AtDGK2, a novel diacylglycerol kinase from Arabidopsis thaliana, phosphorylates 1-stearoyl-2-arachidonoyl-sn-glycerol and 1,2-dioleoyl-sn-glycerol and exhibits cold-inducible gene expression

Diacylglycerol kinase (DGK) phosphorylates diacylglycerol (DAG) to generate phosphatidic acid (PA). Both DAG and PA are implicated in signal transduction pathways. DGKs have been widely studied in animals, but their analysis in plants is fragmentary. Here, we report the cloning and biochemical characterization of AtDGK2, encoding DGK from Arabidopsis thaliana. AtDGK2 has a predicted molecular mass of 79.4 kDa and, like AtDGK1 previously reported, harbors two copies of a phorbol ester/DAG-binding domain in its N-terminal region. AtDGK2 belongs to a family of seven DGK genes in A. thaliana. AtDGK3 to AtDGK7 encode approximately 55-kDa DGKs that lack a typical phorbol ester/DAG-binding domain. Phylogenetically, plant DGKs fall into three clusters. Members of all three clusters are widely expressed in vascular plants. Recombinant AtDGK2 was expressed in Escherichia coli and biochemically characterized. The enzyme phosphorylated 1,2-dioleoyl-sn-glycerol to yield PA, exhibiting Michaelis-Menten type kinetics. Estimated K(m) and V(max) values were 125 microm for DAG and 0.25 pmol of PA min(-1) microg(-1), respectively. The enzyme was maximally active at pH 7.2. Its activity was Mg(2+)-dependent and affected by the presence of detergents, salts, and the DGK inhibitor R59022, but not by Ca(2+). AtDGK2 exhibited substrate preference for unsaturated DAG analogues (i.e. 1-stearoyl-2-arachidonoyl-sn-glycerol and 1,2-dioleoyl-sn-glycerol). The AtDGK2 gene is expressed in various tissues of the Arabidopsis plant, including leaves, roots, and flowers, as shown by Northern blot analysis and promoter-reporter gene fusions. We found that AtDGK2 is induced by exposure to low temperature (4 degrees C), pointing to a role in cold signal transduction.     

Diacylglycerol kinase inhibitor R59022-induced autophagy and apoptosis in the neuronal cell line NG108-15

Phosphatidic acid (PA) is a lipid second messenger and is believed to be involved in cell proliferation and survival. PA is mainly produced by phospholipase D (PLD) and diacylglycerol kinase (DGK). Elevated PLD activity is believed to suppress apoptosis via activation of the mammalian target of rapamycin (mTOR). On the other hand, DGK inhibition has been demonstrated to induce apoptosis, but it is unclear whether DGK can regulate mTOR. Here, we investigated whether DGK inhibition can induce apoptosis and autophagy in neuronal cells, since mTOR is a key mediator of autophagy and the simultaneous activation of apoptosis and autophagy has been detected. A DGK inhibitor, R59022 induced autophagy and apoptosis without serum in NG108-15 cells. Autophagy preceded apoptosis, and apoptosis inhibition did not affect R59022-induced autophagy. R59022-induced autophagy was inhibited by exogenous PA, and protein kinase C activation and increases in intracellular Ca²⁺ levels, which are assumed to be caused by diacylglycerol accumulation, did not appear to be involved in R59022-induced autophagy. We also investigated the effects of R59022 on mTOR signaling pathway, and found that the pathway was not inhibited by R59022. These results imply that DGK plays an important role in cell survival via mTOR-independent mechanism.     

Diacylglycerol kinase alpha regulates globular adiponectin-induced reactive oxygen species

It has previously been reported that the globular form of adiponectin (gAd), mature adipocyte-derived cytokine, induced generation of reactive oxygen species (ROS) and nitric oxide (NO) in the murine macrophage cell line RAW 264. This study investigated whether diacylglycerol kinases (DGKs), enzymes functioning in sub-cellular signalling pathways, had a role on gAd-induced ROS generation in RAW 264 cells. Administration of R59022, a specific inhibitor for DGK, reduced gAd-induced ROS generation and NO release. RAW 264 cell expressed DGKα mRNA. Depression of DGKα mRNA by RNA interference significantly reduced the ROS generation in response to gAd treatment. Interestingly, transfection with the DGKα-specific small interfering RNA attenuated the expression level of Nox1 mRNA in gAd-treated RAW 264 cells. In addition, the DGKα knockdown with siRNA suppressed gAd-induced NO release.     

