Two distinct membrane-bound phosphatidylinositol-4-phosphate phosphatases in bovine brain.

Solubilized phosphatidylinositol-4-phosphate 4-phosphatase from bovine brain resolved into two peaks of activity by ion exchange chromatography. Both exhibited substantial detergent binding characteristic of integral membrane proteins, and both appear specific for phosphatidylinositol-4-phosphate, but their pH optima differ: the earlier eluting fraction (peak 1) is optimally active between pH 5.5 and 6, whereas the later eluting fraction (peak 2) is most active around pH 8.5. Detergent inhibition studies suggest that peak 2, but not peak 1, interacts with phosphatidylinositol-4-phosphate in the context of a single mixed micelle. Further characterization of these activities should help shed light on the biological function of polyphosphoinositide phosphatases.

Roles of lipid turnover in transmembrane signal transduction.

Cells of higher organisms respond to external stimuli with a cascade of intracellular biochemical events initiated by binding of a hormone, growth factor, or neurotransmitter to a specific cell surface receptor. Previously well-characterized signal transduction pathways involve cyclic nucleotides as intracellular second messengers. Over the past decade, increasing attention has been focused on other signaling pathways in which membrane lipids serve as second messengers or their precursors. This review describes current understanding of these pathways and points to recent discoveries likely to open new frontiers in the coming decade.

Phase separation temperatures of mixtures of Triton X-114 and Triton X-45: application to protein separation.

Triton X-114 solutions separate above 22 degrees C into two immiscible aqueous phases. The more dense phase is enriched in detergent, and the less dense phase is depleted of detergent, relative to the original single phase. This phenomenon has been used to partition proteins according to hydrophobicity. The phase separation temperature is sensitive to the length of the polyoxyethylene headgroup. When Triton X-45, with a shorter headgroup, is mixed with Triton X-114 in various proportions, the phase transition temperature can be adjusted anywhere between 0 and 22 degrees C. Partitioning properties of the resulting mixtures are similar to those of Triton X-114 alone.

Bimodal lipid substrate dependence of phosphatidylinositol kinase.

Phosphatidylinositol (PI) kinase activity was solubilized from rat liver microsomes and partially purified by chromatography on hydroxyapatite and Reactive Green 19-Superose. Examination of the ATP dependence using a mixed micellar assay gave a Km of 120 microM. The dependence of reaction rate on PI was more complicated. PI kinase bound a large amount of Triton X-100, and as expected for a micelle-associated enzyme utilizing a micelle-associated lipid substrate, the reaction rate was dependent on the micellar mole fraction, PI/(PI + Triton X-100), with a Km of 0.02 (unitless). Activity showed an additional dependence on bulk PI concentration at high micelle dilution. These results demonstrated two kinetically distinguishable steps leading to formation of a productive PI/enzyme(/ATP) complex. The rate of the first step, which probably represents exchange of PI from the bulk micellar pool into enzyme-containing micelles, depends on bulk PI concentration. The rate of the second step, association of PI with enzyme within a single micelle, depends on the micellar mole fraction of PI. Depression of the apparent Vmax at low ionic strength suggested that electrostatic repulsion between negatively charged PI/Triton X-100 mixed micelles inhibits PI exchange, consistent with a model in which intermicellar PI exchange depends on micellar collisions.

Application of nonionic polyoxyethylene detergents in studies of phosphatidylinositol kinase.

Four commercial nonionic polyoxyethylene detergents were compared with Triton X-100 in terms of physicochemical properties and interaction with an integral membrane protein, phosphatidylinositol kinase. Tergitol 15-S-9, Tergitol TMN-10, polyoxyethylene 10 lauryl ether, and polyoxyethylene 10 tridecyl ether possess aliphatic hydrophobic moieties and therefore do not absorb significantly in the ultraviolet region. Like Triton X-100, these detergents have cloud points well above the physiological temperature range and partial specific volumes greater than 0.9 cm3/g. Critical micelle concentrations range from 0.02 to 0.03% with the exception of Tergitol TMN-10, for which the value is about 0.15%. All five detergents support activity of phosphatidylinositol kinase from rat liver in a mixed micellar assay and show similar ionic strength dependences for solubilization of the enzyme. Enzyme/detergent mixed micelles are somewhat smaller with Triton X-100 than for the other detergents. The data presented suggest that these detergents may be generally substituted for Triton X-100 in applications where uv invisibility is important.

