Analysis of ASK1 and ASK2 as stress signaling intermediates.

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Although the molecular mechanisms of execution phase of apoptosis has extensively been studied, very little is known about the induction phase or signal transduction of apoptosis. Here we investigated the molecular mechanisms of apoptosis through identifying the regulatory mechanisms of ASK1. Based on our previous findings that thioredoxin (Trx) and TRAF2 acts on ASK1 as an inhibitor and activator, respectively, we analyzed the relationship of these molecules on the ASK1 molecule. TRAF2 induced the dissociation of Trx from ASK1 in a reactive oxygen species (ROS)-dependent manner. We found that prior dissociation of Trx from ASK1 is required for TRAF2 binding to ASK1 and thereby activation of ASK1, and TRAF2 bound to ASK1 induced oligomerization of ASK1 and autophosphorylation. In addition, phosphorylation of Bcl-2 by JNK was found to be important for the ASK1-induced apoptosis. Moreover, expression of constitutively active ASK1 was found to induce neurite outgrowth in PC12 cells. We found that p38 and to a lesser extent JNK, but not ERK, were activated by the expression of active ASK1. By the treatment with a p38 inhibitor SB203580, ASK1-induced neurite outgrowth was strongly inhibited, suggesting that the activation of p38 is required for the neurite-inducing activity of ASK1. We also observed that ASK1 induced expression of several neuron-specific proteins and phophorylation of neurofilament proteins, confirming that PC12 cells differentiated into mature neuronal cells by ASK1. Moreover, ASK1-expressing PC12 cells could survive in a serum-starved condition. Therefore, ASK1 appears to mediate signals leading to both differentiation and survival in PC12 cells. Together with the previous reports indicating that ASK1 functions as a pro-apoptotic signaling intermediate, these results suggest that ASK1 has much broader range of biological activities in a cell-type specific manner than we expected before.