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Diacylglycerol kinases (DGKs) are important enzymes in molecular membrane biology, as they can lower the concentration of diacylglycerol through phosphorylation while at the same time producing phosphatidic acid. Dysfunction of DGK is linked with multiple diseases including cancer and autoimmune disorders. Currently, the high-resolution structures have not been determined for any of the 10 human DGK paralogs, which has made it difficult to gain a more complete understanding of the enzyme's mechanism of action and regulation. In the present study, we have taken advantage of the significant developments in protein structural prediction technology by artificial intelligence (i.e., Alphafold 2.0), to conduct a comprehensive investigation on the properties of all 10 human DGK paralogs. Structural alignment of the predictions reveals that the C1, catalytic, and accessory domains are conserved in their spatial arrangement relative to each other, across all paralogs. This suggests a critical role played by this domain architecture in DGK function. Moreover, docking studies corroborate the existence of a conserved ATP-binding site between the catalytic and accessory domains. Interestingly, the ATP bound to the interdomain cleft was also found to be in proximity of the conserved glycine-rich motif, which in protein kinases has been suggested to function in ATP binding. Lastly, the spatial arrangement of DGK, with respect to the membrane, reveals that most paralogs possess a more energetically favorable interaction with curved membranes. In conclusion, AlphaFold predictions of human DGKs provide novel insights into the enzyme's structural and functional properties while also paving the way for future experimentation.

Diacylglycerol kinase ε (DGKε), an enzyme of the phosphatidylinositol (PI) cycle, bears a highly conserved hydrophobic N-terminal segment, which was proposed to anchor the enzyme into the membrane. However, the importance of this segment to the DGKε function remains to be determined. To address this question, it is here reported an in silico and in vitro combined research strategy. Capitalizing on the AlphaFold 2.0 predicted structure of human DGKε, it is shown that its hydrophobic N-terminal segment anchors it into the membrane via a transmembrane α-helix. Coarse-grained based elastic network model studies showed that a conformational change in the hydrophobic N-terminal segment determines the proximity between the active site of DGKε and the membrane-water interface, likely regulating its kinase activity. In vitro studies with a purified DGKε construct lacking the hydrophobic N-terminal segment (His-SUMO*-Δ 50 -DGKε) corroborated the role of the N-terminus in regulating DGKε enzymatic properties. The comparison between the enzymatic properties of DGKε and His-SUMO*-Δ 50 -DGKε showed that the conserved N-terminal segment markedly inhibits the enzyme activity and its sensitivity to membrane intrinsic negative curvature, while also playing a role in the modulation of the enzyme by phosphatidylserine. On the other hand, this segment did not strongly affect its diacylglycerol acyl chain specificity, the modulation of the enzyme by membrane morphological changes, or the activation by phosphatidic acid-rich lipid domains. Hence, these results suggest that the conservation of the hydrophobic N-terminal segment of DGKε throughout evolution guaranteed not only membrane anchorage but also an efficient and elegant manner to regulate the rate of the PI cycle.

Diacylglycerol kinases catalyze the ATP-dependent phosphorylation of diacylglycerol (DAG) to produce phosphatidic acid (PA). In humans, the alpha isoform (DGKα) has emerged as a potential target in the treatment of cancer due to its anti-tumor and pro-immune responses. However, its mechanism of action at a molecular level is not fully understood. In this work, a systematic investigation of the role played by the membrane in the regulation of the enzymatic properties of human DGKα is presented. By using a cell-free system with purified DGKα and model membranes of variable physical and chemical properties, it is shown that membrane physical properties determine human DGKα substrate acyl chain specificity. In model membranes with a flat morphology; DGKα presents high enzymatic activity, but it is not able to differentiate DAG molecular species. Furthermore, DGKα enzymatic properties are insensitive to membrane intrinsic curvature. However, in the presence of model membranes with altered morphology, specifically the presence of physically curved membrane structures, DGKα bears substrate acyl chain specificity for palmitic acid-containing DAG. The present results identify changes in membrane morphology as one possible mechanism for the depletion of specific pools of DAG as well as the production of specific pools of PA by DGKα, adding an extra layer of regulation on the interconversion of these two potent lipid-signaling molecules. It is proposed that the interplay between membrane physical (shape) and chemical (lipid composition) properties guarantee a fine-tuned signal transduction system dependent on the levels and molecular species of DAG and PA., (© 2021 Federation of American Societies for Experimental Biology.)

The diacylglycerol kinase family, which can attenuate diacylglycerol signaling and activate phosphatidic acid signaling, regulates various signaling transductions in the mammalian cells. Studies on the regulation of diacylglycerol and phosphatidic acid levels by various enzymes, the identification and characterization of various diacylglycerol and phosphatidic acid-regulated proteins, and the overlap of different diacylglycerol and phosphatidic acid metabolic and signaling processes have revealed the complex and non-redundant roles of diacylglycerol kinases in regulating multiple biochemical and biological networks. In this review article, we summarized recent progress in the complex and non-redundant roles of diacylglycerol kinases, which is expected to aid in restoring dysregulated biochemical and biological networks in various pathological conditions at the bed side.

