Diacylglycerol kinase in receptor-stimulated cells converts its substrate in a topologically restricted manner.
The regulation of diacylglycerol (DG) kinase activity was studied in fibroblasts and Jurkat T cells. We questioned whether enzyme activity only depends on substrate availability or whether it requires receptor stimulation. To this end, we raised DG levels up to 15-fold by treatment of cells with bacterial phosphatidylinositol-specific phospholipase C (PLC). In detergent cell lysates, DG kinase was readily capable of converting this surplus of DG to phosphatidic acid (PA), but in intact cells the enzyme remained inactive. Stimulation of fibroblasts with bradykinin or endothelin and Jurkat cells with anti-CD3 resulted in DG kinase-mediated formation of PA, but its level was unaffected by PLC pretreatment. Likewise, in streptolysin O-permeabilized fibroblasts, where bradykinin stimulation in the presence of [gamma-32P]ATP and guanosine 5'-O-(thiotriphosphate) generates [32P]PA exclusively via DG kinase, PLC pretreatment did not affect the amount of [32P]PA formed. We conclude that DG kinase acts on DG generated by receptor stimulation, but not on DG generated by exogenous PLC. We propose a model in which DG kinase physically associates with endogenous PLC. Within this complex, receptor-induced DG would then be transmitted ("channeled") from endogenous PLC to the active site of DG kinase, whereas excess DG generated randomly in the plasma membrane by bacterial PLC is inaccessible to this catalytic site.
van der Bend
R L
RL
The Netherlands Cancer Institute, Division of Cellular Biochemistry, Amsterdam.
de Widt
J
J
Hilkmann
H
H
van Blitterswijk
W J
WJ
D000818:Animals; D001920:Bradykinin / Q000494:pharmacology; D002478:Cells, Cultured; D019852:Diacylglycerol Kinase; D004075:Diglycerides / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D008563:Membrane Lipids / Q000378:metabolism; D010712:Phosphatidic Acids / Q000378:metabolism; D010739:Phospholipase D / Q000502:physiology; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000378:metabolism*; D011493:Protein Kinase C / Q000502:physiology; D051381:Rats; D011956:Receptors, Cell Surface / Q000378:metabolism*; D015398:Signal Transduction; D010738:Type C Phospholipases / Q000494:pharmacology
D004075:Diglycerides; D008563:Membrane Lipids; D010712:Phosphatidic Acids; D011956:Receptors, Cell Surface; D017853:Phosphotransferases (Alcohol Group Acceptor); D019852:Diacylglycerol Kinase; D011493:Protein Kinase C; D010738:Type C Phospholipases; D010739:Phospholipase D; D001920:Bradykinin