Analytical Method for Diacylglycerol Kinase ζ Activity in Cells Using Protein Myristoylation and Liquid Chromatography–Tandem Mass Spectrometry

Abstract

Specific inhibitors of diacylglycerol kinase (DGK) ζ can be promising anticancer medications via the activation of cancer immunity. Although the detection of cellular activities of target enzymes is essential for drug screening in addition to in vitro assays, it is difficult to detect the activity of DGKζ in cells. In the present study, we generated AcGFP‐DGKζ cDNA with a consensus N‐myristoylation sequence at the 5′ end (Myr‐AcGFP‐DGKζ) to target DGKζ to membranes. Using liquid chromatography (LC)‐tandem mass spectrometry (MS/MS) (LC–MS/MS), we showed that Myr‐AcGFP‐DGKζ, but not AcGFP‐DGKζ without the myristoylation sequence, substantially augmented the levels of several phosphatidic acid (PtdOH) species. In contrast to Myr‐AcGFP‐DGKζ, its inactive mutant did not exhibit an increase in PtdOH production, indicating that the increase in PtdOH production was DGK activity‐dependent. This method will be useful in chemical compound selection for the development of drugs targeting DGKζ and can be applicable to various soluble (nonmembrane bound) lipid‐metabolizing enzymes, including other DGK isozymes.

Diacylglycerol kinase δ and sphingomyelin synthase–related protein functionally interact via their sterile α motif domains

“…Total lipids were extracted from the samples according to the method of Bligh and Dyer (43) as described previously (36,44). Briefly, 2 ml of methanol and 1 ml of chloroform were added to 700 l of sample.…”

Diacylglycerol Kinase η Activity in Cells Using Protein Myristoylation and Cellular Phosphatidic Acid Sensor

“…We recently established an LC–MS/MS‐based DGK assay (Mizuno et al, 2012; Murakami et al, 2020; Sakai et al, 2014). Moreover, we previously reported that N‐myristoylation of DGKζ facilitated the detection of its intracellular activity by LC–MS/MS (Honda et al, 2019). Thus, we first tried to detect the activity of DGKη in cells using the combination of LC–MS/MS and N‐myristoylation of DGKη.…”

“…Moreover, DGKη‐KO mice indeed exhibited BPD remedy‐sensitive mania‐like behaviors (Isozaki et al, 2016). Although DGKη is pathophysiologically important, it is difficult to detect cellular DGKη activity even using protein myristoylation (Honda et al, 2019; Yamada et al, 2020b) and an LC–MS/MS‐based DGK assay (Mizuno et al, 2012; Murakami et al, 2020; Sakai et al, 2014). In contrast to DGKη, the intracellular activity of Myr‐DGKζ was detected by LC–MS/MS (Honda et al, 2019).…”

“…Moreover, to explore the biological functions of DGKη in more detail, determination of its cellular activity is essential. However, it is difficult to measure the cellular activity of DGKη, even using a recently established liquid chromatography-tandem mass spectrometry (LC-MS/MS)based DGK assay (Honda et al, 2019;Mizuno et al, 2012;Murakami et al, 2020;Sakai et al, 2014). In the present study, we attempted to detect the activity of myristoylated (Myr) DGKη, which was forcibly translocated to membranes where its substrate DAG exists, in cells using a cellular PtdOH sensor, the N-terminal region of α-synuclein (α-Syn-N) that we recently developed (Mizuno et al, 2017;Yamada et al, 2020aYamada et al, , 2020b.…”

1‐Stearoyl‐2‐docosahexaenoyl‐phosphatidic acid interacts with and activates Praja‐1, the E3 ubiquitin ligase acting on the serotonin transporter in the brain

“…Phosphatidic acid species in the extracted lipids (10 μL) were analyzed by LC‐MS and LC‐MS/MS as previously described [16‐19]. Diacylglycerol species in the extracted lipids (10 μL) were analyzed by LC‐MS and LC‐MS/MS as previously described [20].…”