坂根 郁夫

Sphingomyelin (SM) synthase 1 (SMS1), which is involved in lipodystrophy, deafness, and thrombasthenia, generates diacylglycerol (DG) and SM using phosphatidylcholine (PC) and ceramide as substrates. Here, we found that SMS1 possesses DG‐generating activities via hydrolysis of PC and phosphatidylethanolamine (PE) in the absence of ceramide and ceramide phosphoethanolamine synthase (CPES) activity. In the presence of the same concentration (4.7 mol%) of PC and ceramide, the amounts of DG produced by SMS and PC‐phospholipase C (PLC) activities of SMS1 were approximately 65% and 35% of total DG production, respectively. PC‐PLC activity showed substrate selectivity for saturated and/or monounsaturated fatty acid‐containing PC species. A PC‐PLC/SMS inhibitor, D609, inhibited only SMS activity. Mn²⁺ inhibited only PC‐PLC activity. Intriguingly, DG attenuated SMS/CPES activities. Our study indicates that SMS1 is a unique enzyme with PC‐PLC/PE‐PLC/SMS/CPES activities.

We previously reported that diacylglycerol (DG) kinase (DGK) δ interacts with DG-generating sphingomyelin synthase (SMS)-related protein (SMSr), but not SMS1 or SMS2, via their sterile α motif domains (SAMDs). However, it remains unclear whether other DGK isozymes interact with SMSs. Here, we found that DGKζ, which does not contain SAMD, interacts with SMSr and SMS1, but not SMS2. Deletion mutant analyses demonstrated that SAMD in the N-terminal cytosolic region of SMSr binds to the N-terminal half catalytic domain of DGKζ. However, the C-terminal cytosolic region of SMS1 interacts with the catalytic domain of DGKζ. Taken together, these results indicate that DGKζ associates with SMSr and SMS1 in different manners and suggest that they compose new DG signaling pathways.

Diacylglycerol kinase (DGK) α, which is a key enzyme in the progression of cancer and, in contrast, in T cell activity attenuation, preferentially produces saturated fatty acid (SFA)- and/or monounsaturated fatty acid (MUFA)-containing phosphatidic acids (PAs), such as 16:0/16:0-, 16:0/18:0- and 16:1/16:1-PA, in melanoma cells. In the present study, we searched for the target proteins of 16:0/16:0-PA in melanoma cells and identified heat shock protein (HSP) 27, which acts as a molecular chaperone and contributes to cancer progression. HSP27 more strongly interacted with PA than other phospholipids, including phosphatidylcholine, phosphatidylserine, phosphatidylglycerol, cardiolipin, phosphatidylinositol, phosphatidylinositol 4-monophosphate and phosphatidylinositol 4,5-bisphosphate. Moreover, HSP27 more preferentially bound to SFA- and/or MUFA-containing PAs, including 16:0/16:0- and 16:0/18:1-PAs, than PUFA-containing PAs, including 18:0/20:4- and 18:0/22:6-PA. Furthermore, HSP27 and constitutively active DGKα expressed in COS-7 cells colocalized in a DGK activity-dependent manner. Notably, 16:0/16:0-PA, but not phosphatidylcholine or 16:0/16:0-phosphatidylserine, induced oligomer dissociation of HSP27, which enhances its chaperone activity. Intriguingly, HSP27 protein was barely detectable in Jurkat T cells, while the protein band was intensely detected in AKI melanoma cells. Taken together, these results strongly suggest that SFA- and/or MUFA-containing PAs produced by DGKα selectively target HSP27 and regulate its cancer-progressive function in melanoma cells, but not in T cells.

