Esposito F, Cuccovillo F, Russo L, Casella F, Russo T, Cimino F
Dipartimento di Biochimica e Biotecnologie Mediche, Università di Napoli Federico II, Naples, Italy.
Cell Death Differ. 1998 Nov;5(11):940-5. doi: 10.1038/sj.cdd.4400427.
p21waf1/cip1 mRNA and protein accumulate in intact cells exposed to oxidizing agents through a p53-independent, MAPK-dependent mechanism. Treatment with oxidizing agents also yields a second form of this protein (FM p21), characterized by a faster migration on SDS-PAGE. This phenomenon depends on the modification of intracellular redox conditions induced by diethylmaleate, a glutathione-depleting agent, being prevented by the pretreatment with the glutathione precursor N-acetylcysteine. The appearance of this FM p21 form is very early, being observed 5 min after exposure to diethylmaleate, long before the already observed accumulation of p21 induced by oxidative stress. Furthermore, experiments with dominant negative mutants of MEK demonstrate that, in contrast with that observed for the oxidative stress-induced accumulation of p21 mRNA and protein, the appearance of FM p21 form is not dependent from the activation of the MAPK pathway. It was previously observed (Tchou et al, 1996) that in some lung carcinoma cells long exposure to high doses of phorbol esters also induces the appearance of a faster-migrating p21 electrophoretic band and it was suggested that this could result from a different phosphorylation or from a proteolytic processing at the C-terminus of the protein. The latter is not the case for the diethylmaleate-induced FM p21 whose C-terminus is intact, as demonstrated by the expression of a C-terminus tagged p21 cDNA. On the contrary, the observed migration shift seems to be dependent on the hypophosphorylation of the protein; in fact, a pretreatment of cells with okadaic acid, an inhibitor of (serine/threonine) phosphatases, inhibits the oxidation-dependent appearance of the FM p21 and the block of protein synthesis, caused by cycloeximide, does not affect the appearance of FM p21, that thus could derive from the dephosphorylation of preexisting protein.
p21waf1/cip1信使核糖核酸和蛋白质通过一种不依赖p53、依赖丝裂原活化蛋白激酶（MAPK）的机制在暴露于氧化剂的完整细胞中积累。用氧化剂处理还会产生这种蛋白质的第二种形式（FM p21），其特点是在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）上迁移速度更快。这种现象取决于由谷胱甘肽耗竭剂马来酸二乙酯诱导的细胞内氧化还原状态的改变，而用谷胱甘肽前体N-乙酰半胱氨酸预处理可防止这种改变。这种FM p21形式的出现非常早，在暴露于马来酸二乙酯5分钟后即可观察到，远早于已经观察到的氧化应激诱导的p21积累。此外，用MEK显性负性突变体进行的实验表明，与氧化应激诱导的p21信使核糖核酸和蛋白质积累所观察到的情况相反，FM p21形式的出现不依赖于MAPK途径的激活。先前观察到（Tchou等人，1996年），在一些肺癌细胞中，长时间暴露于高剂量佛波酯也会诱导出现迁移速度更快的p21电泳条带，有人认为这可能是由于不同的磷酸化或蛋白质C末端的蛋白水解加工所致。对于马来酸二乙酯诱导的FM p21并非如此，其C末端是完整的，如C末端标记的p21互补脱氧核糖核酸（cDNA）的表达所示。相反，观察到的迁移迁移似乎依赖于蛋白质的低磷酸化；事实上，用冈田酸（一种（丝氨酸/苏氨酸）磷酸酶抑制剂）预处理细胞会抑制FM p21的氧化依赖性出现，而环己酰亚胺引起的蛋白质合成阻断并不影响FM p21的出现，因此FM p21可能源自预先存在的蛋白质的去磷酸化。
