Tumor Suppressor Protein, p53

肿瘤抑制蛋白，p53

• 批准号: 6433043
• 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/6433043
• 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

Phosphorylation of p53 is a mechanism for responding to signaling events initialized by DNA damage or other stresses. To characterize the sites in human p53 that become phosphorylated in response to DNA damage, we have developed polyclonal antibodies that recognize p53 only when it is phosphorylated at specific sites. Several attempts to generate an antibody to p53 phosphorylated at Ser 6 using a phosphoserine-containing peptide as an immunogen were unsuccessful; however, phosphorylation-specific antibodies were produced by using the phosphoserine mimetic, l-2-amino-4-phosphono-4, 4-difluorobutanoic acid (F(2)Pab), in place of phosphoserine. Affinity-purified antibodies elicited by immunizing rabbits with an F(2)Pab peptide coupled to keyhole limpet hemocyanin recognized a p53(1-39) peptide phosphorylated only at Ser 6. Untreated A549 cells exhibited a background of constitutive phosphorylation at Ser 6 that increased approximately 10-fold upon exposure to either ionizing radiation or UV light. Similar results were obtained for Ser 9 using antibodies raised against a conventional phosphopeptide. Ser 9 was phosphorylated by casein kinase 1 in vitro in a phospho Ser 6-dependent manner. Our data identify two additional DNA damage-induced phosphorylations in human p53 and show that F(2)Pab-derivatized peptides can be used to develop phosphorylation site-specific polyclonal antibodies. Ser15 in human p53 (corresponding to Ser18 of mouse p53) is phosphorylated by ATM family kinases in response to ionizing radiation (IR) and UV light. To determine the effects of phosphorylation of endogenous murine p53 at Ser18 on biological responses to DNA damage, we introduced a missense mutation (Ser18 to Ala) by homologous recombination into both alleles of the endogenous p53 gene in mouse embryonic stem (ES) cells. Our analyses showed that phosphorylation of murine p53 at Ser18 in response to IR or UV radiation was required for a full p53-mediated response to these DNA damage-inducing agents. In contrast, phosphorylation of p53 at Ser18 was not required for ATM-dependent cellular resistance following exposure to IR. Additionally, efficient acetylation of the C-terminus of p53 in response to DNA damage did not require phosphorylation of murine p53 at Ser18. Activation of p53-mediated transcription is essential for cell cycle arrest, but its importance for apoptosis remains controversial. To address this question, we employed homologous recombination and LoxP/Cre-mediated deletion to produce mutant murine embryonic stem cells that express p53 with Gln and Ser in place of Leu 25 and Trp 26, respectively. p53(Gln25Ser26) was stable and nuclear and did not accumulate after DNA damage; p21/Waf1 expression was not induced. After exposure of mutant ES cells to UV light, p53(Gln25Ser26) was not acetylated and its DNA binding activity did not increase. More importantly, p53(Gln25Ser26) ES cells, like p53-/- cells, did not undergo DNA damage-induced apoptosis. The tumor suppressor p53 induces cellular senescence in response to oncogenic signals. p53 activity is modulated by protein stability and post-translational modifications, including phosphorylation and acetylation. The mechanism of p53 activation by oncogenes remains largely unknown. Recently, we have reported that the tumor suppressor PML regulates the p53 response to oncogenic signals. We found that oncogenic Ras upregulates PML expression, and overexpression of PML induces senescence in a p53-dependent manner. p53 is acetylated at Lysine 382 upon Ras expression, an event that is essential for its biological function. Ras induces re-localization of p53 and the CBP acetyltransferase within the PML nuclear bodies and induces the formation of a trimeric p53-PML-CBP complex. Lastly, Ras-induced p53 acetylation, p53-CBP complex stabilization and senescence are lost in PML-/- fibroblasts. These data establish a link between PML and p53 and indicate that integrity of the PML bodies is required for p53 acetylation and senescence upon oncogene expression.

p53的磷酸化是对DNA损伤或其他应激初始化的信号事件作出反应的机制。为了表征人类p53中因DNA损伤而磷酸化的位点，我们开发了多克隆抗体，仅在特定位点磷酸化时才能识别p53。使用含丝氨酸的肽作为免疫原，多次尝试产生在丝氨酸6位点磷酸化的p53抗体，均未成功；然而，磷酸化特异性抗体是用磷酸丝氨酸模拟物l-2-氨基-4-磷酸- 4,4 -二氟丁酸（F(2)Pab）代替磷酸丝氨酸产生的。用F(2)Pab肽偶联到keyhole帽贝血青蛋白免疫家兔获得的亲和纯化抗体识别出仅在丝氨酸6位点磷酸化的p53（1-39）肽。未经处理的A549细胞在暴露于电离辐射或紫外光时显示出Ser 6的构成性磷酸化的背景，其增加了约10倍。使用针对常规磷酸肽的抗体对丝氨酸9也获得了类似的结果。酪蛋白激酶1在体外以磷酸化丝氨酸6依赖的方式磷酸化丝氨酸9。我们的数据确定了另外两种DNA损伤诱导的人类p53磷酸化，并表明F(2) bab衍生肽可用于开发磷酸化位点特异性多克隆抗体。人类p53的Ser15（对应小鼠p53的Ser18）在电离辐射（IR）和紫外光的作用下被ATM家族激酶磷酸化。为了确定内源性小鼠p53的Ser18磷酸化对DNA损伤的生物学反应的影响，我们通过同源重组将一个错义突变（Ser18到Ala）引入小鼠胚胎干（ES）细胞的内源性p53基因的两个等位基因中。我们的分析表明，在红外或紫外线辐射下，小鼠p53的Ser18位点磷酸化是p53介导的对这些DNA损伤诱导剂的完整反应所必需的。相反，暴露于IR后，atm依赖性细胞耐药不需要p53的Ser18磷酸化。此外，在DNA损伤的反应中，p53 c末端的有效乙酰化并不需要小鼠p53的Ser18磷酸化。p53介导的转录激活对细胞周期阻滞至关重要，但其对细胞凋亡的重要性仍存在争议。为了解决这个问题，我们采用同源重组和LoxP/ cre介导的缺失来产生突变的小鼠胚胎干细胞，它们分别用Gln和Ser代替Leu 25和Trp 26来表达p53。p53（Gln25Ser26）是稳定的核细胞，DNA损伤后不积累；未诱导p21/Waf1表达。突变ES细胞暴露在紫外线下后，p53（Gln25Ser26）没有乙酰化，其DNA结合活性也没有增加。更重要的是，p53(Gln25Ser26) ES细胞与p53-/-细胞一样，没有发生DNA损伤诱导的凋亡。肿瘤抑制因子p53响应致癌信号诱导细胞衰老。P53活性受蛋白稳定性和翻译后修饰（包括磷酸化和乙酰化）调节。p53被癌基因激活的机制在很大程度上仍然未知。最近，我们报道了肿瘤抑制因子PML调节p53对致癌信号的反应。我们发现致癌Ras上调PML的表达，PML的过表达以p53依赖的方式诱导衰老。p53在Ras表达时在赖氨酸382位点乙酰化，这一事件对其生物学功能至关重要。Ras诱导p53和CBP乙酰转移酶在PML核体内的再定位，并诱导三聚体p53-PML-CBP复合物的形成。最后，ras诱导的p53乙酰化、p53- cbp复合物稳定和衰老在PML-/-成纤维细胞中丧失。这些数据建立了PML和p53之间的联系，并表明PML小体的完整性是p53乙酰化和癌基因表达引起的衰老所必需的。