Differentiation of HL-60 cells to granulocytes involves regulation of select diacylglycerol kinases (DGKs)

Diacylglycerol Kinases (DGKs) are a family of enzymes that regulate the levels of different pools of diacylglycerol (DAG), affecting DAG-mediated signal transduction. Since DAG is known to play several important regulatory roles in granulocyte physiology, we investigated the expression pattern of DGK isoforms throughout differentiation of HL-60 cells to granulocytes. HL-60 cells were incubated with 1.25% dimethyl-sulfoxide (DMSO) to initiate differentiation and total RNA isolated at different time points. DGK expression was assessed through Northern blot, end-point PCR, and real-time PCR. The non-selective inhibitors R59022 and R59949 were used to block DGK at different time points throughout differentiation. CD11b and GPI-80, reactive oxygen species (ROS) generation, changes in the cell cycle, and apoptosis were used as markers of differentiation. Of the nine isoforms of DGK evaluated (alpha, delta, epsilon, gamma, zeta, beta, theta;, iota, eta), only five (alpha, delta, epsilon, gamma, and zeta) were expressed in HL-60 cells. DGKalpha was virtually absent in non-differentiated cells, but was markedly upregulated throughout differentiation. The other isoforms (delta, epsilon, gamma, and zeta) were expressed in undifferentiated HL-60 cells but were substantially decreased throughout differentiation. Non-selective blocking of DGK with R59022 and R59949 led to acceleration of differentiation, reducing the time necessary to observe upregulation of CD11b, GPI-80 and generation of ROS by 50%. Likewise, the cell cycle was disrupted when DGK isoforms were inhibited. These results provide evidence that DGK levels are dynamically regulated throughout differentiation and that expression of DGKs play an important regulatory function during the differentiation of neutrophils.     

Diacylglycerol kinase alpha regulates globular adiponectin-induced reactive oxygen species

Abstract:

It has previously been reported that the globular form of adiponectin (gAd), mature adipocyte-derived cytokine, induced generation of reactive oxygen species (ROS) and nitric oxide (NO) in the murine macrophage cell line RAW 264. This study investigated whether diacylglycerol kinases (DGKs), enzymes functioning in sub-cellular signalling pathways, had a role on gAd-induced ROS generation in RAW 264 cells. Administration of R59022, a specific inhibitor for DGK, reduced gAd-induced ROS generation and NO release. RAW 264 cell expressed DGKα mRNA. Depression of DGKα mRNA by RNA interference significantly reduced the ROS generation in response to gAd treatment. Interestingly, transfection with the DGKα-specific small interfering RNA attenuated the expression level of Nox1 mRNA in gAd-treated RAW 264 cells. In addition, the DGKα knockdown with siRNA suppressed gAd-induced NO release.     

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.     

Diacylglycerol kinase inhibitor R59022 attenuates conjugated linoleic acid-mediated inflammation in human adipocytes

Diacylglycerol kinases (DGK) convert diacylglycerol to phosphatidic acid, which has been reported to stimulate calcium release from the endoplasmic reticulum. Based on our published data showing that trans-10, cis-12 conjugated linoleic acid (t10,c12 CLA)-mediated intracellular calcium accumulation is linked to inflammation and insulin resistance, we hypothesized that inhibiting DGKs with {"type":"entrez-nucleotide","attrs":{"text":"R59022","term_id":"829717","term_text":"R59022"}}R59022 would prevent t10,c12 CLA-mediated inflammatory signaling and insulin resistance in human adipocytes. Consistent with our hypothesis, {"type":"entrez-nucleotide","attrs":{"text":"R59022","term_id":"829717","term_text":"R59022"}}R59022 attenuated t10,c12 CLA-mediated i) increased gene expression and protein secretion of interleukin (IL)-8, IL-6, and monocyte chemoattractant protein-1 (MCP-1); ii) increased activation of extracellular signal-related kinase (ERK), cJun-NH2-terminal kinase (JNK), and cJun; iii) increased intracellular calcium levels; iv) suppressed mRNA or protein levels of peroxisome proliferator activated receptor γ, adiponectin, and insulin-dependent glucose transporter 4; and v) decreased fatty acid and glucose uptake and triglyceride content. DGKη was targeted for investigation based on our findings that i) DGKη was highly expressed in primary human adipocytes and time-dependently induced by t10,c12 CLA and that ii) t10,c12 CLA-induced DGKη expression was dose-dependently decreased with {"type":"entrez-nucleotide","attrs":{"text":"R59022","term_id":"829717","term_text":"R59022"}}R59022. Small interfering RNA (siRNA) targeting DGKη decreased t10,c12 CLA-induced DGKη, IL-8, and MCP-1 gene expression, as well as activation of JNK and cJun. Taken together, these data suggest that DGKs mediate, in part, t10,c12 CLA-induced inflammatory signaling in primary human adipocytes.     