Knockout mice of diacylglycerol kinase (DGK) η, which has been repeatedly suggested to be associated with bipolar disorder (BPD) by genome-wide association studies, exhibited abnormal behaviors similar to the manic phase of BPD. Chronic stress is also linked to changes in mood symptoms, including BPD. In the present study, we analyzed the effects of the glucocorticoid stress hormones, triamcinolone acetonide (TAA) and dexamethasone (DEX), on DGKη protein levels in neuroblastoma cell lines, Neuro-2a and SH-SY5Y. The protein levels of DGKη were significantly increased in the undifferentiated Neuro-2a and SH-SY5Y cells by TAA and DEX, but not in the differentiated neuroblastoma cells. To assess the functions of DGKη in undifferentiated neuroblastoma cells, we established DGKη-deficient SH-SY5Y cells using the clustered regularly interspaced palindromic repeat/caspase 9 system. Notably, proliferation of DGKη-deficient SH-SY5Y cells was markedly attenuated, concomitant with the decrease in levels of phosphorylated extracellular signal-regulated kinase. Taken together, these results suggest that DGKη levels are increased in undifferentiated neuroblastoma cells by glucocorticoid stress hormones and regulate cell proliferation.

Diacylglycerol kinase (DGK) is a lipid kinase that converts diacylglycerol (DG) to phosphatidic acid (PA). Since both DG and PA serve as intracellular second messenger molecules, DGK plays a pivotal role in balancing these two signaling pathways. Of the DGK family, DGKη is classified as a type II DGK. Reportedly, DGKη is expressed ubiquitously through mammalian tissues and cells. Previous studies using cDNA transfection methods reported cytoplasmic localization of DGKη in cultured human cells. However, subcellular localization of native protein is still unknown. Recently, we established a human DGKη-specific monoclonal antibody, DhMab-4. In this study, we examined subcellular localization of native protein of DGKη using DhMab-4 by immunocytochemistry in human cultured cells.

The clathrin coat assembly protein AP180 drives endocytosis, which is crucial for numerous physiological events, such as the internalization and recycling of receptors, uptake of neurotransmitters and entry of viruses, including SARS-CoV-2, by interacting with clathrin. Moreover, dysfunction of AP180 underlies the pathogenesis of Alzheimer's disease. Therefore, it is important to understand the mechanisms of assembly and, especially, disassembly of AP180/clathrin-containing cages. Here, we identified AP180 as a novel phosphatidic acid (PA)-binding protein from the mouse brain. Intriguingly, liposome binding assays using various phospholipids and PA species revealed that AP180 most strongly bound to 1-stearoyl-2-docosahexaenoyl-PA (18:0/22:6-PA) to a comparable extent as phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), which is known to associate with AP180. An AP180 N-terminal homology domain (1-289 aa) interacted with 18:0/22:6-PA, and a lysine-rich motif (K38-K39-K40) was essential for binding. The 18:0/22:6-PA in liposomes in 100 nm diameter showed strong AP180-binding activity at neutral pH. Notably, 18:0/22:6-PA significantly attenuated the interaction of AP180 with clathrin. However, PI(4,5)P2 did not show such an effect. Taken together, these results indicate the novel mechanism by which 18:0/22:6-PA selectively regulates the disassembly of AP180/clathrin-containing cages.

Diacylglycerol (DG) kinase (DGK) phosphorylates DG to generate phosphatidic acid (PA). The α isozyme is activated by Ca2+ through its EF-hand motifs and tyrosine phosphorylation. DGKα is highly expressed in several refractory cancer cells including melanoma, hepatocellular carcinoma, and glioblastoma cells. In melanoma cells, DGKα is an antiapoptotic factor that activates nuclear factor-κB (NF-κB) through the atypical protein kinase C (PKC) ζ-mediated phosphorylation of NF-κB. DGKα acts as an enhancer of proliferative activity through the Raf-MEK-ERK pathway and consequently exacerbates hepatocellular carcinoma progression. In glioblastoma and melanoma cells, DGKα attenuates apoptosis by enhancing the phosphodiesterase (PDE)-4A1-mammalian target of the rapamycin pathway. As PA activates PKCζ, Raf, and PDE, it is likely that PA generated by DGKα plays an important role in the proliferation/antiapoptosis of cancer cells. In addition to cancer cells, DGKα is highly abundant in T cells and induces a nonresponsive state (anergy), which represents the main mechanism by which advanced cancers escape immune action. In T cells, DGKα attenuates the activity of Ras-guanyl nucleotide-releasing protein, which is activated by DG and avoids anergy through DG consumption. Therefore, a DGKα-specific inhibitor is expected to be a dual effective anticancer treatment that inhibits cancer cell proliferation and simultaneously enhances T cell functions. Moreover, the inhibition of DGKα synergistically enhances the anticancer effects of programmed cell death-1/programmed cell death ligand 1 blockade. Taken together, DGKα inhibition provides a promising new treatment strategy for refractory cancers.

