Diacylglycerol kinase: a key modulator of signal transduction?

Diacylglycerol kinase (DGK) plays a central role in the metabolism of diacylglycerol released as a second messenger in agonist-stimulated cells. The major purified form of the enzyme (80 kDa DGK) is highly abundant in lymphocyte cytosol and may become membrane-associated via phosphorylation by protein kinase C. In addition, there are several kinase subspecies immunologically distinct from the 80 kDa enzyme, which differ markedly in their responses to several compounds such as sphingosine and R59022. Thus, further work on each enzyme species is needed to define the function of DGK in stimulated cells.

The different effects of sphingosine on diacylglycerol kinase isozymes in Jurkat cells, a human T-cell line.

We studied the effect of sphingosine on the activities of soluble and membrane-bound isozymes from Jurkat cells using combinations of different substrates (arachidonoyl- and didecanoyl DGs) and assay methods (octylglucoside mixed micellar and deoxycholate suspension assays). The results suggested the presence of at least four DGK isoforms, which could be distinguished from each other with respect to intracellular localization, specificity to DG molecular species, responsiveness to sphingosine, and reactivity to anti-80 kDa DGK antibody. We confirmed the presence of arachidonoyl DG-specific DGK in membranes, though this isozyme was not activated by sphingosine. We detected in the cytosol at least two species of sphingosine-activatable and non-activatable DGK isoforms, the major species being the 80 kDa DGK. We postulate that both or either of the two soluble DGKs may be the target of the sphingosine action.

Sphingosine activates cellular diacylglycerol kinase in intact Jurkat cells, a human T-cell line.

Sphingosine is known to regulate a variety of cellular functions through protein kinase C-dependent or independent pathways. In an attempt to investigate differential functions of diacylglycerol kinase (DGK) isozymes, we tested the effect of sphingosine on DGK operating in intact Jurkat cells, a human T-cell line. We found that phosphatidic acid (PA) synthesized from endogenous diacylglycerol (DG) and exogenously added short-chain DGs like dioctanoylglycerol were markedly enhanced by approx. 20 microM sphingosine. Further studies such as the use of protein kinase C down-regulated cells, mass measurements of cellular DGs, analysis of molecular species of PA and the effect of exogenous DG on the conversion of endogenous DG to PA suggested that sphingosine directly activated cellular DGK having a broad specificity toward DG molecular species.

Distinct specificity in the binding of inositol phosphates by pleckstrin homology domains of pleckstrin, RAC-protein kinase, diacylglycerol kinase and a new 130 kDa protein.

The pleckstrin homology domains (PH domains) derived from four different proteins, the N-terminal part of pleckstrin, RAC-protein kinase, diacylglycerol kinase and the 130 kDa protein originally cloned as an inositol 1,4,5-trisphosphate binding protein, were analysed for binding of inositol phosphates and derivatives of inositol lipids. The PH domain from pleckstrin bound inositol phosphates according to a number of phosphates on the inositol ring, i.e. more phosphate groups, stronger the binding, but a very limited specificity due to the 2-phosphate was also observed. On the other hand, the PH domains from RAC-protein kinase and diacylglycerol kinase specifically bound inositol 1,3,4,5,6-pentakisphosphate and inositol 1,4,5,6-tetrakisphosphate most strongly. The PH domain from the 130 kDa protein, however, had a preference for inositol 1,4,5-trisphosphate and 1,4,5,6-tetrakisphosphate. Comparison was also made between binding of inositol 1,4,5-trisphosphate, inositol 1,3,4,5-tetrakisphosphate and soluble derivatives of their corresponding phospholipids. The PH domains examined, except that from pleckstrin, showed a 8- to 42-times higher affinity for inositol 1,4,5-trisphosphate than that for corresponding phosphoinositide derivative. However, all PH domains had similar affinity for inositol 1,3,4,5-tetrakisphosphate compared to the corresponding lipid derivative. The present study supports our previous proposal that inositol phosphates and/or inositol lipids could be important ligands for the PH domain, and therefore inositol phosphates/inositol lipids may have the considerable versatility in the control of diverse cellular function. Which of these potential ligands are physiologically relevant would depend on the binding affinities and their cellular abundance.

Molecules in focus: diacylglycerol kinase.

