坂根 郁夫

Diacylglycerol kinase (DGK) plays an important role in signal transduction through modulating the balance between two signaling lipids, diacylglycerol and phosphatidic acid. Here we identified a tenth member of the DGK family designated DGKκ. The κ-isozyme (1271 amino acids, calculated molecular mass, 142 kDa) contains a pleckstrin homology domain, two cysteine-rich zinc finger-like structures, and a separated catalytic region as have been found commonly for the type II isozymes previously cloned (DGKδ and DGKη). The new DGK isozyme has additionally 33 tandem repeats of Glu-Pro-Ala-Pro at the N terminus. Reverse transcriptase-PCR showed that the DGKκ mRNA is most abundant in the testis, and to a lesser extent in the placenta. DGKκ, when expressed in HEK293 cells, was persistently localized at the plasma membrane even in the absence of cell stimuli. Deletion analysis revealed that the short C-terminal sequence (amino acid residues 1199-1268) is necessary and sufficient for the plasma membrane localization. Interestingly, DGKκ, but not other type II DGKs, was specifically tyrosine-phosphorylated at Tyr78 through the Src family kinase pathway in H 2O2-treated cells. Moreover, H2O2 selectively inhibited DGKκ activity in a Src family kinase-independent manner, suggesting that the isozyme changes the balance of signaling lipids in the plasma membrane in response to oxidative stress. The expression patterns, subcellular distribution, and regulatory mechanisms of DGKκ are distinct from those of DGKκ and DGKκ despite high structural similarity, suggesting unique functions of the individual type II isozymes. © 2005 by The American Society for Biochemistry and Molecular Biology, Inc.

Diacylglycerol kinase (DGK) catalyzes phosphorylation of diacylglycerol to generate phosphatidic acid, and both molecules are known to serve as second messengers as well as important intermediates for the synthesis of various lipids. In this study, we investigated the spatiotemporal expression patterns of DGK isozymes together with the developmental changes of the mRNA expression and enzymatic property in rat lung. Northern blot and RT-PCR analyses showed that mRNAs for DGKalpha, -epsilon, and -zeta were detected in the lung. By immunohistochemical examination, DGKalpha and -zeta were shown to be coexpressed in alveolar type II cells and macrophages. Interestingly, these isozymes were localized at distinct subcellular locations, i.e., DGKalpha in the cytoplasm and DGKzeta in the nucleus, suggesting different roles for these isozymes. In the developing lung, the expression for DGKalpha and -zeta was transiently elevated on embryonic day 21 (E21) to levels approximately two- to threefold higher than on postnatal day 0 (P0). On the other hand, the expression for DGKepsilon was inversely elevated approximately twofold on P0 compared with that on E21. These unique changes in the expression pattern during the perinatal period suggest that each isozyme may play a distinct role in the adaptation of the lung to air or oxygen breathing at birth.

Female reproductive organs show remarkable cyclic changes in morphology and function in response to a combination of hormones. Evidence has accumulated suggesting that phosphoinositide turnover and the consequent diacylglycerol (DG) protein kinase C (PKC) pathway are intimately involved in these mechanisms. The present study has been performed to investigate the gene expression, cellular localization, and enzymatic activity of the DG kinase (DGK) isozymes that control the DG-PKC pathway. Gene expression for DGKalpha, -epsilon, -zeta, and -iota was detected in the ovary and placenta. Intense expression signals for DGKzeta and -alpha were observed in the theca cells and moderate signals in the interstitium and corpora lutea of the ovary. On the other hand, signals for DGKepsilon were seen more intensely in granulosa cells. In the placenta, signals for DGKalpha and -iota were observed in the junctional zone, whereas those for DGKzeta were detected in the labyrinthine zone. At higher magnification, the signals for DGKalpha were mainly discerned in giant cytotrophoblasts, and those for DGKiota were found in small cytotrophoblasts of the junctional zone. DGKzeta signals were observed in all cellular components of the labyrinthine zone, including mesenchyme, trabecular trophoblasts, and cytotrophoblasts. DGKepsilon signals were detected in the junctional zone on day 13 and 15 of pregnancy and were diffusely distributed both in the labyrinthine and junctional zones at later stages. The present study reveals distinct patterns of mRNA localization for DGK isozymes in the rat ovary and placenta, suggesting that each isozyme plays a unique role in distinct cell types in these organs.

To study the physiological function of diacylglycerol (DAG) kinase ι (DGKι), which converts DAG to phosphatidic acid, we deleted this gene in mice. In contrast to previous studies showing that DGK isoforms decrease Ras activity, signaling downstream of Ras in embryonic fibroblasts was significantly reduced in cells lacking DGKι. DGKs regulate Ras signaling by attenuating the function of the DAG-dependent Ras guanyl nucleotide-releasing proteins (RasGRPs). We tested whether DGKι inhibited the four known RasGRPs and found that it inhibited only RasGRP3. In addition to activating Ras, RasGRP3 also activates Rap1, which in some cases can antagonize the function of Ras. We demonstrate that DGKι bound to RasGRP3 and inhibited its activation of Rap1 by metabolizing DAG. This inhibition consequently affected Ras signaling. We tested the physiological consequence of deleting DGKι by crossing wild-type or DGKι-deficient mice with mice carrying a v-Ha-Ras transgene, and then we assessed tumor formation. We observed significantly fewer tumors in DGKι-deficient mice. Because Rap1 can antagonize the function of Ras, our data are consistent with a model in which DGKι regulates RasGRP3 with a predominant effect on Rap1 activity. Additionally, we found that DGKζ, which is structurally similar to DGKι, inhibited RasGRPs 1, 3, and 4 and predominantly affected Ras signaling. Thus, type IV DGKs regulate RasGRPs, but the downstream effects differ depending on the DGK. © 2005 by The National Academy of Sciences of the USA.