Differential stimulation of the respiratory burst and lysosomal enzyme secretion in human polymorphonuclear leukocytes by synthetic diacylglycerols.

Binding of chemoattractants to receptors on human polymorphonuclear leukocytes (PMN) stimulates the phosphodiesteric cleavage of phosphatidylinositol 4,5-bisphosphate to produce inositol 1,4,5-trisphosphate and 1,2-diacylglycerols. To investigate the possible second messenger function of diacylglycerols in PMN activation, we tested the ability of a series of synthetic sn 1,2-diacylglycerols, known to stimulate protein kinase C in other systems, to promote superoxide anion release, oxygen consumption, lysosomal enzyme secretion, and chemotaxis. None of the diacylglycerols initiated the chemotactic migration of PMN. Several of the diacylglycerols however, were, active in stimulating superoxide anion release and lysozyme secretion, with dioctanoylglycerol (diC8) being the most potent. Unexpectedly, didecanoylglycerol (diC10) induced lysosomal enzyme secretion, but failed to stimulate superoxide production or oxygen consumption. All other biologically active diacylglycerols tested displayed similar EC50 for stimulating lysozyme secretion and superoxide production. The ability of the diacylglycerols to compete for phorbol dibutyrate (PDBu) binding in intact PMN suggested a mechanism for the divergent biological activity of diC10. Although the compounds that stimulated both superoxide production and lysosomal enzyme secretion competed for essentially all [3H]PDBu binding from its receptor, diC10, which only stimulated secretion, competed for 45% of the bound [3H]PDBu. Thus diacylglycerols can selectively activate certain functions of leukocyte chemoattractant receptor. The data suggest that a discrete pool of protein kinase C may mediate activation of the respiratory burst in PMN.

Attenuation of sn-1,2-diacylglycerol second messengers by diacylglycerol kinase. Inhibition by diacylglycerol analogs in vitro and in human platelets.

Diacylglycerol kinase is though to play a central role in the metabolism of diacylglycerol second messengers in agonist-stimulated cells. A series of diacylglycerol analogs were tested for their ability to act as substrates or inhibitors of diacylglycerol kinase with the goal of determining the substrate specificity of the enzyme, and of discovering inhibitors. Screening of these compounds was performed using a partially purified diacylglycerol kinase from pig brain. Modified assays for this enzyme using co-sonicated mixtures of diacylglycerol and anionic phospholipids were developed. This enzyme was found to be quite specific for sn-1,2-diacylglycerol (KM 24 microM for dioctanoyl-glycerol). Among the analogs investigated, only 1,2-dioctanoyl-2-amino-1,3-propanediol was utilized at a significant rate. Two analogs, dioctanoylethylene glycol (KI 58 microM) and 1-monooleoylglycerol (KI 91 microM), were potent inhibitors in vitro. These compounds were tested for effects on diacylglycerol formation and metabolism in thrombin-stimulated human platelets. Dioctanoylethylene glycol inhibited diacylglycerol phosphorylation in platelets (70-100% at 100 microM) leading to a longer-lived diacylglycerol signal. This compound may be a useful tool for studies of diacylglycerol kinase in other cell types. 1-Monooleoylglycerol treatment elevated diacylglycerol levels up to 4-fold in unstimulated platelets and up to 10-fold in thrombin-stimulated platelets. The implications with regard to the pathways of diacylglycerol metabolism in human platelets are discussed.

Specificity and mechanism of protein kinase C activation by sn-1,2-diacylglycerols.

