Type I alpha phosphatidylinositol 4-phosphate 5-kinase is a putative target for increased intracellular phosphatidic acid

Despite the fact that phosphatidic acid (PtdOH) has been implicated as a li pid second messenger for nearly a decade, its intracellular targets have re mained unclear. We sought to investigate how an increase in the level of Pt dOH could modulate phosphatidylinositol 4-phosphate 5-kinase (PIPkin), an e nzyme involved in phosphatidylinositol 4,5-bisphosphate synthesis. Transfec tion of porcine aortic endothelial (PAE) cells with haemagglutinin (HA)-tag ged type I alpha PIPkin followed by immunofluorescence confocal microscopy revealed the enzyme to be localised to the plasma membrane. When the transf ected PAE cells were stimulated with lyso-PtdOH, increased PIPkin activity was found to be associated with HA immunoprecipitates in an in vitro assay. This PIPkin activation was found to be greatly reduced by prior treatment of the cells with 1-butanol, thereby implicating phospholipase D (PLD) as t he in vivo generator of PtdOH. In order to determine if the PtdOH-dependent activation of type I alpha PIPkin was dictated by a specific molecular com position of PtdOH, the wild type murine and porcine alpha isoforms of diacy lglycerol kinase (DGK) were individually co-transfected along with type I a lpha PIPkin. Under these conditions an increase in tape I alpha PIPkin lipi d kinase activity was found in HA immunoprecipitates in an in vitro assay. No increases in lipid kinase activity were observed when type I alpha PIPki n was co-transfected with either the human DGK epsilon isoform or a kinase- dead mutant of the murine DGK alpha isoform. These results provide the firs t direct evidence for the unification of the production of saturated/monoun saturated PtdOH (through two different routes, PLD and DGK) and the in vivo activation of type I alpha PIPkin by this lipid second messenger. (C) 2000 Federation of European Biochemical Societies. Published by Elsevier Scienc e B.V. All rights reserved.