Diacylglycerol-induced translocation of diacylglycerol kinase: use of affinity-purified enzyme in a reconstitution system.

Abstract

Diacylglycerol-induced translocation of diacylglycerol kinase (ATP:1,2-diacylglycerol 3-phosphotransferase, EC 2.7.1.107) from the soluble to the membrane-bound compartments was demonstrated both in crude tissue homogenates and in a reconstituted enzyme-membrane model system. In homogenates of either rat brain or liver, incubation with diacylglycerol or phospholipase C, but not phospholipase A2 or phospholipase D, resulted in the translocation of diacylglycerol kinase activity from the soluble to the particulate fraction. This observation formed the basis for the first step in a two-step purification of diacylglycerol kinase. Enzyme extracted in 1 M salt from membranes of rat brain homogenates made in the presence of phospholipase C was purified further by affinity chromatography on a column containing phosphatidylserine, diacylglycerol, and cholesterol immobilized in polyacrylamide. This step yielded an enzyme preparation (step 2 enzyme) that was 500-to 750-fold purified (relative to the tissue homogenate) and required phosphatidylserine for stability. All other lipids tested failed to stabilize the enzyme. The properties of the enzyme preparation were similar to those of mammalian diacylglycerol kinases described by others. Reconstitution experiments showed that the soluble step 2 enzyme bound to inside-out vesicles of human erythrocytes only in the presence of diacylglycerol or phospholipase C but not phospholipase A2 or D. Redistribution of the kinase from soluble to vesicle-bound forms occurred rapidly and was dependent on the concentration of phospholipase C used to treat the vesicles. Physiological concentrations of calcium (50-1000 nM) did not enhance the phospholipase C-mediated translocation of the kinase. Thus, diacylglycerol kinase can translocate from cytosol to membranes in a manner dependent on the content of membrane-bound diacylglycerol but independent of the ambient concentration of calcium.The recent advent of diacylglycerol as the endogenous activator ofprotein kinase C has propelled diacylglycerol into the limelight as an important second messenger, coupling receptor-mediated hydrolysis of phosphatidylinositols to activation of protein kinase C. It has been known for some time that diacylglycerol is produced as a result of the hydrolysis of phosphatidylinositols by phospholipase C (1-3). Many reports (4-8) indicate that protein kinase C can translocate from cytosol to membranes as a result of the formation of diacylglycerol and the increased cytosolic calcium concentration resulting from inositol trisphosphate formation. However, diacylglycerol is rapidly converted to phosphatidate by diacylglycerol kinase (ATP:1,2-diacylglycerol 3-phosphotransferase, EC 2.7.1.107) (9, 10). Like protein kinase C, diacylglycerol kinase requires phospholipid for catalytic function (11, 12). Moreover, because diacylglycerol kinase is at least partially responsible for the half-life of diacylglycerol, it may play an important role in regulating the coupling of agonist-induced generation of membrane diacy...

Purification and characterization of sn-1-stearoyl-2-arachidonoylglycerol kinase from pig testes

“…An immunologically and electrophoretically identical isoform of the 80 kDa DAG kinase has been detected in particulate fractions [22]. This may be explained by the report that the 80 kDa DAG kinase translocates to the membrane [16,23,26] or cytoskeleton of cells [8], in response to epidermal growth factor stimulation [26]. It has also been shown that protein kinase C isoforms phosphorylate DAG kinase in itro [26,27].…”

“…DAG kinase activities have been characterized in rat brain [17,18], baboon brain [19], Drosophila heads [20] and bovine testes [21]. Mammalian DAG kinase activities have been found to be cytosolic [16,18,22], membrane-associated or membranebound [19,[21][22][23], cytoskeletal [8] or nuclear [24]. The first DAG kinase to be purified, characterized and cloned was an 80 kDa protein from pig lymphocyte cytosol [25].…”

Immunoquantitation of 80 kDa diacylglycerol kinase in pig and human lymphocytes and several other cells

“…The 80 kDa isozyme requires phospholipids as activators [6,22], and its substrate, diacylglycerol, is believed to be generated in the membrane of agonist-stimulated cells. This raises the possibility that cytosolic DGK is an inactive (or less active) reservoir form.…”

Lipid Messenger, Diacylglycerol, and its Regulator, Diacylglycerol Kinase, in Cells, Organs, and Animals: History and Perspective

“…In the case of DGK, we should consider the fact that the substrate DG is membrane-bound and therefore DGK must bind to the membrane, at least transiently, to reach the substrate. In this regard, DGK was shown to translocate from the cytosol to the membrane in homogenates of rat brain and liver, dependent on the content of membrane-bound DG, although which isozyme is responsible for the translocation remained undetermined (Besterman et al 1986). This suggests that DGK may be partially, if not completely, activated by its own substrate DG and that DGK itself might be one of the targets of a second messenger DG.…”