DGK (diacylglycerol kinase) regulates the concentration of two bioactive lipids, diacylglycerol and phosphatidic acid. DGKδ1 or its PH (pleckstrin homology) domain alone has been shown to be translocated to the plasma membrane from the cytoplasm in PMA-treated cells. In the present study, we identified Ser-22 and Ser-26 within the PH domain as the PMA- and epidermal-growth-factor- dependent phosphorylation sites of DGKδ1. Experiments in vitro and with intact cells suggested that the cPKC (conventional protein kinase C) phosphorylated these Ser residues directly. Puzzlingly, alanine/asparagine mutants at Ser-22 and Ser-26 of DGKδ1 and its PH domain are still persistently translocated by PMA treatment, suggesting that the PH domain phosphorylation is not responsible for the enzyme translocation and that the translocation was caused by a PMA-dependent, but cPKC-independent, process yet to be identified. Interestingly, the aspartate mutation, which mimics phosphoserine, at Ser-22 or Ser-26, inhibited the translocation of full-length DGKδ1 and the PH domain markedly, suggesting that the phosphorylation regulates negatively the enzyme translocation. Our results provide evidence of the phosphorylation of the DGKδ1 PH domain by cPKC, and suggest that the phosphorylation is involved in the control of subcellular localization of DGKδ1.

Nine diacylglycerol kinase (DGK) isozymes have been identified. However, our knowledge of their individual functions is still limited. Here, we demonstrate the role of DGKγ in regulating Rac1-governed cell morphology. We found that the expression of kinase-dead DGKγ, which acts as a dominant-negative mutant, and inhibition of endogenous DGKγ activity with R59949 induced lamellipodium and membrane ruffle formation in NIH3T3 fibroblasts in the absence of growth factor stimulation. Reciprocally, lamellipodium formation induced by platelet-derived growth factor was significantly inhibited upon expression of constitutively active DGKγ. Moreover, the constitutively active DGKγ mutant suppressed integrin-mediated cell spreading. These effects are isoform-specific because, in the same experiments, none of the corresponding mutants of DGKα and DGKβ, closely related isoforms, affected cell morphology. These results suggest that DGKγ specifically participates in the Rac1-mediated signaling pathway leading to cytoskeletal reorganization. In support of this, DGKγ co-localized with dominant-active Rac1 especially in lamellipodia. Moreover, we found that endogenous DGKγ was physically associated with cellular Rac1. Dominant-negative Rac1 expression blocked the lamellipodium formation induced by kinase-dead DGKγ, indicating that DGKγ acts upstream of Rac1. This model is supported by studies demonstrating that kinase-dead DGKγ selectively activated Rac1, but not Cdc42. Taken togeher, these results strongly suggest that DGKγ functions through its catalytic action as an upstream suppressor of Rac1 and, consequently, lamellipodium/ruffle formation.

Minor cellular lipids such as diacylglycerol, phosphatidic acid and lysophosphatidic acid are now established to act as lipid mediators regulating a wide range of cellular functions. To maintain orchestrated cellular functions, the concentration of such lipids needs to be strictly regulated by the balance of production and attenuation achieved by the action of metabolic enzymes. We are the first to report successful molecular cloning of two classes of enzymes, i. e. diacylglycerol kinase (DGK) and phosphatidic acid phosphatase (PAP). DGK has been disclosed to be involved in the attenuation of diacylglycerol-signal occurring within spatio-temporally regulated signaling complexes. PAP, on the other hand, acts as an ecto-enzyme degrading extracellular lipid signals like lysophosphatidic acid and sphingosine-1-phosphate, thus resulting in the attenuation of cell surface receptor-mediated lipid signaling. This review summarizes our recent work on the implications of DGK and PAP.

Diacylglycerol kinase (DGK) catalyzes phosphorylation of a second messenger diacylglycerol (DG) to phosphatidic acid in cellular signal transduction. Previous studies have revealed that DGK consists of a family of isozymes including our rat clones. In this study we isolated from rat brain cDNA library the cDNA clones for a rat homologue of DGKι (rDGKι-1) that contains two zinc finger-like sequences, the highly conserved DGK catalytic domain, a bipartite nuclear localization signal, and four ankyrin repeats at the carboxyl terminus. In addition, we found novel splice variants, which contain either insertion 1 (71 bp) or insertion 2 (19 bp) or both in the carboxyl-terminal portion. Each of the insertions causes a frameshift, and the resultant premature stop codons produce two truncated forms (termed rDGKι-2 and -ι-3), the former lacking the ankyrin repeats at the carboxyl terminus and the latter lacking a part of the catalytic domain and the ankyrin repeats. Truncation of the carboxyl-terminal portion clearly exerts effects on the detergent solubility and enzymatic activity of the splice variants, although all three variants showed similar cytoplasmic localization in cDNA-transfected cultured neurons despite the continued presence of the nuclear localization signal sequence. Immunoblot analysis using anti-rDGι antibody raised against the common amino-terminal portion clearly shows that these rDGKι variants are indeed expressed in the brain. These results suggest that the carboxyl-terminal truncated forms of rDGKι-2 and -ι-3 that exhibit reduced enzymatic activities might show a dominant negative effect against the intact rDGHι-1, and that the modulation of signal transduction by the splice variants may play some roles in the physiologic and/or pathologic conditions of neurons.

