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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., Graphical abstract Image 1

The molecular mechanisms generating the mania-like abnormal behaviors caused by diacylglycerol (DG) kinase (DGK) η deficiency remain unclear. Here, we found that DGKη knockout markedly increased dopamine (DA) levels in the midbrain (DA-producing region, 2.8-fold) and cerebral cortex (DA projection region, 1.2-fold). Moreover, DGKη deficiency significantly augmented phosphorylated DA transporter (DAT) levels (1.4-fold increase), which induce DA efflux to the synaptic cleft, in the cerebral cortex. Moreover, phosphorylation levels of protein kinase C-β, which is activated by DG and involved in DAT phosphorylation, were also increased. DAT expressed in Neuro-2a cells recruited DGKη to the plasma membrane and colocalized with it. These results strongly suggest that dopaminergic hyperfunction caused by DGKη deficiency in the brain leads to mania-like behaviors.

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 (DG) is a well-established lipid second messenger. Sphingomyelin synthase (SMS)-related protein (SMSr) produces DG and ceramide phosphoethanolamine (CPE) by the transfer of phosphoethanolamine from phosphatidylethanolamine (PE) to ceramide. We previously reported that human SMSr overexpressed in COS-7 cells significantly increased DG levels, particularly saturated and/or monounsaturated fatty acid–containing DG molecular species, and provided DG to DG kinase (DGK) δ, which regulates various pathophysiological events, including epidermal growth factor–dependent cell proliferation, type 2 diabetes, and obsessive–compulsive disorder. However, mammalian SMSr puzzlingly produces only trace amounts of CPE/DG. To clarify this discrepancy, we highly purified SMSr and examined its activities other than CPE synthase. Intriguingly, purified SMSr showed a DG-generating activity via hydrolysis of PE, phosphatidic acid (PA), phosphatidylinositol (PI), and phosphatidylcholine (PC) in the absence of ceramide. DG generation through the PA phosphatase (PAP) activity of SMSr was approximately 300-fold higher than that with PE and ceramide. SMSr hydrolyzed PI ten times stronger than PI(4,5)bisphosphate (PI(4,5)P2). The PAP and PC-phospholipase C (PLC) activities of SMSr were inhibited by propranolol, a PAP inhibitor, and by D609, an SMS/PC-PLC inhibitor. Moreover, SMSr showed substrate selectivity for saturated and/or monounsaturated fatty acid–containing PA molecular species, but not arachidonic-acid-containing PA, which is exclusively generated in the PI(4,5)P2 cycle. We confirmed that SMSr expressed in COS-7 cells showed PAP and PI-PLC activities. Taken together, our study indicated that SMSr possesses previously unrecognized enzyme activities, PAP and PI/PE/PC-PLC, and constitutes a novel DG/PA signaling pathway together with DGKδ, which is independent of the PI(4,5)P2 cycle.

The β-isoform of diacylglycerol kinase (DGK) localizes predominantly to neurons and induces neurite outgrowth and spine formation. However, the detailed molecular mechanisms underlying the functions of DGKβ remain elusive. During the course of studies on other DGK isozymes, we unexpectedly found that the overexpression of wild-type DGKβ in COS-7 cells markedly induced filopodium formation. Because filopodium formation is closely related to neurite outgrowth and spine formation, we constructed various DGKβ mutants and compared their abilities to induce filopodium formation in order to elucidate the structure-function relationships of DGKβ. We found that the C-terminal, C1 and catalytic domains and catalytic activity were indispensable for filopodium formation, but the recoverin homology domain and EF-hand motifs were not. Moreover, the extent of plasma membrane localization and F-actin colocalization were positively correlated with filopodium formation. Intriguingly, DGKβ selectively interacted and colocalized at the plasma membrane with a Rac1-GTPase-activating protein, β2-chimaerin, which is an inducer of filopodia; it also interacted, to lesser extent, with α2-chimaerin, but not with α1- or β1-chimaerin. Moreover, DGKβ enhanced the plasma membrane localization of β2-chimaerin. These results suggest that DGKβ plays an important role in neurite outgrowth and spine formation in neurons via its ability to induce filopodium formation.

We previously reported that brain-specific diacylglycerol kinase (DGK) δ-knockout (KO) mice showed obsessive-compulsive disorder (OCD)-like behaviors, which were alleviated by a serotonin (5-HT) transporter (SERT) inhibitor. However, the molecular mechanisms causing the OCD-like abnormal behaviors remain unclear. In the present study, we found that DGKδ deficiency increased SERT protein levels in the mouse cerebral cortex. Moreover, DGKδ interacted and co-localized with SERT. Furthermore, DGKδ-KO decreased tryptophan hydroxylase-2 expression and increased monoamine oxidase-A expression. Indeed, the amount of 5-HT in the cerebral cortex was significantly decreased in DGKδ-KO mice. These data strongly suggest that OCD-like behaviors in the DGKδ-KO mice are caused by comprehensive and composite serotonergic hypofunction.

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. 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. 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. 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.

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.

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) 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.

