The relationship between the bilayer to hexagonal phase transition temperature in membranes and protein kinase C activity

A number of substances affect the activity of protein kinase C. Among uncharged and zwitterionic compounds, those which activate protein kinase C also lower the bilayer to hexagonal phase transition temperature of dielaidoylphosphatidylethanolamine while substances which inhibit protein kinase C raise this transition temperature. Using this criteria, we have identified 3-chloro-5-cholestene, 5-cholan-24-ol and eicosane as new protein kinase C activators and have shown that Z-Ser-Leu-NH₂, Z-Gly-Leu-NH₂, Z-Tyr-Leu-NH₂, cyclosporin A and cholestan-3, 5, 6-triol are protein kinase C inhibitors.     

Evidence for the regulation of the activity of protein kinase C through changes in membrane properties

We measured the effects of two branched-chain analogs of distearoyl-phosphatidylcholine, containing either a methyl or an n-butyl group at the 8 position, on the bilayer to hexagonal phase transition temperature of dielaidoylphosphatidylethanolamine. The former compound raised the bilayer to hexagonal phase transition temperature while the latter compound lowered it. The opposite effects of these amphiphiles on protein kinase C activity (inhibition and activation, respectively) correlated with their effects on lipid polymorphism. Because of the similarity of the structures of these two compounds, it seems likely that their opposite effects on the activity of protein kinase C is a result of their alteration of the lipid environment of the membrane rather than to binding to a specific site on the protein.We also compared the effects of hexachlorophene on lipid polymorphism and protein kinase C activity at high and at low calcium concentrations. We also found that the effect of hexachlorophene forming a complex with Ca²⁺ is to increase both the hexagonal phase forming propensity of the membrane as well as to increase the activity of protein kinase C, again demonstrating the correlation between lipid phase propensity and effects on protein kinase C activity.Abbreviations DSPC distearoylphosphatidylcholine - DSPC-8M and DSPC-8B the 8-methyl and 8-n-butyl derivatives of DSPC, respectively - PKC protein kinase C - DSC differential scanning calorimetry     

Effects of viral chemotherapeutic agents on membrane properties. Studies of cyclosporin A, benzyloxycarbonyl-D-Phe-L-Phe-Gly and amantadine

Cyclosporin A, benzyloxycarbonyl-D-Phe-L-Phe-Gly, and amantadine inhibit the dilution of fluorescently labeled lipids, as measured with the resonance energy exchange assay for membrane fusion. The fusion was studied using sonicated vesicles containing 1,2-dioleoyl-sn-glycero(3)phosphoethanolamine, egg (3-sn-phosphatidyl)choline, and cholesterol in a 1:1:1.3 molar ratio. All three antiviral agents inhibited myelin basic protein-induced membrane fusion when present at low concentrations in the membrane. The mechanism by which these agents affect membrane properties was investigated. The effect of these agents on the bilayer to hexagonal phase transition of 1,2-dielaidoyl-sn-glycero(3)phosphoethanolamine was determined using both differential scanning calorimetry and 31P NMR. Benzyloxycarbonyl-D-Phe-L-Phe-Gly is particularly effective in raising the bilayer to hexagonal phase transition temperature while cyclosporin promotes the greatest amount of broadening of the 31P NMR signal. Both effects are suggested to be related to the inhibitory activity of these substances on membrane fusion and possibly also to their antiviral activity.     

Effect of lipid structural modifications on their intermolecular hydrogen bonding interactions and membrane functions

The large number of different membrane lipids with various structural modifications and properties and the characteristic lipid composition of different types of membranes suggest that different lipids have specific functions in the membrane. Many of the varying properties of lipids with different polar head groups and in different ionization states can be attributed to the presence of interactive or repulsive forces between the head groups in the bilayer. The interactive forces are hydrogen bonds between hydrogen bond donating groups such as --P--OH,--OH, and--NH3+ and hydrogen bond accepting groups such as --P--O- and --COO-. These interactions increase the lipid phase transition temperature and can account for the tendency of certain lipids to go into the hexagonal phase and the dependence of this tendency on the pH and ionization state of the lipid. The presence or absence of these interactions can also affect the penetration of hydrophobic substances into the bilayer, including hydrophobic residues of membrane proteins. Evidence for this suggestion has been gathered from studies of the myelin basic protein, a water-soluble protein with a number of hydrophobic residues. In this way the lipid composition can affect the conformation and activity of membrane proteins. Since hydrogen-bonding interactions depend on the ionization state of the lipid, they can be altered by changes in the environment which affect the pK of the ionizable groups. The formation of the hexagonal phase or inverted micelles, the conformation and activity of membrane proteins, and other functions mediated by lipids could thus be regulated in this way.     

