Diacylglycerol kinase θ is translocated and phosphoinositide 3-kinase-dependently activated by noradrenaline but not angiotensin II in intact small arteries

Abstract

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 30 s, followed by a return to near-basal levels at 5 min ; AII was without effect. Translocation to the membrane was to both Triton-soluble and Abbreviations used : DGK, diacylglycerol kinase ; DG, 1,2-diacylglycerol ; PA, phosphatidic acid ; NA, noradrenaline ; AII, angiotensin II ; PKC, protein kinase C ; PH, pleckstrin homology ; OBG, octyl-β-glucoside ; PI 3-K, phosphoinositide 3-kinase ; PKB, protein kinase B. 1 To whom correspondence should be addressed (e-mail johanian!man.ac.uk).-insoluble fractions. NA, but not AII, transiently increased DGKθ activity in immunoprecipitates (126 % at 60 s). 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 agonistspecific activation of DGKθ suggests that this pathway may have a physiological role in vascular smooth-muscle responses.

Norepinephrine and endothelin activate diacylglycerol kinases in caveolae/rafts of rat mesenteric arteries: agonist-specific role of PI3-kinase

“…The initial transient increase in [ 33 P]PA in caveolae/rafts induced by NE was consistent with the transient activation of DGK-by this agonist (49). Recently, we showed that NE activates PI3-kinase/PKB signaling in RMSAs and that this pathway is essential for activation of DGK- (49). Here we show that caveolae/rafts are sites where PI3-kinase/PKB regulates DGK activity and provides evidence that DGK-is involved in the response.…”

“…DGKs exist as multiple isoforms, and in RMSAs, NE activates DGK-in a PI3-kinase-dependent manner (49). In this study we confirmed the presence of DGK-in caveolae/rafts (Fig.…”

“…To further explore the PI3-kinase-dependent regulation of DGK activity in caveolae/rafts, we studied the localization of PKB since we previously demonstrated a NE-stimulated DGK activity associated with PKB (49). In nonstimulated arteries, 5.3 Ϯ 0.5% of PKB localized to caveolae/raft fractions.…”

Diacylglycerol kinases in nuclear lipid-dependent signal transduction pathways

“…DAG kinase-q q and -d d DAG kinase-q is another isozyme which has been found in the nucleus of several cell types, including rat arterial smooth muscle and endothelial cells, human MelJuso melanoma cells, COS-7 cells and IIC9 cells [12,13,40]. Quiescent IIC9 fibroblasts also express DAG kinase-d in the nucleus, but only the -q was activated in response to a-thrombin (see later) [13].…”

“…Since the isotypes possess different domains in their primary structure, the mechanism of activation is also likely to differ among the isozymes. Membrane association (for example of DAG kinase-q) is necessary but not sufficient for the activation [40]. Most likely there must be other activation steps, such as phophorylation and/or interaction with small GTP-binding proteins [12].…”