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  • br Competing interests br Acknowledgements br Introduction D

    2019-11-28


    Competing interests
    Acknowledgements
    Introduction Diacylglycerol kinase (DGK) phosphorylates diacylglycerol (DG) in biomembranes to produce phosphatidic Calcium Gluceptate (PA) [1], [2], [3], [4], [5], [6]. To date, ten mammalian DGK isozymes (α, β, γ, δ, ε, ζ, η, θ, ι and κ) have been identified. These DGK isozymes are divided into five groups (type I: α, β and γ; type II: δ, η and κ; type III: ε; type IV: ζ and ι; type V: θ) according to their structural features [1], [2], [3], [4], [5], [6]. DGK isozymes are known to be involved in a wide variety of patho-physiological functions. For example, DGKα (type I) induces clonal anergy [7], [8]. On the other hand, this isozyme also prevents melanoma apoptosis [9] and promotes hepatocellular carcinoma proliferation [10], 3D cancer cell growth [11] and angiogenesis [12]. DGKβ (type I) is an important regulator in neurite spine formation [13]. DGKγ (type I) serves as an upstream suppressor of Rac1 and lamellipodium formation [14]. This isozyme regulates allergic reactions [15] and insulin secretion [16]. In addition to epidermal growth factor signaling [17], DGKδ (type II) is an important factor in hyperglycemia-induced peripheral insulin resistance, thereby exacerbating the severity of type-2 diabetes [18], [19]. DGKη (type II) enhances C-Raf activity and B-Raf/C-Raf heterodimerization in cancer cells [20]. It was also reported that the gene encoding DGKη is implicated in the etiology of bipolar disorder [21]. DGKκ (type II) is associated with risk of hypospadias [22]. DGKε (type III) controls seizure susceptibility and long-term potentiation through modulating arachidonoyl-inositol lipid signaling [23]. DGKζ (type IV) is an important regulator of dendritic spine maintenance [24]. This isozyme is also known to regulate endothelin-1-induced cardiomyocyte hypertrophy [25]. DGKι (type IV) regulates Ras guanyl-releasing protein 3 and inhibits Rap1 signaling [26] and presynaptic release during metabotropic glutamate receptor-dependent long-term depression [27]. DGKθ (type V) has been implicated in familial Parkinson disease [28] and bile acid signaling [29]. Although DGKs have been established as important biomembrane-related modulators as described above, their enzymological properties have not been fully elucidated. 30years ago, Kanoh et al. purified DGKα from porcine liver and demonstrated that the enzyme had 10–20% 2-monoacylglycerol (MG) kinase (MGK) activity compared with its DGK activity [30]. However, its 1-MGK activity was <4% relative to its DGK activity. Gantayet et al. reported that DGKε also exhibited 6.4% 2-MGK activity compared with its DGK activity [31]. However, because a comprehensive analysis has not been done, the MGK activities of the other isozymes are presently unknown. In this study, we measured the 1-MGK and 2-MGK activities of all ten DGK isozymes. We revealed that the type I DGKs (α, β and γ), type II DGKs (δ, η and κ) and type III DGK (ε) have 8–19% 2-MGK activity compared to their DGK activities, whereas their 1-MGK activities were <3%. Both the 2-MGK and 1-MGK activities of the type IV DGKs (ζ and ι) were <1% relative to their DGK activity. Interestingly, the type V DGKθ has approximately 6% 1-MGK activity and <2% 2-MGK activity compared to its DGK activity. These results indicate that the ten DGK isozymes have enzymological diversity beyond our expectation.
    Materials and methods
    Results
    Discussion 30years ago, Kanoh et al. demonstrated that purified DGKα had high 2-MGK activity (10–20% compared to DGK activity), whereas its 1-MGK activity was <4% [30]. DGKε was also reported to have 2-MGK activity (6.4% of DGK activity) by Gantayet et al. [31]. In the present study, DGKα and ε exhibited approximately 12 and 8% 2-MGK activities compared to their DGK activities (Fig. 1, Fig. 3 and Table 1). Therefore, we reproduced their results in this study. Moreover, we newly revealed that DGKβ, γ, δ, η and κ are also 2-MGK-positive and 1-MGK-negative enzymes (Fig. 1, Fig. 2 and Table 1) like DGKα and ε. Among them, DGKγ has the highest 2-MGK activity (19.2%) relative to its DGK activity (Fig. 1). Intriguingly, we demonstrated for the first time that DGKθ has a high 1-MGK activity (approximately 5–6% of DGK activity), but not 2-MGK activity (Fig. 5, Fig. 6 and Table 1). Notably, DGKζ and ι are both 1-MGK- and 2-MGK-negative (Fig. 4 and Table 1). The existence of 1-MGK- and 2-MGK-negative isozymes emphasizes the importance of 1-MGK and 2-MGK-positive isozymes. These results indicate that the ten DGK isozymes have great enzymological diversity beyond our expectation.