Abstract
Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) 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)P(2) 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 PIP(2) (PI(4,5)P(2), PI(3,5)P(2), or PI(3,4)P(2) isomers) at the expense of PIP when added back to permeabilized cells. The PIP(2) could be hydrolyzed by phospholipase C, identifying it as PI(4,5)P(2). However, the ARF1-stimulated pool of PI(4,5)P(2) was accessible to the phospholipase C more efficiently in the presence of phosphatidylinositol transfer protein-alpha. To examine the role of PLD in the regulation of PI(4,5)P(2) synthesis, we used butanol to diminish the PLD-derived PA. PI(4,5)P(2) 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)P(2) levels. To circumvent the use of alcohols, an ARF1 point mutant was identified (N52R-ARF1) that could selectively activate PIP 5-kinase alpha activity but not PLD activity. N52R-ARF1 was still able to increase PI(4,5)P(2) 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)P(2) synthesis at the plasma membrane in HL60 cells.
Highlights
Synthesis of PI[4,5]P2 at the plasma membrane requires both activation of the lipid kinases as well as a supply of PI, which is synthesized at the endoplasmic reticulum
The level of ARF6 expressed in HL60 cells is 10 –20-fold less than that of ARF1 calculated using recombinant ADP ribosylation factor (ARF) proteins as standards. 20 ng of ARF1 was detected compared with 1–2 ng of ARF6 in 3 ϫ 105 HL60 cells
PIP2 functions at the plasma membrane serving as a substrate for phospholipase C and PI 3-kinase as well as an intact signaling molecule for endocytosis, exocytosis, and for the reorganization of the actin cytoskeleton
Summary
Materials—[␥-32P]ATP, [3H]inositol, [3H]choline, and [3H]alkyl-lysoPC were obtained from Amersham Biosciences, Inc. HL60 cells (5 ϫ 107) were permeabilized with 0.4 IU/ml streptolysin O in PIPES buffer, pH 6.8 (20 mM PIPES, 137 mM NaCl, 3 mM KCl, 2 mM MgCl2, 5.6 mM glucose, and 1 mg/ml bovine serum albumin), in the presence of 1 mM MgATP and 100 nM Ca2ϩ for 10 min. Under these conditions the majority of PITP␣ and ARF proteins leaked out of the cells [7, 21]. At the end of this time samples were quenched with chloroform, methanol, concentrated HCl (100:200:1.5), and the production of PIP2 was analyzed by TLC as described above
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