Activation of diacylglycerol kinase alpha (DGKα) augments proliferation and suppresses apoptosis of cancer cells and induces T lymphocyte anergy. We investigated the dual effects of DGKα inhibition on tumorigenesis and anti-tumor immunity with the aim of establishing a novel therapeutic strategy for cancer. We examined the effects of a DGKα inhibitor (DGKAI) on liver cancer cell proliferation and cytokine production by immune cells in vitro and on tumorigenesis and host immunity in a hepatocellular carcinoma (HCC) mouse model. Oral DGKAI significantly suppressed tumor growth and prolonged survival in model mice. Tumor infiltration of T cells and dendritic cells was also enhanced in mice treated with DGKAI, and the production of cytokines and cytotoxic molecules by CD4+ and CD8+ T cells was increased. Depletion of CD8+ T cells reduced the effect of DGKAI. Furthermore, interferon-γ stimulation augmented the expression of programmed cell death-1 ligand (PD-L1) on cancer cells, and DGKAI plus an anti-PD-L1 antibody strongly suppressed the tumor growth. These results suggest that DGKα inhibition may be a promising new treatment strategy for HCC.

Diacylglycerol kinase (DGK) η translocates from the cytoplasm to punctate vehicles via osmotic shock. Apoptosis signal-regulating kinase (ASK) 3 (MAP kinase kinase kinase (MAPKKK) 15) is also reported to respond to osmotic shock. Therefore, in the present study, we examined the subcellular localization of DGKη and ASK3 expressed in COS-7 cells under osmotic stress. We found that DGKη was almost completely colocalized with ASK3 in punctate structures in response to osmotic shock. In contrast, DGKδ, which is closely related to DGKη structurally, was not colocalized with ASK3, and DGKη failed to colocalize with another MAPKKK, C-Raf, even under osmotic stress. The structures in which DGKη and ASK3 localized were not stained with stress granule makers. Notably, DGKη strongly interacted with ASK3 in an osmotic shock-dependent manner. These results indicate that DGKη and ASK3 undergo osmotic shock-dependent colocalization and associate with each other in specialized structures.

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.

Although an aberrant reduction in pancreatic β-cell mass contributes to the pathogenesis of diabetes, the mechanism underlying the regulation of β-cell mass is poorly understood. Here, we show that diacylglycerol kinase δ (DGKδ) is a key enzyme in the regulation of β-cell mass. DGKδ expression was detected in the nucleus of β-cells. We developed β-cell-specific DGKδ knockout (βDGKδ KO) mice, which showed lower blood glucose, higher plasma insulin levels, and better glucose tolerance compared to control mice. Moreover, an increased number of small islets and Ki-67-positive islet cells, as well as elevated cyclin B1 expression in the islets, were detected in the pancreas of βDGKδ KO mice. DGKδ knockdown in the β-cell line MIN6 induced significant increases in bromodeoxyuridine (BrdU) incorporation and cyclin B1 expression. Finally, we confirmed that streptozotocin-induced hyperglycemia and β-cell loss were alleviated in βDGKδ KO mice. Thus, suppressing the expression or enzymatic activity of DGKδ that functions as a suppressor of β-cell proliferation could be a novel therapeutic approach to increase β-cell mass for the treatment of diabetes.