Diacylglycerol kinase gamma is one of the specific receptors of tumor-promoting phorbol esters.

Diacylglycerol kinase (DGK) and protein kinase C (PKC) are two different enzyme families that interact with diacylglycerol. Both enzymes contain cysteine-rich C1 domains with a zinc finger-like structure. Most of the C1 domains of PKCs show strong phorbol-12,13-dibutyrate (PDBu) binding with nanomolar dissociation constants (K(d)'s). However, there has been no experimental evidence that phorbol esters bind to the C1 domains of DGKs. We focused on DGK gamma because its C1A domain has a high degree of sequence homology to those of PKCs, and because DGK gamma translocates from the cytoplasm to the plasma membrane following 12-O-tetradecanoylphorbol-13-acetate treatment similar to PKCs. Two C1 domains of DGK gamma (DGK gamma-C1A and DGK gamma-C1B) were synthesized and tested for their PDBu binding along with whole DGK gamma (Flag-DGK gamma) expressed in COS-7 cells. DGK gamma-C1A and Flag-DGK gamma showed strong PDBu binding affinity, while DGK gamma-C1B was completely inactive. Scatchard analysis of DGK gamma-C1A and Flag-DGK gamma gave K(d)'s of 3.1 and 4.4 nM, respectively, indicating that the major PDBu binding site of DGK gamma is C1A. This is the first evidence that DGK gamma is a specific receptor of tumor-promoting phorbol esters.     

Molecular cloning of a cDNA encoding diacylglycerol kinase (DGK) in Arabidopsis thaliana

Diacylglycerol kinase (DGK) synthesizes phosphatidic acid from diacylglycerol, an activator of protein kinase C (PKC), to resynthesize phosphatidylinositols. The structure of DGK has not been characterized in plants. We report the cloning of a cDNA, cATDGK1, encoding DGK from Arabidopsis thaliana. The cATDGK1 cDNA contains an open reading frame of 2184 bp, and encodes a putative protein of 728 amino acids with a predicted molecular mass of 79.4 kDa. The deduced ATDGK1 amino acid sequence exhibits significant similarity to that of rat, pig, and Drosophila DGKs. The ATDGK1 mRNA was detected in roots, shoots, and leaves. Southern blot analysis suggests that the ATDGK1 gene is a single-copy gene. The existence of DGK as well as phospholipase C suggests the existence of PKC in plants.     

Domains, amino acid residues, and new isoforms of Caenorhabditis elegans diacylglycerol kinase 1 (DGK-1) important for terminating diacylglycerol signaling in vivo

Diacylglycerol kinases (DGKs) inhibit diacylglycerol (DAG) signaling by phosphorylating DAG. DGK-1, the Caenorhabditis elegans ortholog of human neuronal DGK, inhibits neurotransmission to control behavior. DGK-1, like DGK, has three cysteine-rich domains (CRDs), a pleckstrin homology domain, and a kinase domain. To identify DGK domains and amino acid residues critical for terminating DAG signaling in vivo, we analyzed 20 dgk-1 mutants defective in DGK-1-controlled behaviors. We found by sequencing that the mutations included nine amino acid substitutions and seven premature stop codons that impair the physiological functions of DGK-1. All nine amino acid substitutions are in the second CRD, the third CRD, or the kinase domain. Thus, these domains are important for the termination of DAG signaling by DGK-1 in vivo. Seven of the substituted amino acid residues are present in all human DGKs and likely define key residues required for the function of all DGKs. An ATP-binding site mutation expected to inactivate the kinase domain retained very little physiological function, but we found two stop codon mutants predicted to truncate DGK-1 before its kinase domain that retained significantly more function. We detected novel splice forms of dgk-1 that can reconcile this apparent conflict, as they skip exons containing the stop codons to produce DGK-1 isoforms that contain the kinase domain. Two of these isoforms lack an intact pleckstrin homology domain and yet appear to have significant function. Additional novel isoform(s) account for all of the DGK-1 function necessary for one behavior, dopamine response.     

