An intact zinc finger motif of the C1B domain is critical for stability and activity of diacylglycerol kinase-ε.

Diacylglycerol kinase-ε (DGKε) phosphorylates DAG to phosphatidic acid with unique specificity toward 18:0/20:4 DAG (SAG). SAG is a typical backbone of phosphatidylinositol and its derivatives, therefore DGKε activity is crucial for the turnover of these signaling lipids. Malfunction of DGKε contributes to several pathophysiological conditions, including atypical hemolytic uremic syndrome (aHUS) linked with DGKE mutations. In the present study we analyzed the role of a zinc finger motif of the C1B domain of DGKε, as some aHUS-linked mutations affect this ill-defined part of the kinase. For this, we introduce a novel fluorescent assay for determination of DGKε activity which relies on the use of NBD-SAG in mixed micelles as a substrate, followed by TLC separation of NBD-phosphatidic acid formed. The assay reliably determines the activity of purified human GST-DGKε, also endogenous DGKε or overexpressed mouse DGKε-Myc in cell lysates, homogenates, and kinase immunoprecipitates. Using the above assay we found that four amino acids, Cys135, Cys138, His161 and Cys164, forming the zinc finger motif in the C1B domain are required for the DGKε-Myc activity and stability. Substitution of any of these amino acids with Ala or Trp in DGKε-Myc abolished its activity and led to its proteasomal degradation, possibly assisted by Hsp70/90/40 chaperones. Inhibition of the 26S proteasome prevented the degradation but the mutated proteins were inactive. The present data on the deleterious effect of the zinc finger motif disruption contribute to the understanding of the DGKε-linked aHUS, as the Cys164Trp substitution in mouse DGKε corresponds to the Cys167Trp one in human DGKε found in some aHUS patients.

Exploring the AlphaFold Predicted Conformational Properties of Human Diacylglycerol Kinases.

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.

Human Diacylglycerol Kinase ε N-Terminal Segment Regulates the Phosphatidylinositol Cycle, Controlling the Rate but Not the Acyl Chain Composition of Its Lipid Intermediates.

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*-Δ50-DGKε) corroborated the role of the N-terminus in regulating DGKε enzymatic properties. The comparison between the enzymatic properties of DGKε and His-SUMO*-Δ50-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.

Biomimetic Silica Encapsulation of Lipid Nanodiscs and ß-Sheet-Stabilized Diacylglycerol Kinase.

Integral membrane proteins (IMPs) comprise highly important classes of proteins such as transporters, sensors, and channels, but their investigation and biotechnological application are complicated by the difficulty to stabilize them in solution. We set out to develop a biomimetic procedure to encapsulate functional integral membrane proteins in silica to facilitate their handling under otherwise detrimental conditions and thereby extend their applicability. To this end, we designed and expressed new fusion constructs of the membrane scaffold protein MSP with silica-precipitating peptides based on the R5 sequence from the diatom Cylindrotheca fusiformis. Transmission electron microscopy (TEM) and atomic force microscopy (AFM) revealed that membrane lipid nanodiscs surrounded by our MSP variants fused to an R5 peptide, so-called nanodiscs, were formed. Exposing them to silicic acid led to silica-encapsulated nanodiscs, a new material for stabilizing membrane structures and a first step toward incorporating membrane proteins in such structures. In an alternative approach, four fusion constructs based on the amphiphilic ß-sheet peptide BP-1 and the R5 peptide were generated and successfully employed toward silica encapsulation of functional diacylglycerol kinase (DGK). Silica-encapsulated DGK was significantly more stable against protease exposure and incubation with simulated gastric fluid (SGF) and intestinal fluid (SIF).

Advances in chemical proteomic evaluation of lipid kinases-DAG kinases as a case study.

Advancements in chemical proteomics and mass spectrometry lipidomics are providing new opportunities to understand lipid kinase activity, specificity, and regulation on a global cellular scale. Here, we describe recent developments in chemical biology of lipid kinases with a focus on those members that phosphorylate diacylglycerols. We further discuss future implications of how these mass spectrometry-based approaches can be adapted for studies of additional lipid kinase members with the aim of bridging the gap between protein and lipid kinase-focused investigations.

