Diacylglycerol kinase modulates the levels of diacylglycerol and phosphatidic acid, two critical lipid second messengers, yet little is known about the effects of cellular stimulation on the kinetic behavior of this enzyme. We examined the effects of α-thrombin and activating phospholipids on the activity and substrate affinity of a soluble diacylglycerol kinase, DGKθ. Our data demonstrate that the apparent binding parameters of DGKθ increase following thrombin stimulation, suggesting that α-thrombin antagonizes DGKθ activity. Interestingly, this effect is obscured in the presence of high bulk substrate concentrations. Given the known stimulatory effects of phosphatidylserine on many diacylglycerol kinases, we examined the effects of various phospholipids on DGKθ and found that phosphatidic acid is a more effective activator than phosphatidylserine. Phosphatidic acid decreased the apparent surface KM (K M(surf)app) of DGKθ for dioleoylglycerol (DOG) and promoted binding to vesicles in a dose-dependent manner. Phosphatidylserine also lowered the KM(surf)app of DGKθ, though higher concentrations were required to achieve the same effect. Interestingly, PS promoted binding to vesicles only when present at levels beyond that required to saturate enzyme activity, suggesting that PS and PA activate DGKθ through different mechanisms. The potential physiological implications of these findings are discussed.
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