Diacylglycerol kinase (DGK) phosphorylates diacylglycerol to produce phosphatidic acid (PA). The η isozyme of DGK is abundantly expressed in C2C12 myoblasts. However, the role of DGKη in skeletal muscle cells remains unknown. In the present study, we showed that DGKη was downregulated at an early stage of myogenic differentiation. The knockdown of DGKη by siRNAs significantly inhibited C2C12 myoblast proliferation but did not inhibit differentiation. Moreover, the suppression of DGKη expression decreased the expression levels of mammalian target of rapamycin (mTOR), which is a key regulator of cell proliferation, and fatty acid synthase (FASN), which catalyzes the de novo synthesis of fatty acids for cell proliferation and is transcriptionally regulated via mTOR signaling. Furthermore, the knockdown of mTOR or raptor, which is a component of mTOR complex 1 (mTORC1), decreased the amount of FASN. These results indicate that DGKη regulates myoblast proliferation through the mTOR (mTORC1)-FASN pathway. Interestingly, the knockdown of mTOR reduced the expression levels of DGKη, implying mutual regulation between DGKη and mTOR. In DGKη-knockdown myoblasts, C30-C36-PA species, mTOR activators, were decreased, suggesting that the modulation of mTOR activity through these PA species also plays an important role in myoblast proliferation.

Phosphatidic acid (PA) consists of various molecular species that have different fatty acyl chains at the sn-1 and sn-2 positions; and consequently, mammalian cells contain at least 50 structurally distinct PA molecular species. However, the different roles of each PA species are poorly understood. In the present study, we attempted to identify dipalmitoyl (16:0/16:0)-PA-binding proteins from mouse skeletal muscle using liposome precipitation and tandem mass spectrometry analysis. We identified L-lactate dehydrogenase (LDH) A, which catalyzes conversion of pyruvate to lactate and is a key checkpoint of anaerobic glycolysis critical for tumor growth, as a 16:0/16:0-PA-binding protein. LDHA did not substantially associate with other phospholipids, such as phosphatidylcholine, phosphatidylserine, phosphatidylglycerol, phosphatidylinositol, phosphoinositides and cardiolipin at physiological pH (7.4), indicating that LDHA specifically bound to PA. Interestingly, 18:0/18:0-, 18:0/20:4- and 18:0/22:6-PA also interacted with LDHA, and their binding activities were stronger than 16:0/16:0-PA at pH 7.4. Moreover, circular dichroism spectrometry showed that 18:0/20:4- and 18:0/22:6-PA, but not 16:0/16:0- or 18:0/18:0-PA, significantly reduced the α-helical structure of LDHA. Furthermore, 18:0/20:4- and 18:0/22:6-PA attenuated LDH activity. Taken together, we demonstrated for the first time that LDHA is a PA-binding protein and is a unique PA-binding protein that is structurally and functionally controlled by associating with 18:0/20:4- and 18:0/22:6-PA.

Phosphatidic acid (PA) is the simplest phospholipid and is involved in the regulation of various cellular events. Recently, we developed a new PA sensor, the N-terminal region of α-synuclein (α-Syn-N). However, whether α-Syn-N can sense physiologically produced, endogenous PA remains unclear. We first established an inactive PA sensor (α-Syn-N-KQ) as a negative control by replacing all eleven lysine residues with glutamine residues. Using confocal microscopy, we next verified that α-Syn-N, but not α-Syn-N-KQ, detected PA in macrophagic phagosomes in which PA is known to be enriched, further indicating that α-Syn-N can be used as a reliable PA sensor in cells. Finally, because PA generated during neuronal differentiation is critical for neurite outgrowth, we investigated the subcellular distribution of PA using α-Syn-N. We found that α-Syn-N, but not α-Syn-N-KQ, accumulated at the peripheral regions (close to the plasma membrane) of neuronal growth cones. Experiments using a phospholipase D (PLD) inhibitor strongly suggested that PA in the peripheral regions of the growth cone was primarily produced by PLD. Our findings provide a reliable sensor of endogenous PA and novel insights into the distribution of PA during neuronal differentiation.

