DgMab-6: Antihuman DGKγ Monoclonal Antibody for Immunocytochemistry.

Diacylglycerol kinase (DGK) phosphorylates diacylglycerol (DG) to produce phosphatidic acid (PA). Since both DG and PA serve as lipidic second messengers, DGK plays a pivotal role in regulating the balance of two signaling pathways mediated by DG and PA in cellular functions. Reportedly, DGKγ, one of the 10 mammalian DGK isozymes, is involved in leukemic cell differentiation, mast cell function, and membrane traffic. Transfection studies using tagged expression vectors and immunohistochemistry on rat tissues revealed that DGKγ localizes to the cytoplasm, plasma membrane, and Golgi apparatus. However, a limited number of studies reported the detailed localization of native protein of DGKγ in human tissues and cells. In this study, we developed a novel anti-DGKγ monoclonal antibody, DgMab-6, which is very useful in immunocytochemistry of human cultured cells.

Expression, Purification, and Properties of a Human Arachidonoyl-Specific Isoform of Diacylglycerol Kinase.

Diacylglycerol kinase ε (DGKε) catalyzes the phosphorylation of diacylglycerol, producing phosphatidic acid. DGKε demonstrates exquisite specificity for the acyl chains of diacylglycerol. This contributes to the enrichment of particular acyl chains within the lipids of the phosphatidylinositol cycle. Phosphatidylinositol is highly enriched with 1-stearoyl-2-arachidonoyl, which is important for maintaining cellular health. Dysregulation of DGKε perturbs lipid signaling and biosynthesis, which has been linked to epilepsy, Huntington's disease, and heart disease. Recessive loss-of-function mutations in the DGKε gene cause atypical hemolytic uremic syndrome. Because DGKε has never been purified, little is known about its molecular properties. We expressed human DGKε and a truncated version lacking the first 40 residues (DGKεΔ40) and purified both proteins to near homogeneity using nickel affinity chromatography. Kinase activity measurements showed that both purified constructs retained their acyl chain specificity for diacylglycerol with an activity level comparable to that of N-terminally FLAG epitope-tagged forms of these proteins expressed in COS7 cells. Both constructs lost activity upon being stored, particularly upon freezing and thawing, which was minimized by the addition of glycerol. Circular dichroism revealed that DGKε and DGKεΔ40 both contain significant amounts of α-helical and ß structure and exhibit biphasic thermal denaturations. The loss of secondary structure upon heating was irreversible for both constructs, with relatively little effect of added dioleoylphosphatidylcholine. The addition of 50% glycerol stabilized both constructs and facilitated refolding of their secondary structures after heating. This is the first successful purification and characterization of DGKε's enzymatic and conformational properties. The purification of DGKε permits detailed analyses of this unique enzyme and will improve our understanding of DGKε-related diseases.

Measuring Diacylglycerol Kinase-θ Activity and Binding.

This section provides detailed protocols for the analysis of a mammalian diacylglycerol kinase, DGKθ, including an activity assay, a kinetic analysis, preparation of small unilamellar vesicles, and a vesicle pulldown assay. The goal of this section is to provide an overview of the unique challenges inherent in the study of an interfacial enzyme such as DGKθ and to outline methods useful for analysis. We include a short tutorial on selecting lipids for forming the interface since this is critical for a successful in vitro assay, and lipids are important regulators of this enzyme. The general principles can be applied to the study of other interfacial enzymes.

Molecular cloning and expression patterns of a putative olfactory diacylglycerol kinase from the noctuid moth Spodoptera littoralis.

In the aim of the characterization of the molecular actors of insect olfactory transduction, we have cloned the full cDNA encoding a Spodoptera littoralis diacylglycerol kinase (DGK) named SlDGK. In male adults, SlDGK transcript was detected predominantly in the brain and in the olfactory sensilla trichodea located on the antennae. SlDGK expression was first detected at day 3 of the pupal stage, then reached a maximum at the end of this stage and was maintained at this level during the adult period. These data provide the first molecular characterization of a DGK potentially involved in the regulation of signalling pathways responsible for the establishment and/or the functioning of the olfactory system in Lepidoptera.

