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Analytical Method for Diacylglycerol Kinase ζ Activity in Cells Using Protein Myristoylation and Liquid Chromatography–Tandem Mass Spectrometry
Author(s) -
Honda Shotaro,
Murakami Chiaki,
Yamada Haruka,
Murakami Yuki,
Ishizaki Ayuka,
Sakane Fumio
Publication year - 2019
Publication title -
lipids
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.601
H-Index - 120
eISSN - 1558-9307
pISSN - 0024-4201
DOI - 10.1002/lipd.12201
Subject(s) - diacylglycerol kinase , phosphatidic acid , myristoylation , phosphatidylethanol , chemistry , biochemistry , liquid chromatography–mass spectrometry , phospholipase d , kinase , enzyme , biology , mass spectrometry , protein kinase c , phosphorylation , chromatography , phospholipid , membrane
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.