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.     

Diacylglycerol kinase zeta regulates Ras activation by a novel mechanism

Guanine nucleotide exchange factors (GEFs) activate Ras by facilitating its GTP binding. Ras guanyl nucleotide-releasing protein (GRP) was recently identified as a Ras GEF that has a diacylglycerol (DAG)-binding C1 domain. Its exchange factor activity is regulated by local availability of signaling DAG. DAG kinases (DGKs) metabolize DAG by converting it to phosphatidic acid. Because they can attenuate local accumulation of signaling DAG, DGKs may regulate RasGRP activity and, consequently, activation of Ras. DGK zeta, but not other DGKs, completely eliminated Ras activation induced by RasGRP, and DGK activity was required for this mechanism. DGK zeta also coimmunoprecipitated and colocalized with RasGRP, indicating that these proteins associate in a signaling complex. Coimmunoprecipitation of DGK zeta and RasGRP was enhanced in the presence of phorbol esters, which are DAG analogues that cannot be metabolized by DGKs, suggesting that DAG signaling can induce their interaction. Finally, overexpression of kinase-dead DGK zeta in Jurkat cells prolonged Ras activation after ligation of the T cell receptor. Thus, we have identified a novel way to regulate Ras activation: through DGK zeta, which controls local accumulation of DAG that would otherwise activate RasGRP.     

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.     

Developmentally expressed myosin heavy-chain kinase possesses a diacylglycerol kinase domain.

In Dictyostelium, an ordered actin and myosin assembly-disassembly process is necessary for proper development, differentiation, and motility (Yumura S, Fukui F, 1985, Nature 314(6007): 194-196; Ravid S, Spudich JA, 1989, J Biol Chem 264(25): 15144-15150), and phosphorylation of myosin heavy chains has been implicated in the myosin assembly-disassembly process (Egelhoff TT, Lee RJ, Spudich JA, 1993, Cell 75(2):363-371). The developmentally expressed 84-kDa myosin heavy-chain kinase (MHCK) from Dictyostelium (Ravid S, Spudich JA, 1992, Proc Natl Acad Sci USA 89(13):5877-5881) is known to be a member of the protein kinase C (PKC) family. We have observed a rather striking homology between the large central domain of MHCK and the catalytic domain of diacylglycerol kinase (DGK), indicating that MHCK is in fact a gene fusion between a DGK and a PKC, possessing two separate kinase domains. The combined diacylglycerol kinase/myosin heavy-chain kinase (DGK/MHCK) may therefore have dual functionality, possessing the ability to phosphorylate both protein and lipid. We present a hypothesis that DGK/MHCK can antagonize both actin and myosin assembly, as well as other cellular processes, by coordinated down regulation of signaling via myosin heavy-chain kinase activity and diacylglycerol kinase activity.     

A domain with homology to neuronal calcium sensors is required for calcium-dependent activation of diacylglycerol kinase alpha