Lipid phosphate phosphatases (LPPs) are integral membrane proteins with six transmembrane domains that act as ecto-enzymes dephosphorylating a variety of extracellular lipid phosphates. Using polarized MDCK cells stably expressing human LPP1 and LPP3, we found that LPP1 was located exclusively at the apical surface whereas LPP3 was distributed mostly in the basolateral subdomain. We identified a novel apical sorting signal at the N-terminus of LPP1 composed of F(2)DKTRL(7). In the case of LPP3, a dityrosine motif present in the second cytoplasmic portion was identified as basolateral targeting signal. Our work shows that LPP1 and LPP3 are equipped with distinct sorting signals that cause them to differentially localize to the apical vs. the basolateral subdomain, respectively.

Diacylglycerol kinase (DGK) participates in regulating the intracellular concentrations of two bioactive lipids, diacylglycerol and phosphatidic acid. DGKη(η1, 128 kDa) is a type II isozyme containing a pleckstrin homology domain at the amino terminus. Here we identified another DGKη isoform (η2, 135 kDa) that shared the same sequence with DGKη1 except for a sterile α motif (SAM) domain added at the carboxyl terminus. The DGKη1 mRNA was ubiquitously distributed in various tissues, whereas the DGKη2 mRNA was detected only in testis, kidney, and colon. The expression of DGKη2 was suppressed by glucocorticoid in contrast to the marked induction of DGKη1. DGKη2 was shown to form through its SAM domain homo-oligomers as well as hetero-oligomers with other SAM-containing DGKs (δ1 and δ2). Interestingly, DGKη1 and DGKη2 were rapidly translocated from the cytoplasm to endosomes in response to stress stimuli. In this case, DGKη1 was rapidly relocated back to the cytoplasm upon removal of stress stimuli, whereas DGKη2 exhibited sustained endosomal association. The experiments using DGKη mutants suggested that the oligomerization of DGKη2 mediated by its SAM domain was largely responsible for its sustained endosomal localization. Similarly, the oligomerization of DGKη2 was suggested to result in negative regulation of its catalytic activity. Taken together, alternative splicing of the human DGKη gene generates at least two isoforms with distinct biochemical and cell biological properties responding to different cellular metabolic requirements.

Although nine diacylglycerol kinase (DGK) isozymes have been identified, our knowledge of their individual functions is still limited. Here we report that the levels of DGKγ mRNA/protein in human leukemia HL-60 and U937 cells were rapidly and markedly decreased upon cellular differentiation into macrophages. In contrast, the enzyme expression remained almost unchanged in granulocytic differentiation pathway. Interestingly, the overexpression of wild-type or constitutively active DGKγ, but not its kinase-dead mutant, markedly inhibited phorbol ester-induced cell attachment and nonspecific esterase activity, which are hallmarks of macrophage differentiation. We noted in this case that no effects were observed for the corresponding constructs of a closely related isozyme, DGKα. Prior to the cell attachment, phorbol ester induced translocation of DGKγ from the cytoplasm to the cell periphery, resulting in its co-localization with F-actin together with protein kinase Cδ. The results suggest that DGKγ negatively regulates macrophage differentiation through its catalytic action operating on the cytoskeleton. © 2003 Elsevier Science (USA). All rights reserved.

Diacylglycerol kinase (DGK) plays an important role in signal transduction through modulating the balance between two signaling lipids, diacylglycerol and phosphatidic acid. DGKδ (type II isozyme) contains a pleckstrin homology domain at the N terminus and a sterile α motif domain at the C terminus. We identified another DGKδ isoform (DGKδ2, 135 kDa) that shared the same sequence with DGKδ previously cloned (DGKδ1, 130 kDa) except for the 52 residues N-terminally extended. Analysis of panels of human normal and tumor tissue cDNAs revealed that DGKδ2 was ubiquitously expressed in all normal and tumor tissues examined, whereas the transcript of DGKδ1 was detected only in ovary and spleen, and in a limited set of tumor-derived cells. The expression of DGKδ2 was induced by treating cells with epidermal growth factor and tumor-promoting phorbol ester. In contrast, the levels of mRNA and protein of DGKδ1 were suppressed by phorbol ester treatment. It thus becomes clear that the two DGKδ isoforms are expressed under distinct regulatory mechanisms. DGKδ1 was translocated through its pleckstrin homology domain from the cytoplasm to the plasma membrane in response to phorbol ester stimulation, whereas DGKδ2 remained in the cytoplasm even after stimulation. Further experiments showed that the δ2-specific N-terminal sequence blocks the phorbol ester-dependent translocation of this isoform. Co-immunoprecipitation analysis of differently tagged DGKδ1 and DGKδ2 proteins showed that they were able to form homo- as well as hetero-oligomers. Taken together, alternative splicing of the human DGKδ gene generates at least two isoforms, differing in their expressions and regulatory functions.