The specificity of protein kinase C activation by sn-1,2-diacylglycerols and analogues was investigated by using a Triton X-100 mixed micellar assay [Hannun, Y. A., Loomis, C. R. & Bell, R. M. (1985) J. Biol. Chem. 260, 10039-10043]. Analogues containing acyl or alkyl chains eight carbons in length were synthesized because sn-1,2-dioctanoylglycerol is an effective cell-permeant activator of protein kinase C. These analogues were tested as activators and antagonists of rat brain protein kinase C to determine the exact structural features important for activity. The analogues established that activation of protein kinase C by diacylglycerols is highly specific. Several analogues established that both carbonyl moieties of the oxygen esters are required for maximal activity and that the 3-hydroxyl moiety is also required. None of the analogues were antagonists. These data, combined with previous investigations, permitted formulation of a model of protein kinase C activation. A three-point attachment of sn-1,2-diacylglycerol to the surface-bound protein kinase C-phosphatidylserine-Ca2+ complex is envisioned to cause activation. Direct ligation of diacylglycerol to Ca2+ is proposed to be an essential step in the mechanism of activation of protein kinase C.

Structural requirements for diacylglycerols to mimic tumor-promoting phobol diester action on the epidermal growth factor receptor.

The structural requirements for diacylglycerols to mimic the action of tumor-promoting phorbol diesters on the epidermal growth factor (EGF) receptor of A431 human epidermoid carcinoma cells were investigated. Five biological effects were considered: inhibition of high affinity 125I-EGF binding, change in the phosphorylation state of the EGF receptor, inhibition of the EGF-dependent tyrosine phosphorylation of the EGF receptor, inhibition of [3H]phorbol 12 beta, 13 alpha-dibutyrate binding, and stimulation of calcium- and phospholipid-dependent protein kinase (C-kinase) in vitro. A marked effect of the acyl chain length, 3-10 carbons, of symmetric sn-1,2-diacylglycerols was observed on their ability to mimic the effect of 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA). sn-1,2-Dipropanoylglycerol did not mimic the effects of PMA, but sn-1,2-didecanoylglycerol potently mimicked PMA action. A correlation was found between the ability of these diacylglycerols to stimulate the activity of C-kinase in vitro and to mimic the effects of PMA on the EGF receptor in intact cells. Analogues of sn-1,2-dioctanoylglycerol in which the 3' hydroxyl group was substituted with hydrogen, thio or chloro moieties were inactive when assayed for their ability to stimulate C-kinase in vitro and mimic PMA action in intact cells. We conclude that the hydroxyl group of a diacylglycerol is vital for the interaction with the phorbol diester receptor. The stringent correlation between the potency of the 11 diacylglycerol analogues tested to modulate C-kinase in vitro and to mimic PMA action in vivo provides strong evidence for the hypothesis that C-kinase plays a central role in the regulation of A431 cell EGF receptors by tumor-promoting phorbol diesters.

Role of protein kinase C in diacylglycerol-mediated induction of ornithine decarboxylase and reduction of epidermal growth factor binding.

Tumor-promoting phorbol esters induce ornithine decarboxylase (ODCase) activity and reduce epidermal growth factor (EGF) binding in rat tracheal epithelial 2C5 cells. Phorbol esters activate protein kinase C by interacting at the same site as sn-1,2-diacylglycerols, the presumed physiological regulators. The effects of added sn-1,2-diacylglycerols and those generated by phospholipase C treatment of 2C5 cells on ODCase induction and EGF binding were investigated to establish a role for protein kinase C in these cellular responses. Treatment of 2C5 cells with phospholipase C induced ODCase activity and reduced EGF binding, whereas phospholipases A2 and D were inactive. When sn-1,2-diacylglycerols containing fatty acids 3-10 carbons in length were added to 2C5 cells, those diacylglycerols containing fatty acids 5-10 carbons in length caused ODCase induction and reduction in EGF binding. sn-1,2-Dioctanoylglycerol was one of the most active compounds tested. It induced ODCase in a dose- (50-500 microM) and time-dependent manner. The reduction of binding of 125I-labeled EGF by sn-1,2-dioctanoylglycerol was also time and dose dependent and appeared to result from a change in EGF affinity and not the number of receptor sites. This series of sn-1,2-diacylglycerols showed similar structure-function relationships in their ability to induce ODCase activity, to decrease EGF binding, to stimulate protein kinase C, and to inhibit [3H]phorbol dibutyrate binding to the phorbol ester receptor. These data demonstrate biological activities for a number of diacylglycerols and indicate that protein kinase C activation is implicated in ODCase induction and decreased EGF binding.