Differential activation and inhibition of lymphocyte proliferation by modulators of protein kinase C: diacylglycerols, "rationally designed" activators and inhibitors of protein kinase C

The tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA) can enhance or inhibit lymphocyte proliferation. Enhancement correlated with increased interleukin 2 (IL-2) production and activation of protein kinase C while inhibition correlated with decreased IL-2 and downregulation of protein kinase C activity (D.S. Grove and A.M. Mastro, Cancer Res. 51, 82-88). In this study, various activators and inhibitors of protein kinase C were used in order to try to separate the effects of TPA on this enzyme from its effects on IL-2 production and determine if protein kinase C activity was directly or indirectly related to IL-2 production. 1,2-Dioctanoylglycerol, 1-oleoyl-2-acetyl-glycerol, phospholipase C, and two "rationally designed" activators, 6-(N-decylamino)-4-hydroxy-methylindole and 3-(N-acetylamino)-5-(N-decyl-N-methylamino)-benzyl alcohol, were tested. Some activators enhanced proliferation in the presence of a Ca2+ ionophore, ionomycin, but not concanavalin A. Some activators suppressed proliferation and downregulated protein kinase C. Others neither downregulated protein kinase C nor inhibited IL-2 production and proliferation. However, inhibition or downregulation of protein kinase C activity always correlated with decreased IL-2 and depressed proliferation. Thus, the evidence in this and the previous study suggests that activation of protein kinase C is directly related to IL-2 production in activated T cells.     

Polyamines and polyamino acids regulation of cytosolic tyrosine protein (Tyr-P) kinase from human erythrocytes.

In vitro regulation of cytosolic tyrosine protein (Tyr-P) kinase from human erythrocytes by polyamines, polyamino acids, negative charged compounds or by insulin using angiotensin II or poly (Glu-Tyr)4:1 as substrates was studied. All the three polyamines, putrescine (Put), spermidine (Spd) and spermine (Spm) stimulated the Tyr-P kinase activity in a dose dependent manner. Spm stimulated Tyr-P kinase activity higher than Put and Spd whether the substrate was angiotension II or poly (Glu-Tyr)4:1. Polyamino acids (polyornithine, polyarginine, polyglutamic acid and polyaspartic acid) did not affect significantly the Tyr-P kinase phosphorylation except polylysine which significantly stimulated the Tyr-P kinase activity. Negative charged compounds (chondroitin sulfate A, B and C) and heparin inhibited the Tyr-P kinase phosphorylation while insulin did not influence the enzyme activity in the presence of either substrates.     

Kinases,intracellular calcium,and apolipophorin-III influence the adhesion of larval hemocytes of the lepidopterous insect,Galleria mellonella

Based on the results from the use of selective inhibitors and activators, active protein kinase A, protein tyrosine kinase, and protein kinase C (PKC) isoforms decreased the adhesion of larval Galleria mellonella hemocytes to glass slides. The protein kinase A inhibitor at all concentrations increased granular cell adhesion only whereas protein tyrosine kinase elevated both granular and plasmatocyte attachment at the lowest concentration. Active, Ca(2+)- and lipid-dependent PKC isoforms limited plasmatocyte and granular cell adhesion whereas PKC that was inhibited by selected compounds (with differed modes of PKC inhibition) enhanced hemocyte attachment. The granular cells were more sensitive to the PKC inhibitors than were plasmatocytes. Phospholipase C and its diacylglyceride product were necessary to reduce hemocyte adhesion and maintain PKC activity. Extracellular Ca(2+), possibly transported through L-channels, was required for plasmatocyte attachment. In contrast, lowering the levels of cytosolic Ca(2+) was associated with decreased PKC activity and was required for hemocyte adhesion.     