Diacylglycerol kinase (DGK) plays an important role in signal transduction through modulating the balance between two signaling lipids, diacylglycerol and phosphatidic acid. In yeast two-hybrid screening, we unexpectedly found a self-association of the C-terminal part of DGKδ containing a sterile α-motif (SAM) domain. We then bacterially expressed the SAM domain fused with maltose-binding protein and confirmed the formation of dimeric and tetrameric structures. Moreover, gel filtration and co-immunoprecipitation analyses demonstrated that DGKδ formed homo-oligomeric structures in intact cells and that the SAM domain was critically involved in the oligomerization. Interestingly, phorbol ester stimulation induced dissociation of the oligomeric structures with concomitant phosphorylation of DGKδ. Furthermore, we found that DGKδ was translocated from cytoplasmic vesicles to the plasma membrane upon phorbol ester stimulation. In this case, DGKδ mutants lacking the ability of self-association were localized at the plasma membranes even in the absence of phorbol ester. A protein kinase C inhibitor, staurosporine, blocked all of the effects of phorbol ester, i.e. oligomer dissociation, phosphorylation, and translocation. We confirmed that tumor-promoting phorbol esters did not directly bind to DGKδ. The present studies demonstrated that the formation and dissociation of oligomers serve as the regulatory mechanisms of DGKδ and that DGKδ is a novel downstream effector of phorbol ester/protein kinase C signaling pathway.

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.

Arachidonoyldiacylglycerol (20:4-DAG) is a second messenger derived from phosphatidylinositol 4,5-bisphosphate and generated by stimulation of glutamate metabotropic receptors linked to G proteins and activation of phospholipase C. 20:4-DAG signaling is terminated by its phosphorylation to phosphatidic acid, catalyzed by diacylglycerol kinase (DGK). We have cloned the murine DGKε gene that showed, when expressed in COS-7 cells, selectivity for 20:4-DAG. The significance of DGKε in synaptic function was investigated in mice with targeted disruption of the DGKε. DGKε-/- mice showed a higher resistance to eletroconvulsive shock with shorter tonic seizures and faster recovery than DGKε+/+ mice. The phosphatidylinositol 4,5-bisphosphate-signaling pathway in cerebral cortex was greatly affected, leading to lower accumulation of 20:4-DAG and free 20:4. Also, long-term potentiation was attenuated in perforant path-dentate granular cell synapses. We propose that DGKε contributes to modulate neuronal signaling pathways linked to synaptic activity, neuronal plasticity, and epileptogenesis.

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-methyl-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 DGK alpha 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 DGK alpha by binding to its catalytic domain. BIOCHEM PHARMACOL 59;7:763-772, 2000. (C) 2000 Elsevier Science Inc.

Myosin II was identified as a binding protein to the pleckstrin homology (PH) domain of protein kinase B (PKB) in CHO cell extract by using the glutathione S-transferase-fusion protein as a probe. When myosin II purified from rabbit skeletal muscle was employed, myosin II was shown to bind almost exclusively to the PH domain of PKB among the PH domain fusion proteins examined. The purified myosin II bound to the PH domain of PKB with a K-d value of 1.1 x 10(-7) M. Studies with a series of truncated molecules indicated that the whole structure of the PH domain is required for the binding of myosin II, and the binding to the PH domain was inhibited by phosphatidylinositol 4,5-bisphosphate. These results suggest that myosin II is a specific binding protein to the PH domain of particular proteins including PKB. (C) 1999 Academic Press.

The pleckstrin homology domains (PH domains) derived from four different proteins, the N-terminal part of pleckstrin, RAG-protein kinase, diacylglycerol kinase and the 130kDa 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 RAG-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,il,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 1,3,3,5-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. (C) 1997 Elsevier Science B.V.

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. (C) 1997 Elsevier Science Ltd.

Calnexin, an abundant membrane protein, and its lumenal homolog calreticulin interact with nascent proteins in the endoplasmic reticulum, Because they have an affinity for monoglucosylated N-linked oligosaccharides which can be regenerated from the aglucosylated sugar, it has been speculated that this repeated oligosaccharide binding may play a role in nascent chain folding, To investigate the process, we have developed a novel assay system using microsomes freshly prepared from pulse labeled HepG2 cells, Unlike the previously described oxidative folding systems which required rabbit reticulocyte lysates, the oxidative folding of transferrin in isolated microsomes could be carried out in a defined solution, In this system, addition of a glucose donor, UDP-glucose, to the microsomes triggered glucosylation of transferrin and resulted in its cyclic interaction with calnexin and calreticulin. When the folding of transferrin in microsomes was analyzed, UDP-glucose enhanced the amount of folded transferrin and reduced the disulfide-linked aggregates, Analysis of transferrin folding in briefly heat-treated microsomes revealed that UDP-glucose was also effective in elimination of heat-induced misfolding, Incubation of the microsomes with an alpha-glucosidase inhibitor, castanospermine, prolonged the association of transferrin with the chaperones and prevented completion of folding and, importantly, aggregate formation, particularly in the calnexin complex, Accordingly, we demonstrate that repeated binding of the chaperones to the glucose of the transferrin sugar moiety prevents and corrects misfolding of the protein.

