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.

Introduction Diacyglycerol kinase δ isozyme (DGKδ) plays critical roles in lipid signaling by phosphorylating diacylglycerol (DG) into phosphatidic acid (PA). DGKδ regulates a wide variety of physiological and pathological events, such as type II diabetes and obsessive compulsive disorder. Because DGK is one of the components of phosphatidylinositol (PI) turnover, it is thought that DGKδ also utilizes mainly 18:0/20:4‐DG (X:Y; the total number of carbon atoms: the total number of double bonds) derived from PI turnover. Interestingly, we recently demonstrated that DGKδ preferably metabolized palmitic acid (16:0)‐containing DG molecular species, but not arachidonic acid (20:4)‐containing DG species, in response to high glucose stimulation. However, it is still unclear what kind of DG‐generating enzyme provides palmitic acid‐containing DG species. Sphingomyelin synthase (SMS) 1, SMS2 and SMS‐related protein (SMSr) are DG‐generating enzymes utilizing phosphatidylcholine/phosphatidylethanolamine and ceramide. SMS1 and SMSr contain a sterile α motif domain (SAM), which is a protein‐protein interaction module, at their N‐termini. DGKδ also possesses SAM at its C‐terminus, and DGKδ‐SAM is highly homologous to SMSr‐SAM. Therefore, we hypothesised that DGKδ interacts with SMSr through their SAMs. In the present study, we investigated the interaction between DGKδ‐SAM and SMSr‐SAM. Results We first examined whether DGKδ‐SAM interacts with SMSr‐SAM by immunoprecipitation analysis. We found that SMSr‐SAM, but not SMS1‐SAM, was co‐immunoprecipitated with DGKδ‐SAM. Full‐length DGKδ (DGKδ‐FL) was also co‐immunoprecipitated with SMSr‐FL more strongly than with SMS1‐FL and with SMS2‐FL. To examine whether SMSr‐SAM contribute to the interaction, we performed immunoprecipitation analysis using SMSr‐FL and a SAM‐deletion mutant (SMSr‐DSAM). DGKδ‐FL was strongly co‐immunoprecipitated with SMSr, whereas SMSr‐DSAM only weakly interacted with DGKδ‐FL. Immunostaining analysis demonstrated that DGKδ‐FL co‐localized partly with SMSr‐FL in COS‐7 cells overexpressing these proteins. These results strongly suggest that DGKδ interacts with SMSr through their SAMs. Conclusion In summary, the present study for the first time showed that DGKδ interacted with SMSr mainly through their SAMs. It is possible that SMSr is one of the candidates of up stream DG‐providing enzymes of DGKδ, which composes a new pathway independent of PI turnover. Support or Funding Information This work was supported by JSPS KAKENHI (Grant Number JP18J20003) and supported in part by Venture Business Laboratory in Chiba University (Nanohana Competition 2018 Award). This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

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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.

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We have revealed that diacylglycerol kinase η (DGKη)-knockout (KO) mice display bipolar disorder (BPD) remedy-sensitive mania-like behaviors. However, the molecular mechanisms causing the mania-like abnormal behaviors remain unclear. In the present study, microarray analysis was performed to determine global changes in gene expression in the DGKη-KO mouse brain. We found that the DGKη-KO brain had 43 differentially expressed genes and the following five affected biological pathways: “neuroactive ligand-receptor interaction”, “transcription by RNA polymerase II”, “cytosolic calcium ion concentration”, “Jak-STAT signaling pathway” and “ERK1/2 cascade”. Interestingly, mRNA levels of prolactin and growth hormone, which are augmented in BPD patients and model animals, were most strongly increased. Notably, all five biological pathways include at least one gene among prolactin, growth hormone, forkhead box P3, glucagon-like peptide 1 receptor and interleukin 1β, which were previously implicated in BPD. Consistent with the microarray data, phosphorylated ERK1/2 levels were decreased in the DGKη-KO brain. Microarray analysis showed that the expression levels of several glycerolipid metabolism-related genes were also changed. Liquid chromatography-mass spectrometry revealed that several polyunsaturated fatty acid (PUFA)-containing phosphatidic acid (PA) molecular species were significantly decreased as a result of DGKη deficiency, suggesting that the decrease affects PUFA metabolism. Intriguingly, the PUFA-containing lysoPA species were markedly decreased in DGKη-KO mouse blood. Taken together, our study provides not only key broad knowledge to gain novel insights into the underlying mechanisms for the mania-like behaviors but also information for developing BPD diagnostics.

Edited by Sandro SonninoSerotonin 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) d 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 DGKd. 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 DGKd-knockout mouse brain, indicating that DGKd 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 DGKd activates Praja-1 to degrade SERT in the brain.Keywords: diacylglycerol kinase; docosahexaenoic acid; E3 ubiquitinprotein ligase; obsessive-compulsive disorder; Praja-1; serotonin transporter Serotonin/5-hydroxytryptamine (5-HT) is known to be involved in anxiety, depression, compulsiveness, and impulsivity [1]. The serotonin/5-HT transporter (SERT) reuptakes 5-HT from the synaptic cleft into the presynaptic neuron for recycling and metabolic degradation [2,3]. Selective serotonin reuptake (SERT) inhibitors (SSRIs) are used for the treatment of obsessive-compulsive disorder (OCD) [2,3] and major depressive disorder [4]. Several studies using SERTdeficient rodents have demonstrated that SERT is implicated in neurodevelopmental disorders, such as depression, anxiety, and autism [5]. Therefore, hyperfunction of SERT, which reduces the amount of 5-HT in the synaptic cleft, causes various psychiatric disorders mentioned above. However, the regulatory mechanisms of SERT function are still obscure.Diacylglycerol (DG) kinase (DGK), which consists of ten isozymes (a-j), is a lipid-metabolizing enzyme that phosphorylates DG to generate phosphatidic acid (PA) [6][7][8][9][10]. DG and PA are established lipid second messengers that regulate a wide variety of physiological and pathological events.

Although there are many phosphatidic acid (PA) molecular species based on its fatty acyl compositions, their interacting partners have been poorly investigated. Here, we identified synaptojanin‐1 (SYNJ1), Parkinson’s disease‐related protein that is essential for regulating clathrin‐mediated synaptic vesicle endocytosis via dually dephosphorylating D5 and D4 position phosphates from phosphatidylinositol (PI) (4,5)‐bisphosphate, as a 1‐stearoyl‐2‐docosahexaenoyl (18:0/22:6)‐PA‐binding protein. SYNJ1 failed to substantially associate with other acidic phospholipids. Although SYNJ1 interacted with 18:0/20:4‐PA in addition to 18:0/22:6‐PA, the association of the enzyme with 16:0/16:0‐, 16:0/18:1‐, 18:0/18:0‐, or 18:1/18:1‐PA was not considerable. 18:0/20:4‐ and 18:0/22:6‐PAs bound to SYNJ1 via its SAC1 domain, which preferentially hydrolyses D4 position phosphate. Moreover, 18:0/20:4‐ and 18:0/22:6‐PA selectively enhanced the D4‐phosphatase activity, but not the D5‐phosphatase activity, of SYNJ1.