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  • DGK deficiency caused a significant increase in the


    DGKδ deficiency caused a significant increase in the SERT protein level, but its mRNA level was not affected (Fig. 1). These results suggest that SERT protein stability is directly reduced by interaction with the DGKδ protein. In contrast, DGKδ deficiency regulated both TPH-2 and MAO-A protein and mRNA expression levels (Fig. 4), indicating that their expression is indirectly regulated by DGKδ in the transcription step. It is possible that pre-synaptic neuron 5-HT accumulation, caused by the DGKδ deficient-dependent increase of SERT protein levels (Fig. 1), affects transcription of TPH-2 and MAO-A mRNAs through feedback regulation. SERT was reported to undergo acute down-regulation in response to the activation of protein kinase C (PKC) [38]. Overexpressed DGKδ in MCF-7 cells interacted with PKCα, δ, ε and η [14]. Moreover, interaction with RACK1, a receptor for activated C-kinase, was also reported [39]. However, expression and phosphorylation levels of conventional and novel PKCs were not changed in the DGKδ-KO mouse GW311616 hydrochloride (data not shown). We reported that PKC-dependent phosphorylation of Ser-22 and Ser-26 in the pleckstrin homology domain of DGKδ released DGKδ from the plasma membrane [40]. Therefore, PKC may act upstream, but not downstream, of DGKδ in the brain. In summary, the present study strongly suggests that OCD-like behaviors in the DGKδ-KO mice are associated with comprehensive and composite serotonergic dysfunction. These new findings provide novel insight into the molecular mechanisms of OCD pathogenesis and the serotonergic system functions. However, it is still unclear how DGKδ and SERT interact with each other and how the interaction regulates the SERT protein level. Given that SERT is also implicated in autism and depression in addition to OCD [41], it would be interesting to investigate the relationship between DGKδ and these SERT-related psychiatric disorders in future studies.
    Acknowledgments This work was supported in part by grants from MEXT/JSPS KAKENHI Grant Numbers 26291017 (Grant-in-Aid for Scientific Research (B)), 15K14470 (Grant-in-Aid for Challenging Exploratory Research), and 17H03650 (Grant-in-Aid for Scientific Research (B)); the Futaba Electronic Memorial Foundation; the Ono Medical Research Foundation; the Japan Foundation for Applied Enzymology; the Food Science Institute Foundation; the Skylark Food Science Institute; the Asahi Group Foundation and the Japan Milk Academic Alliance (FS).
    Introduction Diacylglycerol kinase (DGK) from different species exhibits different specificities. Thus, bacterial forms of DGK can phosphorylate ceramide as well as diacylglycerol (DAG), while DGK from yeast utilizes CTP, rather than ATP, as the source of phosphate [1]. Mammalian DGKs are a family of enzymes comprised of at least 10 isoforms [2]. We undertook this study to evaluate the interactions of 2-acyl-glycerols with isoforms of DGK in order to assess the possible role of this enzyme family in affecting the concentration of these signaling lipids in cells as well as to further understand the nature of substrate and lipid interactions with binding sites on DGKs. Mammalian isoforms of DGK have only been shown to catalyze the phosphorylation of one class of lipid substrates, DAG, using only ATP as the source of phosphate. In the present study, we determined if structurally related monoacylglycerols are either substrates or inhibitors of mammalian DGKs. A particularly important acyl chain for monoacyl- and diacylglycerols is arachidonic acid. DAG having arachidonic acid at the sn-2 position is an intermediate in phosphatidylinositol cycling. Arachidonoyl-DAG is preferentially phosphorylated by the isoform DGKε [2]. The monoglyceride with arachidonic acid at the sn-2 position is 2-arachidonoyl glycerol (2-AG). This monoglyceride is an important ligand for the CB1 cannabinoid receptor [3]. 2-AG is known to be generated in the brain by the enzyme diacylglycerol (DAG) lipase [4] and is one of the most abundant molecular species of monoacylglycerols in the brain [5]. The concentration of DAG in brain synaptosomes is at least an order of magnitude higher than that of 2-AG [6]. Interestingly, even in organisms lacking known cannabinoid receptors, such as nematodes, 2-AG has been identified [7]. This suggests that 2-AG, in addition to being a cannabinoid receptor ligand, is also an intermediate in lipid metabolism in organisms without developed endocannabinoid systems. The expression of DAG lipase, that converts DAG to a monoglyceride, is required for axonal growth during development and for retrograde synaptic signaling at mature synapses. Endocannabinoid signaling is a key regulator of synaptic communication throughout the central nervous system [8]. The lysolipid 2-Arachidonoyl-sn-glycero-3-phosphate, an arachidonic acid-containing lysophosphatidic acid is found in rat brain and can be rapidly converted to 2-AG. However, the metabolic fate of 2-AG is not known. A principle metabolic fate of this lipid is its hydrolysis by monoacylglycerol lipase. However, an additional possibility is that 2-AG is also a substrate for diacylglycerol kinase (DGK) to reform lysophosphatidic acid that is also a signaling lipid [9], [10], [11], [12]. This possibility was tested in the present study.