sn-1,2-Dioctanoylglycerol. A cell-permeable diacylglycerol that mimics phorbol diester action on the epidermal growth factor receptor and mitogenesis.

The cell-permeable diacylglycerol, sn-1,2-dioctanoylglycerol (DiC8), is shown to mimic the effect of tumor promoting phorbol diesters on epidermal growth factor (EGF) binding and action in intact cells. DiC8 inhibited the binding of [3H]phorbol dibutyrate to A431 cell monolayers indicating that the diacylglycerol interacts with the phorbol diester receptor. At 0.3 microM, DiC8 half-maximally inhibited the high affinity binding of 125I-EGF to A431 human epidermoid carcinoma cells. Scatchard analysis indicated that the inhibition of 125I-EGF binding was very similar to that observed in the presence of 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA). DiC8 also mimicked the action of PMA to increase the phosphorylation state of the EGF receptor in 32P-labeled cells. Phosphoamino acid analysis demonstrated that DiC8 and PMA caused an increase in the level of EGF-receptor phosphoserine and phosphothreonine, whereas EGF caused an increase in the level of phosphoserine, phosphothreonine, and phosphotyrosine. Phosphopeptide mapping of the EGF receptor showed that DiC8 and PMA enhanced the phosphorylation of the same tryptic peptides. DiC8 inhibited the EGF-dependent tyrosine phosphorylation of the EGF receptor in A431 cells in a similar manner to that observed with PMA. In further experiments with quiescent Swiss 3T3 fibroblasts, DiC8 mimicked the ability of PMA to stimulate the incorporation of [methyl-3H]thymidine synergistically with low concentrations of EGF. This result indicates that DiC8 will mimic the long-term effects of PMA to regulate mitogenesis and raises the possibility that it may be active in two stage carcinogenesis. As both DiC8 and PMA stimulate the Ca2+- and phospholipid-dependent protein kinase (C-kinase) in vitro, the results support the hypothesis that the activation of C-kinase is a critical component of phorbol diester action on EGF receptor modulation and cell proliferation.

Exogenous sn-1,2-diacylglycerols containing saturated fatty acids function as bioregulators of protein kinase C in human platelets.

The ability of exogenous sn-1,2-diacylglycerols and analogs to function as bioregulators of protein kinase C in human platelets was investigated. The activation of protein kinase C in platelets is indicated by specific phosphorylation of a 40,000-dalton protein. Dihexanoylglycerol, dioctanoylglycerol (diC8), didecanoylglycerol, and sn-1-oleoyl-2-acetylglycerol were active in stimulating 40,000-dalton protein phosphorylation. Only a trace of phosphorylation was elicited by dibutyrylglycerol. Phosphorylation was not induced by analogs of diC8 in which an -H, -SH, or -Cl group replaced the free -OH, nor by monoacylglycerols or long chain diacylglycerols. Maximum phosphorylation was induced by dihexanoylglycerol, diC8, and didecanoylglycerol at concentrations from 5 to 20 microM and between 5 and 30 S after exposure of platelets to these diacylglycerols. Under conditions of maximal phosphorylation of the 40,000-dalton protein, these diacylglycerols did not induce phosphatidylinositol turnover, or platelet aggregation, or stimulate release of ATP or serotonin. A small degree of aggregation was evident with platelets isolated in the absence of prostacyclin, and release of serotonin was observed when 1 mM Ca2+ or submaximal concentrations of ionophore A23187 were included. These results are consistent with a model in which platelet activation requires the simultaneous formation of two intracellular signals, diacylglycerols and Ca2+. These diacylglycerols and diacylglycerol analogs provide useful tools to investigate the function of diacylglycerols as bioregulators in intact cells.

