Long Jaclyn, Darroch Peter, Wan Kah Fei, Kong Kok Choi, Ktistakis Nicholas, Pyne Nigel J, Pyne Susan
Department of Physiology and Pharmacology, Strathclyde Institute for Biomedical Sciences, University of Strathclyde, 27 Taylor Street, Glasgow G4 0NR, UK.
Biochem J. 2005 Oct 1;391(Pt 1):25-32. doi: 10.1042/BJ20050342.
We have shown previously that LPPs (lipid phosphate phosphatases) reduce the stimulation of the p42/p44 MAPK (p42/p44 mitogen-activated protein kinase) pathway by the GPCR (G-protein-coupled receptor) agonists S1P (sphingosine 1-phosphate) and LPA (lysophosphatidic acid) in serum-deprived HEK-293 cells [Alderton, Darroch, Sambi, McKie, Ahmed, N. J. Pyne and S. Pyne (2001) J. Biol. Chem. 276, 13452-13460]. In the present study, we now show that this can be blocked by pretreating HEK-293 cells with the caspase 3/7 inhibitor, Ac-DEVD-CHO [N-acetyl-Asp-Glu-Val-Asp-CHO (aldehyde)]. Therefore LPP2 and LPP3 appear to regulate the apoptotic status of serum-deprived HEK-293 cells. This was supported further by: (i) caspase 3/7-catalysed cleavage of PARP [poly(ADP-ribose) polymerase] was increased in serum-deprived LPP2-overexpressing compared with vector-transfected HEK-293 cells; and (ii) serum-deprived LPP2- and LPP3-overexpressing cells exhibited limited intranucleosomal DNA laddering, which was absent in vector-transfected cells. Moreover, LPP2 reduced basal intracellular phosphatidic acid levels, whereas LPP3 decreased intracellular S1P in serum-deprived HEK-293 cells. LPP2 and LPP3 are constitutively co-localized with SK1 (sphingosine kinase 1) in cytoplasmic vesicles in HEK-293 cells. Moreover, LPP2 but not LPP3 prevents SK1 from being recruited to a perinuclear compartment upon induction of PLD1 (phospholipase D1) in CHO (Chinese-hamster ovary) cells. Taken together, these data are consistent with an important role for LPP2 and LPP3 in regulating an intracellular pool of PA and S1P respectively, that may govern the apoptotic status of the cell upon serum deprivation.
我们之前已经表明，在血清饥饿的HEK - 293细胞中，脂质磷酸磷酸酶（LPPs）可降低G蛋白偶联受体（GPCR）激动剂鞘氨醇-1-磷酸（S1P）和溶血磷脂酸（LPA）对p42/p44丝裂原活化蛋白激酶（p42/p44 MAPK）途径的刺激作用[奥尔德顿、达罗克、桑比、麦基、艾哈迈德、N. J. 派恩和S. 派恩（2001年）《生物化学杂志》276卷，13452 - 13460页]。在本研究中，我们现在表明，用半胱天冬酶3/7抑制剂Ac - DEVD - CHO [N - 乙酰 - 天冬氨酸 - 谷氨酸 - 缬氨酸 - 天冬氨酸 - 醛（醛）]预处理HEK - 293细胞可阻断这种作用。因此，LPP2和LPP3似乎调节血清饥饿的HEK - 293细胞的凋亡状态。这进一步得到以下支持：（i）与载体转染的HEK - 293细胞相比，血清饥饿的过表达LPP2的细胞中，半胱天冬酶3/7催化的聚（ADP - 核糖）聚合酶（PARP）裂解增加；（ii）血清饥饿的过表达LPP2和LPP3的细胞表现出有限的核小体间DNA梯状条带，而载体转染的细胞中不存在这种条带。此外，LPP2降低了基础细胞内磷脂酸水平，而LPP3降低了血清饥饿的HEK - 293细胞中的细胞内S1P水平。LPP2和LPP3在HEK - 293细胞的细胞质小泡中与鞘氨醇激酶1（SK1）组成性共定位。此外，在诱导中国仓鼠卵巢（CHO）细胞中的磷脂酶D1（PLD1）后，LPP2而非LPP3可阻止SK1被募集到核周区室。综上所述，这些数据与LPP2和LPP3分别在调节细胞内PA和S1P池方面的重要作用一致，这可能决定细胞在血清饥饿时的凋亡状态。
