Cell-permeable ceramides prevent the activation of phospholipase D by ADP-ribosylation factor and RhoA
Cell-permeable ceramides prevent the activation of phospholipase D by ADP-ribosylation factor and RhoA
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DOI：
10.1074/jbc.272.2.1069
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发表时间：
1997-01-10
影响因子：
4.8
通讯作者：
Brindley, DN
中科院分区：
文献类型：
--
作者：
Abousalham, A;Liossis, C;Brindley, DN
使用从人早幼粒细胞白血病 (HL-60) 细胞分化的粒细胞确定了神经酰胺抑制磷脂酶 D (PLD) 的机制。在无细胞系统中，膜结合 PLD 对磷脂酰胆碱的水解取决于磷脂酰肌醇 4,5-二磷酸、鸟苷 5'-3-O-(硫代)三磷酸 (GTP gamma S) 以及包括 ADP-核糖基化因子 (ARF) 和 RhoA 在内的胞质因子。 C-2(N-乙酰基-)、C-8-(N-辛酰基-)和长链神经酰胺抑制PLD活性，但二氢-C-2-神经酰胺不抑制PLD活性。三磷酸双磷酸酶或大田酸不会改变神经酰胺对 PLD 的抑制作用，表明无细胞系统中的效果不太可能依赖于神经酰胺刺激的激酶或磷蛋白磷酸酶。 C-2- 和 C-8- 神经酰胺可防止 GTP gamma S 诱导的 ARF1 和 RhoA 从细胞质转移到膜部分。在整个细胞中，C-2-神经酰胺（而非二氢-C-2-神经酰胺）抑制 N-甲酰甲硫氨酰亮氨酰苯丙氨酸对 PLD 的刺激，并减少与膜部分相关的 ARF1、RhoA、CDC42、Rab4 以及蛋白激酶 C-α 和 -β(1) 的量，但不改变蛋白激酶 C-ε 和 -zeta 的分布。结论是神经酰胺防止 PLD 激活的一种机制是抑制 PLD 活性所需的 G 蛋白和蛋白激酶 C 亚型向膜的易位。
The mechanism of inhibition of phospholipase D (PLD) by ceramides was determined using granulocytes differentiated from human promyelocytic leukemic (HL-60) cells. In a cell free system, hydrolysis of phosphatidylcholine by membrane-bound PLD depended upon phosphatidylinositol 4,5-bisphosphate, guanosine 5'-3-O-(thio)triphosphate) (GTP gamma S), and cytosolic factors including ADP-ribosylating factor (ARF) and RhoA. C-2(N-acetyl-), C-8-(N-octanoyl-), and long-chain ceramides, but not dihydro-C-2-ceramide inhibited PLD activity. Apyrase or okadaic acid did not modify the inhibition of PLD by ceramides, indicating that the effect in the cell-free system was unlikely to be dependent upon a ceramide-stimulated kinase or phosphoprotein phosphatases. C-2- and C-8-ceramides prevented the GTP gamma S-induced translocation of ARF1 and RhoA from the cytosol to the membrane fraction. In whole cells, C-2-ceramide, but not dihydro-C-2-ceramide, inhibited the stimulation of PLD by N-formylmethionylleucylphenylalanine and decreased the amounts of ARF1, RhoA, CDC42, Rab4, and protein kinase C-alpha and -beta(1) that were associated with the membrane fraction, but did not alter the distribution of protein kinase C-epsilon and -zeta. It is concluded that one mechanism by which ceramides prevent the activation of PLD is inhibition of the translocation to membranes of G-proteins and protein kinase C isoforms that are required for PLD activity.