Recent observations suggest that diacylglycerol kinase (DGK) is one of the key enzymes involved in the regulation of signal transduction. It attenuates protein kinase C activity and cell cycle progression of T-lymphocytes, through controlling the intracellular levels of the second messengers, diacylglycerol and phosphatidic acid. To date, eight DGK isozymes containing characteristic zinc finger structures in common have been identified. Type I DGKs (alpha, beta and gamma) contain EF-hand motifs that contribute to the calcium-dependent activities of this type of DGK. A pleckstrin homology and/or an EPH C-terminal tail homology domains are found in type II isozymes (DGK delta and eta). DGK epsilon represents a third type of DGK that selectively phosphorylates arachidonate-containing diacylglycerol. DGK zeta (type IV) and DGK theta (type V) contain four tandem ankyrin repeats and a Ras-associating domain, respectively.

Immunoquantitation of 80 kDa diacylglycerol kinase in pig and human lymphocytes and several other cells.

80 kDa diacylglycerol kinase (DGK) was immunoquantitated in cell homogenates and subcellular fractions. It was extremely abundant in the cytosol of various lymphocytes and comprised, in the highest case, more than 0.2% of the total soluble protein in T cell-enriched pig splenocytes. The lymphocyte membrane contained less than 10% of the total cellular DGK protein. The content of 80 kDa DGK in the human T cell leukemic cell line, Jurkat (360 ng/mg homogenate protein), was similar to those in pig and human peripheral blood lymphocytes. In contrast, the enzyme level was very low in the human promyeloblastic cell line, HL-60 (less than 10 ng/mg homogenate protein), and was undetectable in human polymorphonuclear leukocytes. These findings indicate that the content of 80 kDa DGK is markedly variable depending on the type of cells, even though all these cells are known to accumulate phosphatidate rapidly upon cell stimulation.

Different effects of sphingosine, R59022 and anionic amphiphiles on two diacylglycerol kinase isozymes purified from porcine thymus cytosol.

Porcine thymus cytosol contains two immunologically distinct forms of diacylglycerol kinase (DGK) [Yamada, K. and Kanoh, H. (1988) Biochem. J. 255, 601-608]. These 2 DGK species, having apparent molecular masses of 80 and 150 kDa, were purified from the thymus cytosol. Upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the 150-kDa DGK gave 2 polypeptide bands of 50 and 75 kDa, whereas the 80-kDa DGK yielded a single protein band. The 80-kDa DGK was markedly activated by 10-20 microM sphingosine as well as by the known anionic activators such as phosphatidylserine and deoxycholate. In contrast, the 150-kDa DGK was fully active in the absence of the anionic activators and was strongly inhibited by sphingosine (IC50, 20 microM). The putative DGK inhibitor R59022 inhibited the 80-kDa DGK (IC50, 10 microM), but had little effect on the 150-kDa form. It is therefore clear that in the thymus cytosol there are at least 2 DGK isozymes operating under different control mechanisms.

Translocation of diacylglycerol kinase alpha to the nuclear matrix of rat thymocytes and peripheral T-lymphocytes.

The cytosolic alpha-diacylglycerol kinase (DGK) was translocated to and tightly associated with the nuclear matrix when rat thymocytes and peripheral T-lymphocytes were stimulated with concanavalin A or anti-T-cell receptor antibody. This translocation occurred rather slowly and was completed in 3-4 h after cell stimulation. We also detected significant accumulation of nuclear phosphatidic acid interpreted as being formed by the translocated enzyme. The enzyme translocation is not directly linked to phosphoinositide turnover and protein phosphorylation, since phorbol myristate acetate and calcium ionophore did not affect the cellular DGK alpha and since we detected no covalent modification of the enzyme molecule. Although the mechanisms underlying the enzyme translocation remain unknown, our results indicate that DGK alpha participates in nuclear phospholipid metabolism occurring at the intermediate stage of lymphocyte activation.

Selectivity of the diacylglycerol kinase inhibitor 3-[2-(4-[bis-(4-fluorophenyl)methylene]-1-piperidinyl)ethyl]-2, 3-dihydro-2-thioxo-4(1H)quinazolinone (R59949) among diacylglycerol kinase subtypes.