We obtained two human cDNA clones encoding phosphatidic acid phosphatase (PAP) isozymes named PAP-2a (M-r = 32,158) and -2b (M-r = 35, 119), both of which contained six putative transmembrane domains, Both enzymes were glycosylated and cleaved by N glycanase and endo-beta-galactosidase, thus suggesting their post-Golgi localization. PAP-Za and -2b shared 47% identical sequence and were judged to be the human counterparts of the previously sequenced mouse 35-kDa PAP(83% identity) and rat Dri42 protein (94% identity), respectively, Furthermore, the sequences of both PAPs were 34-39% identical to that of Drosophila Wunen protein. In view of the functions ascribed to Wunen and Dri42 in germ cell migration and epithelial differentiation, respectively, these findings unexpectedly suggest critical roles of PAP isoforms in cell growth and differentiation, Although the two PAPs hydrolyzed lysophosphatidate and ceramide-l-phosphate. in addition to phosphatidate, the hydrolysis of sphingosine-l-phosphate was detected only for PAP-2b, PAP-2b was expressed almost ubiquitously in all human tissues examined, whereas the expression of PAP-2a was relatively variable, being extremely low in the placenta and thymus, In HeLa cells, the transcription of PAP-2a was not affected by different stimuli, whereas PAP-2b was induced (up to 3-fold) by epidermal growth factor. These findings indicate that despite structural similarities, the two PAP isozymes may play distinct functions through their different patterns of substrate utilization and transcriptional regulation.

The three diacylglycerol kinase isoenzymes (DGKα, DGKβ and DGKγ) cloned so far contain in common a tandem repeat of EF-hand motifs. However, the Ca2+ dependences of the DGK activities are known to be variable between isoenzymes, and the Ca2+-binding activities of these motifs have not been tested except for those present in DGKα. We therefore attempted to define the intrinsic properties of EF-hands occurring in the DGK isoenzymes. For this purpose we bacterially expressed and purified the EF-hand motifs (termed DKE forms) of the three DGKs. Equilibrium dialysis with the purified DKE forms showed that all of the expressed proteins could bind approx. 2 mol of Ca2+ per mol. However, the apparent dissociation constant (K(d)) for calcium binding to α-DKE (9.9 μM) was an order of magnitude greater than those estimated for β-DKE (0.89 μM) and γ-DKE (0.40 μM). Experiments with 2-p-toluidinyl-naphthalene 6-sulphonate, a probe for hydrophobic regions of proteins, showed that the binding of Ca2+ to β-DKE resulted in the exposure of hydrophobic amino acids, whereas hydrophobic regions of α-DKE and γ-DKE were masked by the addition of Ca2+. Taken together, these results indicate that DGKα, DGKβ and DGKγ possess EF-hand structures with intrinsic properties different from each other with respect to affinities for Ca2+ and Ca2+-induced conformational changes.

All mammalian diacylglycerol kinase (DGK) isoenzymes so far cloned consist of four conserved regions, namely C1, C2 (tandem EF-hand structures), C3 (tandem cysteine-rich zinc finger sequences) and the C-terminal C4 domains. To determine the catalytic domain we expressed in COS-7 cells various truncation mutants of pig DGKα and assessed their enzyme activities. We found that the C4 domain lacking the whole N-terminal region including the zinc fingers possessed DGK activity that was dependent on the concentrations of diacylglycerol and ATP very similarly, as did the wild-type DGKα. Furthermore the DGK activity of the wild-type DGK and that expressed by the C4 domain were similarly activated by anionic amphiphiles such as phosphatidylserine, phosphatidylinositol and deoxycholate. It was also shown that a DGK mutant consisting of the zinc fingers and the C4 domain has enzymological properties very similar to those expressed by the C4 domain alone. We also confirmed that the intact DGKs α, β and γ expressed in COS-7 cells displayed no detectable phorbol ester binding. These results show that the C4 domain of DGK is the catalytic region that is responsible for the enzyme activities sensitive to different activators. We cannot exclude the possibility that the N-terminal portion including the zinc fingers can still interact with diacylgIycerol and activators without affecting the enzyme activity measured in vitro. However, it is quite likely that the DGK zinc fingers do not serve as diacylglycerol-binding sites, in contrast with those present in other proteins such as protein kinases C and n-chimaerin. Site-directed mutagenesis of all six putative ATP binding sites (Lys248, Lys383, Lys395, Lys483, Lys492, and Lys554) did not significantly affect the enzyme activity. We therefore suggest that DGK does not contain a typical P-loop of ATP binding sites.

The cytosolic α-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 DGKα and since we detected no covalent modification of the enzyme molecule. Although the mechanisms underlying the enzyme translocation remain unknown, our results indicate that DGKα participates in nuclear phospholipid metabolism occurring at the intermediate stage of lymphocyte activation.