Lipid kinases regulate a wide variety of cellular functions and have emerged as one the most promising targets for drug design. Diacylglycerol kinases (DGKs) are a family of enzymes that catalyze the ATP-dependent phosphorylation of diacylglycerol (DAG) to phosphatidic acid (PtdOH). Despite the critical role in lipid biosynthesis, both DAG and PtdOH have been shown as bioactive lipids mediating a number of signaling pathways. Although there is increasing recognition of their role in signaling systems, our understanding of the key enzyme which regulate the balance of these two lipid messages remain limited. Solved structures provide a wealth of information for understanding the function and regulation of these enzymes. Solving the structures of mammalian DGKs by traditional NMR and X-ray crystallography approaches have been challenging and so far, there are still no three-dimensional structures of these DGKs. Despite this, some insights may be gained by examining the similarities and differences between prokaryotic DGKs and other mammalian lipid kinases. This review focuses on summarizing and comparing the structure of prokaryotic and mammalian DGKs as well as two other lipid kinases: sphingosine kinase and phosphatidylinositol-3-kinase. How these known lipid kinases structures relate to mammalian DGKs will also be discussed., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The epsilon isoform of mammalian diacylglycerol kinase (DGKϵ) is an enzyme that associates strongly with membranes and acts on a lipid substrate, diacylglycerol. The protein has one segment that is predicted to be a transmembrane helix, but appears to interconvert between a transmembrane helix and a re-entrant helix. Despite the hydrophobicity of this segment and the fact that the lipid substrate is also hydrophobic, removal of this hydrophobic segment by truncating the protein at the amino terminus has no effect on its enzymatic activity. The amino acid sequence of the catalytic segment of DGKϵ is highly homologous to that of a bacterial DGK, DgkB. This has allowed us to predict a conformation of DGKϵ based on the known crystal structure of DgkB. An important property of DGKϵ is that it is specific for diacylglycerol species containing an arachidonoyl group. The region of DGKϵ that interacts with this group is found within the accessory domain of the protein and not in the active site nor in the hydrophobic amino terminus. The nature of the acyl chain specificity of the enzyme indicates that DGKϵ is associated with the synthesis of phosphatidylinositol. Defects or deletion of the enzyme give rise to several disease states., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Diacylglycerol kinase (DGK) η plays important roles in various patho-physiological events such as oncogenesis. In this study, we performed an enzymological characterization of DGKη splice variant 1 (DGKη1). The Km value for diacylglycerol was 0.14 mol%. Intriguingly, the Km value of DGKη1 for diacylglycerol was at least 9-fold lower than those of other DGK isozymes including DGKα, indicating that DGKη1 is a high affinity isozyme for diacylglycerol. Therefore, DGKη1 is a unique DGK isozyme, which may function at particular membrane sites where only low concentrations of diacylglycerol are supplied., (Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Decreased expression of diacylglycerol (DG) kinase (DGK) δ in skeletal muscles is closely related to the pathogenesis of type 2 diabetes. To identify DG species that are phosphorylated by DGKδ in response to high glucose stimulation, we investigated high glucose-dependent changes in phosphatidic acid (PA) molecular species in mouse C2C12 myoblasts using a newly established liquid chromatography/MS method. We found that the suppression of DGKδ2 expression by DGKδ-specific siRNAs significantly inhibited glucose-dependent increases in 30:0-, 32:0-, and 34:0-PA and moderately attenuated 30:1-, 32:1-, and 34:1-PA. Moreover, overexpression of DGKδ2 also enhanced the production of these PA species. MS/MS analysis revealed that these PA species commonly contain palmitic acid (16:0). D609, an inhibitor of phosphatidylcholine-specific phospholipase C (PC-PLC), significantly inhibited the glucose-stimulated production of the palmitic acid-containing PA species. Moreover, PC-PLC was co-immunoprecipitated with DGKδ2. These results strongly suggest that DGKδ preferably metabolizes palmitic acid-containing DG species supplied from the PC-PLC pathway, but not arachidonic acid (20:4)-containing DG species derived from the phosphatidylinositol turnover, in response to high glucose levels., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

The diacylglycerol kinase (DGK) family of enzymes plays critical roles in lipid signaling pathways by converting diacylglycerol to phosphatidic acid, thereby downregulating signaling by the former and upregulating signaling by the latter second messenger. Ten DGK family isozymes have been identified to date, which possess different interaction motifs imparting distinct temporal and spatial control of DGK activity to each isozyme. Two DGK family members, δ and η, contain a sterile alpha motif (SAM) domain. The SAM domain of DGKδ1 forms helical polymers that are important for retaining the enzyme in cytoplasmic puncta, thereby inhibiting activity at the plasma membrane until pathway activation. Because zinc was found to be important for stabilizing the similar SAM polymers of the scaffolding protein Shank-3, we investigated the potential role of zinc in DGKδ SAM domain (DGKδSAM) assembly. We find that DGKδSAM binds zinc at multiple sites, driving the organization of the DGKδSAM into large sheets of polymers. Moreover, a mutant DGKδ containing a SAM domain refractory to zinc binding diminishes the formation of cytoplasmic puncta, shows partially impaired regulation of transport to the plasma membrane, and lacks the ability to inhibit the formation of CopII coated vesicles. These results suggest that zinc may play an important role in the assembly and physiology of the DGKδ isozyme.