Diacylglycerol kinases (DGKs) attenuate diacylglycerol-induced protein kinase C activation during stimulated phosphatidylinositol turnover. This reaction also initiates phosphatidylinositol resynthesis. Two agents, 3-(2-(4-[bis-(4-fluorophenyl)methylene]-1-piperidinyl)ethyl)-2,3-dihydro -2-thioxo-4(1H)quinazolinone (R59949) and 6-(2-(4-[(4-fluorophenyl)phenylmethylene]-1-piperidinyl)ethyl)-7-m ethyl-5H-thiazolo(3,2-a)pyrimidin-5-one (R59022), inhibit diacylglycerol phosphorylation in several systems. To examine the mechanism of this effect, we developed a mixed micelle method suitable for in vitro study of DGK inhibition. Animal cells express multiple DGK isoforms. In a survey of DGK isotypes, these agents selectively inhibited Ca2+-activated DGKs. R59949 was the more selective of the two. To map the site of interaction with the enzyme, a series of DGKalpha deletion mutants were prepared and examined. Deletion of the Ca2+-binding EF hand motif, which is shared by Ca2+-activated DGKs, had no effect on inhibition. Consistent with this observation, inhibition kinetics were noncompetitive with Ca2+. A construct expressing only the catalytic domain was also inhibited by R59949. Studies of substrate kinetics demonstrated that MgATP potentiated R59949 inhibition, indicating synergy of inhibitor and MgATP binding. These results indicate that R59949 inhibits DGKalpha by binding to its catalytic domain.

Gene cloning, sequence, expression and in situ localization of 80 kDa diacylglycerol kinase specific to oligodendrocyte of rat brain.

A 3.1 kbp cDNA clone encoding diacylglycerol (DG) kinase of 80 kDa (80K-DG kinase) was isolated from a rat brain cDNA library. The deduced amino acid sequence was 82% homologous to previously identified porcine 80K-DG kinase and contained zinc finger-like sequences, E-F hand motifs and ATP-binding sites similar to the porcine counterpart. By in situ hybridization histochemistry of rat brain at postnatal week 3, the expression signals for 80K-DG kinase mRNA appeared predominantly on somata of discrete cells in the white matter, and the expression pattern was similar to that of the myelin-specific proteins. In immunohistochemistry using the antibody against bacterially expressed DG kinase-fusion protein, numerous fibrous or dot-like structures exhibiting the immunoreactivity were concentrated in the white matter and they were arranged to radiate in the cerebral cortex and the cerebellar granular layer in a pattern almost identical to that of oligodendrocytes. No neuronal cells exhibited the immunoreactivity. The present finding thus strongly suggests that 80K-DG kinase is expressed specifically in the oligodendrocytes, but not neurons, and may be involved in the myelin formation and metabolism. In addition, the intense hybridization signals and the immunoreactivity for this protein were detected in the entire medulla of the thymus and the periarterial lymphatic area of the splenic white pulp both of which represent T-cell-dependent areas.

Purification and characterization of a membrane-bound NADPH-cytochrome c reductase capable of catalyzing menadione-dependent O2- formation in guinea pig polymorphonuclear leukocytes.

A membrane-bound NADPH-cytochrome c reductase, which is capable of forming the superoxide anion (O2-) in the presence of menadione, was highly purified from membrane fractions of disrupted guinea pig polymorphonuclear leukocytes by solubilization with 0.2% Triton X-100 and chromatographies on Sephacryl S-300 and 2',5'-ADP-agarose. The overall purification from the membrane fraction was over 110-fold, with a yield of about 6%. The purified preparation did not contain two other pyridine nucleotide-oxidizing enzymes: NADH- and NAD(P)H-oxidizing enzymes (J. Biochem. 94, 931-936, 1983). Besides cytochrome c, the purified enzyme was able to reduce menadione, Nitroblue tetrazolium (NBT) and 2,6-dichlorophenolindophenol. The reduction of menadione alone resulted in the formation of O2-. The purified enzyme preparation contained FAD. When assayed by measuring O2--generation in the presence of menadione, the enzyme showed an optimum pH at 7.0-7.4, and Km values for NADPH, NADH, and menadione were 25, 230, and 5.3 microM, respectively. The enzyme activity was not inhibited by NaN3 or dicumarol, but was by N-ethylmaleimide, EDTA, and quercetin; these inhibition profiles agree with those observed for the NADPH oxidase in the membrane fraction of phorbol-myristate acetate-stimulated leukocytes. Furthermore, when compared by means of the NBT-staining method combined with disc gel electrophoresis, the purified enzyme was electrophoretically indistinguishable from the NADPH-NBT reductase in the plasma membrane as well as phagosomes of the leukocytes. These results suggest that the purified NADPH-cytochrome c reductase is the putative flavoprotein of the NADPH oxidase system responsible for the respiratory burst.

Separation of three menadione-dependent, O2--generating, pyridine nucleotide-oxidizing enzymes in guinea pig polymorphonuclear leukocytes.

