Tracking the localization and dynamics of the intracellular bioactive lipid phosphatidic acid (PA) is important for understanding diverse biological phenomena. Although several PA sensors have been developed, better ones are still needed for comprehensive PA detection in cells. We recently found that a-synuclein (a-Syn) selectively and strongly bound to PA in vitro. Here, we revealed that the N-terminal region of a-Syn (a-Syn-N) specifically bound to PA, with a dissociation constant of 6.6 lM. a-Syn-N colocalized with PAproducing enzymes, diacylglycerol kinase (DGK) b at the plasma membrane (PM), myristoylated DGKf at the Golgi apparatus, phorbol ester-stimulated DGKc at the PM, and phospholipase D2 at the PM and Golgi but not with the phosphatidylinositol-4,5-bisphosphate-producing enzyme in COS-7 cells. However, a-Syn-N failed to colocalize with them in the presence of their inhibitors and/or their inactive mutants. These results indicate that a-Syn-N specifically binds to cellular PA and can be applied as an excellent PA sensor.
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 (DGK) phosphorylates diacylglycerol to produce phosphatidic acid (PtdOH) and regulates the balance between two lipid second messengers: diacylglycerol and PtdOH. Several lines of evidence suggest that the η isozyme of DGK is involved in the pathogenesis of bipolar disorder. However, the detailed molecular mechanisms regulating the pathophysiological functions remain unclear. One reason is that it is difficult to detect the cellular activity of DGKη. To overcome this difficulty, we utilized protein myristoylation and a cellular PtdOH sensor, the N-terminal region of α-synuclein (α-Syn-N). Although DGKη expressed in COS-7 cells was broadly distributed in the cytoplasm, myristoylated (Myr)-AcGFP-DGKη and Myr-AcGFP-DGKη-KD (inactive (kinase-dead) mutant) were substantially localized in the plasma membrane. Moreover, DsRed monomer-α-Syn-N significantly colocalized with Myr-AcGFP-DGKη but not Myr-AcGFP-DGKη-KD at the plasma membrane. When COS-7 cells were osmotically shocked, all DGKη constructs were exclusively translocated to osmotic shock-responsive granules (OSRG). DsRed monomerα-Syn-N markedly colocalized with only Myr-AcGFP-DGKη at OSRG and exhibited a higher signal/background ratio (3.4) than Myr-AcGFP-DGKη at the plasma membrane in unstimulated COS-7 cells (2.5), indicating that α-Syn-N more effectively detects Myr-AcGFP-DGKη activity in OSRG. Therefore, these results demonstrated that the combination of myristoylation and the PtdOH sensor effectively detects DGKη activity in cells and that this method is convenient to examine the molecular functions of DGKη. Moreover, this method will be useful for the development of drugs targeting DGKη. Furthermore, the combination of myristoylation (intensive accumulation in membranes) and α-Syn-N can be applicable to assays for various cytosolic PtdOH-generating enzymes.
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