Abstract
The role of phospholipase D (PLD) activation in hydrogen peroxide (H(2)O(2))-induced signal transduction and cellular responses is not completely understood. Here we present evidence that Ca(2+)-dependent tyrosine kinase, Pyk2, requires PLD activation to mediate survival pathways in rat pheochromocytoma PC12 cells under oxidative stress. The H(2)O(2)-induced phosphorylation of two Pyk2 sites (Tyr(580), and Tyr(881)) 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(2)O(2)-enhanced association of Src with Pyk2 leading to full activation of Pyk2. H(2)O(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(2)O(2) was reduced by 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 (Tyr(580) and Tyr(881)) by promoting the complex formation between Pyk2 and activated Src in PC12 cells exposed to H(2)O(2), thereby resulting in activation of the survival signaling pathway PI3K/Akt/p70S6K.
Highlights
It is known that the cellular redox state is an important mediator of various signaling systems [1,2,3]
This study is the first demonstration that PLD2 activation is implicated in Src-dependent phosphorylation of Pyk2 (Tyr580 and Tyr881) by promoting the complex formation between Pyk2 and activated Src in PC12 cells exposed to H2O2, thereby resulting in activation of the survival signaling pathway phosphatidylinositol 3-kinase (PI3K)/Akt/p70S6K
We have previously shown that the phospholipase D (PLD) activation induced by sphingosine 1-phosphate (S1P), but not insulin-like growth factor-I, was implicated in the S1P3-mediated PI3K and Akt activation in Chinese hamster ovary cells [26, 27]
Summary
It is known that the cellular redox state is an important mediator of various signaling systems [1,2,3]. This study is the first demonstration that PLD2 activation is implicated in Src-dependent phosphorylation of Pyk2 (Tyr580 and Tyr881) by promoting the complex formation between Pyk2 and activated Src in PC12 cells exposed to H2O2, thereby resulting in activation of the survival signaling pathway PI3K/Akt/p70S6K.
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