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

为了明确磷脂酶D （PLD）同工酶PLD1和PLD2在细胞功能中的作用，我们将PLD1和PLD2 cDNA转染到不同的细胞中，研究其对信号转导和细胞功能的影响。在H_2O_2或低氧诱导PC12细胞凋亡的早期，PLD活性瞬间升高。H_2O_2对PLD的激活归因于PLD2，并通过MAP激酶家族（ERK和p38MAP激酶）激活。此外，将PLD2转染到PC12细胞中可以抑制低氧诱导的细胞凋亡，提示PLD具有预防细胞凋亡的作用。鞘氨醇1-磷酸（S1P）是一种有丝分裂原，参与细胞存活。我们利用过度表达S1P受体EDG3的CHO细胞检测了PLD在PI3激酶和Akt存活信号通路中的作用。S1P刺激EDG3-CHO细胞，而不是载体转染的细胞，诱导PLD、pi3激酶和Akt的激活。pi3激酶抑制剂wortmannin和LY294002可阻止Akt磷酸化，表明Akt的活化依赖于pi3激酶。1-丁醇可以抑制s1p诱导的磷脂酸（PA）的积累，而2-丁醇则不能抑制s1p诱导的pi3激酶和Akt的活化。野生型PLD2与myc-Akt的共表达导致Akt对S1P的激活增加。相反，PLD2催化失活突变体的共表达消除了s1p诱导的Akt活化。外源性色褐链霉菌PLD处理EDG3-CHO细胞，导致PA积累，导致PI 3-激酶活性增加，Akt磷酸化，后者被LY294002完全消除。此外，sip诱导的膜皱化依赖于PI 3-激酶和Rac， 1-丁醇可以抑制sip诱导的膜皱化，而2-丁醇则不能。这些结果表明，PLD参与了PI 3-激酶和Akt的激活，刺激了EDG3受体。此外，在人类肾癌中发现了显著的高PLD2活性。与正常组织相比，PLD2抗体免疫染色在肾癌细胞核中显示出较强的阳性染色，提示PLD2可能参与肿瘤发生。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