Type Ialpha phosphatidylinositol 4-phosphate 5-kinase is a putative target for increased intracellular phosphatidic...

Despite the fact that phosphatidic acid (PtdOH) has been implicated as a lipid second messenger for nearly a decade, its intracellular targets have remained unclear. We sought to investigate how an increase in the level of PtdOH could modulate phosphatidylinositol 4-phosphate 5-kinase (PIPkin), an enzyme involved in phosphatidylinositol 4,5-bisphosphate synthesis. Transfection of porcine aortic endothelial (PAE) cells with haemagglutinin (HA)-tagged type Ialpha PIPkin followed by immunofluorescence confocal microscopy revealed the enzyme to be localised to the plasma membrane. When the transfected 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 the in vivo generator of PtdOH. In order to determine if the PtdOH-dependent activation of type Ialpha PIPkin was dictated by a specific molecular composition of PtdOH, the wild type murine and porcine alpha isoforms of diacylglycerol kinase (DGK) were individually co-transfected along with type Ialpha PIPkin. Under these conditions an increase in type Ialpha PIPkin lipid kinase activity was found in HA immunoprecipitates in an in vitro assay. No increases in lipid kinase activity were observed when type Ialpha PIPkin was co-transfected with either the human DGKepsilon isoform or a kinase-dead mutant of the murine DGKalpha isoform. These results provide the first direct evidence for the unification of the production of saturated/monounsaturated PtdOH (through two different routes, PLD and DGK) and the in vivo activation of type Ialpha PIPkin by this lipid second messenger.