Sphingomyelin synthase, a potential regulator of intracellular levels of ceramide and diacylglycerol during SV40 transformation - Does sphingomyelin synthase account for the putative, phosphatidylcholine-specific phospholipase C?
Sphingomyelin synthase, a potential regulator of intracellular levels of ceramide and diacylglycerol during SV40 transformation - Does sphingomyelin synthase account for the putative, phosphatidylcholine-specific phospholipase C?
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DOI：
10.1074/jbc.273.23.14550
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发表时间：
1998-06-05
影响因子：
4.8
通讯作者：
Hannun, YA
中科院分区：
文献类型：
--
作者：
Luberto, C;Hannun, YA
鞘磷脂合成酶（SMS），一种参与鞘磷脂（SM）和神经酰胺代谢的酶，可以潜在地以相反的方向调节神经酰胺和甘油二酯的水平。在该研究中，在正常和SV 40转化的人肺成纤维细胞（WI 38）中研究SMS活性。向细胞中加入[H-3]C-2-神经酰胺导致[H-3]C-2-SM的时间依赖性形成。在处理后24 h，正常WI 38细胞清除了17%的产生[H-3] C-2-SM的[H-3]C-2-神经酰胺，占总放射性的13%。另一方面，SV 40转化细胞清除了45%的[H-3]C-2-神经酰胺并产生C-2-SM，占总放射性的24%。这种C-2-SM产量的提高也得到了细胞总SMS活性（体外测定）增加的支持，使得SV 40转化细胞的SMS活性为222 pmol/mg蛋白/h，而野生型细胞的SMS活性为78 pmol/mg蛋白/h。旨在检查针对质膜中产生的神经酰胺的SMS活性的其他研究。用外源性细菌鞘磷脂酶（SMase）处理细胞25分钟导致总SM的90-95%的裂解和伴随的神经酰胺的产生。在细菌SMase处理后，野生型WI 38细胞非常缓慢地清除神经酰胺（孵育6小时后19.2pmol神经酰胺/nmol磷脂P-i）并且几乎不再生slay SM。另一方面，SV 40转化的细胞清除神经酰胺快得多（孵育6小时后41.1 pmol/nmol的P-i），并再生约80%的原始SM。这些结果表明，当神经酰胺在质膜中产生时，转化细胞的SMS活性增强特别明显。最后，一些观察使我们考虑SMS与“假定的”磷脂酰胆碱特异性磷脂酶C（PC-PLC）的关系。因此，我们测试了D 609（一种假定的PC-PLC特异性抑制剂）对SMS活性的影响，D 609在体外抑制SMS活性。此外，细胞研究表明，SMS活性被先前用于研究PC-PLC的浓度（10-50 μ g/ml）的D 609显著抑制。这些结果表明SMS作为D 609的重要生化靶标，并且它们提高了PC-PLC的许多作用，特别是在细胞转化中，可能归因于SMS的明显可能性。
Sphingomyelin synthase (SMS), an enzyme involved in sphingomyelin (SM) and ceramide metabolism, can potentially regulate, in opposite directions, the levels of ceramide and diacylglycerol. in this study SMS activity was investigated in normal and SV40-transformed human lung fibroblasts (WI38). The addition of [H-3]C-2-ceramide to cells resulted in a time-dependent formation of [H-3]C-2-SM. At 24 h after treatment, normal WI38 cells cleared 17% of [H-3]C-2-ceramide producing [H-3]C-2-SM, which accounted for 13% of total radioactivity. On the other hand, SV40-transformed cells cleared 45% of [H-3]C-2-ceramide and produced C-2-SM, which accounted for 24% of total radioactivity. This enhanced production of C-2-SM was also supported by an increase in the total SMS activity of cells (measured in vitro), such that SV40-transformed cells had SMS activity of 222 pmol/mg of protein/h, whereas wild type cells had 78 pmol/mg of protein/h of activity. Additional studies aimed at examining the SMS activity directed at ceramide produced in the plasma membrane. Treatment of cells with exogenous bacterial sphingomyelinase (SMase) for 25 min resulted in cleavage of 90-95% of total SM and the concomitant generation of ceramide, After bacterial SMase treatment, wild type WI38 cells cleared ceramide very slowly (19.2 pmol of ceramide/nmol of phosholipid P-i after 6 h of incubation) and hardly regenerated slay SM. On the other hand, SV40-transformed cells cleared ceramide much faster (41.1 pmol/nmol of P-i after 6 h of incubation) and regenerated approximately 80% of the original SM. These results show that the enhanced SMS activity of transformed cells is particularly pronounced when ceramide is produced in the plasma membrane.Finally, several observations led us to consider the relationship of SMS to the "putative" phosphatidylcholine-specific phospholipase C (PC-PLC), We, therefore, tested the effects of D609, a pur-ported PC-PLC-specific inhibitor on the activity of SMS, D609 inhibited SMS activity in vitro. In addition, cellular studies showed that SMS activity was dramatically inhibited by concentrations of D609 used previously to study PC-PLC (10-50 mu g/ml). These results suggest SMS as an important biochemical target for D609, and they raise the distinct possibility that many of the roles of PC-PLC, especially in cell transformation, may be attributable to SMS.