Tracking the localization and dynamics of the intracellular bioactive lipid phosphatidic acid (PA) is important for understanding diverse biological phenomena. Although several PA sensors have been developed, better ones are still needed for comprehensive PA detection in cells. We recently found that α-synuclein (α-Syn) selectively and strongly bound to PA in vitro. Here, we revealed that the N-terminal region of α-Syn (α-Syn-N) specifically bound to PA, with a dissociation constant of 6.6 μm. α-Syn-N colocalized with PA-producing enzymes, diacylglycerol kinase (DGK) β at the plasma membrane (PM), myristoylated DGKζ at the Golgi apparatus, phorbol ester-stimulated DGKγ at the PM, and phospholipase D2 at the PM and Golgi but not with the phosphatidylinositol-4,5-bisphosphate-producing enzyme in COS-7 cells. However, α-Syn-N failed to colocalize with them in the presence of their inhibitors and/or their inactive mutants. These results indicate that α-Syn-N specifically binds to cellular PA and can be applied as an excellent PA sensor.

Serotonin transporter (SERT) is involved in serotonergic system regulation and in the pathophysiology/therapeutics of serotonin-/SERT-related diseases such as obsessive-compulsive disorder, depression, autism, and schizophrenia. We recently revealed that diacylglycerol (DG) kinase (DGK) δ induces ubiquitination/degradation of SERT in a DGK activity-dependent manner through Praja-1 E3 ubiquitin-protein ligase. However, it is still unclear how Praja-1 activity is regulated by DGKδ. Here, we reveal that 1-stearoyl-2-docosahexaenoyl (18:0/22:6)-phosphatidic acid (PA) and 18:0/22:6-DG are simultaneously decreased and accumulated, respectively, in the DGKδ-knockout mouse brain, indicating that DGKδ selectively phosphorylates 18:0/22:6-DG to generate 18:0/22:6-PA. Moreover, we find that 18:0/22:6-PA selectively binds to Praja-1 and enhances its activity. These results strongly suggest that 18:0/22:6-PA generated by DGKδ activates Praja-1 to degrade SERT in the brain.

Diacylglycerol kinase (DGK) α enhances the proliferation of melanoma and hepatocellular carcinoma cells whereas, in contrast, DGKα induces a nonproliferative state in T cells. We previously found that DGKα produces palmitic acid (16:0)-containing PA species, such as 16:0/16:0- and 16:0/18:0-PA, in melanoma cells under serum-starved (nonproliferative) conditions. In the present study, we identified the PA species generated by DGKα in T cells under serum-starved (nonproliferative) conditions. We found that serum starvation markedly increased the levels of many PA species, such as 14:1/16:1-, 14:0/16:1-, 14:0/16:0-, 16:1/16:2-, 16:1/16:1-, 16:0/16:1-, 16:0/16:0-, 16:1/18:2-, 16:1/18:1-, 16:0/18:1-, 16:0/18:0-, 18:1/18:2-, 18:1/18:1- and 18:0/18:1-PA, in Jurkat T cells. In lysates from serum-starved Jurkat T cells, DGKα activity, which was Ca2+-dependent and sensitive to a DGKα-specific inhibitor (CU-3), was substantially increased, indicating its activation. Moreover, CU-3 (1-10 μM) significantly reduced the amounts of palmitic acid- and/or palmitoleic acid (16:1)-containing PA species, such as 14:1/16:1-, 14:0/16:1-, 14:0/16:0-, 16:1/16:2-, 16:1/16:1-, 16:0/16:1-, 16:0/16:0-, 16:0/18:1- and 16:0/18:0-PA, which were increased by serum starvation. These results indicate that DGKα generates different PA species in starved melanoma cells (palmitic acid-containing PA species) and T cells (palmitic acid- and/or palmitoleic acid (16:1)-containing PA species). Therefore, the differences in the PA molecular species may account for the opposing functions of DGKα in melanoma and T cells.

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.