Diacylglycerol kinases (DGKs) phosphorylate diacylglycerol produced during stimulus-induced phosphoinositide turnover and attenuate protein kinase C activation. Diacylglycerol kinase alpha is an 82-kDa DGK isoform that is activated in vitro by Ca(2+). The DGK alpha regulatory region includes tandem C1 protein kinase C homology domains and Ca(2+)-binding EF hand motifs. It also contains an N-terminal recoverin homology (RVH) domain that is related to the N termini of the recoverin family of neuronal calcium sensors. To probe the structural basis of Ca(2+) regulation, we expressed a series of DGK alpha deletions spanning its regulatory domain in COS-1 cells. Deletion of the RVH domain resulted in loss of Ca(2+)-dependent activation. Further deletion of the EF hands resulted in a constitutively active enzyme, suggesting that sequences in or near the EF hands are sufficient for autoinhibition. Binding of Ca(2+) to the EF hands protected sites within both the RVH domain and EF hands from trypsin cleavage and increased the phenyl-Sepharose binding of a recombinant DGK alpha fragment that included both the RVH domain and EF hands. These observations suggested that Ca(2+) elicits a concerted conformational change of these two domains. A cationic amphiphile, octadecyltrimethylammonium chloride, also activated DGK alpha. As with Ca(2+), this activation required the RVH domain. However, this agent did not protect the EF hands and RVH domain from trypsin cleavage. These findings indicate that the EF hands and RVH domain act as a functional unit during Ca(2+)-induced DGK alpha activation.     

Diacylglycerol kinase activity in purified basolateral membranes of kidney tubules. I. Evidence for coupling with phospholipase C

The diacylglycerol kinase (DGK) catalyzes the phosphorylation of diacylglycerol (DAG) yielding phosphatidic acid (PA) signaling molecules which are involved in the modulation of different cell responses. The aim of this work was to characterize the DGK activity associated to the basolateral membranes (BLM) of kidney proximal tubules, in a native preparation that preserves the membrane microenvironment. The Arrhenius plot of DGK activity was non-linear, indicating a complex influence of the lipid environment of the native membrane. The formation of PA was strongly impaired by U73122, an inhibitor of PLC, whereas remained unmodified when exogenous DAG or PLC were added. The Mg.ATP2- complex is the true phosphoryl-donor substrate, and the very narrow peak of activation at pH 7.0 suggests that amino acids that dissociate at this pH, i.e. hystidine residues, play a role by acting in the coordination of the Mg2+ atoms. The renal DGK is almost completely blocked by 0.1 mM sphingosine, but it is insensitive to micromolar free Ca2+ concentrations and to R59499, the most potent inhibitor of the classical DGKs. Taken as a whole, these data suggest that the DGK isoform present in BLM of proximal tubules is different from those included in the type I family, and that membranous PLC could be the main source of DAG for DGK catalysis.     

Diacylglycerol kinase δ associates with receptor for activated C kinase 1, RACK1

The δ-isozyme (type II) of diacylglycerol kinase (DGK) is known to positively regulate growth factor receptor signaling. DGKδ, which is distributed to clathrin-coated vesicles, interacts with DGKδ itself, protein kinase C and AP2α. To search for additional DGKδ-interacting proteins, we screened a yeast two-hybrid cDNA library from HepG2 cells using aa 896–1097 of DGKδ as a bait. We identified aa 184–317 (WD40 repeats 5–7) of receptor for activated C kinase 1 (RACK1), which interacts with various important signaling molecules, as a novel binding partner of DGKδ. Co-immunoprecipitation analysis, using COS-7 cells co-expressing RACK1 and DGKδ, revealed that RACK1 selectively interacted with DGKδ, but not with type I DGKs, in mammalian cells. The interaction was dynamically regulated by phorbol ester. Intriguingly, DGKδ appeared to recruit RACK1 to clathrin-coated vesicles and co-localized with RACK1. These results suggest that DGKδ serves as an adaptor protein to regulate the localization of the versatile scaffold protein, RACK1.     

Plant PA signaling via diacylglycerol kinase

Accumulating evidence suggests that phosphatidic acid (PA) plays a pivotal role in the plant's response to environmental signals. Besides phospholipase D (PLD) activity, PA can also be generated by diacylglycerol kinase (DGK). To establish which metabolic route is activated, a differential ³²P-radiolabelling protocol can be used. Based on this, and more recently on reverse-genetic approaches, DGK has taken center stage, next to PLD, as a generator of PA in biotic and abiotic stress responses. The DAG substrate is generally thought to be derived from PI-PLC activity. The model plant system Arabidopsis thaliana has 7 DGK isozymes, two of which, AtDGK1 and AtDGK2, resemble mammalian DGK?, containing a conserved kinase domain, a transmembrane domain and two C1 domains. The other ones have a much simpler structure, lacking the C1 domains, not matched in animals. Several protein targets have now been discovered that bind PA. Whether the PA molecules engaged in these interactions come from PLD or DGK remains to be elucidated.