Pyridine nucleotide-oxidizing enzymes in guinea pig polymorphonuclear leukocytes were separated by Sephacryl S-300 gel filtration of the sonicated cells in the presence of 0.2% Triton X-100. Two peaks of NADPH-dependent cytochrome c reductase activities with apparent molecular weights of 400,000 and 120,000 were detected. The replacement of NADPH by NADH, on the other hand, revealed two NADH-dependent cytochrome c reductases with apparent molecular weights of 300,000 and 120,000. The addition of 40 microM menadione to assay mixtures considerably enhanced all the cytochrome c-reducing activities, and the enhancement was accompanied by the formation of superoxide anion (O2-). Analysis of the subcellular localizations of these enzymes by fractional centrifugation demonstrated that the NADPH-dependent enzyme (400,000 daltons) was membrane-bound in nature, and that the NADH-dependent enzyme (300,000 daltons) and the NADPH- and NADH-dependent enzyme (120,000 daltons) existed in the cytosol of leukocytes. Thus, the leukocytes contained at least three types of menadione-dependent, O2--forming enzymes: a membrane-bound NADPH-oxidizing enzyme, and soluble NADH-oxidizing and NAD(P)H-oxidizing enzymes.

Stabilizing effect of glutaraldehyde on the respiratory burst NADPH oxidase of guinea pig polymorphonuclear leukocytes.

The membrane fraction of guinea pig polymorphonuclear leukocytes stimulated with phorbol myristate acetate exhibits the respiratory burst NADPH oxidase activity. This activity is markedly unstable at 37 degrees C, disappearing with a half-life of 11.0 min. When the membrane fraction was pretreated with 0.1% glutaraldehyde, the NADPH oxidase was found to become more stable; its half-life increased about sixfold without any enhancement of the initial activity. The glutaraldehyde treatment of the membrane fraction also protected the NADPH oxidase against inactivation with 0.1-0.2% Triton X-100. These stabilizing effects of glutaraldehyde on the NADPH oxidase seem to be due to its protein cross-linking ability, since its monovalent analogue, butyraldehyde, did not show any effect on the NADPH oxidase activity. In fact, sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that glutaraldehyde cross-linked many proteins constituting the membrane.

Purification and characterization of NADPH-cytochrome c reductase from porcine polymorphonuclear leukocytes.

NADPH-cytochrome c reductase [NADPH: ferricytochrome oxidoreductase, EC 1.6.2.4] was highly purified from the membrane fraction of porcine polymorphonuclear leukocytes by column chromatographies on DEAE cellulose DE-52, 2',5'-ADP-agarose, Sephacryl S-300, and Bio-gel HTP. Upon sodium dodecyl sulfate polyacrylamide gel electrophoresis, the purified preparation gave a main band with a molecular weight of 80,000. The enzyme contained 0.79 mol of FAD and 0.88 mol of FMN per mol, and was capable of exhibiting a benzphetamine N-demethylation activity in the presence of cytochrome P-450 purified from rabbit liver microsomes and dilauroylphosphatidylcholine, as is the case with liver NADPH-cytochrome P-450 reductase. The cytochrome c reductase activity of the polymorphonuclear leukocytes (PMN) enzyme was precipitated with rabbit anti-guinea pig liver NADPH-cytochrome P-450 reductase IgG followed by addition of guinea pig anti-rabbit IgG antibody. The biochemical and immunological properties of the PMN enzyme so far examined were similar to those of the liver enzyme, although its function in leukocytes has not yet been determined.

Phosphorylation of diacylglycerol kinase in vitro by protein kinase C.

We investigated the effects of enzyme phosphorylation in vitro on the properties of diacylglycerol kinase. Diacylglycerol kinase and protein kinase C, both present as Mr-80,000 proteins, were highly purified from pig thymus cytosol. Protein kinase C phosphorylated diacylglycerol kinase (up to 1 mol of 32P/mol of enzyme) much more actively than did cyclic AMP-dependent protein kinase. Phosphorylated and non-phosphorylated diacylglycerol kinase showed a similar pI, approx. 6.8. Diacylglycerol kinase phosphorylated by either protein kinase C or cyclic AMP-dependent protein kinase was almost exclusively associated with phosphatidylserine membranes. In contrast, soluble kinase consisted of the non-phosphorylated form. The catalytic properties of the lipid kinase were not much affected by phosphorylation, although phosphorylation-linked binding with phosphatidylserine vesicles resulted in stabilization of the enzyme activity.