Myeloid cell leukemia 1 is phosphorylated through two distinct pathways, one associated with extracellular signal-regulated kinase activation and the other with G2/M accumulation or protein phosphatase 1/2A inhibition

Protein kinase C activators and microtubule-damaging drugs stimulate BCL2 phosphorylation, which has been associated with either enhancement or inhibition of cell viability. In a Burkitt lymphoma cell line, both types of agents likewise stimulated phosphorylation of myeloid cell leukemia 1 (MCL1), another viability-promoting BCL2 family member. However, while MCL1 phosphorylation induced by the protein kinase C activator, 12-O-tetradecanoylphorbol-13-acetate (TPA), did not affect its electrophoretic mobility, microtubule-damaging agents, such as taxol, induced MCL1 phosphorylation associated with a band shift to decreased mobility. Inhibitors of extracellular signal-regulated kinase (ERK) activation blocked TPA-induced MCL1 phosphorylation but not the taxol-induced band shift. TPA-induced MCL1 phosphorylation occurred rapidly and was not associated with decreased viability, while the taxol-induced band shift occurred upon extended exposure as cells accumulated in G(2)/M followed by cell death. Protein phosphatase 1/2A inhibitors also induced the MCL1 band shift/phosphorylation. Thus, MCL1 undergoes two distinct types of phosphorylation: (i) TPA-induced, ERK-associated phosphorylation, which does not alter the electrophoretic mobility of MCL1, and (ii) ERK-independent phosphorylation, which results in an MCL1 band shift and is induced by events in G(2)/M or protein phosphatase 1/2A inhibitors.     

Cell regulation by sphingosine and more complex sphingolipids

Sphingolipids have the potential to regulate cell behavior at essentially all levels of signal transduction. They serve as cell surface receptors for cytoskeletal proteins, immunoglobulins, and some bacteria; as modifiers of the properties of cell receptors for growth factors (and perhaps other agents); and as activators and inhibitors of protein kinases, ion transporters, and other proteins. Furthermore, the biological activity of these compounds resides not only in the more complex species (e.g., sphingomyelin, cerebrosides, gangliosides, and sulfatides), but also in their turnover products, such as the sphingosine backbone which inhibits protein kinase C and activates the EGF-receptor kinase,inter alia. Since sphingolipids change with cell growth, differentiation, and neoplastic transformation, they could be vital participants in the regulation of these processes.This review is dedicated to Professor Herbert E. Carter on the occasion of his 80th birthday.     

Diacylglycerols, lysolecithin, or hydrocarbons markedly alter the bilayer to hexagonal phase transition temperature of phosphatidylethanolamines

The bilayer to hexagonal phase transition temperatures of dielaidoylphosphatidylethanolamine and 1-palmitoyl-2-oleoylphosphatidylethanolamine are 65.6 and 71.4 degrees C, respectively. Using high-sensitivity differential scanning calorimetry, I have shown that these transition temperatures are extremely sensitive to the presence of small amounts of other lipid components. For example, at a mole fraction of only 0.01, dilinolenin lowers the bilayer to hexagonal phase transition temperature of 1-palmitoyl-2-oleoyl-phosphatidylethanolamine by 8.5 degrees C. Other diacylglycerols have similar effects on this transition temperature, although the degree of unsaturation of the acyl chains has some effect, with distearin being less potent. In comparison, the 20-carbon alkane eicosane lowers this transition temperature by 5 degrees C, while palmitoyl-lysolecithin raises it by 2.5 degrees C. Similar effects of these additives on the bilayer to to hexagonal phase transition temperature are observed with dielaidoylphosphatidylethanolamine. At these concentrations of additive, there is no effect on the gel-state to liquid-crystalline-state transition temperature. The observed shifts in the temperature of the bilayer to the hexagonal phase transition can be qualitatively interpreted in terms of the effects of these additives on the hydrophilic surface area and on the hydrophobic volume. Substances expanding the hydrophobic domain promote hexagonal phase formation and lower the bilayer to hexagonal phase transition temperature. The sensitivity of the bilayer to hexagonal phase transition temperature to the presence of additives is at least as great as that which has been observed for any other lipid phase transition.     