We previously described the purification of an 83-kDa phosphatidic acid phosphatase (PAP) from the porcine thymus membranes (Kanoh, H., Imai, S.-i., Yamada, K. and Sakane, F. (1992) J. Biol. Chem. 267, 25309-25314). However, we found that a minor 35-kDa protein could account for the PAP activity when the purified enzyme preparation was further analyzed. We thus determined the N-terminal sequence of the 35-kDa candidate protein and prepared antipeptide antibody against the determined sequence, MFDKTRLPYVALDVL. The antibody almost completely precipitated the purified enzyme activity. Furthermore, the antibody precipitated from the radioiodinated enzyme preparation a single 35- kDa protein, which was converted to a 29-kDa form when treated with N- glycanase. We also found that the immunoprecipitable PAP activity was exclusively associated with the plasma membranes of porcine thymocytes. These results indicated that the 35-kDa glycosylated protein represents the plasma membrane-bound (type 2) PAP. We surprisingly noted that the N-terminal sequence of the porcine PAP was almost completely conserved in the internal sequence encoded by a mouse partial cDNA clone, hic53, reported as a H2O2- inducible gene (Egawa, K., Yoshiwara, M., Shibanuma, M., and Nose, K. (1995) FEBS Lett. 372, 74-77). We thus amplified from the mouse kidney RNA the hic53 clone by polymerase chain reaction, and obtained a cDNA encoding a novel protein of 283 amino acid residues with a calculated M(r) of 31,894. Methionine reported as an internal residue was found to serve as an initiator, and the C-terminal 64 residues were lacking in hic53. The protein contains several putative membrane-spanning domains and two N-glycosylation sites. When transfected into 293 cells, the cDNA gave more than 10-fold increase of the membrane-bound PAP activity, which could be precipitated by the antipeptide antibody. In [35S]methionine-labeled cells, the translational product was confirmed to be a 35-kDa protein, which became 30 kDa in cells treated with tunicamycin, an inhibitor of N-glycosylation. We thus succeeded first in identifying the porcine type 2 PAP and subsequently in determining the primary structure of a mouse homolog of the PAP.

A fourth member of the diacylglycerol kinase (DGK) gene family termed DGK delta was cloned from the human testis cDNA library. The cDNA sequence contains an open reading frame of 3,507 nucleotides encoding a putative DGK protein of 130,006 Da. Interestingly, the new DGK isozyme contains a pleckstrin homology domain found in a number of proteins involved in signal transduction. Furthermore, the C-terminal tail of this isozyme is very similar to those of the EPH family of receptor tyrosine kinases. The primary structure of the delta-isozyme also has two cysteine-rich zinc finger-like structures (C3 region) and the C-terminal C4 region, both of which have been commonly found in the three isozymes previously cloned (DGKs alpha, beta and gamma). However, DGK delta lacks the EF-hand motifs (C2) and contains a long Glu- and Ser-rich insertion (317 residues), which divides the C4 region into two portions. Taken together, these structural features of DGK delta indicate that this isozyme belongs to a DGK subfamily distinct from that consisting of DGKs alpha, beta, and gamma. Increased DGK activity without marked preference to arachidonoyl type of diacylglycerol was detected in the particulate fraction of COS-7 cells expressing the transfected DGK delta cDNA. The enzyme activity was independent of phosphatidylserine, which is a common activator for the previously sequenced DGKs. Northern blot analysis showed that the DGK delta mRNA (similar to 6.3 kilobases) is most abundant in human skeletal muscle but undetectable in the brain, thymus, and retina. This expression pattern is different from those of the previously cloned DGKs. Our results show that the DGK gene family consists of at least two subfamilies consisting of enzymes with distinct structural characteristics and that each cell type probably expresses its own characteristic repertoire of DGKs whose functions may be regulated through different signal transduction pathways.

A unique type of chaperone that requires glucose trimming of the target proteins has been shown to be important for their maturation in the endoplasmic reticulum (ER). Calnexin, an ER membrane chaperone, is the first example of such a class. Here, we focus on calreticulin, a major ER luminal protein, which shares with calnexin two sets of characteristic sequence repeat. We evaluated the chaperone function of calreticulin by expressing it on the ER luminal membrane surface. We constructed a membrane-anchored calreticulin chimera by fusing truncated calreticulin to the membrane- anchoring tagged segment of calnexin. When expressed in HepG2 cells, the calreticulin chimera transiently interacted with a set of nascent secretory proteins in a castanospermine-sensitive manner. The spectrum of proteins recognized by the membrane-anchored calreticulin was remarkably similar to that observed with calnexin. Next, we tested if such a chaperone function of calreticulin is expressed at its physiological location. Luminally expressed calreticulin preferentially bound to nascent transferrin and released it upon chase. Association with ether calnexin ligands was observed, however, at low efficiencies. Interactions were abrogated by castanospermine treatment. We conclude that calreticulin per se is another chaperone with apparently the same characteristics as calnexin and selectively interacts with nascent transferrin in the lumen, suggesting that calreticulin may cover the diversity of maturations.

