TUMOR SUPPRESSOR PROTEIN, P53

肿瘤抑制蛋白，P53

• 批准号: 6289128
• 负责人: ETTORE APPELLA
• 金额: --
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6289128
• 关键词: DNA binding protein; cell growth regulation; chemical binding; chemical fingerprinting; conformation; dimer; gene expression; genetic regulatory element; growth inhibitors; human tissue; molecular shape; oncoprotein p21; phosphorylation; protein engineering; protein structure function; sedimentation equilibrium; transcription factor; tumor suppressor proteins

The p53 tumor suppressor protein is a critical regulator of cell cycle progression that responds to DNA damage and other cellular stresses by arresting cell cycle progression or by inducing apoptosis. The p53 protein is stabilized in response to DNA damage, which also activates p53 as a transcription factor. Both stabilization of the p53 protein and activation of its sequence-specific DNA binding ability are widely believed to be mediated, at least in part, by post-translational modifications to the p53 protein. p53 is phosphorylated on several sites (15,33 and 37) in its N-terminal transactivation domain as well as on several sites in the C-terminal tetramerization/regulatory domain after cells are exposed either to ionizing radiation (IR) or to UV light. The N-terminal segment of p53 also binds Mdm2, a protein that targets p53 for degradation through a ubiquitin-mediated mechanism. Recently, we have shown that the direct association between p53 and Mdm2 is disrupted by phosphorylation of p53 on Thr-18 but not by phosphorylation at other N-terminal sites. Thr-18 was phosphorylated in vitro by casein kinase 1 (CK1); phosphorylation by CK1 required prior phosphorylation of Ser-15. Thr-18 was phosphorylated in vivo in response to DNA damage, and phosphorylation required Ser-15. These results suggest that the initial stabilization of p53 after ionizing radiation may result from inhibiting Mdm2 binding through a DNA damage- activated phosphorylation-phosphorylation cascade. Expression of oncogenic ras is associated with activation of cell cycle arrest and senescence in a p53-dependent manner. We have analyzed various downstream components of the ras signaling pathway for their contribution to the activation of cellular p53 . In collaboration with Drs.D. Bulavin and A. Fornace, our results indicate that p38 kinase had a pronounced effect on p53 activation in comparison to other members of the ras pathway. In vitro, p38 kinase phosphorylated p53 at serines 33 and 46. Mutation of these sites resulted in a significant decrease in the ability of p53 to trigger apoptosis. These results indicate that p38 kinase has a prominent role in the regulation of p53-dependent apoptosis after UV radiation. DNA binding activity of p53 is crucial for its tumor suppressor function. Our recent studies have indicated that four molecules of the DNA binding domain of human p53 (p53DBD) bind a response element with high cooperativity and bend the DNA. However, using A-tract phasing experiments, we find significant differences in the bending and twisting of DNA upon binding by p53DBD and full length human wild type p53 (WT p53). Our data show that four subunits of p53DBD and WT p53 bend the DNA by 30-35o and 50-55o respectively, and that the directionality of bending is consistent with major groove bends at the two pentamer junctions in the consensus DNA response element. Phasing analyses also demonstrate that p53DBD and WT p53 overtwist the DNA response element by ~35o and ~70o, respectively and these results are in accord with molecular modeling studies of the tetrameric complex. Overall, the four p53 moieties are associated laterally in a staggered array at the external side of the DNA loop with numerous inter-protein interfaces increasing the stability and cooperativity of binding. This novel architectural organization of the p53 tetrameric complex may have important functional implications including possible p53 interactions with chromatin. The activities of p53 leading to cell cycle arrest or apoptosis are restricted in unstressed cells both by the low level of p53 and by negative regulation through its carboxy terminal domain. The carboxy-terminal domain of p53 binds to non-sequence specific DNA as well as some unusual DNA structures. We have recently observed that p53 binds preferentially to supercoiled DNA via its C-terminal domain. This preferential binding does not result from an unwinding of the DNA by p53 nor a chiral wrapping of the DNA around the p53 and occurs with highly positively as well as highly negatively supercoiled DNA. These observations suggest that p53 preferentially interacts with DNA crossovers and may provide a mechanism for retaining latent p53 near actively transcribed DNA. - DNA binding domain, equilibrium centrifugation, modeling system, p, protein, phosphorylated peptides, - Neither Human Subjects nor Human Tissues