The effects of hydrophobic mismatch between phosphatidylcholine bilayers and transmembrane alpha-helical peptides depend on the nature of interfacially exposed aromatic and charged residues

In this study, we investigated the extent to which different aromatic and positively charged side chains, which often flank transmembrane segments of proteins, can influence lipid-peptide interactions. Model systems consisting of phosphatidylcholine and hydrophobic alpha-helical peptides with different flanking residues were investigated. The peptides were incorporated in relatively thick and in relatively thin lipid bilayers to create a peptide-bilayer hydrophobic mismatch, and the compensating effects on lipid structure were analyzed. When relatively long with respect to the thickness of the bilayer, the peptides that are flanked by the aromatic side chains, Trp, Tyr, and Phe, all induce a significant ordering of the lipid acyl chains, while the peptides flanked by the charged residues Lys, Arg, and His do not. However, when the peptides are relatively short with respect to the thickness of the bilayer, their effect on lipid organization does not depend primarily on their aromatic or charged character. Peptides flanked by Trp, Tyr, Lys, or (at low pH) His residues are effective in inducing mismatch-relieving cubic and inverted hexagonal phases, while analogues flanked by Phe, Arg, or (at neutral pH) His residues cannot induce an inverted hexagonal phase. The different responses to mismatch might reflect the different interfacial affinities of the residues that were investigated.     

A role of protein kinase C in signal reactions in outer segments of the bovine retinal rods

The effect of modulators of protein kinase C activity on Ca2+ translocation in dark-adapted and bleached retinal rod outer segments (ROS) was studied. The activators (1,2-diacyl glycerol and phorbol-12-myristate-13-acetate) and the inhibitor (chelerythrine chloride) of protein kinase C were shown to stimulate and inhibit the ATP-dependent Ca(2+)-uptake in dark-adapted retinal ROS, correspondingly. Apparently, this action is due to the influence of protein kinase C on Ca(2+)-ATPase activity in these vesicular structures. No involvement of modulators of protein kinase C activity on ATP-dependent Ca(2+)-uptake in bleached retinal ROS was found. The influence of protein kinase C on Ca(2+)-release from retinal ROS was observed. It was shown that the activators and inhibitors of protein kinase C increased the efficiency of this process both in dark-adapted and bleached retinal ROS. The mechanisms of action of the protein kinase C activity modulators on the Ca(2+)-uptake and Ca(2+)-release in retinal ROS are discussed.     

The multifunctional Ca2+/calmodulin-dependent protein kinase mediates Ca2+-dependent phosphorylation of tyrosine hydroxylase

Stimulation of rat pheochromocytoma PC12 cells with ionophore A23187, carbachol, or high K+ medium, agents which increase intracellular Ca2+, results in the phosphorylation and activation of tyrosine hydroxylase (Nose, P., Griffith, L. C., and Schulman, H. (1985) J. Cell Biol. 101, 1182-1190). We have identified three major protein kinases in PC12 cells and investigated their roles in the Ca2+-dependent phosphorylation of tyrosine hydroxylase and other cytosolic proteins. A set of PC12 proteins were phosphorylated in response to both elevation of intracellular Ca2+ and to protein kinase C (Ca2+/phospholipid-dependent protein kinase) activators. In addition, distinct sets of proteins responded to either one or the other stimulus. The three major regulatory kinases, the multifunctional Ca2+/calmodulin-dependent protein kinase, the cAMP-dependent protein kinase, and protein kinase C all phosphorylate tyrosine hydroxylase in vitro. Neither the agents which increase Ca2+ nor the agents which directly activate kinase C (12-O-tetradecanoylphorbol-13-acetate or 1-oleyl-2-acetylglycerol) increase cAMP or activate the cAMP-dependent protein kinase, thereby excluding this pathway as a mediator of these stimuli. The role of protein kinase C was assessed by long term treatment of PC12 cells with 12-O-tetradecanoylphorbol-13-acetate, which causes its "desensitization." In cells pretreated in this manner, agents which increase Ca2+ influx continue to stimulate tyrosine hydroxylase phosphorylation maximally, while protein kinase C activators are completely ineffective. Comparison of tryptic peptide maps of tyrosine hydroxylase phosphorylated by the three protein kinases in vitro with phosphopeptide maps generated from tyrosine hydroxylase phosphorylated in vivo indicates that phosphorylation by the Ca2+/calmodulin-dependent kinase most closely mirrors the in vivo phosphorylation pattern. These results indicate that the multifunctional Ca2+/calmodulin-dependent protein kinase mediates phosphorylation of tyrosine hydroxylase by hormonal and electrical stimuli which elevate intracellular Ca2+ in PC12 cells.     

