Phosphorylation of Thr18 and Ser20 of p53 in Ad-p53-induced apoptosis
Phosphorylation of Thr18 and Ser20 of p53 in Ad-p53-induced apoptosis
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DOI：
10.1215/15228517-2008-015
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发表时间：
2008-06-01
期刊：
影响因子：
15.9
通讯作者：
Lang, Frederick F.
中科院分区：
文献类型：
--
作者：
Nakamizo, Akira;Amano, Toshiyuko;Lang, Frederick F.
p53蛋白在诱导细胞周期停滞或凋亡中起关键作用。由于p53在人脑胶质瘤中失活，因此恢复p53功能是胶质瘤治疗的主要焦点。最临床测试的替代p53的策略是腺病毒介导的p53基因治疗（Ad-p53）。除了它们的治疗意义之外，对Ad-p53的研究提供了用于理解p53诱导细胞周期停滞与凋亡的能力的模型系统，特别是因为野生型p53细胞对Ad-p53诱导的凋亡具有抗性。在这里，我们使用Ad-p53结构来测试的假设，即同时磷酸化的p53在苏氨酸18（Thr 18）和丝氨酸20（Ser 20）是因果关系与p53介导的细胞凋亡。使用磷酸化特异性抗体的研究表明，p53诱导的细胞凋亡与p53在Thr 18和Ser 20的磷酸化相关，但与羧基末端磷酸化（Ser 392）无关。为了证明细胞凋亡与p53的Thr 18和Ser 20磷酸化之间的因果关系，在野生型p53神经胶质瘤中比较了未磷酸化的腺病毒p53构建体（Ad-p53）与Thr 18/Ser 20磷酸化模拟构建体（Ad-p53- 18 D2 D）的作用。而用Ad-p53处理仅导致细胞周期停滞，用Ad-p53- 18 D20 D处理诱导显著的凋亡。基因芯片和蛋白质印迹分析表明，只有Ad-p53- 18 D20 D能够诱导凋亡诱导蛋白的表达。染色质免疫沉淀实验表明，Ad-p53- 18 D20 D蛋白产物能够与前列腺增生相关基因结合，而Ad-p53蛋白产物不能与前列腺增生相关基因结合。因此，我们得出结论，Thr 18和Ser 20的磷酸化足以诱导p53介导的胶质瘤细胞凋亡。这些结果对p53基因治疗具有重要意义，并为旨在恢复p53功能的其他策略提供了信息。
The p53 protein plays a critical role in inducing cell cycle arrest or apoptosis. Because p53 is inactivated in human gliomas, restoring p53 function is a major focus of glioma therapy. The most clinically tested strategy for replacing p53 has been adenoviral-mediated p53 gene therapy (Ad-p53). In addition to their therapeutic implications, investigations into Ad-p53 provide model systems for understanding p53's ability to induce cell cycle arrest versus apoptosis, particularly because wild-type p53 cells are resistant to Ad-p53-induced apoptosis. Here we use Ad-p53 constructs to test the hypothesis that simultaneous phosphorylation of p53 at threonine 18 (Thr18) and serine 20 (Ser20) is causally associated with p53-mediated apoptosis. Studies using phosphorylation-specific antibodies demonstrated that p53-induced apoptosis correlates with phosphorylation of p53 at Thr18 and Ser20 but not with carboxy-terminal phosphorylation (Ser392). To prove a causal relationship between apoptosis and Thr18 and Ser20 phosphorylation of p53, the effects of an adenoviral p53 construct that was not phosphorylated (Ad-p53) was compared with a Thr18/Ser20 phosphomimetic construct (Ad-p53-18D2D)) in wild-type p53 gliomas. Whereas treatment with Ad-p53 resulted only in cell cycle arrest, treatment with Ad-p53-18D20D induced dramatic apoptosis. Microarray and Western blot analyses showed that only Ad-p53-18D20D) was capable of inducing expression of apoptosis-inducing proteins. Chromatin immunoprecipitation assays indicated that the protein product of Ad-p53-18D20D, but not Ad-p53, was capable of binding to apoptosis-related genes. We thus conclude that phosphorylation of Thr18 and Ser20 is sufficient for inducing p53-mediated apoptosis in glioma cells. These results have implications for p53 gene therapy and inform other strategies that aim to restore p53 function.