Functional analysis of the new signal transduction enzyme PLD by the molecular genetic technique

为了阐明磷脂酶D（PLD）同工酶PLD 1和PLD 2在细胞功能中的作用，将PLD 1和PLD 2 cDNA转染到不同的细胞中，观察其对信号转导和细胞功能的影响。在H_2O_2或低氧诱导的PC 12细胞凋亡早期，PLD活性短暂升高。H_2O_2对PLD的激活主要是通过PLD 2，并通过MAP激酶家族（ERK和p38 MAP激酶）激活PLD。此外，PLD 2转染PC 12细胞可抑制低氧诱导的细胞凋亡，表明PLD具有防止细胞凋亡的作用。已知鞘氨醇1-磷酸（S1 P）是促有丝分裂剂并参与细胞存活。我们使用过表达S1 P受体EDG 3的CHO细胞研究PLD在存活信号PI 3激酶和Akt中的作用。S1 P刺激EDG 3-CHO细胞，而不是载体转染的细胞，诱导PLD，PI 3-激酶和Akt的激活。Akt磷酸化 ...更多信息 PI 3-激酶抑制剂wortmannin和LY 294002阻止Akt的激活，表明Akt的激活依赖于PI 3-激酶。S1 P诱导的PI 3-激酶和Akt的激活被1-丁醇消除，1-丁醇抑制PLD诱导的磷脂酸（PA）的积累，而2-丁醇则不抑制。野生型PLD 2与myc-Akt的共表达导致Akt对S1 P的激活增加。相反，PLD 2的催化失活突变体的共表达消除了S1 P诱导的Akt激活。外源性链霉菌chromofuscus PLD处理EDG 3-CHO细胞，引起PA的积累，导致PI 3-激酶活性和Akt磷酸化的增加，后者被LY 294002完全废除。此外，SIP诱导的膜皱褶，这是依赖于PI 3-激酶和Rac，被抑制1-丁醇，但不2-丁醇。这些结果表明，PLD参与了PI 3-激酶和Akt的激活，从而刺激EDG 3受体。此外，在人肾癌中发现了显著高的PLD 2活性。PLD 2抗体免疫组化染色显示，与正常组织相比，PLD 2在肾癌细胞核中呈强阳性染色，提示PLD 2可能参与了肿瘤的发生。少

In order to clarify the role of the phospholipase D (PLD) isozyme PLD1 and PLD2 in cell functions, PLD1 and PLD2 cDNA were transfected into various cells, and the influences on the signal transduction and cell functions were examined. At the early stages of apotosis induced by H_2O_2 or low oxygen in PC12 cell, PLD activity was increased transiently. The activation of PLD by H_2O_2 was attributed to PLD2, and was activated through the MAP kinase family (ERK and p38MAP kinase). Moreover, the apotosis induced by low oxygen was suppressed by transfection of PLD2 into PC12 cells, suggesting that PLD acts to prevent apotosis. It has been known that sphingosine 1-phosphate (S1P) is mitogen and participates in a cell survival. We examined a role of PLD in the survival signaling PI3 kinase and Akt using the CHO cell overexpressed with the S1P receptor EDG3. S1P stimulation of EDG3-CHO cells, but not vector-transfected cells, induced activation of PLD, PI3-kinase, and Akt. The Akt phosphorylati … More on was prevented by the PI3-kinase inhibitors wortmannin and LY294002, indicating that the Akt activation was dependent on PI3-kinase. S1P-induced activation of PI3-kinase and Akt was abrogated by 1-butanol, which inhibited S1P-induced accumulation of phosphatidic acid (PA) by PLD, whereas it was not inhibited by 2-butanol without such an action. Coexpression of the wild-type PLD2 with myc-Akt resulted in increased Akt activation in response to S1P. In contrast, co-expression of a catalytically inactive mutant of PLD2 eliminated the S1P-induced Akt activation. The treatment of EDG3-CHO cells with exogenous Streptomyces chromofuscus PLD, which caused an accumulation of PA, resulted in increases in PI 3-kinase activity and the phosphorylation of Akt, the latter of which was completely abolished by LY294002. Furthermore, SIP-induced membrane ruffling, which was dependent on PI 3-kinase and Rac, was inhibited by 1-butanol, but not 2-butanol. These results demonstrate that PLD participates in the activation of PI 3-kinase and Akt in stimulation of EDG3 receptor. Moreover, the remarkable high PLD2 activity was found in a human renal cancer. Immuno-stainining with the antibody of PLD2 revealed strong positive stain in the nuclei of a renal cancer compared with the normal tissue, suggesting that PLD2 may be associated with the participation in tumorigenesis. Less