The effect of new potent selective inhibitors of protein kinase C on the neutrophil respiratory burst

New potent inhibitors of protein kinase C were found to inhibit protein kinase C isolated from rat brain and human neutrophils, with a large degree of selectivity over cAMP-dependent kinase and Ca2+/calmodulin-dependent kinase. These novel compounds were potent inhibitors of the fluoride, diC8- and formyl-methionyl-leucyl-phenylalanine-mediated respiratory bursts in intact neutrophils. The opsonized zymosan-stimulated burst was only marginally affected by the compounds. These results differ from those obtained in studies with H7 and CI, (which are less potent and less specific protein kinase C inhibitors) and are consistent with the hypothesis that protein kinase C has a role in the transduction mechanism for the neutrophil oxidative burst stimulated with fluoride, formyl-methionyl-leucyl-phenylalanine and diC8.     

Modulation of the bilayer to hexagonal phase transition and solvation of phosphatidylethanolamines in aqueous salt solutions

Several salts affect the temperature of the bilayer to hexagonal phase transition of phosphatidylethanolamines. Their effects are dependent on the anion as well as the cation of the salt. Salt effects on this transition can be explained by preferential hydration and ion binding. Those salts which are excluded from the solvation sphere of the membrane promote hexagonal phase formation. For example, Na2SO4 promotes preferential hydration and is a hexagonal phase promoter while NaSCN does not do this and is a bilayer stabilizer. Unlike amphiphiles and hydrocarbons, salts can shift the bilayer to hexagonal phase transition temperature without altering the cooperativity of the transition. The effect of these salts on the gel to liquid-crystal transition is opposite to their effect on the bilayer to hexagonal phase transition. We also find that MnCl2 markedly raises the gel to liquid-crystal transition temperature. This effect is due to binding of the cation to the membrane surface. The effect is reduced with MnSO4 because of preferential hydration. Our results demonstrate that the nature of the anion as well as the cation can alter the effect of salts on lipid phase transition properties. The observed effects can be explained as resulting from preferential hydration and ion binding.     

Stimulation of Cell Elongation in Teleost Rod Photoreceptors by Distinct Protein Kinase Inhibitors

Abstract: The rod photoreceptors of teleost retinas elongate in the light. To characterize the role of protein kinases in elongation, pharmacological studies were carried out with rod fragments consisting of the motile inner segment and photosensory outer segment (RIS-ROS). Isolated RIS-ROS were cultured in the presence of membrane-permeant inhibitors that exhibit selective activity toward specific serine/threonine protein kinases. We report that three distinct classes of protein kinase inhibitors stimulated elongation in darkness: (1) cyclic AMP-dependent protein kinase (PKA)-selective inhibitors (H-89 and KT5720), (2) a protein kinase C (PKC)-selective inhibitor (GF 109203X) that affects most PKC isoforms, and (3) a kinase inhibitor (H-85) that does not affect PKC and PKA in vitro. Other kinase inhibitors tested neither stimulated elongation in darkness nor inhibited light-induced elongation; these include the myosin light chain kinase inhibitors ML-7 and ML-9, the calcium-calmodulin kinase II inhibitor KN-62, and inhibitors or activators of diacylglycerol-dependent PKCs (sphingosine, calphostin C, chelerythrine, and phorbol esters). The myosin light chain kinase inhibitors as well as the PKA and PKC inhibitors H-89 and GF 109203X all enhanced light-induced elongation. These observations suggest that light-induced RIS-ROS elongation is inhibited by both PKA and an unidentified kinase or kinases, possibly a diacylglycerol-independent form of PKC.     