Brain-specific diacylglycerol kinase (DGK) δ-knockout mice exhibited serotonin transporter (SERT) inhibitor-sen- sitive obsessive-compulsive disorder-like behaviors. Moreover, SERT protein levels were markedly increased in the DGKδ-deficient brain. However, its molecular mechanisms remain unclear. We found that the catalytic subdo- main-a and the coiled-coil structure-containing region of DGKδ interacted with the C-terminal cytoplasmic region (CTC) of SERT. Moreover, the protein levels of full-length SERT and SERT-CTC alone were significantly decreased by DGKδ in a catalytic activity-dependent manner. A proteasome inhibitor, MG-132, inhibited DGKδ-dependent SERT degradation. Notably, DGKδ interacted with MAGE-D1 adaptor protein and Praja-1 E3 ubiquitin-protein lig- ase, and enhanced the ubiquitination of SERT through Praja-1. Taken together, these results indicate that DGKδ interacts with SERT and induces SERT degradation in an activity-dependent manner through the Praja-1 ubiqui- tin ligase-proteasome system. These new findings provide novel insights into serotonergic system regulation and the pathophysiology/therapeutics of serotonin-/SERT-related diseases such as obsessive-compulsive disorder, de- pression, autism and schizophrenia.

Diacylglycerol kinase (DGK) δ, which is a key enzyme in the pathogenesis of type 2 diabetes (T2D), preferentially generates saturated fatty acid (SFA)- and/or monounsaturated fatty acid (MUFA)-containing phosphatidic acids (PAs) such as 16:0/16:0-PA and 16:0/18:1-PA, but not polyunsaturated fatty acid (PUFA)-containing PAs, in glucose-stimulated myoblast cells. Here, we searched for the target proteins of 16:0/16:0-PA in the mouse skeletal muscle and identified an energy metabolizing enzyme, creatine kinase muscle type (CKM), which is correlated with T2D. CKM bound to 16:0/16:0-PA with the highest affinity (dissociation constant: 2.0 μM) among all the PA-binding proteins reported thus far. Intriguingly, CKM preferentially interacted with SFA- and/or MUFA-containing PAs, but not with PUFA-containing PAs. Notably, CKM exclusively interacted with PA, whereas the protein did not bind to other lipids such as diacylglycerol, phosphatidylserine, phosphatidylglycerol, phosphatidylinositol, phosphatidylinositol (3,4,5)-trisphosphate and cardiolipin. Taken together, these results demonstrate that CKM is a very unique PA-binding protein that possesses exceedingly high affinity for PA, exceptional preference for SFA/MUFA-PA and extremely high specificity to PA and suggest that SFA/MUFA-PAs produced by DGKδ are novel regulators of CKM function.

Diacylglycerol kinase (DGK) is a lipid kinase that converts diacylglycerol (DG) into phosphatidic acid (PA). DG and PA function as lipid messengers contributing to various signalling pathways. Thus, DGK plays a pivotal role in the signalling pathways by maintaining DG and PA levels. For example, DGKδ is involved in diabetes and DGKβ is important for higher brain function including memory and emotion. Recently, we also revealed that the activation of DGKα ameliorated diabetic nephropathy (DN) in mice, suggesting that DGK can be therapeutic target. However, there is no commercially available DGK subtype-specific inhibitors or activators. Therefore, in a series of experiment to find DGK subtype-specific inhibitors or activators, we tried to screen novel DGKα activators from 9,600 randomly selected compounds by using high-throughput screening we had recently developed. Finally, we obtained two lead compounds for DGKα activators, KU-8 and KU-10. Focusing KU-8, we assessed the effect of KU-8 on all mammalian DGKs activities. Thus, KU-8 activates not only DGKα but also DGKθ by approximately 20%, and strongly inhibited DGKκ. In conclusion, KU-8 would be a good lead compound for DGKα and DGKθ activators, and useful as a DGKκ inhibitor.