Phosphatidic acid (PA) is one of the phospholipids composing the plasma membrane and acts as a second messenger to regulate a wide variety of important cellular events, including mitogenesis, migration and differentiation. PA consists of various molecular species with different acyl chains at the sn-1 and sn-2 positions. However, it has been poorly understood what PA molecular species are produced during such cellular events. Here we identified the PA molecular species generated during retinoic acid (RA)-induced neuroblastoma cell differentiation using a newly established liquid chromatography/mass spectrometry (LC/MS) method. Intriguingly, the amount of 32:0-PA species was dramatically and transiently increased in Neuro-2a neuroblastoma cells 24–48 h after RA-treatment. In addition, 30:0- and 34:0-PA species were also moderately increased. Moreover, similar results were obtained when Neuro-2a cells were differentiated for 24 h by serum starvation. MS/MS analysis revealed that 32:0-PA species contains two palmitic acids (16:0 s). RT-PCR analysis showed that diacylglycerol kinase (DGK) δ and DGKζ were highly expressed in Neuro-2a cells. The silencing of DGKζ expression significantly decreased the production of 32:0-PA species, whereas DGKδ-siRNA did not. Moreover, neurite outgrowth was also markedly attenuated by the deficiency of DGKζ. Taken together, these results indicate that DGKζ exclusively generates very restricted PA species, 16:0/16:0-PA, and up-regulates neurite outgrowth during the initial/early stage of neuroblastoma cell differentiation., Graphical abstract fx1, Highlights • We analyzed phosphatidic acid (PA) molecular species in neuroblastoma cells. • We identified a marked increase of 32:0-PA during neuroblastoma cell differentiation. • 32:0-PA species contained two palmitic acids (16:0 s). • Diacylglycerol kinase (DGK) ζ-siRNA decreased 16:0/16:0-PA production. • Neurite outgrowth was markedly attenuated by deficiency of DGKζ.

A variety of diacylglycerol (DG) molecular species are produced in stimulated cells. Conventional (α, βII and γ) and novel (δ, ε, η and θ) protein kinase C (PKC) isoforms are known to be activated by DG. However, a comprehensive analysis has not been performed. In this study, we analyzed activation of the PKC isozymes in the presence of 2–2000 mmol% 16:0/16:0-, 16:0/18:1-, 18:1/18:1-, 18:0/20:4- or 18:0/22:6-DG species. PKCα activity was strongly increased by DG and exhibited less of a preference for 18:0/22:6-DG at 2 mmol%. PKCβII activity was moderately increased by DG and did not have significant preference for DG species. PKCγ activity was moderately increased by DG and exhibited a moderate preference for 18:0/22:6-DG at 2 mmol%. PKCδ activity was moderately increased by DG and exhibited a preference for 18:0/22:6-DG at 20 and 200 mmol%. PKCε activity moderately increased by DG and showed a moderate preference for 18:0/22:6-DG at 2000 mmol%. PKCη was not markedly activated by DG. PKCθ activity was the most strongly increased by DG and exhibited a preference for 18:0/22:6-DG at 2 and 20 mmol% DG. These results indicate that conventional and novel PKCs have different sensitivities and dependences on DG and a distinct preference for shorter and saturated fatty acid-containing and longer and polyunsaturated fatty acid-containing DG species, respectively. This differential regulation would be important for their physiological functions., Highlights • We comprehensively analyzed activation of c/nPKC isozymes by different DG species. • c/nPKCs have different sensitivities and dependences on DG. • c/nPKCs have a distinct preference for different fatty acid-containing DG species. • This differential regulation would be important for PKCs' physiological functions.

Diacylglycerol kinase (DGK) consists of 10 isozymes. The α-isozyme enhances the proliferation of cancer cells. However, DGKα facilitates the nonresponsive state of immunity known as T-cell anergy; therefore, DGKα enhances malignant traits and suppresses immune surveillance. The aim of this study was to identify a novel small molecule that selectively and potently inhibits DGKα activity. We screened a library containing 9,600 chemical compounds using a newly established high-throughput DGK assay. As a result, we have obtained a promising compound, 5-[(2E)-3-(2-furyl)prop-2-enylidene]-3-[(phenylsulfonyl)amino]2-thioxo-1,3-thiazolidin-4-one) (CU-3), which selectively inhibited DGKα with an IC50 value of 0.6 μM. CU-3 targeted the catalytic region, but not the regulatory region, of DGKα. CU-3 competitively reduced the affinity of DGKα for ATP, but not diacylglycerol or phosphatidylserine. Moreover, this compound induced apoptosis in HepG2 hepatocellular carcinoma and HeLa cervical cancer cells while simultaneously enhancing the interleukin-2 production of Jurkat T cells. Taken together, these results indicate that CU-3 is a selective and potent inhibitor for DGKα and can be an ideal anticancer drug candidate that attenuates cancer cell proliferation and simultaneously enhances immune responses including anticancer immunity.

Diacylglycerol kinase (DGK) phosphorylates diacylglycerol (DG) to generate phosphatidic acid (PA). Mammalian DGK consists of ten isozymes (α–κ) and governs a wide range of physiological and pathological events, including immune responses, neuronal networking, bipolar disorder, obsessive-compulsive disorder, fragile X syndrome, cancer, and type 2 diabetes. DG and PA comprise diverse molecular species that have different acyl chains at the sn-1 and sn-2 positions. Because the DGK activity is essential for phosphatidylinositol turnover, which exclusively produces 1-stearoyl-2-arachidonoyl-DG, it has been generally thought that all DGK isozymes utilize the DG species derived from the turnover. However, it was recently revealed that DGK isozymes, except for DGKε, phosphorylate diverse DG species, which are not derived from phosphatidylinositol turnover. In addition, various PA-binding proteins (PABPs), which have different selectivities for PA species, were recently found. These results suggest that DGK–PA–PABP axes can potentially construct a large and complex signaling network and play physiologically and pathologically important roles in addition to DGK-dependent attenuation of DG–DG-binding protein axes. For example, 1-stearoyl-2-docosahexaenoyl-PA produced by DGKδ interacts with and activates Praja-1, the E3 ubiquitin ligase acting on the serotonin transporter, which is a target of drugs for obsessive-compulsive and major depressive disorders, in the brain. This article reviews recent research progress on PA species produced by DGK isozymes, the selective binding of PABPs to PA species and a phosphatidylinositol turnover-independent DG supply pathway.

