Diacylglycerol kinase θ: regulation and stability

Abstract

Given the well-established roles of diacylglycerol (DAG) and phosphatidic acid (PtdOH) in a variety of signaling cascades, it is not surprising that there is an increasing interest in understanding their physiological roles and mechanisms that regulate their cellular levels. One class of enzymes capable of coordinately regulating the levels of these two lipids is the diacylglycerol kinases (DGKs). These enzymes catalyze the transfer of the γ-phosphate of ATP to the hydroxyl group of DAG, which generates PtdOH while reducing DAG. As these enzymes reciprocally modulate the relative levels of these two signaling lipids, it is essential to understand the regulation and roles of these enzymes in various tissues. One system where these enzymes play important roles is the nervous system. Of the ten mammalian DGKs, eight of them are readily detected in the mammalian central nervous system (CNS): DGK-α, DGK-β, DGK-γ, DGK-η, DGK-ζ, DGK-ι, DGK-ε, and DGK-θ. Despite the increasing interest in DGKs, little is known about their regulation. We have focused some attention on understanding the enzymology and regulation of one of these DGK isoforms, DGK-θ. We recently showed that DGK-θ is regulated by an accessory protein containing polybasic regions. We now report that this accessory protein is required for the previously reported broadening of the pH profile observed in cell lysates in response to phosphatidylserine (PtdSer). Our data further reveal DGK-θ is regulated by magnesium and zinc, and sensitive to the known DGK inhibitor R599022. These data outline new parameters involved in regulating DGK-θ.

Fig. 1

Activators alter the pH profile of DGK-θ. (A) The presence of PtdSer (9 mol%) in vesicles broadens the pH profile for DGK-θ overexpressed in lysates (Tu-Sekine et al., 2006). (B) A similar profile broadening is observed in response to PtdSer using purified DGK-θ, when polylysine is also added to the assay, and (C) to a lesser extent when polylysine is added to vesicles that do not contain PtdSer. Experiments completed in triplicate, Error = SEM.

Fig. 2

Effect of free magnesium on purified DGK-θ activity. (A) Representative profiles from 2 independent experiments in the presence (triangles) or absence (squares) of 9 mol% PtdSer show free magnesium dose-dependence on activity (pH 7.5). Calculated $K_M^{\text{app}}$ values for free Mg in the presence (gray bar) and absence (white bar) of PtdSer (9 mol%). Error = SEM.

Fig. 3

Modulation of DGK-θ activity in vitro. Purified DGK-θ was pretreated for 15 min with (A) ATP (1 mM), ADP (0.25 mM), NaPPI (0.5 mM) or BPG (1 mM) prior to assay. (B) DGK-θ was pretreated for 20 min with R59022 prior to an activity assay at pH 8.0 with R59022. (C) Purified DGK-θ was pretreated with 1 U CIP or CIP buffer only (control) for 10 min at 25 °C prior to an activity assay at pH 7.0. All preincubations contained 0.1% NP40. BPG = betaglycerolphosphate. Error = SD.

Fig. 4

Purified DGK-θ is inhibited by zinc. (A) DGK-θ was incubated with 100 uM zinc for 10 min. 100 μM over a pH range prior to activity assay in the absence of additional zinc. Gray bars: control; black bars: zinc treated. (B) DGK-θ was incubated over a range of zinc concentrations prior to assay in the absence of zinc (black bars); or incubated with vehicle prior to assay in the presence of zinc (white bars). 0.1 uM protamine present in all assays. Error = SD.

Fig. 5

In vivo stability of DGK-θ. (A) Cyclohexamide treated 3T3 and N2a lysates were probed for endogenous DGK-θ by western blot over a 72 h time course (B) DGK-θ is evident as a 104 kD (full-length), 75 kD and 35 kD bands from western blots of primary cortical neurons.

Fig. 6

DGK-θ activity is stabilized by the presence of an interface. Purified DGK-θ was incubated with detergents or PEG3500 for 10 min prior to assay. (A) Specific activity of treated enzyme (B) fold stimulation by histone H1 (0.1 μM) of treated enzyme. (C). DGK-θ was incubated in increasing concentrations of vesicles (9% PtdSer, 0% DAG (DOG), pH 7.5) for 1 h on ice, then for 30 min at room temperature prior to assay (pH 7.5, 0.1 μM Histone H1). Error bars = SD.