p53 肿瘤抑制蛋白是细胞周期进程的关键调节因子，它通过阻止细胞周期进程或诱导细胞凋亡来响应 DNA 损伤和其他细胞应激。 p53 蛋白因 DNA 损伤而稳定，这也激活 p53 作为转录因子。人们普遍认为，p53 蛋白的稳定化及其序列特异性 DNA 结合能力的激活至少部分是由 p53 蛋白的翻译后修饰介导的。当细胞暴露于电离辐射 (IR) 或紫外线后，p53 在其 N 端反式激活结构域的多个位点（15,33 和 37）以及 C 端四聚化/调节结构域的多个位点被磷酸化。 p53 的 N 端片段还结合 Mdm2，Mdm2 是一种通过泛素介导的机制靶向 p53 进行降解的蛋白质。最近，我们发现 p53 和 Mdm2 之间的直接关联会被 p53 Thr-18 上的磷酸化破坏，但不会被其他 N 末端位点的磷酸化破坏。 Thr-18 在体外被酪蛋白激酶 1 (CK1) 磷酸化； CK1 的磷酸化需要事先对 Ser-15 进行磷酸化。 Thr-18 在体内因 DNA 损伤而被磷酸化，而磷酸化需要 Ser-15。这些结果表明，电离辐射后 p53 的初始稳定可能是通过 DNA 损伤激活的磷酸化-磷酸化级联抑制 Mdm2 结合所致。致癌性 ras 的表达以 p53 依赖性方式与细胞周期停滞和衰老的激活相关。我们分析了 ras 信号通路的各种下游成分，了解它们对细胞 p53 激活的贡献。与 Drs.D 合作。 Bulavin 和 A. Fornace，我们的结果表明，与 ras 途径的其他成员相比，p38 激酶对 p53 激活具有显着影响。在体外，p38 激酶在丝氨酸 33 和 46 处磷酸化 p53。这些位点的突变导致 p53 触发细胞凋亡的能力显着降低。这些结果表明p38激酶在UV辐射后p53依赖性细胞凋亡的调节中具有显着作用。 p53 的 DNA 结合活性对其肿瘤抑制功能至关重要。我们最近的研究表明，人 p53 DNA 结合结构域 (p53DBD) 的四个分子以高度协同性结合响应元件并使 DNA 弯曲。然而，使用 A-tract 定相实验，我们发现 p53DBD 和全长人野生型 p53 (WT p53) 结合后 DNA 的弯曲和扭曲存在显着差异。我们的数据表明，p53DBD 和 WT p53 的四个亚基分别使 DNA 弯曲 30-35o 和 50-55o，并且弯曲的方向性与共有 DNA 响应元件中两个五聚体连接处的主要凹槽弯曲一致。定相分析还表明，p53DBD 和 WT p53 分别使 DNA 响应元件过度扭曲约 35° 和约 70°，这些结果与四聚体复合物的分子模型研究一致。总体而言，四个 p53 部分在 DNA 环外侧以交错阵列横向关联，具有大量蛋白质间界面，增加了结合的稳定性和协同性。 p53 四聚体复合物的这种新颖的结构组织可能具有重要的功能意义，包括 p53 与染色质可能的相互作用。 p53 导致细胞周期停滞或凋亡的活性在未受应激的细胞中受到 p53 水平低和通过其羧基末端结构域的负调节的限制。 p53 的羧基末端结构域与非序列特异性 DNA 以及一些不寻常的 DNA 结构结合。我们最近观察到 p53 通过其 C 末端结构域优先结合超螺旋 DNA。这种优先结合不是由 p53 解旋 DNA 引起的，也不是 DNA 围绕 p53 的手性缠绕引起的，并且发生在高度正超螺旋和高度负超螺旋 DNA 中。这些观察结果表明，p53 优先与 DNA 交换相互作用，并可能提供一种将潜在 p53 保留在活跃转录 DNA 附近的机制。 - DNA 结合结构域、平衡离心、建模系统、p、蛋白质、磷酸化肽， - 既不是人类受试者，也不是人类组织