Brain-specific diacylglycerol kinase (DGK) δ-knockout mice exhibited serotonin transporter (SERT) inhibitor-sensitive 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 subdomain-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 ligase, 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 ubiquitin 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, depression, autism and schizophrenia.

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.

Diacylglycerol kinase (DGK) is responsible for the enzymatic conversion of diacylglycerol to phosphatidic acid. Since both diacylglycerol and phosphatidic acid serve as signaling molecules, DGK is regarded as a hub between diacylglycerol-mediated and phosphatidic acid-mediated signaling. One of the 10 DGK isozymes, DGKα, is shown to be involved in T cell function. Transfection studies using tagged expression vectors revealed that DGKα localizes to the cytoplasm and nucleus and translocates to the plasma membrane in response to T cell receptor stimulation. However, a limited number of studies reported the localization of native protein of DGKα in tissues and cells. In this study, we immunized mice with recombinant DGKα and developed several anti-DGKα monoclonal antibodies (mAbs). One of the established anti-DGKα mAbs is a clone DaMab-2 (mouse IgG1, kappa). In enzyme-linked immunosorbent assay, DaMab-2 recognized only DGKα, and did not react with the other isozymes, such as DGKγ, DGKζ, DGKη, and DGKδ. Importantly, DaMab-2 is very useful in immunocytochemical analysis of human cultured cells, indicating that DaMab-2 is advantageous to analyze the localization and function of DGKα.

There are ten isozymes of diacylglycerol kinase (DGK), and they regulate diverse patho-physiological functions. Here, we investigated the lipid-binding properties of DGK isozymes using protein–lipid overlay and liposome-binding assays. DGKγ showed a strong binding activity compared with other DGK isozymes for phosphatidic acid (PA) among the various glycerophospholipids tested. However, DGKγ failed to interact with DG and lyso-PA. Moreover, the isozyme was capable of binding to ceramide-1-phosphate but not to ceramide or sphingosine-1-phosphate. The isozyme bound more strongly to PA containing unsaturated fatty acid than to PA having only saturated fatty acid. An analysis using a series of deletion mutants of DGKγ revealed that the N-terminal region, which contains a recoverin homology domain and EF-hand motifs, is responsible for the PA binding activity of DGKγ. Taken together, these results indicate that DGKγ is an anionic phospholipid binding protein that preferably interacts with a small highly charged head group that is very close to the glycerol or sphingosine backbone.

Graphical abstract, Highlights • We revealed the intra-molecular interaction of DGKα. • The EF-hand motifs of DGKα directly binds to its C1 domains. • The intra-molecular interaction was negatively regulated by Ca2+. • The intra-molecular interaction is important for the activation mechanism of DGKα., Diacylglycerol kinase (DGK) α, which is activated by Ca2+, contains a recoverin homology (RVH) domain, tandem repeats of two Ca2+-binding EF-hand motifs, two cysteine-rich C1 domains and the catalytic domain. We previously found that a DGKα mutant lacking the RVH domain and EF-hands was constitutively active and that the N-terminal region of DGKα, consisting of the RVH domain and EF-hand motifs, interacted intra-molecularly with the C-terminal region containing the C1 and catalytic domains. In this study, we narrowed down the interaction regions of DGKα. At the C-terminal region, the C1 domains are responsible for the intra-molecular interaction. At the N-terminal region, the EF-hand motifs mainly contribute to the interaction. Moreover, using highly purified EF-hand motifs and C1 domains, we demonstrate that they directly bind to each other. The co-precipitation of these two domains was clearly attenuated by the addition of Ca2+. These results indicate that the Ca2+-induced dissociation of the intra-molecular interaction between the EF-hand motifs and the C1 domains of DGKα is the key event that regulates the activity of the enzyme.

Diacylglycerol kinase (DGK) catalyzes the conversion of diacylglycerol (DAG) to phosphatidic acid. This study investigated the expression and function of DGK in pancreatic β-cells. mRNA expression of type I DGK isoforms (α, β, γ) was detected in mouse pancreatic islets and the β-cell line MIN6. Protein expression of DGKα and DGKγ was also detected in mouse β-cells and MIN6 cells. The type I DGK inhibitor R59949 inhibited high K(+)- and glucose-induced insulin secretion in MIN6 cells. Moreover, single knockdown of DGKα or DGKγ by small interfering RNA slightly but significantly decreased glucose- and high K(+)-induced insulin secretions, and the double knockdown further decreased them to the levels comparable with those induced by R59949. R59949 and DiC8, a membrane permeable DAG analog, decreased intracellular Ca(2+) concentration elevated by glucose and high K(+) in MIN6 cells. Real-time imaging in MIN6 cells expressing green fluorescent protein-tagged DGKα or DGKγ showed that the DGK activator phorbol 12-myristate 13-acetate rapidly induced translocation of DGKγ to the plasma membrane, whereas high K(+) slowly translocated DGKα and DGKγ to the plasma membrane. R59949 increased the DAG content in MIN6 cells when stimulated with high KCl, whereas it did not increase the DAG content but decreased the phosphatidic acid content when stimulated with high glucose. Finally, R59949 was confirmed to inhibit high K(+)-induced insulin secretion from mouse islets and glucose-induced insulin secretion from rat islets. These results suggest that DGKα and DGKγ are present in β-cells and that the depression of these DGKs causes a decrease in intracellular Ca(2+) concentration, thereby reducing insulin secretion.