Measurement of mammalian diacylglycerol kinase activity in vitro and in cells.

Diacylglycerol kinase (DGK) catalyzes the conversion of diacylglycerol to phosphatidic acid. Because both the lipid substrate and the product are important in regulation, this enzyme plays an important role in signal transduction. In mammals there are several isoforms of diacylglycerol kinase. Their activities can be evaluated in vitro as well as in intact cells. In vitro assays are based on measuring the incorporation of (32)P from ATP into diacylglycerol, resulting in the formation of labeled phosphatidic acid. Diacylglycerol with long acyl chains is insoluble in water and must be dispersed with detergent or incorporated into liposomes. Detergent-based assays are easier to perform and generally more precise; however, liposomes more closely resemble the organization of biological membranes and also allow for the testing of the modulation of enzyme activity by changes in the physical or chemical properties of the membrane. The micelle assay can also be used to measure DGK activity in cellular organelles after stimulation of intact cells to activate particular DGK isoforms. This will assess the translocation of DGK among different subcellular compartments. In this regard the plasma membrane and nucleus appear to be particularly important for the regulatory actions of these enzymes. Finally, one can also measure the DGK activity in whole cells that have been prelabeled with [(32)P]phosphate and determine the incorporation of label into phosphatidic acid that can be extracted from the whole cell or from cellular organelles.

Identification of a soluble diacylglycerol kinase required for lipoteichoic acid production in Bacillus subtilis.

Diacylglycerol kinases (DagKs) are key enzymes in lipid metabolism that function to reintroduce diacylglycerol formed from the hydrolysis of phospholipids into the biosynthetic pathway. Bacillus subtilis is a prototypical Gram-positive bacterium with a lipoteichoic acid structure containing repeating units of sn-glycerol-1-P groups derived from phosphatidylglycerol head groups. The B. subtilis homolog of the prokaryotic DagK gene family (dgkA; Pfam01219) was not a DagK but rather was an undecaprenol kinase. The three members of the soluble DagK protein family (Pfam00781) in B. subtilis were tested by complementation of an E. coli dgkA mutant, and only the essential yerQ gene possessed DagK activity. This gene was dubbed dgkB, and the soluble protein product was purified, and its DagK activity was verified in vitro. Conditional inactivation of dgkB led to the accumulation of diacylglycerol and the cessation of lipoteichoic acid formation in B. subtilis. This study identifies a soluble protein encoded by the dgkB (yerQ) gene as an essential kinase in the diacylglycerol cycle that drives lipoteichoic acid production.

Expression, purification, crystallization and preliminary diffraction studies of the mammalian DAG kinase homologue YegS from Escherichia coli.

yegS is a gene encoding a 32 kDa cytosolic protein with unknown function but with strong sequence homology to a family of structurally uncharacterized eukaryotic non-protein kinases: diacylglycerol kinases, sphingosine kinases and ceramide kinases. Here, the overexpression, crystallization and preliminary diffraction analysis of Escherichia coli YegS are reported. The crystals belong to space group P2(1), with unit-cell parameters a = 42.4, b = 166.1, c = 48.5 A, beta = 96.97 degrees. The presence of a dimer in the asymmetric unit was estimated to give a Matthews coefficient (VM) of 2.5 A3 Da(-1) and a solvent content of 50.8%(v/v). Single-wavelength diffraction data were collected to a resolution of 1.9 A using synchrotron radiation.

Purification of diacylglycerol kinase from Microsporum gypseum and its phosphorylation by the catalytic subunit of protein kinase A.

