Mechanism of ADP ribosylation factor-stimulated phosphatidylinositol 4,5-bisphosphate synthesis in HL60 cells

Abstract

Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) is required both as a substrate for the generation of lipid-derived second messengers as well as an intact lipid for many aspects of cell signaling, endo- and exocytosis, and reorganization of the cytoskeleton. ADP ribosylation factor (ARF) proteins regulate PI(4,5)P2 synthesis, and here we have examined whether this is due to direct activation of Type I phosphatidylinositol 4-phosphate (PIP) 5-kinase or indirectly by phosphatidate (PA) derived from phospholipase D (PLD) in HL60 cells. ARF1 and ARF6 are both expressed in HL60 cells and can be depleted from the cells by permeabilization. Both ARFs increased the levels of PIP2 (PI(4,5)P2, PI(3,5)P2, or PI(3,4)P2 isomers) at the expense of PIP when added back to permeabilized cells. The PIP2 could be hydrolyzed by phospholipase C, identifying it as PI(4,5)P2. However, the ARF1-stimulated pool of PI(4,5)P2 was accessible to the phospholipase C more efficiently in the presence of phosphatidylinositol transfer protein-a. To examine the role of PLD in the regulation of PI(4,5)P2 synthesis, we used butanol to diminish the PLD-derived PA. PI(4,5)P2 synthesis stimulated by ARF1 was not blocked by 0.5% butanol but could be blocked by 1.5% butanol. Although 0.5% butanol was optimal for maximal transphosphatidylation, PA production was still detectable. In contrast, 1.5% butanol was found to inhibit the activation of PLD by ARF1 and also decrease PIP levels by 50%. Thus the toxicity of 1.5% butanol prevented us from concluding whether PA was an important factor in raising PI(4,5)P2 levels. To circumvent the use of alcohols, an ARF1 point mutant was identified (N52R-ARF1) that could selectively activate PIP 5-kinase a activity but not PLD activity. N52R-ARF1 was still able to increase PI(4,5)P2 levels but at reduced efficiency. We therefore conclude that both PA derived from the PLD pathway and ARF proteins, by directly activating PIP 5-kinase, contribute to the regulation of PI(4,5)P2 synthesis at the plasma membrane in HL60 cells.