Ten mammalian diacylglycerol kinase (DGK) isozymes (a-γ) have been identified. Recent studies have revealed that DGK isozymes play pivotal roles in a wide variety of pathophysiological functions. Thus, it is important to be able to easily check DGK activity in each pathophysiological event. Moreover, the conventional DGK assay is quite laborious because it requires the use of a radioisotope and thin-layer chromatography including multiple extraction steps. In order to minimize the laborious procedures, we established a non-radioactive, single well, two-step DGK assay system. We demonstrated that, compared to the conventional method, the new assay system has comparable sensitivity and much higher efficiency, and is effective in detecting potential agents with high reliability (Z'-factor = 0.69 ± 0.12; n = 3). Using the newly developed assay, we comprehensively evaluated the DGK isozyme selectivities of commercially available DGK inhibitors, R59022 and R59949, in vitro. We found that among 10 isozymes, R59022 strongly inhibited type I DGKa and moderately attenuated type III DGKe and type V DGKθ, and that R59949 strongly inhibited type I DGK a and γ, and moderately attenuated type II DGK d and γ.

Ten mammalian diacylglycerol kinase (DGK) isozymes (alpha–kappa) have been identified to date. Our previous review noted that several DGK isozymes can serve as potential drug targets for cancer, epilepsy, autoimmunity, cardiac hypertrophy, hypertension and type II diabetes (Curr. Drug Targets 9, 626–640 (2008)). Since then, recent genome-wide association studies have implied several new possible relationships between DGK isozymes and diseases. For example, DGKtheta and DGKkappa have been suggested to be associated with susceptibility to Parkinson’s disease and hypospadias, respectively. In addition, the DGKeta gene has been repeatedly identified as a bipolar disorder (BPD) susceptibility gene. Intriguingly, we found that DGKeta-knockout mice showed lithium (BPD remedy)-sensitive mania-like behaviors, suggesting that DGKeta is one of key enzymes of the etiology of BPD. Because DGKs are potential drug targets for a wide variety of diseases, the development of DGK isozyme-specific inhibitors/activators has been eagerly awaited. Recently, we have identified DGKalpha-selective inhibitors. Because DGKalpha has both pro-tumoral and anti-immunogenic properties, the DGKalpha-selective inhibitors would simultaneously have anti-tumoral and pro-immunogenic (anti-tumor immunogenic) effects. Although the ten DGK isozymes are highly similar to each other, our current results have encouraged us to identify and develop specific inhibitors/activators against every DGK isozyme that can be effective regulators and drugs against a wide variety of physiological events and diseases.

The type II diacylglycerol kinases (DGKs) contain several functional domains such as a pleckstrin homology (PH) domain, two C1 domains and a sterile α-motif (SAM) domain. It was previously revealed that DGKδ contributes to hyperglycemia-induced peripheral insulin resistance and thereby exacerbate the severity of type 2 diabetes. Moreover, a high extracellular concentration of glucose activated DGKδ in skeletal muscle cells, which was followed by a reduction in the intracellular diacylglycerol levels and the inactivation of protein kinase Cα, the enzyme that phosphorylates and inactivates the insulin receptor. However, the intracellular behavior of DGKδ upon high glucose stimulation remains unclear. In this study, we found that DGKδ1, but not a splice variant DGKδ2 or the other type II DGKη1/2, translocated from the cytoplasm to the plasma membrane in human embryonic kidney HEK293 and mouse myoblast C2C12 cells within 5min in response to high glucose levels. The translocation was inhibited by phosphatidylinositol 3-kinase inhibitors, LY294002 and GDC-0941, suggesting that the event is regulated via the phosphatidylinositol 3-kinase pathway. Moreover, we revealed that the PH and C1 domains are responsible for the plasma membrane translocation and that the SAM domain negatively regulates the translocation. These results indicate that DGKδ1 is the sole type II DGK isoform that responds rapidly and dynamically to high glucose levels.

Type I diacylglycerol kinase (DGK) isozymes (α, β, and γ) contain recoverin homology domains and calcium-binding EF-hand motifs at their N-termini. The γ-isoform of DGK is abundantly expressed in retinal and Purkinje cells; however, its function in neuronal cells remains unknown. Here, we report that the mRNA and protein levels of DGKγ, but not DGKα or β, were markedly increased in N1E-115 neuroblastoma cells upon cellular differentiation by serum starvation. Interestingly, overexpression of wild-type DGKγ, which was partially located at the plasma membrane, considerably induced the formation of slender, filopodia-like cytoplasmic projections from N1E-115 cell bodies. Deletion of the recoverin homology domain and the EF-hand motifs, which potentiated the plasma membrane localization of the isozyme, significantly enhanced the formation of the filopodia-like protrusions. Intriguingly, the catalytic activity of the isozyme is not essential for the protrusion formation. The N-terminal half of the catalytic domain and a short stretch of amino acid residues at the C-terminus are responsible for plasma membrane localization and filopodia-like process formation. Taken together, we have described a potentially novel morphological function of the C-terminal DGKγ catalytic region that is independent of its enzymatic activity.

The type I diacylglycerol kinase (DGK) isozymes (α, β and γ) contain a shared recoverin homology (RVH) domain, a tandem repeat of Ca2+-binding EF-hand motifs, two cysteine-rich C1 domains, and the catalytic domain. We previously reported that a DGKα mutant lacking the RVH domain and EF-hands was constitutively active, implying that the N-terminal region (NTR) of DGKα, consisting of the RVH domain and EF-hand motifs, intramolecularly interacts with and masks the activity of the C-terminal region (CTR), containing the C1 and catalytic domains. In this study, we demonstrate that a glutathione S-transferase (GST)-fused DGKα-NTR construct physically binds to a green fluorescent protein (GFP)-fused DGKα-CTR construct. Moreover, co-precipitation of GFP-DGKα-CTR with GST-DGKα-NTR was clearly attenuated by the addition of 1 μM Ca2+. This result indicates that Ca2+ induces dissociation of the physical interaction between DGKα-NTR and DGKα-CTR. In addition to previously reported calcium-dependent changes in the hydrophobicity and net surface charge, Ca2+ also appeared to induce a decrease in the α-helical content of DGKα-NTR. These results suggest that Ca2+-induced conformational changes in the NTR release the intramolecular association between the NTR and the CTR of DGKα.