Diacylglycerol (DG) kinase (EC 2.7.1.107) was purified to homogeneity from the soluble extract of Microsporum gypseum, a dermatophyte. Purified enzyme showed a final specific activity of 2172 pmol/min/mg protein and its apparent molecular weight on SDS-PAGE was found to be 93 kDa. The activity of purified enzyme was inhibited in a dose-dependent manner in the presence of DG-kinase inhibitor (D5919, Sigma). DG-kinase activity was found to be stimulated in the presence of phosphatidylcholine, phosphatidylethanolamine, and cardiolipin while the activity was alleviated in the presence of phosphatidic acid and arachidonic acid. Kinase activity was partially inhibited when assayed after prior treatment with alkaline phosphatase. Treatment of DG-kinase with the catalytic subunit of protein kinase A (PKA)-stimulated DG-kinase activity in a dose-dependent manner. Incubation of DG-kinase with the catalytic subunit of PKA led to the phosphorylation of DG-kinase as revealed by autoradiography. The phosphorylated band disappeared completely in the presence of specific PKA inhibitor. Increased activity of DG-kinase on incubation with the catalytic subunit of PKA was possibly due to the phosphorylation of the former by the latter. Whether this in vitro phosphorylation and activation of DG-kinase occurs under physiological conditions remains to be elucidated.

Use of amphipathic polymers to deliver a membrane protein to lipid bilayers.

Data are presented which suggest that a class of amphiphilic polymers known as 'amphipols' may serve as a vehicle for delivering complex integral membrane proteins into membranes. The integral membrane protein diacylglycerol kinase (DAGK) was maintained in soluble form by either of two different amphipols. Small aliquots of these solutions were added to pre-formed lipid vesicles and the appearance of DAGK catalytic activity was monitored as an indicator of the progress of productive protein insertion into the bilayers. For one of the two amphipols tested, DAGK was observed to productively transfer from its amphipol complex into vesicles with moderate efficiency. Results were not completely clear for the other amphipol.

Fatty acids inhibit growth-factor-induced diacylglycerol kinase alpha activation in vascular smooth-muscle cells.

We have previously shown that unsaturated fatty acids amplify platelet-derived-growth-factor (PDGF)-induced protein kinase C (PKC) activation in vascular smooth-muscle cells (VSMCs). Diacylglycerol-induced PKC activation is normally terminated by diacylglycerol kinases (DGKs). We thus hypothesized that fatty acids act by inhibiting a DGK. Fractionation of VSMC extracts demonstrated that the DGK alpha isoform was the major DGK activity present. PDGF markedly increased the DGK activity of cultured cells. An inhibitor selective for the DGK alpha isoform, R59949 [3-[2-[4-(bis-(4-fluorophenyl)methylene]piperidin-1-yl)ethyl]-2,3-dihydro-2-thioxo-4(1H)-quinazolinone], abolished the growth-factor-induced increase in DGK activity, but had little effect on basal activity. PDGF thus selectively activates DGKalpha. Epidermal growth factor and alpha-thrombin stimulated total DGK activity similarly to PDGF. Activation by epidermal growth factor was sensitive to R59949, again suggesting involvement of DGKalpha. However, the alpha-thrombin-induced activity was unaffected by this agent. Unsaturated fatty acids inhibited growth-factor-induced DGKalpha activation, but had no effect on basal activity. Fatty acids also amplified the PDGF-induced increase in cell diacylglycerol content. These results indicate that inhibition of DGKalpha contributes to fatty-acid-induced amplification of PKC activation. Increased levels of fatty acids in diabetes may thus contribute to chronic PKC activation associated with this disorder.

Diacylglycerol kinase zeta in hypothalamus interacts with long form leptin receptor. Relation to dietary fat and body weight regulation.