Copyright © 2019 American Chemical Society. Diacylglycerol kinase ζ (DGKζ) phosphorylates diacylglycerol (DG) to generate phosphatidic acid. The dysfunction of DGKζ has been linked to several diseases, such as cardiac hypertrophy, ischemia, and seizures. Moreover, much attention has been paid to DGKζ, together with DGKα, as a potential target for cancer immunotherapy. However, DGKζ has never been purified and, thus, neither its enzymatic properties nor its structure has yet been reported, hindering our understanding of the catalytic mechanism of DGKζ and the development of a reasonable structure-based drug design. In the present study, we generated a full-length DGKζ using a baculovirus-insect cell expression system for enzymological and structural studies. Full-length DGKζ remained soluble and was purified to near homogeneity as a monomer with yields suitable for protein crystallization (0.63 mg/1 L culture). Enzymatic characterization showed that the purified DGKζ is in a fully functional state. The K m values for adenosine triphosphate (ATP) and DG were 0.05 mM and 1.5 mol %, respectively, and the EC50 for the activator phosphatidylserine was 8.6 mol %, indicating that its affinity for ATP is moderately higher than those of DGKα and DGKϵ, and its affinities for DG and phosphatidylserine are comparable to those of DGKα/DGKϵ. We further confirmed that the purified enzyme could be concentrated without any significant aggregation. Circular dichroism revealed that DGKζ is comprised of 25% α-helices and 18% β-strands. This is the first successful purification and characterization of the enzymatic and conformational properties of DGKζ. The purification of DGKζ allows detailed analyses of this important enzyme and will advance our understanding of DGKζ-related diseases and therapies.

Diacylglycerol kinase (DGK) phosphorylates diacylglycerol (DG) to produce phosphatidic acid (PA). Mammalian DGK comprises ten isozymes (α-κ) and regulates a wide variety of physiological and pathological events, such as cancer, type II diabetes, neuronal disorders and immune responses. DG and PA consist of various molecular species that have different acyl chains at the sn-1 and sn-2 positions, and consequently, mammalian cells contain at least 50 structurally distinct DG/PA species. Because DGK is one of the components of phosphatidylinositol (PI) turnover, the generally accepted dogma is that all DGK isozymes utilize 18:0/20:4-DG derived from PI turnover. We recently established a specific liquid chromatography-mass spectrometry method to analyze which PA species were generated by DGK isozymes in a cell stimulation-dependent manner. Interestingly, we determined that DGKδ, which is closely related to the pathogenesis of type II diabetes, preferentially utilized 14:0/16:0-, 14:0/16:1-, 16:0/16:0-, 16:0/16:1-, 16:0/18:0- and 16:0/18:1-DG species (X:Y = the total number of carbon atoms: the total number of double bonds) supplied from the phosphatidylcholine-specific phospholipase C pathway, but not 18:0/20:4-DG, in high glucose-stimulated C2C12 myoblasts. Moreover, DGKα mainly consumed 14:0/16:0-, 16:0/18:1-, 18:0/18:1- and 18:1/18:1-DG species during cell proliferation in AKI melanoma cells. Furthermore, we found that 16:0/16:0-PA was specifically produced by DGKζ in Neuro-2a cells during retinoic acid- and serum starvation-induced neuronal differentiation. These results indicate that DGK isozymes utilize a variety of DG molecular species derived from PI turnover-independent pathways as substrates in different stimuli and cells. DGK isozymes phosphorylate various DG species to generate various PA species. It was revealed that the modes of activation of conventional and novel protein kinase isoforms by DG molecular species varied considerably. However, PA species-selective binding proteins have not been found to date. Therefore, we next attempted to identify PA species-selective binding proteins from the mouse brain and identified α-synuclein, which has causal links to Parkinson's disease. Intriguingly, we determined that among phospholipids, including several PA species (16:0/16:0-PA, 16:0/18:1-PA, 18:1/18:1-PA, 18:0/18:0-PA and 18:0/20:4-PA); 18:1/18:1-PA was the most strongly bound PA to α-synuclein. Moreover, 18:1/18:1-PA strongly enhanced secondary structural changes from the random coil form to the α-helix form and generated a multimeric and proteinase K-resistant α-synuclein protein. In contrast with the dogma described above, our recent studies strongly suggest that PI turnover-derived DG species and also various DG species derived from PI turnover-independent pathways are utilized by DGK isozymes. DG species supplied from distinct pathways may be utilized by DGK isozymes based on different stimuli present in different types of cells, and individual PA molecular species would have specific targets and exert their own physiological functions.