c-Abl is a tyrosine kinase involved in many cellular processes, including cell cycle control and proliferation. However, little is known about its substrates. Here, we show that c-Abl directly phosphorylates diacylglycerol kinase α (DGKα), an important regulator of many cellular events through its conversion of diacylglycerol to phosphatidic acid. We found that DGKα was transported from the cytoplasm to the nucleus in response to serum starvation, and serum restoration induced the nuclear export of the enzyme to the cytoplasm. This serum-induced export involves two tyrosine kinases, c-Src and c-Abl. The latter, c-Abl, is activated by c-Src, phosphorylates DGKα, and shuttles between the nucleus and the cytoplasm in a direction opposite to that of DGKα in response to serum restoration. Moreover, an in vitro phosphorylation assay using purified mutants of DGKα identified Tyr-218 as a site of phosphorylation by c-Abl. We confirmed these results for endogenous DGKα using an antibody specific for phospho-Tyr-218, and this phosphorylation was necessary for the serum-induced export of DGKα. These results demonstrate that the nucleo-cytoplasmic shuttling of DGKα is orchestrated by tyrosine phosphorylation by the Src-activated tyrosine kinase c-Abl and that this phosphorylation is important for regulating the function of cytoplasmic and/or nuclear DGKα.

Diacylglycerol (DAG) kinase (DGK) modulates the balance between the two signaling lipids, DAG and phosphatidic acid (PA), by phosphorylating (consuming) DAG to yield PA. Ten mammalian DGK isozymes have been identified to date. In addition to two or three cysteine-rich C1 domains (protein kinase C-like zinc finger structures) commonly conserved in all DGKs, these isoforms possess a variety of regulatory domains of known and/or predicted functions, such as a pair of EF-hand motifs, a pleckstrin homology domain, a sterile alpha motif domain, a MARCKS (myristoylated alanine-rich C kinase substrate) phosphorylation site domain and ankyrin repeats. Recent studies have revealed that DGK isozymes play pivotal roles in a wide variety of mammalian signal transduction pathways conducting growth factor/cytokine-dependent cell proliferation and motility, seizure activity, immune responses, cardiovascular responses and insulin receptor-mediated glucose metabolism. It is suggested that several DGK isozymes can serve as potential drug targets for cancer, epilepsy, autoimmunity, cardiac hypertrophy, hypertension and type II diabetes. Unfortunately, there are no DGK isozyme-specific inhibitors/activators at present. Development of these compounds is eagerly awaited for the development of novel drugs targeting DGKs.

Type 2 (non-insulin-dependent) diabetes mellitus is a progressive metabolic disorder arising from genetic and environmental factors that impair beta cell function and insulin action in peripheral tissues. We identified reduced diacylglycerol kinase delta (DGKdelta) expression and DGK activity in skeletal muscle from type 2 diabetic patients. In diabetic animals, reduced DGKdelta protein and DGK kinase activity were restored upon correction of glycemia. DGKdelta haploinsufficiency increased diacylglycerol content, reduced peripheral insulin sensitivity, insulin signaling, and glucose transport, and led to age-dependent obesity. Metabolic flexibility, evident by the transition between lipid and carbohydrate utilization during fasted and fed conditions, was impaired in DGKdelta haploinsufficient mice. We reveal a previously unrecognized role for DGKdelta in contributing to hyperglycemia-induced peripheral insulin resistance and thereby exacerbating the severity of type 2 diabetes. DGKdelta deficiency causes peripheral insulin resistance and metabolic inflexibility. These defects in glucose and energy homeostasis contribute to mild obesity later in life.

beta2-Chimaerin, an intracellular receptor for the second messenger diacylglycerol and phorbol esters, is a GTPase-activating protein (GAP) specific for Rac. beta2-Chimaerin negatively controls many Rac-dependent pathophysiological events including tumor development. However, the regulatory mechanism of beta2-chimaerin remains largely unknown. Here we report that beta2-chimaerin is tyrosine-phosphorylated by Src-family kinases (SFKs) upon cell stimulation with epidermal growth factor (EGF). Mutational analysis identified Tyr-21 in the N-terminal regulatory region as a major phosphorylation site. Intriguingly, the addition of SFK inhibitor and the replacement of Tyr-21 with Phe (Y21F) markedly enhanced Rac-GAP activity of beta2-chimaerin in EGF-treated cells. Moreover, the Y21F mutant inhibited integrin-dependent cell spreading, in which Rac1 plays a critical role, more strongly than wild-type beta2-chimaerin. These results suggest Tyr-21 phosphorylation as a novel, SFK-dependent mechanism that negatively regulates beta2-chimaerin Rac-GAP activity.

Diacylglycerol (DAG) kinase (DGK) modulates the balance between the two signaling lipids, DAG and phosphatidic acid (PA), by phosphorylating DAG to yield PA. To date, ten mammalian DGK isozymes have been identified. In addition to the C1 domains (protein kinase C-like zinc finger structures) conserved commonly in all DGKs, these isoforms possess a variety of regulatory domains of known and/or predicted functions, such as a pair of EF-hand motifs, a pleckstrin homology domain, a sterile alpha motif domain and ankyrin repeats. Beyond our expectations, recent studies have revealed that DGK isozymes play pivotal roles in a wide variety of signal transduction pathways conducting development, neural and immune responses, cytoskeleton reorganization and carcinogenesis. Moreover, there has been rapidly growing evidence indicating that individual DGK isoforms exert their specific roles through interactions with unique partner proteins such as protein kinase Cs, Ras guanyl nucleotide-releasing protein, chimaerins and phosphatidylinositol-4-phosphate 5-kinase. Therefore, an emerging paradigm for DGK is that the individual DGK isoforms assembled in their own signaling complexes should carry out spatio-temporally segregated tasks for a wide range of biological processes via regulating local, but not global, concentrations of DAG and/or PA.