Diacylglycerol Kinase alpha in X Linked Lymphoproliferative Disease Type 1.

Diacylglycerol kinases are intracellular enzymes that control the balance between the secondary messengers diacylglycerol and phosphatidic acid. DGKα and DGKζ are the prominent isoforms that restrain the intensity of T cell receptor signalling by metabolizing PLCγ generated diacylglycerol. Thus, their activity must be tightly controlled to grant cellular homeostasis and refine immune responses. DGKα is specifically inhibited by strong T cell activating signals to allow for full diacylglycerol signalling which mediates T cell response. In X-linked lymphoproliferative disease 1, deficiency of the adaptor protein SAP results in altered T cell receptor signalling, due in part to persistent DGKα activity. This activity constrains diacylglycerol levels, attenuating downstream pathways such as PKCθ and Ras/MAPK and decreasing T cell restimulation induced cell death. This is a form of apoptosis triggered by prolonged T cell activation that is indeed defective in CD8+ cells of X-linked lymphoproliferative disease type 1 patients. Accordingly, inhibition or downregulation of DGKα activity restores in vitro a correct diacylglycerol dependent signal transduction, cytokines production and restimulation induced apoptosis. In animal disease models, DGKα inhibitors limit CD8+ expansion and immune-mediated tissue damage, suggesting the possibility of using inhibitors of diacylglycerol kinase as a new therapeutic approach.

Membrane morphology determines diacylglycerol kinase α substrate acyl chain specificity.

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.

Membrane shape as determinant of protein properties.

Membrane lipids play a role in the modulation of a variety of biological processes. This is often achieved through fine-tuned changes in membrane physical and chemical properties. While some membrane physical properties (e.g., curvature, lipid domains, fluidity) have received increased scientific attention over the years, only recently has membrane shape emerged as an active modulator of protein properties. Biological membranes are mostly found organized into a lipid bilayer arrangement, in which the spontaneous shape is an intrinsically flat, planar morphology (in relation to the size of proteins). However, it is known that many cells and organelles have non-planar morphologies. In addition, perturbations in membrane morphology occur in a variety of biological processes. Recent studies have shown that membrane shape can modulate a variety of biological processes by determining protein properties. While membrane shape generation modulates proteins via changes in membrane mechanical properties, membrane shape recognition regulates proteins by providing the optimal surface for interaction. Hence, membranes have evolved an elegant mechanism to couple mesoscopic perturbations to molecular properties and vice-versa. In this review, the regulation of the enzymatic properties of two isoforms of mammalian diacylglycerol kinase, which play important roles in cellular signal transductions, will be used to exemplify the recent advancements in the field of membrane shape recognition, as well as future challenges and perspectives.

Structure of membrane diacylglycerol kinase in lipid bilayers.

Diacylglycerol kinase (DgkA) is a small integral membrane protein, responsible for the ATP-dependent phosphorylation of diacylglycerol to phosphatidic acid. Its structures reported in previous studies, determined in detergent micelles by solution NMR and in monoolein cubic phase by X-ray crystallography, differ significantly. These differences point to the need to validate these detergent-based structures in phospholipid bilayers. Here, we present a well-defined homo-trimeric structure of DgkA in phospholipid bilayers determined by magic angle spinning solid-state NMR (ssNMR) spectroscopy, using an approach combining intra-, inter-molecular paramagnetic relaxation enhancement (PRE)-derived distance restraints and CS-Rosetta calculations. The DgkA structure determined in lipid bilayers is different from the solution NMR structure. In addition, although ssNMR structure of DgkA shows a global folding similar to that determined by X-ray, these two structures differ in monomeric symmetry and dynamics. A comparative analysis of DgkA structures determined in three different detergent/lipid environments provides a meaningful demonstration of the influence of membrane mimetic environments on the structure and dynamics of membrane proteins.