AIMS/HYPOTHESIS: Previously, we demonstrated that myristic acid (14:0) increases levels of diacylglycerol kinase (DGK) δ, a key enzyme involved in type 2 diabetes exacerbation, and enhances glucose uptake in C2C12 myotube cells. Moreover, results from a population-based cohort study suggest that consumption of high-fat dairy products, which contain high amounts of myristic acid, is associated with a lower risk of developing type 2 diabetes. Taken together, we hypothesised that intake of myristic acid reduces type 2 diabetes risk in vivo. The aim of this study was to examine the glucose-lowering effect of myristic acid in Nagoya-Shibata-Yasuda (NSY) mice, a spontaneous model for studying obesity-related type 2 diabetes. METHODS: Male NSY mice were orally administered vehicle (n = 9), 300 mg/kg of myristic acid (n = 14) or 300 mg/kg of palmitic acid (16:0) (n = 9) every other day from 4 weeks of age. Glucose and insulin tolerance tests were performed at weeks 18, 24 and 30, and weeks 20 and 26, respectively. DGKδ levels were measured in skeletal muscle from 32-36-week-old NSY mice via western blot. RESULTS: Chronic oral administration of myristic acid ameliorated glucose tolerance (24-28% decrease in blood glucose levels during glucose tolerance tests) and reduced insulin-responsive blood glucose levels (~20% decrease) in male NSY mice compared with vehicle and palmitic acid groups at 24-30 weeks of age (the age at which the severity of type 2 diabetes is exacerbated in NSY mice). Myristic acid also attenuated the increase in body weight seen in NSY mice. Furthermore, the fatty acid increased DGKδ levels (~1.6-fold) in skeletal muscle of NSY mice. CONCLUSIONS/INTERPRETATION: These results suggest that the chronic oral administration of myristic acid improves hyperglycaemia by decreasing insulin-responsive glucose levels and reducing body weight, and that the fatty acid accounts for the diabetes protective properties of high-fat dairy products. Myristic acid is a potential candidate for the prevention and treatment of type 2 diabetes mellitus and its related diseases.

Phosphatidylcholine (PC)-specific phospholipase C (PC-PLC) hydrolyzes PC to generate the second messenger 1,2-diacylglycerol (DG) and phosphocholine. PC-PLC plays pivotal roles in inflammation, carcinogenesis, tumor progression, atherogenesis, and subarachnoid hemorrhage. Although the activity of PC-PLC in mammalian tissues was discovered approximately 40 years ago, neither the protein nor its gene has been identified. In the present study, we developed a non-radioactive enzyme activity assay for PC-PLC based on mass spectrometric detection of DG following HPLC separation. This new liquid chromatography-mass spectrometry (LC-MS) assay directly determines a specific reaction product, 1-palmitoyl-2-oleoyl-DG, that is generated from 1-palmitoyl-2-oleoyl-PC by purified Bacillus cereus PC-PLC. The LC-MS assay offers several advantages including a lower background (0.02% versus 91%), higher signal background ratio (4242 versus 1.06)/signal noise ratio (7494 versus 4.4), higher sensitivity (≥32-fold), and lower limit of quantitation (0.04 pmol versus 0.69 pmol of PC-PLC), than a conventional fluorometric assay, which indirectly detects phosphocholine produced in the reaction. In addition to Bacillus cereus PC-PLC, the LC-MS assay was applicable to the measurement of mammalian PC-PLC prepared from the mouse brain. The radioisotope-free, highly sensitive and precise LC-MS assay for PC-PLC would be useful for the purification and identification of PC-PLC protein.