Diacylglycerols mimic phorbol diester induction of leukemic cell differentiation.

Activation of cellular protein kinase C appears to be involved in the mechanism by which phorbol diesters induce differentiation of human myeloid leukemia cells (HL-60). Protein kinase C is thought to be physiologically activated by diacylglycerol derived from receptor-mediated phosphatidylinositol hydrolysis. sn-1,2-diacylglycerols with short saturated acyl side chains (C4-C10) were synthesized and found to be potent activators of protein kinase C partially purified from HL-60 cells. These diacylglycerols were also competitive inhibitors of [3H]phorbol dibutyrate binding to the soluble phorbol diester receptor. The most potent diacylglycerol, sn-1,2-dioctanoylglycerol, displaced greater than 90% of [3H]phorbol dibutyrate from the phorbol diester receptor of intact HL-60 cells. Because of probable cellular metabolism of sn-1,2-dioctanoylglycerol, hourly doses were required to maintain persistent occupancy of the phorbol diester binding site. Treatment of HL-60 cells with either phorbol 12-myristate 13-acetate or sn-1,2-dioctanoylglycerol produced identical phosphoprotein changes. Finally, sn-1,2-dioctanoylglycerol induced differentiation of the HL-60 cells into cells with morphologic characteristics of macrophages. Substitution of the hydroxyl group at position 3 with a hydrogen, chloro, or sulfhydryl moiety inactivated sn-1,2-dioctanoylglycerol. These data strengthen the hypothesis that protein kinase C activation plays a role in macrophage differentiation.

Diacylglycerols release LH: structure-activity relations reveal a role for protein kinase C.

A series of diacylglycerols were synthesized with varying lengths and substituents in order to establish the structure-activity relationship between each with activation of protein kinase C and stimulation of a biological response system (pituitary luteinizing hormone release). This approach enables distinction between actions mediated by direct activation of protein kinase C and those due to other, presumably nonspecific, actions. The ability of diacylglycerols to function as regulators of a biological response system (pituitary luteinizing hormone release) and of protein kinase C was investigated with a series of sn-1,2 diacylglycerols containing fatty acids 4-10 carbons in length and with analogs in which the 3' hydroxyl was replaced with a chloro, hydrogen, or sulfhydryl moiety. Several diacylglycerols stimulated LH release in a saturable, time and dose dependent manner that was independent of extra-cellular calcium. Dioctanoylglycerol (diC8) was the most effective of the diacylglycerols tested; 3' analogs lacking the hydroxyl were inactive. The diacylglycerols activated protein kinase C in vitro whereas the 3' analogs did not. These data implicate protein kinase C in the mechanism of LH release, demonstrate that unsaturated fatty acyl moieties within the diacylglycerol are not required for protein kinase C activation, and establish diacylglycerol-protein kinase C structure-function relationships that should prove useful for investigations in other systems.

Escherichia coli membrane vesicles with elevated phosphatidic acid levels. A detergent-free system for in vitro phospholipid synthesis.

A new method for studying phospholipid biosynthesis in Escherichia coli is described. The method makes use of the previously reported observation that E. coli cytidine auxotrophs accumulate phosphatidic acid when starved of cytidine (Ganong, B. and Raetz, C.R.H. (1982) J. Biol. Chem. 257, 389-394). We now show that phosphatidic acid that accumulates in these cells is competent for further biosynthetic use in vivo, if cytidine is re-supplied to the cells. Furthermore, phosphatidic acid-rich membranes prepared from such cells can be used for in situ assays of the later steps of phospholipid biosynthesis. Since this system does not require detergent, our in situ assays more accurately reflect the conditions of an intact membrane. We have used this system to probe the regulation of the branch-point of the biosynthetic pathway for phospholipid polar headgroups. Phosphatidic acid-rich membranes prepared from cells that overproduce either phosphatidylserine synthase or phosphatidylglycerolphosphate synthase do not have increased rates of lipid synthesis in our in situ assays. This correlates with synthetic rates measured in vivo and, thus, our in situ assays accurately reflect conditions in a growing cell's membrane.

