Diacylglycerol kinases: emerging downstream regulators in cell signaling systems

Diacylglycerol kinase (DGK) regulates signal transduction by modulating the balance between the two signaling lipids, diacylglycerol and phosphatidic acid. DGK and its homologs occur in a wide range of multicellular organisms and the mammalian DGK is known to consist of nine members with a considerable incidence of alternative splicing. Recent work has established that DGK serves as a key attenuator of diacylglycerol of signaling functions and that the mammalian isozymes are equipped with molecular machineries which enable them to act in specific intracellular sites and/or in signaling protein complexes.     

Effects of CuCl2 on the dimer-tetramer equilibrium of concanavalin A

Tetrameric concanavalin A at neutral pH dissociated into the dimer when CuCl2 was added in a concentration range comparable to protomer concentration. This effect of CuCl2 was largely suppressed when NaCl concentration was increased. Neither the conformation of the protein nor its binding activity to 4-methylumbelliferyl alpha-D-mannopyranoside was affected on addition of CuCl2.     

Purification and characterization of cytosolic diacylglycerol kinases of human platelets

Three isozymes of diacylglycerol kinase (DGK), DGK-I, DGK-II, and DGK-III, were purified from the cytosol of human platelets by successive chromatography on DEAE-cellulose, Ultrogel AcA34, heparin-Sepharose, ATP-agarose, Mono Q, phenyl-Superose, HCA-hydroxyapatite, Wakopak G40, and TSK-3000SW columns. Two DGK species (DGK-I and DGK-III) were purified to apparent homogeneity, and upon SDS-polyacrylamide gel electrophoresis, they showed a single band of apparent molecular mass of 152 kDa (DGK-I) or 58 kDa (DGK-III). The peptide mapping analysis showed that DGK-I and DGK-III are structurally different. DGK-II was only partially purified, and its apparent Mr was estimated to be 75,000 by gel filtration. The specific enzyme activities of the three isozymes were increased 1,480-fold (DGK-I), 690-fold (DGK-II) and 2,100-fold (DGK-III) over original platelet cytosol. The activities of DGK-II and DGK-III were markedly enhanced by the presence of deoxycholate or phosphatidylserine, whereas DGK-I activity was not much affected by the anionic compounds. All of the three activities were strongly suppressed by phosphatidylcholine. Triton X-100 and octyl glucoside were strongly inhibitory to all of the enzymes, although to different extents. The DGK inhibitor, R59022, inhibited DGK-II and to a lesser extent DGK-III, but little affected DGK-I activity. DGK-I was much more heat-stable than DGK-II and DGK-III. The Km values for ATP were 150 microM for DGK-I, 245 microM for DGK-II, and 450 microM for DGK-III. The apparent Km values for suspended diolein were not much different among the DGKs and were in the range of 50-80 microM. These observations indicate that human platelet cytosol contains DGK isozymes with different enzymological properties. Furthermore, the three DGKs isolated from human platelets were found not to cross-react with the antibody raised against porcine brain 80-kDa DGK, thus indicating that human platelets contain novel species of DGK.     

Structure-Function Analyses of Cytochrome P450revI Involved in Reveromycin A Biosynthesis and Evaluation of the Biological Activity of Its Substrate,Reveromycin T