In order to clone novel diacylglycerol kinase (DGK) isozymes, we first obtained a DGK-related cDNA fragment by polymerase chain reaction using the human hepatoma cell line HepG2 mRNA and degenerated primers. The amplified fragment was subsequently used as a probe for screening the cDNA library from HepG2 cells. We obtained a cDNA clone coding for a novel DGK isozyme (designated DGK gamma) comprised of 791 amino acid residues. The amino acid sequence of DGK gamma was 52 and 62% identical to those of previously sequenced porcine 80-kDa and rat 90-kDa enzymes, respectively. DGK gamma, although initially cloned from the HepG2 cDNA libraries, was unexpectedly expressed in the human retina abundantly and to a much lesser extent in the brain. Other human tissues, including the liver and HepG2 cells, contained extremely low levels of DGK gamma mRNA. Furthermore, HepG2 cells and most of the human tissues except for the retina and brain expressed a truncated DGK gamma with an internal deletion of 25 amino acid residues (Ile(451)-Gly(475)). When transfected into COS-7 cells, the nontruncated cDNA gave phosphatidylserine-dependent DGK activity with no apparent specificity with regard to the acyl compositions of diacylglycerol. In contrast the truncated cDNA failed to give DGK activity in spite of the expression of its mRNA and enzyme protein in COS cells, thus demonstrating that the truncated DGK gamma is catalytically inactive. The sequence comparison of the three cloned DGKs revealed the presence of four highly conserved regions including the two sets each of EF-hand and zinc finger structures. Although the implication of the catalytically inactive form of DGK gamma remains unknown, this work further demonstrates the occurrence of multiple animal DGK isozymes with a conserved basic structure but with markedly different expression patterns depending on the cell types.

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. © 1993.

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 μM 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. © 1993.

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 muM 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.

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.

The 80 kDa diacylglycerol kinase (DGK) is abundantly expressed in oligodendrocytes and lymphocytes but not to a detectable extent in other cells such as neurons and hepatocytes. As an initial attempt to delineate the mechanism of the transcriptional control of the DGK gene, we have cloned from a human genomic library a 22 kb genomic fragment. The genomic clone consists of the 5'-flanking region and 17 exons coding for approx. 53% of the total exons of human DGK, including those encoding EF-hand and zinc-finger regions. The translation initiation site is located in the second exon. S1 nuclease mapping and primer extension analysis of the human DGK mRNA identified a major transcription initiation site (position +1) at 264 bp upstream from the initiator ATG. In the 5'-flanking sequence we detected a single GC box at -35 but no canonical TATA and CAAT sequences. However, the sequence starting from the cap site (AGTTCCTGCCA) is very similar to the initiator element that specifies the transcription initiation site of some housekeeping genes. In addition, the 5'-upstream region contains several putative cis-elements. Jurkat and HepG2 cells were transfected with various 5'-deletion mutants of the upstream region fused to the structural gene of chloramphenicol acetyltransferase (CAT). The CAT assay revealed that among constructs containing up to 3.4 kb of the 5'-flanking region, a fragment of 263 bp from the transcription initiation site contains a basic promoter that is active in both types of cells. Moreover, the region between -263 and -850 contains a negative element that is active in HepG2 but not in Jurkat cells. This negative element may, at least in part, be responsible for the cell type-specific expression of the DGK gene.

We purified phosphatidic acid phosphatase (EC 3.1.3.4) 2300-fold from porcine thymus membranes. The enzyme was solubilized with beta-octyl glucoside and Triton X-100 and fractionated with ammonium sulfate. The purification was then achieved by chromatography in the presence of Triton X-100 with Sephacryl S-300, hydroxylapatite, heparin-Sepharose, and Affi-Gel Blue. The final enzyme preparation gave a single band of M(r) = 83,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing and nonreducing conditions. The native enzyme, on the other hand, was eluted at M(r) = 218,000 in gel filtration chromatography with Superose 12 in the presence of Triton X-100. The enzyme was judged to be specific to phosphatidic acid, since excess amounts of dicetylphosphate or lysophosphatidic acid did not inhibit the enzyme activity. In this respect, the enzyme was inhibited by 1,2-diacylglycerol but not by 1- or 2-monoacylglycerol and triacylglycerol. The enzyme required Triton X-100 or deoxycholate for its activity. Although the enzyme appeared to be an integral membrane protein, we could not detect its phospholipid dependencies. The activity was independent of Mg2+, and other cations were strongly inhibitory. The specific enzyme activity was 15 mumol/min/mg of protein when assayed using phosphatidic acid as Triton X-100 mixed micelles. The K(m) for the surface concentration of phosphatidic acid was 0.30 mol %. The enzyme was inhibited by sphingosine and chlorpromazine, and less potently, by propranolol and NaF. The enzyme was insensitive to thio-reactive reagents like N-ethylmaleimide.

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. © 1992.