Type II diacylglycerol kinase (DGK) isozymes (δ, η, and κ) have a pleckstrin homology domain (PH) at their N termini. Here, we investigated the lipid binding properties of the PHs of type II DGK isozymes using protein-lipid overlay and liposome binding assays. The PH of DGKη showed the most pronounced binding activity to phosphatidylinositol (PI) 4,5-bisphosphate (PI(4,5)P2) among the various glycero- and sphingolipids including PI 3,4,5-trisphosphate, PI 3,4-bisphosphate, PI 3-phosphate, PI 4-phosphate, and PI 5-phosphate. Moreover, the PI(4,5)P2 binding activity of the DGKη-PH was significantly stronger than that of other type II DGK isozymes. Notably, compared with the PH of phospholipase C (PLC) δ1, which is generally utilized as a cellular PI(4,5)P2- probe, the DGKη-PH is equal to or superior than the PLCδ1-PH in terms of affinity and selectivity for PI(4,5)P2. Furthermore, in COS-7 cells, GFP-fused wild-type DGKη1 and its PH partly translocated from the cytoplasm to the plasma membrane where the PLCδ1-PH was co-localized in response to hyperosmotic stress in an inositol 5-phosphatase-sensitive manner, whereas a PH deletion mutant did not. Moreover, K74A and R85A mutants of DGKη-PH, which lack the conserved basic amino acids thought to ligate PI(4,5)P2, were indeed unable to bind to PI(4,5)P2 and co-localize with the PLCδ1-PH even in osmotically shocked cells. Overexpression of wild-type DGKη1 enhanced EGF-dependent phosphorylation of ERK, whereas either K74A or R85A mutant did not. Taken together, these results indicate that the DGKη-PH preferentially interacts with PI(4,5)P2 and has crucial roles in regulating the subcellular localization and physiological function of DGKη. Moreover, the DGKη-PH could serve as an excellent cellular sensor for PI(4,5)P2.

Lipid phosphate phosphatases (LPPs), integral membrane proteins with six transmembrane domains, dephosphorylate a variety of extracellular lipid phosphates. Although LPP3 is already known to bind to Triton X-100-insoluble rafts, we here report that LPP1 is also associated with lipid rafts distinct from those harboring LPP3. We found that LPP1 was Triton X-100-soluble, but CHAPS-insoluble in LNCaP cells endogenously expressing LPP1 and several LPP1 cDNA-transfected cells including NIH3T3 fibroblasts. In addition to the non-ionic detergent insolubility, LPP1 further possessed several properties formulated for raft-localizing proteins as follows: first, the CHAPS-insolubility was resistant to the actin-disrupting drug cytochalasin D; second, the CHAPS-insoluble LPP1 floated in an Optiprep density gradient; third, the CHAPS insolubility of LPP1 was lost by cholesterol depletion; and finally, the subcellular distribution pattern of LPP1 exclusively overlapped with that of a raft marker, cholera toxin B subunit. Interestingly, confocal microscopic analysis showed that LPP1 was distributed to membrane compartments distinct from those of LPP3. Analysis using various LPP1/LPP3 chimeras revealed that their first extracellular regions determine the different Triton X-100 solubilities. These results indicate that LPP1 and LPP3 are distributed in distinct lipid rafts that may provide unique microenvironments defining their non-redundant physiological functions.

Diacylglycerol kinases (DGKs) phosphorylate diacylglycerol (DAG) to terminate its signaling. To study DGKdelta, we disrupted its gene in mice and found that DGKdelta deficiency reduced EGF receptor (EGFR) protein expression and activity. Similar to EGFR knockout mice, DGKdelta-deficient pups were born with open eyelids and died shortly after birth. PKCs are activated by DAG and phosphorylate EGFR to reduce its expression and activity. We found DAG accumulation, increased threonine phosphorylation of EGFR, enhanced phosphorylation of other PKC substrates, and increased PKC autophosphorylation in DGKdelta knockout cells, indicating that DGKdelta regulates EGFR by modulating PKC signaling.

DGK (diacylglycerol kinase) regulates the concentration of two bioactive lipids, diacylglycerol and phosphatidic acid. DGKdelta1 or its PH (pleckstrin homology) domain alone has been shown to be translocated to the plasma membrane from the cytoplasm in PMA-treated cells. In the present study, we identified Ser-22 and Ser-26 within the PH domain as the PMA- and epidermal-growth-factor-dependent phosphorylation sites of DGKdelta1. Experiments in vitro and with intact cells suggested that the cPKC (conventional protein kinase C) phosphorylated these Ser residues directly. Puzzlingly, alanine/asparagine mutants at Ser-22 and Ser-26 of DGKdelta1 and its PH domain are still persistently translocated by PMA treatment, suggesting that the PH domain phosphorylation is not responsible for the enzyme translocation and that the translocation was caused by a PMA-dependent, but cPKC-independent, process yet to be identified. Interestingly, the aspartate mutation, which mimics phosphoserine, at Ser-22 or Ser-26, inhibited the translocation of full-length DGKdelta1 and the PH domain markedly, suggesting that the phosphorylation regulates negatively the enzyme translocation. Our results provide evidence of the phosphorylation of the DGKdelta1 PH domain by cPKC, and suggest that the phosphorylation is involved in the control of subcellular localization of DGKdelta1.