Numerous cytochrome P450s are involved in secondary metabolite biosynthesis. The biosynthetic gene cluster for reveromycin A (RM-A), which is a promising lead compound with anti-osteoclastic activity, also includes a P450 gene, revI. To understand the roles of P450revI, we comprehensively characterized the enzyme by genetic, kinetic, and structural studies. The revI gene disruptants (ΔrevI) resulted in accumulation of reveromycin T (RM-T), and revI gene complementation restored RM-A production, indicating that the physiological substrate of P450revI is RM-T. Indeed, the purified P450revI catalyzed the C18-hydroxylation of RM-T more efficiently than the other RM derivatives tested. Moreover, the 1.4 Å resolution co-crystal structure of P450revI with RM-T revealed that the substrate binds the enzyme with a folded compact conformation for C18-hydroxylation. To address the structure-enzyme activity relationship, site-directed mutagenesis was performed in P450revI. R190A and R81A mutations, which abolished salt bridge formation with C1 and C24 carboxyl groups of RM-T, respectively, resulted in significant loss of enzyme activity. The interaction between Arg¹⁹⁰ and the C1 carboxyl group of RM-T elucidated why P450revI was unable to catalyze both RM-T 1-methyl ester and RM-T 1-ethyl ester. Moreover, the accumulation of RM-T in ΔrevI mutants enabled us to characterize its biological activity. Our results show that RM-T had stronger anticancer activity and isoleucyl-tRNA synthetase inhibition than RM-A. However, RM-T showed much less anti-osteoclastic activity than RM-A, indicating that hemisuccinate moiety is important for the activity. Structure-based P450revI engineering for novel hydroxylation and subsequent hemisuccinylation will help facilitate the development of RM derivatives with anti-osteoclast activity.     

Solubilization and purification of rat liver microsomal 1,2-diacylglycerol: CDP-choline cholinephosphotransferase and 1,2-diacylglycerol: CDP-ethanolamine ethanolaminephosphotransferase.

1. The procedure, which involved 2-step sonication of microsomes at pH 7.4 and then at pH 8.5 in the presence of sodium deoxycholate and subsequent dialysis, resulted in 4-5-fold purification of choline-phosphotransferase and ethanolaminephosphotransferase with the yield of 40-50%. 2. Ethanolaminephosphotransferase was further purified 8.5-fold over microsomes by sucrose density gradient centrifugation of the partially purified preparation, while cholinephosphotransferase activity was considerably lost during this procedure. No separation of the two transferases from each other was achieved at this step. 3. Cholinephosphotransferase required Mg2+ as cofactor, and microsomal phospholipids for its maximal activity. On the other hand, Mn2+ was more effective than Mg2+ as cofactor for ethanol aminephosphotransferase, and this enzyme was inhibited by microsomal phospholipids. 4. Both transferases were stimulated several-fold by sodium deoxycholate and also showed similar optimal pH ranging from pH 8.0 to 8.5. 5. Km values for 1,2-diacylglycerol emulsion were 81.0 muM for cholinephosphotransferase and 63.0 muM for ethanolaminephosphotransferase, respectively. CDP-choline and CDP-ethanolamine competitively inhibited, with the same Ki value (both 350 muM), ethanolaminephosphotransferase and cholinephosphotransferase, respectively. The Ki values obtained were much greater than the corresponding Km values for the cytidine substrates (36.4 muM for CDP-choline and 22.0 muM for CDP-ethanolamine). 6. The partially purified enzymes were further treated with Triton X-100. When enzyme activities were assayed with Mg2+, cholinephosphotransferase, although considerably inactivated, was partially separated from ethanolaminephosphotransferase by sucrose density gradient centrifugation of Triton-treated preparations. Furthermore, cholinephosphotransferase (but not ethanol-aminephosphotransferase) itself was partially separated into Mg2+ -requiring and Mn2+ -requiring components. In contrast, ethanolaminephosphotransferase assayed with either Mg2+ or Mn2+ formed a single peak together with Mn2+ -requiring cholinephosphotransferase.     

Thermostability of Rhodopseudomonas viridis and Rhodospirillum rubrum chromatophores reflecting physiological conditions

Relationships between growth conditions and thermostability were examined for photosynthetic inner membranes (chromatophores) from Rhodopseudomonas viridis and Rhodospirillum rubrum of which morphology, lipid composition, and protein/lipid rate are rather mutually different. Signals observed by differential scanning calorimetry of the chromatophores were correlated with thermal state transitions of the membrane components by reference to temperature dependencies of circular dichroism and absorption spectra of the purified supramolecule comprising a photoreaction center and surrounding light-harvesting pigment-protein complexes that are the prominent proteins in both membranes. The differential scanning calorimetry curves of those chromatophores exhibited different dependencies on growth stages and environmental temperatures. The obtained result appeared to reflect the differences in the protein/lipid rate and protein-lipid specificity between the two chromatophores.     