This chapter discusses the properties of 80K diacylglycerol kinase (DGK) and another 150K DGK, both of which have been purified from porcine thymus cytosol. To introduce DG, a water-insoluble substrate, into the reaction mixture, several detergents have been employed, among which deoxycholate and β-octylglucoside has been used. The purification method is based on finding that porcine thymus is highly enriched with the 80K DGK. The separation and purification of diacylglycerol kinases is discussed in the chapter. As an inhibitor of DGK from human erythrocytes and platelets, R59022 has been actively tested in a variety of cells to assess the function of DGK. When tested in vitro with purified enzymes, R59022 inhibits the 80K DGK but not the 150K DGK. Such differences for this inhibitor are also noted for DGK isozymes from human platelets. Interpretation of these data obtained under artificial assay conditions is difficult, but care should be taken to see whether the particular type of cells contains R59022-sensitive DGK species. © 1992, Elsevier Inc. All rights reserved.

To elucidate the regulatory function of EF-hand motifs of pig 80K diacylglycerol (DG) kinase, we constructed and expressed several truncation and deletion mutants of the enzyme in E. coli or COS-7 cells. The bacterially expressed EF-hand region could bind Ca2+ and was suggested to undergo conformational change like calmodulin. A mutant enzyme lacking EF-hands lost Ca2+-binding activity, but could be fully activated by phosphatidylserine (PS) or deoxycholate in the absence of Ca2+. The full activation of the wild-type enzyme by PS, on the other hand, was totally dependent on Ca2+. Further, the wild-type enzyme expressed in COS-7 cells was exclusively soluble, whereas the EF-hand-deleted mutant was considerably associated with the membranes. The results suggest that under Ca2+-free condition, the EF-hand masks the PS-binding site of the DG kinase, and that the Ca2+-binding results in the exposure of the PS-binding site through the conformational change of the EF-hand region. © 1991.

We attempted to assess the regulatory role of EF-hand motifs recently detected in the primary structure of porcine 80-kDa diacylglycerol kinase (DGK) (Sakane, F., Yamada, K., Kanoh, H., Yokoyama, C., and Tanabe, T. (1990) Nature 344, 345-348). By using 80-kDa DGK purified from porcine thymus cytosol, we found that this isozyme indeed bound 2 mol Ca2+ per mol enzyme with high affinity (apparent dissociation constant, Kd = 0.3 /UM). The Ca2+ binding was cooperative with a Hill coefficient of 1.4. We next studied the effect of 1 × 10-5 M Ca2+ on the kinetic properties of DGK employing a β-octyl glucoside mixed micellar assay system. In the absence of Ca2+, phosphatidylserine, so far used as an enzyme activator in various assay systems, was rather inhibitory, and Ca2+ alone activated enzyme to a limited extent. However, phosphatidylserine plus Ca2+ markedly activated the enzyme, giving ∼4-fold higher Vmax and 10-fold less Km values for ATP. In contrast, the apparent Km values for diacylglycerol were not significantly affected (∼3 mol %). Furthermore, by immunoblotting using anti-80 kDa DGK antibodies we found that the soluble DGK in the homogenate of porcine thymocytes was translocated to membranes in a Ca2+-dependent manner. Indeed we noted the presence of a 33-residue amphipathic α-helix in the DGK sequence, which may account for the protein-lipid interaction. The results demonstrate that Ca2+ plays a key role in the regulation of DGK action by controlling enzyme interaction with membrane phospholipids.

CELL stimulation causes diacylglycerol kinase (DGK) to convert the second messenger diacylglycerol into phosphatidate, thus initiating the resynthesis of phosphatidylinositols and attenuating protein kinase C activity1. Of the DGK isoforms so far reported2-4, only porcine DGK from lymphocytes5 has been characterized in detail3,5-7. Here we report the isolation and sequencing of complementary DNA clones that together cover the entire region encoding porcine DGK (relative molecular mass 80,000 (80K)). The deduced primary structure of this DGK contains the putative ATP-binding sites, two cysteine-rich zinc finger-like sequences similar to those found in protein kinase C8, and two E-F hand motifs, typical of Ca2+-binding proteins like calmodulin9. Indeed, we find that the activity of this DGK isoform is enhanced by micromolar concentrations of Ca2+ in the presence of deoxycholate or sphingosine. These properties of 80K DGK indicate that its action is probably linked with both of the second messengers diacylglycerol10 and inositol 1,4,5-trisphosphate11.

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. © 1990.

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 μM 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 μM). The putative DGK inhibitor R59022 inhibited the 80-kDa DGK (IC50, 10 μM), 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. © 1989.

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 (< 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. © 1989.

We investigated the effects of enzyme phosphorylation in vitro on the properties of diacylglycerol kinase. Diacylglycerol kinase and protein kinase C, both present as M(r)-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.

NADPH-cytochrome c reductase [NADPH: ferricytochrome oxidoreductase, EC 1.6.2.4] was highly purified from the membrane fraction of porcine polymorphonudear 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-demeth-ylation 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. © 1987, by the Japanese Biochemical Society.

NADPH-cytochrome c reductase and cytochrome b559 were purified from the membrane fraction of phorbol myristate acetate-stimulated porcine polymorphonuclear leukocytes. The highly purified reductase oxidized NADPH and generated superoxide when combined with partially purified cytochrome b559 in the presence of phosphatidylcholine. In the same system, but under the anaerobic condition, the reductase was found to reduce cytochrome b559. © 1987 Academic Press, Inc.

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°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. © 1987 The Journal of Biochemistry.