Diacylglycerol kinase (DGK) participates in regulating the intracellular concentrations of two bioactive lipids, diacylglycerol and phosphatidic acid. DGK eta (eta 1, 128 kDa) is a type II isozyme containing a pleckstrin homology domain at the amino terminus. Here we identified another DGK eta isoform (eta 2, 135 kDa) that shared the same sequence with DGK eta 1 except for a sterile alpha motif (SAM) domain added at the carboxyl terminus. The DGK eta 1 mRNA was ubiquitously distributed in various tissues, whereas the DGK eta 2 mRNA was detected only in testis, kidney, and colon. The expression of DGK eta 2 was suppressed by glucocorticoid in contrast to the marked induction of DGK eta 1. DGK eta 2 was shown to form through its SAM domain homo-oligomers as well as hetero-oligomers with other SAM-containing DGKs (delta 1 and delta 2). Interestingly, DGK eta 1 and DGK eta 2 were rapidly translocated from the cytoplasm to endosomes in response to stress stimuli. In this case, DGK eta 1 was rapidly relocated back to the cytoplasm upon removal of stress stimuli, whereas DGK eta 2 exhibited sustained endosomal association. The experiments using DGK eta mutants suggested that the oligomerization of DGK eta 2 mediated by its SAM domain was largely responsible for its sustained endosomal localization. Similarly, the oligomerization of DGK eta 2 was suggested to result in negative regulation of its catalytic activity. Taken together, alternative splicing of the human DGK eta gene generates at least two isoforms with distinct biochemical and cell biological properties responding to different cellular metabolic requirements.

Background We have revealed that the type II diacylglycerol kinases (DGKs) δ, η and κ were expressed in the testis and ovary. However, these enzymes’ functions in the reproductive organs remain unknown. Results In this study, we first identified the expression sites of type II DGKs in the mouse reproductive organs in detail. Reverse transcription-polymerase chain reaction and Western blotting confirmed that DGKδ2 (splicing variant 2) but not DGKδ1 (splicing variant 1) and DGKκ were expressed in the testis, ovary and uterus. DGKη1 (splicing variant 1) but not DGKη2 (splicing variant 2) was strongly detected in the ovary and uterus. Interestingly, we found that a new alternative splicing product of the DGKη gene, DGKη3, which lacks exon 26 encoding 31 amino acid residues, was expressed only in the testis. Moreover, we investigated the distribution of type II DGKs in the testis, ovary and uterus through in situ hybridization. DGKδ2 was distributed in the primary spermatocytes of the testis and ovarian follicles. DGKη1 was distributed in the oviductal epithelium of the ovary and the luminal epithelium of the uterus. Intriguingly, DGKη3 was strongly expressed in the secondary spermatocytes and round spermatids of the testis. DGKκ was distributed in the primary and secondary spermatocyte of the testis. Conclusion These results indicate that the expression patterns of the type II DGK isoforms δ2, η1, η3 and κ differ from each other, suggesting that these DGK isoforms play specific roles in distinct compartments and developmental stages of the reproductive organs, especially in the processes of spermatogenesis and oocyte maturation.

Diacylglycerol kinase (DGK) plays an important role in signal transduction through modulating the balance between two signaling lipids, diacylglycerol and phosphatidic acid. DGKdelta (type II isozyme) contains a pleckstrin homology domain at the N terminus and a sterile alpha motif domain at the C terminus. We identified another DGKdelta isoform (DGKdelta2, 135 kDa) that shared the same sequence with DGKdelta previously cloned (DGKdelta1, 130 kDa) except for the 52 residues N-terminally extended. Analysis of panels of human normal and tumor tissue cDNAs revealed that DGKdelta2 was ubiquitously expressed in all normal and tumor tissues examined, whereas the transcript of DGKdelta1 was detected only in ovary and spleen, and in a limited set of tumor-derived cells. The expression of DGKdelta2 was induced by treating cells with epidermal growth factor and tumor-promoting phorbol ester. In contrast, the levels of mRNA and protein of DGKdelta1 were suppressed by phorbol ester treatment. It thus becomes clear that the two DGKdelta isoforms are expressed under distinct regulatory mechanisms. DGKdelta1 was translocated through its pleckstrin homology domain from the cytoplasm to the plasma membrane in response to phorbol ester stimulation, whereas DGKdelta2 remained in the cytoplasm even after stimulation. Further experiments showed that the delta2-specific N-terminal sequence blocks the phorbol ester-dependent translocation of this isoform. Co-immunoprecipitation analysis of differently tagged DGKdelta1 and DGKdelta2 proteins showed that they were able to form homo- as well as hetero-oligomers. Taken together, alternative splicing of the human DGKdelta gene generates at least two isoforms, differing in their expressions and regulatory functions.

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 0.14mol%. 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.

The functions of type II diacylglycerol kinase (DGK) δ and -η in the brain are still unclear. As a first step, we investigated the spatial and temporal expression of DGKδ and -η in the brains of mice. DGKδ2, but not DGKδ1, was highly expressed in layers II–VI of the cerebral cortex; CA–CA3 regions and dentate gyrus of hippocampus; mitral cell, glomerular and granule cell layers of the olfactory bulb; and the granule cell layer in the cerebellum in 1- to 32-week-old mice. DGKδ2 was expressed just after birth, and its expression levels dramatically increased from weeks 1 to 4. A substantial amount of DGKη (η1/η2) was detected in layers II–VI of the cerebral cortex, CA1 and CA2 regions and dentate gyrus of the hippocampus, mitral cell and glomerular layers of the olfactory bulb, and Purkinje cells in the cerebellum of 1- to 32-week-old mice. DGKη2 expression reached maximum levels at P5 and decreased by 4 weeks, whereas DGKη1 increased over the same time frame. These results indicate that the expression patterns of DGK isozymes differ from each other and also from other isozymes, and this suggests that DGKδ and -η play distinct and specific roles in the brain.