Beyond Lipid Signaling: Pleiotropic Effects of Diacylglycerol Kinases in Cellular Signaling.

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.

Whole exome sequencing revealed a novel homozygous variant in the DGKE catalytic domain: a case report of familial hemolytic uremic syndrome.

BACKGROUND: Atypical hemolytic uremic syndrome (aHUS) is a rare disease characterized by microangiopathic hemolytic anemia caused by small vessel thrombosis, thrombocytopenia, and renal failure. The common cause of aHUS is a dysregulation in the alternative complement pathway. Mutations in none complement genes such as diacylglycerol kinase epsilon (DGKE) can also result in this syndrome. CASE PRESENTATION: Here, we report on a 19-year-old female with the clinical diagnosis of aHUS, who has unaffected consanguineous parents and an older sibling who was deceased from aHUS when she was seven months old. We performed whole exome sequencing (WES) followed by evaluation of detected variants for functional significance, using several online prediction tools. Next, in order to confirm the detected pathogenic variant in proband and segregation analysis in her family, Sanger sequencing was done. The novel variant was analyzed in terms of its impact on the protein 3-dimensional structure by computational structural modeling. The results revealed that the proband carried a novel homozygous missense variant in DGKE located in exon 6 of the gene (NM_003647.3, c.942C > G [p.Asn314Lys]), and in silico analysis anticipated it as damaging. Protein computational study confirmed the influence of potential pathogenic variant on structural stability and protein function. CONCLUSION: We suggest that some variations in the catalytic domain of DGKE like p.Asn314Lys which can cause alterations in secondary and 3-D structure of protein, might lead to aHUS.

Determinants of lipids acyl chain specificity: A tale of two enzymes.

It is becoming widely acknowledged that many biological processes are dependent on specific lipid molecular species. In healthy humans, two important lipid molecular species for cell physiology are tetralinoleoyl cardiolipin (in the heart) and 1-stearoyl-2-arachidonoyl phosphatidylinositols (throughout the organism). The predominance of these lipid molecular species is in part due to the presence of enzymes along their biosynthetic pathways that favor their enrichment with specific acyl chains. In cardiolipin biosynthesis, one example is the reaction catalyzed by the enzyme tafazzin, while for the biosynthesis of phosphatidylinositols the epsilson isoform of diacylglycerol kinase (DGKε) plays an important role. Here a discussion of the roles played by both enzyme structure and membrane environment on the production of specific lipid molecular species by these two membrane-acting enzymes will be made. It is proposed that the enrichment of certain lipid molecular species within the organism is a result of a fine-tuned interplay between enzyme structure and membrane environment.

Light-Induced Uncaging for Time-Resolved Observations of Biochemical Reactions by MAS NMR Spectroscopy.

Light-induced activation of biomolecules by uncaging of photolabile protection groups has found many applications for triggering biochemical reactions with minimal perturbations directly within cells. Such an approach might also offer unique advantages for solid-state NMR experiments on membrane proteins for initiating reactions within or at the membrane directly within the closed MAS rotor. Herein, we demonstrate that the integral membrane protein E. coli diacylglycerol kinase (DgkA), which catalyzes the phosphorylation of diacylglycerol, can be controlled by light under MAS-NMR conditions. Uncaging of NPE-ATP or of lipid substrate NPE-DOG by in situ illumination triggers its enzymatic activity, which can be monitored by real-time 31 P-MAS NMR. This proof-of-concept illustrates that combining MAS-NMR with uncaging strategies and illumination methods offers new possibilities for controlling biochemical reactions at or within lipid bilayers.

Arabidopsis DIACYLGLYCEROL KINASE4 is involved in nitric oxide-dependent pollen tube guidance and fertilization.

