested by its ability to increase permeability of carboxyfluorescein-filled unilamellar vesicles. The intensity of interaction was dependent on the concentration of phosphatidylserine. The hydrophilic quencher, N-methylpicolinium perchlorate, was used to show that the tryptophan residues affected by ligand-induced conformational changes were in a hydrophobic region(s) of the enzyme. Using quenching of intrinsic tryptophan fluorescence, the enzyme was shown to penetrate the lipid bilayer to the C-16 position of labeled fatty acid probes. The association and subsequent penetration of the enzyme into the lipid bilayer was independent of divalent cations in these systems and had no significant effect on activator-independent substrate phosphorylation.
Adenosine Triphosphate↗
Ligand-induced conformational changes in cytosolic protein kinase C.
The changes in intrinsic spectral properties of protein kinase C were monitored upon association with its divalent cation and lipid activators in a model membrane system. The enzyme demonstrated changes in both its intrinsic fluorescence and far ultraviolet circular dichroism spectra upon association with lipid vesicles in the absence of calcium. The acidic phospholipid, phosphatidylserine, significantly quenched the intrinsic tryptophan fluorescence and was also the most potent lipid support for the phosphorylating activity of the enzyme. The enzyme was fully activated by a number of Ca2(+)-lipid combinations which correlated with maximal fluorescence quenching (40-50%) of available tryptophan residues in hydrophobic domains. The circular dichroism structure of the associated active-protein Ca2(+)-lipid complexes suggested different active enzyme secondary structures. However, the Ca2(+)-dependent changes in fluorescence and circular dichroism spectra were observed only after the enzyme associated with the lipid vesicles. These data suggest that protein kinase C has the properties of a complex multidomain protein and provides an additional perspective into the mechanism of protein kinase C activation.
Animals↗