Diacylglycerol kinase gamma interacts with and activates beta2-chimaerin, a Rac-specific GAP, in response to epidermal growth factor

Diacylglycerol kinase (DGK)gamma was shown to act as an upstream suppressor of Rac1. Here we report that, in COS7 cells stimulated with epidermal growth factor (EGF), DGKgamma specifically interacts and co-localizes at the plasma membrane with beta2-chimaerin, a GTPase-activating protein (GAP) for Rac. Moreover, DGKgamma enhanced EGF-dependent translocation of beta2-chimaerin to the plasma membrane. Interestingly, DGKgamma markedly augmented EGF-dependent GAP activity of beta2-chimaerin through its catalytic action. These results indicate that DGKgamma is a novel regulator of beta2-chimaerin, and thus suggest that beta2-chimaerin is an effector molecule, linking DGKgamma functionally with Rac1.     

Discovery of a marine bacterium producing 4-hydroxybenzoate and its alkyl esters, parabens

Chemically synthesized 4-hydroxybenzoate (4HBA) is widely used in the chemical and electrical industries as a material for producing polymers such as those of the liquid crystal type. Its alkyl esters, called parabens, have been the most widely used preservatives by the food and cosmetic industries. We report here for the first time a microorganism, a marine bacterium, which biosynthesizes these petrochemical products. The marine bacterial strain, A4B-17, which was found to belong to the genus Microbulbifer on the basis of its rRNA and gyrB sequences, was isolated from an ascidian in the coastal waters of Palau. Strain A4B-17 was, surprisingly, found to produce 10 mg/liter of 4HBA, together with its butyl (24 mg/liter), heptyl (0.4 mg/liter), and nonyl (6 mg/liter) esters. We therefore characterized 23 other marine bacteria belonging to the genus Microbulbifer, which our institute had previously isolated from various marine environments, and found that these bacteria also produced 4HBA, although with low production levels (less than one-fifth of that produced by A4B-17). We also show that the alkyl esters of 4HBA produced by strain A4B-17 were effective in preventing the growth of yeasts, molds, and gram-positive bacteria.     

Characterization of four Rhodococcus alcohol dehydrogenase genes responsible for the oxidation of aromatic alcohols

Four genes were isolated and characterized for alcohol dehydrogenases (ADHs) catalyzing the oxidation of aromatic alcohols such as benzyl alcohol to their corresponding aldehydes, one from o-xylene-degrading Rhodococcus opacus TKN14 and the other three from n-alkane-degrading Rhodococcus erythropolis PR4. Various aromatic alcohols were bioconverted to their corresponding carboxylic acids using Escherichia coli cells expressing each of the four ADH genes together with an aromatic aldehyde dehydrogenase gene (phnN) from Sphingomonas sp. strain 14DN61. The ADH gene (designated adhA) from strain TKN14 had the ability to biotransform a wide variety of aromatic alcohols, i.e., 2-hydroxymethyl-6-methylnaphthalene, 2-hydroxymethylnaphthalene, xylene-α,α’-diol, 3-chlorobenzyl alcohol, and vanillyl alcohol, in addition to benzyl alcohol with or without a hydroxyl, methyl, or methoxy substitution. In contrast, the three ADH genes of strain PR4 (designated adhA, adhB, and adhC) exhibited lower ability to degrade these alcohols: these genes stimulated the conversion of the alcohol substrates by only threefold or less of the control value. One exception was the conversion of 3-methoxybenzyl alcohol, which was stimulated sevenfold by adhB. A phylogenetic analysis of the amino acid sequences of these four enzymes indicated that they differed from other Zn-dependent ADHs.The first two authors contributed equally to this work