Rümenapp U, Schmidt M, Wahn F, Tapp E, Grannass A, Jakobs K H
Institut für Pharmakologie, Universitätsklinikum Essen, Germany.
Eur J Biochem. 1997 Sep 1;248(2):407-14. doi: 10.1111/j.1432-1033.1997.00407.x.
Phospholipase D (PLD) activity in human embryonic kidney (HEK) cells is stimulated by phorbol-ester-activated protein kinase C (PKC) and by membrane receptors, the latter apparently acting via the GTP-binding proteins, ADP-ribosylation factor (ARF) and Rho. In the present study, performed in cell-free preparations, we have characterized and compared the regulation of HEK cell PLD activity by the stable GTP analogue, guanosine 5'-O-[gamma-thio]triphosphate (GTP[S]), and the phorbol ester, phorbol 12-myristate 13-acetate (PMA). In digitonin-permeabilized HEK cells, prelabeled with [3H]oleic acid, GTP[S] and PMA caused an approximately threefold concentration-dependent increase in the formation of [3H]phosphatidylethanol, measured in the presence of ethanol. Neomycin, which is known to complex with the PLD cofactor, phosphatidylinositol 4,5-bisphosphate, decreased basal and GTP[S]- or PMA-stimulated PLD activities with similar sensitivity. GDP and its analogue, guanosine 5'-O-[beta-thio]diphosphate, inhibited the stimulatory effect of GTP[S], whereas the PMA response was prevented by the nonselective PKC inhibitor, staurosporine, but not vice versa. PLD stimulation by GTP[S], but not by PMA, was markedly reduced upon cytosol depletion and reconstituted by purified recombinant ARF1. In HEK cell membranes, addition of purified recombinant ARNO, a guanine-nucleotide-exchange factor for ARF1. potentiated the GTP[S]-stimulated PLD activity. PLD stimulation by PMA in HEK cell membranes required MgATP and was largely prevented by the selective PKC inhibitors Goe 6976 and bisindolylmaleimide I. Immunoblot analysis demonstrated that both conventional PKC (alpha, beta, gamma) and atypical PKC isozymes (zeta, tau) were present in HEK cell membranes. The results indicate that phorbol ester stimulation of PLD activity in HEK cells apparently occurs by a phosphorylation-dependent mechanism involving membrane-associated PKC isozymes but not ARF proteins, the major targets of GTP[S]' action.
佛波酯激活的蛋白激酶C（PKC）以及膜受体可刺激人胚肾（HEK）细胞中的磷脂酶D（PLD）活性，后者显然是通过鸟苷三磷酸结合蛋白、ADP核糖基化因子（ARF）和Rho起作用。在本研究中，我们在无细胞制剂中对稳定的鸟苷三磷酸类似物5'-O-[γ-硫代]三磷酸鸟苷（GTP[S]）和佛波酯12-肉豆蔻酸酯13-乙酸酯（PMA）对HEK细胞PLD活性的调节进行了表征和比较。在用[3H]油酸预标记的洋地黄皂苷通透的HEK细胞中，在乙醇存在的情况下测定，GTP[S]和PMA使[3H]磷脂酰乙醇的形成呈约三倍的浓度依赖性增加。已知新霉素可与PLD辅因子磷脂酰肌醇4,5-二磷酸结合，它以相似的敏感性降低基础以及GTP[S]或PMA刺激的PLD活性。鸟苷二磷酸（GDP）及其类似物5'-O-[β-硫代]二磷酸鸟苷可抑制GTP[S]的刺激作用，而非选择性PKC抑制剂星形孢菌素可阻断PMA反应，但反之则不然。GTP[S]而非PMA对PLD的刺激作用在细胞溶质耗尽后显著降低，并可通过纯化的重组ARF1重建。在HEK细胞膜中，添加纯化的重组ARNO（一种ARF1的鸟嘌呤核苷酸交换因子）可增强GTP[S]刺激的PLD活性。PMA对HEK细胞膜中PLD的刺激作用需要MgATP，并且在很大程度上被选择性PKC抑制剂Goe 6976和双吲哚马来酰亚胺I阻断。免疫印迹分析表明，传统PKC（α、β、γ）和非典型PKC同工酶（ζ、τ）均存在于HEK细胞膜中。结果表明，佛波酯对HEK细胞中PLD活性的刺激作用显然是通过一种依赖磷酸化的机制发生的，该机制涉及膜相关PKC同工酶，但不涉及ARF蛋白，而ARF蛋白是GTP[S]作用的主要靶点。
