REGULATORY MECHANISM BY PHOSPHOLIPASE D IN OXIDANT-STRESS INDUCED SURVIVAL SIGNALING

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We examined implication of phospholipase D (PLD) in oxidative stress. The role of PLD activation in hydrogen peroxide (H_20_2)-induced signal transduction and cellular responses are not completely understood. We present evidence that Ca^<2+> tyrosine kinase, Pyk2 requires PLD activation to mediate survival pathways in rat pheochromocytoma PC12 cells under oxidative stress. The H_20_2-induced phosphorylation of Pyk2 was suppressed by 1-butanol, an inhibitor of transphosphatidylation by PLD, and also by transfection of catalytically negative mouse PLD2K758R (PLD2KR). Furthermore, we found that PLD2 was associated with Pyk2 and Src, and that activation of PLD2 was required for H_20_2-enhanced association of Src with Pyk2 leading to full activation of Pyk2. H_20_2-induced phosphorylation of Akt and p70S6K was dependent on phosphatidylinositol 3-kinase (PI3K) activity and was abolished by 1-butanol but not t-butanol. Furthermore, the PI3K/Akt activation in response to H_20_2 was reduced by … More transfection of either PLD2KR or the dominant negative Pyk2DN. This study is the first demonstration that PLD2 activation is implicated in Src-dependent phosphorylation of Pyk2 by promoting the complex formation between Pyk2 and activated Src in PC12 cells exposed to H_20_2, thereby resulting in activation of the survival signaling pathway PI3K/Akt/p70S6K.A human prostate cancer cell line PC3 is resistant to camptothecin (CPU). To elucidate the mechanism of this resistance, we have examined the involvement of sphingosine kinase (SPHK) and sphingosine 1-phosphate (S1P) receptor in CPT-resistant PC3 and -sensitive LNCaP cells. PC3 cells exhibited higher activity accompanied with higher expression levels of protein and mRNA of SPHK1, and also elevated expression of SiP receptors, S1P1 and S1P3, as compared with those of LNCaP cells. The treatment of PC3 cells with CPT was found to induce up-regulation of the SPHK1/S1P signaling by induction of both SPHK1 enzyme and S1PL1/S1P3 receptors. These findings strongly suggest that high expression and up-regulation of SPHK1 and S1P receptors protect PC3 cells from the apoptosis induced by CPT. Less