Leptin and its long form receptor, Ob-Rb, in hypothalamic nuclei play a key role in regulating energy balance. The mutation of Ob-Rb into one of its natural variants, Ob-Ra, results in severe obesity in rodents. We demonstrate here that diacylglycerol kinase zeta (DGKzeta) interacts, via its ankyrin repeats, with the cytoplasmic portion of Ob-Rb in yeast two-hybrid systems, in protein precipitation experiments in vitro and in vivo. It does not interact, however, with the short form, Ob-Ra, which mediates the entry of leptin into the brain. Furthermore, we show by in situ hybridization that DGKzeta is expressed in neurons of hypothalamic nuclei known to synthesize Ob-Rb and to participate in energy homeostasis. The mutant ob-/ob- and db-/db- mice exhibit increased hypothalamic DGKzeta mRNA level compared with their wild-type controls, suggesting a role for the leptin/OB-Rb system in regulating DGKzeta expression. Further experiments show that hypothalamic DGKzeta mRNA level is stimulated by the consumption of a high-fat diet. In addition, DGKzeta mRNA is statistically significantly lower in rats and inbred mice that become obese on a high-fat diet compared with their lean counterparts. In fact, it is strongly, negatively correlated with both body fat and circulating levels of leptin. Taken together, our evidence suggests that DGKzeta constitutes a downstream component of the leptin signaling pathway and that reduced hypothalamic DGKzeta mRNA, and possibly activity, is associated with obesity.

Reconstitutive refolding of diacylglycerol kinase, an integral membrane protein.

While the formation of kinetically trapped misfolded structural states by membrane proteins is related to a number of diseases, relatively few studies of misfolded membrane proteins in their purified state have been carried out and few methods for refolding such proteins have been reported. In this paper, misfolding of the trimeric integral membrane protein diacylglycerol kinase (DAGK) is documented and a method for refolding the protein is presented; 65 single-cysteine mutants of DAGK were examined. A majority were found to have lower-than-expected activities when purified into micellar solutions, with additional losses in activity often being observed following membrane reconstitution. A variety of evidence indicates that the low activities observed for most of these mutants results from kinetically based misfolding of the protein, with misfolding often being manifested by the formation of aberrant oligomeric states. A method referred to as "reconstitutive refolding" for correcting misfolded DAGK is presented. This method is based upon reconstituting DAGK into multilamellar POPC vesicles by dialyzing the detergent dodecylphosphocholine out of mixed micellar mixtures. For 55 of the 65 mutants tested, there was a gain of DAGK activity during reconstitutive refolding. In 33 of these cases, the gain in activity was greater than 2-fold. The refolding results for cysteine replacement mutants at DAGK sites known to be highly conserved provide teleological insight into whether sites are conserved, because they are critical for catalysis, for maintenance of the proper folding pathway, or for some other reason.

Affinity purification and catalytic properties of a soluble, Ca2+-independent, diacylglycerol kinase.

We used a new procedure that involved selective enzyme binding to lipid vesicles to partially purify a soluble diacylglycerol kinase, then studied the relation between enzyme-vesicle binding and activity in vesicle-based assays. The vesicle-binding procedure required about 2 h, increased the enzyme's specific activity 50-fold with a 50% yield of activity, and combined well with additional purification steps. Studies of the activity of the partially purified diacylglycerol kinase toward vesicle-associated diacylglycerols revealed linear reaction kinetics that reflected enzyme binding to the vesicles; factors known to influence enzyme binding to the vesicles affected enzyme activity only indirectly, not by influencing the diacylglycerol kinase reaction itself. On the other hand, special incubation experiments that caused both substrate depletion in vesicles and enzyme stalling provided evidence that the diacylglycerol kinase could desorb from these vesicles, adsorb to freshly added, substrate-containing vesicles, and resume catalysis of phosphorylation reactions. The molecular basis for this enzyme-vesicle "hopping" behavior remains to be clarified. But enzyme-catalyzed conversion of diacylglycerol to phosphatidic acid may not have been a contributing factor because separate, enzyme-vesicle binding experiments showed that the enzyme had only a marginally higher affinity for diacylglycerol-containing vesicles than it did for vesicles that contained comparable amounts of phosphatidic acid. The combined results of our experiments suggest that the linear rates of diacylglycerol phosphorylation observed in standard assays with diacylglycerol-containing vesicles may have been combined functions of both the rate of enzyme hopping among vesicles and the rate of diacylglycerol phosphorylation by enzyme that was bound transiently on substrate-containing vesicles.