Zinc homeostasis in C6 glioma cells

Zinc homeostasis in mammalian cells is precisely regulated by cellular signal transduction mechanisms. The main result of this study is the finding that modulators of phospholipase C (PLC) activity affect cellular zinc export. Two different PLC inhibitors caused an increase of the total cellular zinc level whereas two different PLC activators caused a decrease. Furthermore, both the inhibition of cyclic nucleotide phosphodiesterases as well as the administration of 8-bromo-cAMP evoked a drop in the intracellular zinc level, indicating the involvement of cAMP in the control of cellular zinc export. It is concluded that the activity of PLC controls cellular zinc transport and that the effect of elevated zinc concentrations on PLC activity might be mediated by cAMP. However, modulation of other major signaling enzymes did not affect the cellular zinc homeostasis. These include activation and inhibition of guanylate cyclase, activation of protein kinase G, activation of protein kinase A, and activation or inhibition of protein kinase C. Furthermore there was no evidence for the existence of a zinc-sensing receptor in C6 glioma cells, which would stimulate PLC activity and evoke a mobilization of intracellular free-calcium levels.     

Protein kinase C regulates endothelial cell tube formation on basement membrane matrix, Matrigel.

Human umbilical vein endothelial cells differentiate within 12 h to form capillary-like networks of tube structures when the cells are plated on Matrigel, a mixture of basement membrane proteins. Nothing is known about the intracellular signaling events involved in this differentiation. As a first step to define the process, we investigated the possible role of protein kinase C activation by beta-phorbol 12-myristate 13-acetate (PMA) in regulating the formation of the tube structures. In this model, PMA increased tube formation several-fold in a dose-dependent manner with half-maximum stimulation of tube formation at approximately 5 nM PMA. In the absence of serum, essentially little or no tubes were formed on Matrigel unless PMA was added to the medium. Only active phorbol analogs increased tube formation, while the protein kinase C inhibitor, H-7, blocked tube formation. The protein kinase C activators and inhibitors were effective only when added at or just after plating of the cells and did not affect already formed tubes. This study suggests that protein kinase C is involved in the early events of in vitro endothelial cell tube formation on Matrigel.     

Dual modulation of protein kinase C activity by sphingosine.

Sphingosine is one of a number of cationic amphiphiles that inhibit the activity of protein kinase C (PKC) in commonly used assay conditions. This inhibition occurs only at high concentrations of this amphiphile. In the presence of excess negative charge from oleic acid, the addition of sphingosine surprisingly leads to activation of PKC. The results are explicable in terms of the dual role of charge and lipid phase propensity. When the positive charge on sphingosine is compensated by the negative charge on oleic acid, sphingosine, a hexagonal phase promoting amphiphile, becomes an activator of PKC. This does not occur with a bilayer stabilizing cationic amphiphile, N,N,N-Trimethyl-N'-cholesteryl amido-ethyl ammonium which is an inhibitor of PKC at all mol fractions, as well as in the presence of oleic acid. The results indicate that effects of sphingosine on more complex biological systems should be interpreted with caution because of this dual role of the amphiphile.     

Protein Kinase Inhibitors Block Neurite Outgrowth from Explants of Goldfish Retina

A role for protein phosphorylation in the process of neurite outgrowth has been inferred from many studies of the effects of protein kinase inhibitors and activators on cultured neurotumor cells and primary neuronal cells from developing brain or ganglia. Here we re-examine this issue, using a culture system derived from a fully differentiated neuronal system undergoing axonal regeneration—the explanted goldfish retina following optic nerve crush. Of the relatively non-selective protein kinase inhibitors employed, H7, staurosporine and K_252a were found to block neurite outgrowth, whereas HA1004 had no effect, a result which appears to rule out a critical role for protein kinase A. The more selective protein kinase C inhibitors, sphingosine, calphostin C and Ro-31-8220 were all inhibitory, as was prolonged treatment with phorbol ester and the protein phosphatase inhibitor okadaic acid. These results are in support of a role for protein kinase C in axonal regrowth.