PAC1 in Signaling Apoptosis and Tumor Suppression

描述（由申请人提供）：转录因子p53和E2 F-1协调并介导细胞凋亡以响应遗传毒性应激。然而，p53和E2 F-1介导细胞凋亡的机制尚不清楚。我们最近报道，p53是PAC 1的直接转录调节因子，PAC 1是一种对MAP激酶具有严格底物特异性的双特异性苏氨酸和酪氨酸磷酸酶。我们还发现E2 F-1在转录水平上调节PAC 1。PAC 1通过MAPK的去磷酸化作用而被证明是MAP激酶活性的有效抑制剂。在我们的p53诱导系统中，PAC 1在p53激活后被大大上调，导致细胞凋亡。PAC 1在正常细胞中的表达在氧化应激后以p53依赖的方式显著增加。我们发现p53与PAC 1启动子中的回文基序结合并激活PAC 1启动子，导致PAC 1的表达。重要的是，PAC 1仅在引起细胞凋亡的应激条件下诱导，而不是在导致细胞周期停滞的条件下诱导，这表明p53可能在凋亡应激后被修饰。E2 F-1还刺激PAC 1启动子荧光素酶报告基因的活性。我们进一步证明E2 F-1的过表达提高了PAC 1的转录水平，并且E2 F-1是完全诱导PAC 1响应氧化应激所必需的。最后，PAC 1的过表达大大增加了细胞对凋亡的易感性。我们的研究结果表明，p53和E2 F-1是PAC 1的转录调节因子，PAC 1是p53和E2 F-1途径中的细胞死亡介质。这是第一个证据表明p53和E2 F-1在细胞凋亡信号转导中共享共同的靶基因。在本申请中，我们提出了一系列实验方法来证明p53修饰如何影响其结合回文基序和选择性调节PAC 1的选择。我们将确定E2 F1如何调节PAC 1。我们还将确定p53旁抑癌基因p73是否以及如何调节PAC 1，并证明PAC 1在肿瘤发生中的重要性。我们的具体目标的成功实现将为p53，p73和E2 F-1如何协同调节双特异性磷酸酶，进而灭活MAP激酶，细胞生长和增殖的信号转导途径提供强有力的证据。这项资助可能揭示基因治疗的新靶点，或导致癌症治疗中有效的化疗策略。

DESCRIPTION (provided by applicant): Transcription factors p53 and E2F-1 coordinate and mediate apoptosis in response to genotoxic stress. However, the mechanism whereby p53 and E2F-1 mediate apoptosis is unclear. We have recently reported that p53 is a direct transcriptional regulator of PAC1, a dual-specific threonine and tyrosine phosphatase with stringent substrate specificity for MAP kinase. We also found that E2F-1 regulates PAC1 at the transcriptional level. PAC1 has been shown as a potent inhibitor of MAP kinase activity through dephosphorylation of MAPK, In our p53 inducible system, PAC1 is greatly upregulated upon activation of p53 that leads to apoptosis. The expression of PAC1 in normal cells is significant increased following oxidative stress in a p53-dependent manner. We show that p53 binds to a palindromic motif in the PAC1 promoter and activates the PAC1 promoter, leading to the expression of PAC1. Importantly, PAC1 is induced only under the stress conditions that cause apoptosis but not the conditions that lead to cell cycle arrest, suggesting that p53 may be modified following apoptotic stresses. E2F-1 also stimulates the activity of the PAC1 promoter luciferase reporter. We further demonstrate that overexpression of E2F-1 elevates the levels of PAC1 transcription and that E2F-1 is required for full induction of PAC1 in response to oxidative stress. Finally, overexpression of PAC1 greatly increases cellular susceptibility to apoptosis. Our findings indicate that p53 and E2F-1 are transcriptional regulators of PAC1 and that PAC1 is a cell death mediator in the p53 and E2F-1 pathways. This is the first evidence that p53 and E2F-1 share a common target gene in signaling apoptosis. In this grant application, we propose a series of experimental approaches to demonstrate how p53 modifications influence its choice of binding to the palindromic motif and of selectively regulating PAC1. We will determine how PAC1 is regulated by E2F1. We will also determine whether and how p73, a p53 paralog tumor suppressor, regulates PAC1 and we will demonstrate the importance of PAC1 in tumorigenesis. Successful achievement of our specific aims will provide strong evidence for how p53, p73 and E2F-1 cooperatively regulate a dual-specific phosphatase that in turn inactivates MAP kinase, a signal transduction pathway for cell growth and proliferation. This grant may reveal a new target for gene therapy or lead to effective chemotherapeutical strategies in cancer treatment.