The phosphatidylinositol (PI) cycle mediates many cellular events by controlling the metabolism of many lipid second messengers. Diacylglycerol kinase epsilon (DGK epsilon) has an important role in this cycle. DGK epsilon is the only DGK isoform to show inhibition by its product phosphatidic acid (PA) as well as substrate specificity for sn-2 arachidonoyl-diacylglycerol (DAG). Here, we show that this inhibition and substrate specificity are both determined by selectivity for a combination of the sn-1 and sn-2 acyl chains of PA or DAG, respectively, preferring the most prevalent acyl chain composition of lipids involved specifically in the PI cycle, 1-stearoyl-2-arachidonoyl. Although the difference in rate for closely related lipid species is small, there is a significant enrichment of 1-stearoyl-2-arachidonoyl PI because of the cyclical nature of PI turnover. We also show that the inhibition of DGK epsilon by PA is competitive and that the deletion of the hydrophobic segment and cationic cluster of DGK epsilon does not affect its selectivity for the acyl chains of PA or DAG. Thus, this active site not only recognizes the lipid headgroup but also a combination of the two acyl chains in PA or DAG. We propose a mechanism of DGK epsilon regulation where its dual acyl chain selectivity is used to negatively regulate its enzymatic activity in a manner that ensures DGK epsilon remains committed to the PI turnover cycle. This novel mechanism of enzyme regulation within a signaling pathway could serve as a template for the regulation of enzymes in other pathways in the cell.

C1 domains, cysteine-rich modules originally identified in protein kinase C (PKC) isozymes, are present in multiple signaling families, including PKDs, chimaerins, RasGRPs, diacylglycerol kinases (DGKs) and others. Typical C1 domains bind the lipid second messenger diacylglycerol (DAG) and DAG-mimetics such as phorbol esters, and are critical for governing association to membranes. On the contrary, atypical C1 domains possess structural determinants that impede phorbol ester/DAG binding. C1 domains are generally expressed as twin modules (C1A and C1B) or single domains. Biochemical and cellular studies in PKC and PKD isozymes revealed that C1A and C1B domains are non-equivalent as lipid-binding motifs or translocation modules. It has been recently determined that individual C1 domains have unique patterns of ligand recognition, driven in some cases by subtle structural differences. Insights from recent 3-D studies on beta2-chimaerin and Munc13-1 revealed that their single C1 domains are sterically blocked by intramolecular interactions, suggesting that major conformational changes would be required for exposing the site of DAG interaction. Thus, it is clear that the protein context plays a major role in determining whether binding of DAG to the C1 domain would lead to enzyme activation or merely serves as an anchoring mechanism.

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.

We compared the diacylglycerol kinase (DGK) catalyzed phosphorylation of 1-O-hexanoyl-2-oleoylglycerol (HOG) with 1-O-hexanoyl-2-arachidonoylglycerol (HAG). We assayed the activity of DGKalpha and DGKzeta using a liposomal-based assay system. Liposomal assays show that the DGKalpha and, to a lesser extent, DGKzeta preferentially act on substrates containing an arachidonoyl group when this group is incorporated into alkylacylglycerols. The activity of DGKalpha was 82 times greater with HAG compared to HOG. DGKzeta is 10 times more active in catalyzing the phosphorylation of HAG compared to HOG. Although diacylglycerols were better substrates for both DGKalpha and DGKzeta than the alkylacylglycerols, no specificity was exhibited for arachidonoyl-containing diacylglycerols. However, this specificity for HAG over HOG is modulated by the phospholipid composition of the liposome. Addition of cholesterol and/or phosphatidylethanolamine partially reduces the substrate selectivity. We also analyzed the kinetic constants for the phosphorylation of both diacylglycerol and 1-alkyl-2-acylglycerol catalyzed by the alpha, epsilon, or zeta isoforms using a soluble Triton mixed micelle system. We found that all three isoforms of DGK can phosphorylate 1-alkyl-2-acylglycerols but generally at a lower rate than for the corresponding diacylglycerol. The specificity of DGKepsilon for diacylglycerols containing an arachidonoyl group was retained when the ester group in the C-1 position is replaced with an ether linkage. In contrast, DGKalpha and, to a lesser extent, DGKzeta had greater specificity for arachidonoyl-containing 1-alkyl-2-acylglycerols than for arachidonoyl-containing diacylglycerols. This demonstrates that the acyl chain specificity is affected by the structure of the lipid headgroup.

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.