Mills J C, Lee V M, Pittman R N
Cell Biology Graduate Group, School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.
J Cell Sci. 1998 Mar;111 ( Pt 5):625-36. doi: 10.1242/jcs.111.5.625.
The execution phase is an evolutionarily conserved stage of apoptosis that occurs with remarkable temporal and morphological uniformity in most if not all cell types regardless of the condition used to induce death. Characteristic features of apoptosis such as membrane blebbing, DNA fragmentation, chromatin condensation, and cell shrinkage occur during the execution phase; therefore, there is considerable interest in defining biochemical changes and signaling events early in the execution phase. Since onset of the execution phase is asynchronous across a population with only a small fraction of cells in this stage at any given time, characterizing underlying biochemical changes is difficult. An additional complication is recent evidence suggesting that the execution phase occurs after cells commit to die; thus, agents that modulate events in the execution phase may alter the morphological progression of apoptosis but will not affect the time-course of death. In the present study, we use a single cell approach to study and temporally order biochemical and cytoskeletal events that occur specifically in the execution phase. Microtubules de-acetylate and disassemble as terminally differentiated PC12 cells enter the execution phase following removal of nerve growth factor. Using phosphorylation sensitive antibodies to tau, we show that this microtubule-stabilizing protein becomes dephosphorylated near the onset of the execution phase. Low concentrations of okadaic acid inhibit dephosphorylation suggesting a PP2A-like phosphatase is responsible. Transfecting (tau) into CHO cells to act as a 'reporter' protein shows a similar dephosphorylation of (tau) by a PP2A-like phosphatase during the execution phase following induction of apoptosis with UV irradiation. Therefore, activation of PP2A phosphatase occurs at the onset of the execution phase in two very different cell types following different initiators of apoptosis which is consistent with activation of PP2A phosphatase being a common feature of the execution phase of apoptosis. Experiments using either taxol to inhibit microtubule disassembly or okadaic acid to inhibit tau dephosphorylation suggest that microtubule disassembly is necessary for tau dephosphorylation to occur. Thus, we propose that an early step in the execution phase (soon after a cell commits to die) is microtubule disassembly which frees or activates PP2A to dephosphorylate tau as well as other substrates.
执行阶段是凋亡过程中一个进化保守的阶段，在大多数（即便不是所有）细胞类型中，无论诱导死亡的条件如何，该阶段都会以显著的时间和形态一致性发生。凋亡的特征性表现，如细胞膜起泡、DNA片段化、染色质凝聚和细胞皱缩，都发生在执行阶段；因此，人们对确定执行阶段早期的生化变化和信号事件非常感兴趣。由于执行阶段在细胞群体中开始是异步的，在任何给定时间只有一小部分细胞处于这个阶段，所以表征潜在的生化变化很困难。另一个复杂情况是，最近有证据表明执行阶段发生在细胞决定死亡之后；因此，调节执行阶段事件的试剂可能会改变凋亡的形态进展，但不会影响死亡的时间进程。在本研究中，我们采用单细胞方法来研究并按时间顺序排列在执行阶段特异性发生的生化和细胞骨架事件。当终末分化的PC12细胞在去除神经生长因子后进入执行阶段时，微管会去乙酰化并解聚。使用对tau蛋白磷酸化敏感的抗体，我们发现这种微管稳定蛋白在执行阶段开始时会去磷酸化。低浓度的冈田酸抑制去磷酸化，表明一种类似PP2A的磷酸酶起作用。将（tau）转染到CHO细胞中作为“报告”蛋白，结果显示在用紫外线照射诱导凋亡后的执行阶段，（tau）会被一种类似PP2A的磷酸酶进行类似的去磷酸化。因此，在两种非常不同的细胞类型中，在不同的凋亡启动因子作用后，PP2A磷酸酶的激活都发生在执行阶段开始时，这与PP2A磷酸酶的激活是凋亡执行阶段的一个共同特征相一致。使用紫杉醇抑制微管解聚或冈田酸抑制tau去磷酸化的实验表明，微管解聚是tau去磷酸化发生所必需的。因此，我们提出执行阶段的一个早期步骤（在细胞决定死亡后不久）是微管解聚，这会释放或激活PP2A，使其对tau以及其他底物进行去磷酸化。
