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，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蛋白在DNA损伤后稳定下来，这也激活了P53作为转录因子。人们普遍认为，P53蛋白的稳定及其序列特异性DNA结合能力的激活至少部分是通过对P53蛋白的翻译后修饰来实现的。细胞暴露于电离辐射(IR)或紫外光后，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/调节域的多个位点上被磷酸化。P53的N端片段还与MDM2结合，MDM2是一种通过泛素介导的机制针对P53进行降解的蛋白质。最近，我们发现P53与MDM2之间的直接联系被Thr-18上P53的磷酸化所破坏，但不被其他N-末端的磷酸化所破坏。酪蛋白激酶1(CK1)在体外使Thr-18磷酸化；CK1的磷酸化需要Ser-15的磷酸化。苏氨酸-18在体内被磷酸化以响应DNA损伤，而磷酸化需要丝氨酸-15。这些结果表明，电离辐射后P53的初步稳定可能是通过DNA损伤激活的磷酸化-磷酸化级联抑制MDM2结合的结果。癌基因ras的表达与细胞周期停滞和衰老的激活有关，且依赖于P53。我们已经分析了ras信号通路的不同下游成分对细胞P53激活的贡献。在与D.Bulavin博士和A.Fornace博士的合作中，我们的结果表明，与ras途径的其他成员相比，p38激酶对p53的激活有显著的影响。在体外，p38激酶使P53在33和46位丝氨酸发生磷酸化。这些位点的突变导致P53触发细胞凋亡的能力显著降低。这些结果表明，p38激酶在紫外线照射后P53依赖的细胞凋亡的调控中起着重要作用。P53的DNA结合活性是其抑癌功能的关键。我们最近的研究表明，人类p53(P53DBD)的DNA结合域的四个分子与一个高度协同的反应元件结合，使DNA弯曲。然而，利用A-道时相实验，我们发现p53DBD和全长人野生型p53(WT P53)结合时DNA的弯曲和扭转有显著差异。我们的数据显示，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识DNA反应元件中两个五聚体连接处的主要凹槽弯曲是一致的。相位分析还表明，p53DBD和WT p53分别使DNA反应元件扭曲了~35o和~70o，这些结果与四聚体络合物的分子模拟研究相一致。总体而言，四个p53部分在DNA环的外侧以交错阵列的形式横向关联，具有大量的蛋白质间接口，增加了结合的稳定性和协同性。P53四聚体复合体的这种新的结构组织可能具有重要的功能意义，包括可能的P53与染色质的相互作用。在非应激细胞中，导致细胞周期停滞或凋亡的P53活性受到低水平P53及其羧基末端结构域负调控的限制。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结构相结合。我们最近观察到P53通过其C-末端结构域优先与超螺旋DNA结合。这种优先结合既不是由于P53解开DNA，也不是由于DNA手性包裹在P53周围，而是发生在高度正向和高度负向的超螺旋DNA中。这些观察表明，P53优先与DNA交叉作用，并可能提供一种机制，将潜在的P53保留在活跃转录的DNA附近。-DNA结合域，平衡离心法，模拟系统，p，蛋白质，磷酸化肽，-非人类受试者或人类组织