Skippen Alison, Jones David H, Morgan Clive P, Li Michelle, Cockcroft Shamshad
Department of Physiology, University College London, London WC1E 6JJ, United Kingdom.
J Biol Chem. 2002 Feb 22;277(8):5823-31. doi: 10.1074/jbc.M110274200. Epub 2001 Dec 14.
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.
磷脂酰肌醇4,5 - 二磷酸（PI(4,5)P(2)）既是脂质衍生的第二信使生成的底物，也是细胞信号传导、胞吞和胞吐以及细胞骨架重组等许多方面所必需的完整脂质。ADP核糖基化因子（ARF）蛋白调节PI(4,5)P(2)的合成，在此我们研究了这是否是由于I型磷脂酰肌醇4 - 磷酸（PIP）5 - 激酶的直接激活，还是通过HL60细胞中磷脂酶D（PLD）衍生的磷脂酸（PA）间接实现的。ARF1和ARF6均在HL60细胞中表达，并且可以通过透化作用从细胞中去除。当将这两种ARF重新添加到透化细胞中时，它们都以PIP为代价增加了PIP(2)（PI(4,5)P(2)、PI(3,5)P(2)或PI(3,4)P(2)异构体）的水平。PIP(2)可被磷脂酶C水解，确定其为PI(4,5)P(2)。然而，在磷脂酰肌醇转移蛋白 - α存在的情况下，磷脂酶C能更有效地作用于ARF1刺激产生的PI(4,5)P(2)池。为了研究PLD在PI(4,5)P(2)合成调节中的作用，我们使用丁醇来减少PLD衍生的PA。ARF1刺激的PI(4,5)P(2)合成不受0.5%丁醇的阻断，但可被1.5%丁醇阻断。虽然0.5%丁醇最适合最大程度的转磷脂酰基作用，但仍可检测到PA的产生。相比之下，发现1.5%丁醇可抑制ARF1对PLD的激活，并且还使PIP水平降低50%。因此，1.5%丁醇的毒性使我们无法得出PA是否是提高PI(4,5)P(2)水平的重要因素的结论。为了避免使用醇类，我们鉴定了一种ARF1点突变体（N52R - ARF1），它可以选择性激活PIP 5 - 激酶α活性，但不激活PLD活性。N52R - ARF1仍然能够增加PI(4,5)P(2)水平，但效率降低。因此，我们得出结论，PLD途径衍生的PA和ARF蛋白通过直接激活PIP 5 - 激酶，共同参与HL60细胞中质膜上PI(4,5)P(2)合成的调节。