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.

We previously reported that diacylglycerol kinase β (DGKβ) induces neurites and branches, contributing to higher brain function including emotion and memories. However, the detailed molecular mechanism of DGKβ function remains unknown. Therefore, we constructed various mutants of DGKβ and compared their enzyme activity, intracellular localization, and ability to induce neurites and branching in SH-SY5Y cells. Even when RVH-domain and EF-hand motif were deleted, the mutant showed similar plasma membrane localization and neurite induction compared to wild type (WT), although the kinase activity of the mutant was three times higher than that of WT. In contrast, further deletion of C1 domain reduced the activity to 50% and abolished plasma membrane localization and neurite induction ability. When 34 amino acids were deleted from C-terminus, the mutants completely lost enzyme activity, plasma membrane localization, and the ability to induce neurites. A kinase-negative mutant of DGKβ retained plasma membrane localization and induced significant neurites and branches; however, the rate of induction was weaker than that of WT. Furthermore, C1A and C1B mutants, which have a mutation in a cysteine residue in the C1A or C1B domain, and the RK/E mutant, which has substitutions of arginine and lysine to glutamic acid in a cluster of basic amino acids at the C-terminus, lost their plasma membrane localization and neurite induction ability. These results indicate that in addition to kinase activity, plasma membrane localization via the C1 domain and basic amino acids at the C-terminus were indispensable for neurite induction by DGKβ.

The pleckstrin homology domains (PH domains) derived from four different proteins, the N-terminal part of pleckstrin, RAC-protein kinase, diacylglycerol kinase and the 130kDa protein originally cloned as an inositol 1,4,5-trisphosphate binding protein, were analysed for binding of inositol phosphates and derivatives of inositol lipids. The PH domain from pleckstrin bound inositol phosphates according to a number of phosphates on the inositol ring, i.e. more phosphate groups, stronger the binding, but a very limited specificity due to the 2-phosphate was also observed. On the other hand, the PH domains from RAC-protein kinase and diacylglycerol kinase specifically bound inositol 1,3,4,5,6-pentakisphosphate and inositol 1,4,5,6-tetrakisphosphate most strongly. The PH domain from the 130kDa protein, however, had a preference for inositol 1,4,5-trisphosphate and 1,4,5,6-tetrakisphosphate. Comparison was also made between binding of inositol 1,4,5-trisphosphate, inositol 1,3,4,5-tetrakisphosphate and soluble derivatives of their corresponding phospholipids. The PH domains examined, except that from pleckstrin, showed a 8- to 42-times higher affinity for inositol 1,4,5-trisphosphate than that for corresponding phosphoinositide derivative. However, all PH domains had similar affinity for inositol 1,3,4,5-tetrakisphosphate compared to the corresponding lipid derivative. The present study supports our previous proposal that inositol phosphates and/or inositol lipids could be important ligands for the PH domain, and therefore inositol phosphates/inositol lipids may have the considerable versatility in the control of diverse cellular function. Which of these potential ligands are physiologically relevant would depend on the binding affinities and their cellular abundance.

We obtained two human cDNA clones encoding phosphatidic acid phosphatase (PAP) isozymes named PAP-2a (Mr = 32,158) and -2b (Mr = 35, 119), both of which contained six putative transmembrane domains. Both enzymes were glycosylated and cleaved by N-glycanase and endo-beta-galactosidase, thus suggesting their post-Golgi localization. PAP-2a and -2b shared 47% identical sequence and were judged to be the human counterparts of the previously sequenced mouse 35-kDa PAP(83% identity) and rat Dri42 protein (94% identity), respectively. Furthermore, the sequences of both PAPs were 34-39% identical to that of Drosophila Wunen protein. In view of the functions ascribed to Wunen and Dri42 in germ cell migration and epithelial differentiation, respectively, these findings unexpectedly suggest critical roles of PAP isoforms in cell growth and differentiation. Although the two PAPs hydrolyzed lysophosphatidate and ceramide-1-phosphate in addition to phosphatidate, the hydrolysis of sphingosine-1-phosphate was detected only for PAP-2b. PAP-2b was expressed almost ubiquitously in all human tissues examined, whereas the expression of PAP-2a was relatively variable, being extremely low in the placenta and thymus. In HeLa cells, the transcription of PAP-2a was not affected by different stimuli, whereas PAP-2b was induced (up to 3-fold) by epidermal growth factor. These findings indicate that despite structural similarities, the two PAP isozymes may play distinct functions through their different patterns of substrate utilization and transcriptional regulation.

Recent studies have revealed that phosphoinositide (PI) signaling molecules are expressed in mammalian retinas, suggesting their importance in its signal transduction. We previously showed that diacylglycerol kinase (DGK) isozymes are expressed in distinct patterns in rat retina at the mRNA level. However, little is known about the nature and morphological aspects of DGKs in the retina. For this study, we performed immunohistochemical analyses to investigate in the retina the expression and localization of DGK isozymes at the protein level. Here, we show that both DGKβ and DGKι localize in the outer plexiform layer, within which photoreceptor cells make contact with bipolar and horizontal cells. These isozymes exhibit distinct subcellular localization patterns: DGKι localizes to the synaptic area of bipolar cells in a punctate manner, whereas DGKβ distributes diffusely in the subsynaptic and dendritic regions of bipolar and horizontal cells. However, punctate labeling for DGKϵ is evident in the outer limiting membrane. DGKζ and DGKα localize predominantly to the nucleus of ganglion cells. These findings show distinct expression and localization of DGK isozymes in the retina, suggesting a different role of each isozyme.