Nitric oxide (NO) is a key signaling molecule that regulates diverse biological processes in both animals and plants, including important roles in male gamete physiology. In plants, NO is generated in pollen tubes (PTs) and affects intracellular responses through the modulation of Ca2+ signaling, actin organization, vesicle trafficking and cell wall deposition, bearing consequences in pollen-stigma interactions and PT guidance. In contrast, the NO-responsive proteins that mediate these responses remain elusive. Here, we show that PTs of Arabidopsis thaliana mutants impaired in the pollen-specific DIACYLGLYCEROL KINASE4 (DGK4) grow slower and become partially insensitive to NO-dependent growth inhibition and re-orientation responses. Recombinant DGK4 protein yields NO-responsive spectral and catalytic changes in vitro that are compatible with a role in NO perception and signaling in PTs. In addition to the expected phosphatidic acid-producing kinase activity, DGK4 recombinant protein also revealed guanylyl cyclase activity, as inferred by sequence analysis. Our results are compatible with a role for the fast-diffusible NO gas in signaling and cell-cell communication via the modulation of DGK4 activity during the progamic phase of angiosperm reproduction.

Diacylglycerol kinase δ and sphingomyelin synthase-related protein functionally interact via their sterile α motif domains.

The δ isozyme of diacylglycerol kinase (DGKδ) plays critical roles in lipid signaling by converting diacylglycerol (DG) to phosphatidic acid (PA). We previously demonstrated that DGKδ preferably phosphorylates palmitic acid (16:0)- and/or palmitoleic acid (16:1)-containing DG molecular species, but not arachidonic acid (20:4)-containing DG species, which are recognized as DGK substrates derived from phosphatidylinositol turnover, in high glucose-stimulated myoblasts. However, little is known about the origin of these DG molecular species. DGKδ and two DG-generating enzymes, sphingomyelin synthase (SMS) 1 and SMS-related protein (SMSr), contain a sterile α motif domain (SAMD). In this study, we found that SMSr-SAMD, but not SMS1-SAMD, co-immunoprecipitates with DGKδ-SAMD. Full-length DGKδ co-precipitated with full-length SMSr more strongly than with SMS1. However, SAMD-deleted variants of SMSr and DGKδ interacted only weakly with full-length DGKδ and SMSr, respectively. These results strongly suggested that DGKδ interacts with SMSr through their respective SAMDs. To determine the functional outcomes of the relationship between DGKδ and SMSr, we used LC-MS/MS to investigate whether overexpression of DGKδ and/or SMSr in COS-7 cells alters the levels of PA species. We found that SMSr overexpression significantly enhances the production of 16:0- or 16:1-containing PA species such as 14:0/16:0-, 16:0/16:0-, 16:0/18:1-, and/or 16:1/18:1-PA in DGKδ-overexpressing COS-7 cells. Moreover, SMSr enhanced DGKδ activity via their SAMDs in vitro Taken together, these results strongly suggest that SMSr is a candidate DG-providing enzyme upstream of DGKδ and that the two enzymes represent a new pathway independent of phosphatidylinositol turnover.

Reprogramming fatty acyl specificity of lipid kinases via C1 domain engineering.

C1 domains are lipid-binding modules that regulate membrane activation of kinases, nucleotide exchange factors and other C1-containing proteins to trigger signal transduction. Despite annotation of typical C1 domains as diacylglycerol (DAG) and phorbol ester sensors, the function of atypical counterparts remains ill-defined. Here, we assign a key role for atypical C1 domains in mediating DAG fatty acyl specificity of diacylglycerol kinases (DGKs) in live cells. Activity-based proteomics mapped C1 probe binding as a principal differentiator of type 1 DGK active sites that combined with global metabolomics revealed a role for C1s in lipid substrate recognition. Protein engineering by C1 domain swapping demonstrated that exchange of typical and atypical C1s is functionally tolerated and can directly program DAG fatty acyl specificity of type 1 DGKs. Collectively, we describe a protein engineering strategy for studying metabolic specificity of lipid kinases to assign a role for atypical C1 domains in cell metabolism.

Comprehensive Genomic Analysis and Expression Profiling of Diacylglycerol Kinase (DGK) Gene Family in Soybean (Glycine max) under Abiotic Stresses.