Transmembrane movement of phosphatidylglycerol and diacylglycerol sulfhydryl analogues.

Transmembrane movement of phospholipids is a fundamental step in the process of biological membrane assembly and intracellular lipid sorting. To facilitate study of transmembrane movement, we have synthesized analogues of phosphatidylglycerol and diacylglycerol in which a sulfhydryl group replaces a hydroxyl group in the polar head group. A rapid, continuous assay for the movement of phospholipids across single-walled lipid vesicles was developed that exploits the reactivity of these analogues toward 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), a nonpenetrating, colorimetric, sulfhydryl reagent. In the reaction of DTNB with vesicles containing phosphatidylthioglycerol, a phosphatidylglycerol analogue, two kinetic phases were seen, which represent the reaction of DTNB with phosphatidylthioglycerol in the outer and inner leaflets of the bilayer. Analysis of the slow second phase indicated that the half-time for phosphatidylthioglycerol transbilayer movement was in excess of 8 days. In a similar experiment using dioleoylthioglycerol, a diacylglycerol analogue, the reaction was complete within 15 s. The large difference in translocation rates between these two lipids indicates that the primary barrier to transmembrane movement is the polar head group and implies that phospholipid translocation events in biological membranes may not be unlike those for molecules similar to the polar head groups alone.

1,2-Diacylglycerols do not potentiate the action of phospholipases A2 and C in human platelets.

1,2-Diacylglycerol has recently been reported to potentiate the ability of phospholipases A and C to hydrolyze phospholipids in a cell-free system. The present study has been undertaken to investigate whether 1,2-diacylglycerol can also perform this function in intact cells using the platelet as a test system. Exogenous 1-oleoyl-2-acetyl-glycerol ( OAG ) and 1,2- didecanoylglycerol , at concentrations sufficient to produce maximal phosphorylation of a 40,000 dalton protein, caused no significant formation of [3H]inositol phosphates and [32P]phosphatidic acid (products of phospholipase C activation) or [14C]arachidonic acid metabolites and lysophosphatidyl[3H]inositol (products of phospholipase A2 activation). These data therefore imply that 1,2-diacylglycerols do not potentiate the actions of phospholipases A2 and C in intact platelets at concentrations that are physiologically relevant.

pH-sensitive CDP-diglyceride synthetase mutants of Escherichia coli: phenotypic suppression by mutations at a second site.

In Escherichia coli, mutations which lower the level of CDP-diglyceride synthetase are designated cds and map at min 4. The cds-8 mutation resulted in strikingly defective enzyme activity and also rendered cells pH sensitive for growth. Both the inhibition of growth and the massive accumulation of phosphatidic acid which occur in a cds-8 mutant at pH 8 were suppressed by mutations at a second locus, designated cdsS, which mapped between argG and gltB near min 68. The cdsS3 mutation by itself did not affect CDP-diglyceride synthetase activity in wild-type cells, but it caused a twofold stimulation of the residual activity present in strains harboring cds-8. Both the insensitivity to pH and the twofold stimulation of residual activity were lost by introduction of an F' strain carrying cdsS+ into a recA1 cds-8 cdsS3 host. When a culture of a cds-8 cdsS+ strain was shifted to pH 8, the residual specific activity of synthetase dropped by 75% within 100 min. In a cds-8 cdsS3 double mutant under the same conditions, the activity declined appreciably less, about to the level found in the cds-8 cdsS+ strain under permissive conditions (pH 6). Thus, it appears that mutations in the cdsS gene suppress the pH sensitivity of cds mutants by inhibiting the decay of residual CDP-diglyceride synthetase activity at the nonpermissive pH. The cdsS locus appears to be distinct from any known nonsense or missense suppressor.