Diacylglycerol (DG) kinase (DGK) phosphorylates the lipid second messenger DG to phosphatidic acid. We reported previously that noradrenaline (NA), but not angiotensin II (AII), increases membrane-associated DGK activity in rat small arteries [Ohanian and Heagerty (1994) Biochem. J. 300, 51–56]. Here, we have identified this DGK activity as DGKθ, present in both smooth muscle and endothelial cells of these small vessels. Subcellular fractionation of artery homogenates revealed that DGKθ was present in nuclear, plasma membrane (and/or Golgi) and cytosolic fractions. Upon NA stimulation, DGKθ translocated towards the membrane and cytosol (155 and 153% increases relative to the control, respectively) at 30s, followed by a return to near-basal levels at 5min; AII was without effect. Translocation to the membrane was to both Triton-soluble and -insoluble fractions. NA, but not AII, transiently increased DGKθ activity in immunoprecipitates (126% at 60s). Membrane translocation and DGKθ activation were regulated differently: NA-induced DGKθ activation, but not translocation, was dependent on transient activation of phosphoinositide 3-kinase (PI 3-K). In addition, DGK activity co-immunoprecipitated with protein kinase B, a downstream effector of PI 3-K, and was increased greatly by NA stimulation. The rapid and agonist-specific activation of DGKθ suggests that this pathway may have a physiological role in vascular smooth-muscle responses.
- diacylglycerol kinase
- protein kinase B
- smooth muscle
- The Biochemical Society, London © 2001