• 2018-07
  • 2018-10
  • 2018-11
  • 2019-04
  • 2019-05
  • 2019-06
  • 2019-07
  • 2019-08
  • 2019-09
  • 2019-10
  • Probenecid DGK is classified into the type


    DGKη is classified into the type II DGK subfamily [12], [13], [14], [15]. As described above, DGKη has the splice variants η1 and η2 [10]. DGKs η1 and η2 possess in common a pleckstrin homology domain at the N-terminus and a catalytic domain that is divided into two subdomains (catalytic subdomain-a and -b). However, only DGKη2 has a sterile α motif domain at the C-terminus [10]; DGKη1 [10], [13] lacks this domain. The expression of DGKη1 is markedly increased by glucocorticoid [10], [13]. In contrast, the DGKη2 expression is moderately decreased by the hormone [10]. DGKη1 is highly expressed in the Probenecid [10], [13]. Moreover, DGKη1 in the brain is distributed in the cerebral cortex, the hippocampus (CA1 and CA2 regions and dentate gyrus), the olfactory bulb (mitral cell and glomerular layer), and the cerebellum (the Purkinje cells) [16]. In the brain, DGKη2 expression reaches its maximum levels at P5 and decreases by 4weeks, whereas DGKη1 expression increases over the same timeframe. We recently reported that DGKη1 is required for the Ras/B-Raf/C-Raf/MEK/ERK signaling cascade to be activated by epidermal growth factor (EGF) in HeLa cervical cancer cells [17]. Importantly, DGKη regulates the recruitment of B-Raf and C-Raf from the cytosol to membranes and controls their heterodimerization. Moreover, we demonstrated that DGKη activates C-Raf, but not B-Raf, in an EGF-dependent manner. The data show that DGKη1 is a novel key regulator of the Ras/B-Raf/C-Raf/MEK/ERK signaling pathway. In addition, Nakano et al. reported that depleting DGKη in lung cancer cell lines that harbor mutant EGF receptor reduces their growth on plastic and in soft agar, and also augments the effects of afatinib, an EGF receptor inhibitor [18]. Notably, a genome wide association study has recently indicated that the gene encoding DGKη is implicated in the etiology of bipolar disorder [19]. Moreover, it has been reported that DGKη is highly expressed in the brain of bipolar disorder patients [20]. As described above, DGKη1 is physiologically and pathologically important. However, its enzymatic properties are poorly understood. Therefore, in this study, we determined its affinities for ATP and DG, selectivity for DG molecular species and dependency for phosphatidylserine (PS). Intriguingly, the Km value of DGKη1 for DG was significantly lower than those of several DGK isozymes Probenecid reported so far, indicating that DGKη1 is a unique high-affinity isozyme for DG.
    Materials and methods
    Discussion Although DGKη is physiologically and pathologically important, its enzymatic properties have been poorly understood. In this study, we revealed several enzymatic properties of DGKη1. Interestingly, we found that DGKη1 is an isozyme having higher affinity for DG (Km: 0.14mol%) (Fig. 4 and Table 1) compared with other isozymes including DGKα (Km: 1.3–3.4mol% ([22], [23], [24] and this study)), ε (Km: 1.6–2.4mol%, substrate: 1-stearoyl-2-arachidonoyl-DG [22], [29]) and ζ (Km: 1.6mol% [22]). Essentially the same result was obtained using Triton X-100 mixed micelles, instead of octyl glucoside mixed micelles (Supplemental Fig. S3). In addition to DGKη1 expressed in mammalian COS-7 cells, the enzyme that was bacterially expressed and affinity-purified also exhibited nearly the same affinity for DG (Km: 0.19mol%) (Supplemental Fig. S4C and D), indicating that this property is intrinsic. In contrast to DG, the Km value of DGKη1 for ATP (0.052mM) (Fig. 2 and Table 1) is comparable to that of DGKα (0.12mM) and values reported previously for the enzyme (0.10 [24] and 0.13mM [23]). The PS dependency of DGKη1 (8.5mol%) (Fig. 1 and Table 1) is also comparable with that of DGKα (9.1mol%) and values reported previously for the enzyme (14 [24] and 16mol% [23]). Therefore, the affinity of DGKη1 for DG alone is markedly different from that of DGKα. Vmax of DGKη for DG normalized for band intensities in Western blotting was lower than (approximately 1/5) that of DGKα (Fig. 4 and Table 1). It is known that Vmax values of DGK isozymes are varied. For example, the values of DGKζ and ε are approximately 1/3 and 1/16 that of DGKα, respectively [22]. It is possible that in addition to PS, those isozymes including DGKη may require an unidentified activator.