Analysis of ASK1 and ASK2 as stress signaling intermediates.

尽管人们对细胞凋亡执行阶段的分子机制进行了广泛的研究，但对细胞凋亡的诱导阶段或信号转导机制知之甚少。在这里，我们通过鉴定ASK1的调控机制来研究细胞凋亡的分子机制。根据我们以前的发现，硫氧还蛋白(Trx)和TRAF2分别作为抑制物和激活剂作用于ASK1，我们分析了这些分子在ASK1分子上的关系。TRAF2以ROS依赖的方式诱导TRX从ASK1解离。我们发现，TRAF2与ASK1结合从而激活ASK1需要预先将TRX从ASK1解离，而TRAF2与ASK1结合诱导ASK1寡聚和自磷酸化。此外，JNK对Bcl2的磷酸化在ASK1诱导的细胞凋亡中起重要作用。此外，结构性活性ASK1的表达可诱导PC12细胞突起生长。我们发现，活化的ASK1可以激活p38和JNK，但不能激活ERK。用p38抑制剂SB203580处理后，ASK1诱导的轴突生长受到强烈抑制，提示ASK1的轴突诱导活性需要p38的激活。我们还观察到ASK1诱导了几种神经元特异性蛋白的表达和神经丝蛋白的磷酸化，证实了ASK1使PC12细胞分化为成熟的神经细胞。此外，表达ASK1的PC12细胞可以在血清饥饿的条件下存活。因此，ASK1似乎介导了导致PC12细胞分化和存活的信号。这些结果表明，ASK1以一种细胞类型特异性的方式具有比我们之前预期的更广泛的生物学活性。

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.