Morana S J, Wolf C M, Li J, Reynolds J E, Brown M K, Eastman A
Department of Pharmacology and Toxicology and The Norris Cotton Cancer Center, Dartmouth Medical School, Hanover, New Hampshire 03755, USA.
J Biol Chem. 1996 Jul 26;271(30):18263-71. doi: 10.1074/jbc.271.30.18263.
Many events in apoptosis have been identified but their temporal relationships remain obscure. Apoptosis in human ML-1 cells induced by etoposide is characterized by intracellular acidification, enhanced Hoechst 33342 fluorescence, DNA digestion, chromatin condensation, and proteolysis of poly(ADP-ribose) polymerase. This proteolysis is a marker for the action of ICE/CED-3 proteases, which are critical activators of apoptosis. We observed that three serine/threonine protein phosphatase inhibitors, okadaic acid, calyculin A, and cantharidin, prevented all of these apoptotic characteristics. To determine which protein phosphatase was involved, we investigated the dephosphorylation of the retinoblastoma susceptibility protein Rb, a substrate for protein phosphatase 1 but not protein phosphatase 2A. Rb was dephosphorylated during apoptosis, and each inhibitor prevented this dephosphorylation at the same concentrations that prevented apoptosis. No increase in protein phosphatase 1 activity was observed in apoptotic cells suggesting that dephosphorylation of Rb may result from loss of Rb kinase activity in the presence of a constant level of protein phosphatase activity. Long term inhibition of protein phosphatase 1 (>8 h) also led to the appearance of dephosphorylated Rb, cleavage of poly(ADP-ribose) polymerase and apoptosis, suggesting these events are not solely dependent upon protein phosphatase 1. Rb dephosphorylation was also observed in several other models of apoptosis. Hence, an imbalance between protein phosphatase 1 and Rb kinase may be a common means to activate ICE/CED-3 proteases resulting in the subsequent events of apoptosis.
凋亡过程中的许多事件已被识别，但它们的时间关系仍不清楚。依托泊苷诱导的人ML-1细胞凋亡的特征是细胞内酸化、Hoechst 33342荧光增强、DNA消化、染色质凝聚以及聚（ADP-核糖）聚合酶的蛋白水解。这种蛋白水解是ICE/CED-3蛋白酶作用的标志物，而ICE/CED-3蛋白酶是凋亡的关键激活剂。我们观察到三种丝氨酸/苏氨酸蛋白磷酸酶抑制剂，冈田酸、花萼海绵诱癌素A和斑蝥素，可阻止所有这些凋亡特征。为了确定涉及哪种蛋白磷酸酶，我们研究了视网膜母细胞瘤易感蛋白Rb的去磷酸化，Rb是蛋白磷酸酶1而非蛋白磷酸酶2A的底物。Rb在凋亡过程中发生去磷酸化，每种抑制剂在阻止凋亡的相同浓度下可阻止这种去磷酸化。在凋亡细胞中未观察到蛋白磷酸酶1活性增加，这表明在蛋白磷酸酶活性水平恒定的情况下，Rb的去磷酸化可能是由于Rb激酶活性丧失所致。长期抑制蛋白磷酸酶1（>8小时）也导致去磷酸化Rb的出现、聚（ADP-核糖）聚合酶的切割和凋亡，这表明这些事件并非仅依赖于蛋白磷酸酶1。在其他几种凋亡模型中也观察到了Rb去磷酸化。因此，蛋白磷酸酶1和Rb激酶之间的失衡可能是激活ICE/CED-3蛋白酶从而导致后续凋亡事件的常见方式。