Diacylglycerol kinase (DGK) is an enzyme that plays a pivotal role in abiotic and biotic stress responses in plants by transforming the diacylglycerol into phosphatidic acid. However, there is no report on the characterization of soybean DGK genes in spite of the availability of the soybean genome sequence. In this study, we performed genome-wide analysis and expression profiling of the DGK gene family in the soybean genome. We identified 12 DGK genes (namely GmDGK1-12) which all contained conserved catalytic domains with protein lengths and molecular weights ranging from 436 to 727 amino acids (aa) and 48.62 to 80.93 kDa, respectively. Phylogenetic analyses grouped GmDGK genes into three clusters-cluster I, cluster II, and cluster III-which had three, four, and five genes, respectively. The qRT-PCR analysis revealed significant GmDGK gene expression levels in both leaves and roots coping with polyethylene glycol (PEG), salt, alkali, and salt/alkali treatments. This work provides the first characterization of the DGK gene family in soybean and suggests their importance in soybean response to abiotic stress. These results can serve as a guide for future studies on the understanding and functional characterization of this gene family.

Crystal structure and calcium-induced conformational changes of diacylglycerol kinase α EF-hand domains.

Diacylglycerol kinases (DGKs) are multi-domain lipid kinases that phosphorylate diacylglycerol into phosphatidic acid, modulating the levels of these key signaling lipids. Recently, increasing attention has been paid to DGKα isozyme as a potential target for cancer immunotherapy. We have previously shown that DGKα is positively regulated by Ca2+ binding to its N-terminal EF-hand domains (DGKα-EF). However, little progress has been made for the structural biology of mammalian DGKs and the molecular mechanism underlying the Ca2+ -triggered activation remains unclear. Here we report the first crystal structure of Ca2+ -bound DGKα-EF and analyze the structural changes upon binding to Ca2+ . DGKα-EF adopts a canonical EF-hand fold, but unexpectedly, has an additional α-helix (often called a ligand mimic [LM] helix), which is packed into the hydrophobic core. Biophysical and biochemical analyses reveal that DGKα-EF adopts a protease-susceptible "open" conformation without Ca2+ that tends to form a dimer. Cooperative binding of two Ca2+ ions dissociates the dimer into a well-folded monomer, which resists to proteolysis. Taken together, our results provide experimental evidence that Ca2+ binding induces substantial conformational changes in DGKα-EF, which likely regulates intra-molecular interactions responsible for the activation of DGKα and suggest a possible role of the LM helix for the Ca2+ -induced conformational changes. SIGNIFICANCE STATEMENT: Diacylglycerol kinases (DGKs), which modulates the levels of two lipid second messengers, diacylglycerol and phosphatidic acid, is still structurally enigmatic enzymes since its first identification in 1959. We here present the first crystal structure of EF-hand domains of diacylglycerol kinase α in its Ca2+ bound form and characterize Ca2+ -induced conformational changes, which likely regulates intra-molecular interactions. Our study paves the way for future studies to understand the structural basis of DGK isozymes.

Epitope Mapping of Antidiacylglycerol Kinase α Monoclonal Antibody DaMab-2.

Diacylglycerol kinase (DGK) is responsible for the enzymatic conversion of diacylglycerol (DG) to phosphatidic acid (PA). Both DG and PA serve as signaling molecules; therefore, DGK functions as a key enzyme between DG- and PA-mediated signaling. DGKα, one of the 10 DGK isozymes, is involved in T cell function and has been shown to localize in the cytoplasm and nucleus. Furthermore, DGKα translocates to the plasma membrane in response to T cell receptor stimulation. Recently, we developed a specific monoclonal antibody (mAb), DaMab-2 (mouse IgG1, kappa), against DGKα. DaMab-2 is very useful in immunocytochemical analysis using HeLa cells. In this study, we characterized the binding epitope of DaMab-2 using Western blot and revealed that Cys246, Lys249, Pro252, and Cys253 of DGKα are important for DaMab-2 binding to the DGKα protein. Our findings can be applied for the production of more functional anti-DGKα mAbs.

Diacylglycerol kinases: Relationship to other lipid kinases.

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.