PAC1 in Signaling Apoptosis and Tumor Suppression

描述（申请人提供）：转录因子p53和E2F-1在基因毒性应激反应中协调和介导细胞凋亡。然而，p53和E2F-1介导细胞凋亡的机制尚不清楚。我们最近报道了p53是PAC1的直接转录调节因子，PAC1是一种双特异性的苏氨酸和酪氨酸磷酸酶，对MAP激酶具有严格的底物特异性。我们还发现E2F-1在转录水平上调控PAC1。通过MAPK的去磷酸化，PAC1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，当p53激活导致细胞凋亡时，PAC1被大幅上调。在氧化应激后，正常细胞中PAC1的表达以p53依赖的方式显著增加。我们发现p53与PAC1启动子中的一个回文基序结合并激活PAC1启动子，导致PAC1的表达。重要的是，PAC1仅在导致细胞凋亡的应激条件下被诱导，而不是在导致细胞周期停滞的条件下被诱导，这表明p53可能在凋亡应激后被修饰。E2F-1也刺激PAC1启动子荧光素酶报告基因的活性。我们进一步证明，E2F-1的过表达会提高PAC1的转录水平，并且E2F-1是氧化应激反应中PAC1的完全诱导所必需的。最后，PAC1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E2F-1是PAC1的转录调节因子，PAC1是p53和E2F-1通路中的细胞死亡介质。这是p53和E2F-1在细胞凋亡信号传导中具有共同靶基因的第一个证据。在这项拨款申请中，我们提出了一系列实验方法来证明p53修饰如何影响其与回文基序结合的选择和选择性调节PAC1。我们将确定PAC1是如何被E2F1调节的。我们还将确定p73 （p53的副肿瘤抑制因子）是否以及如何调节PAC1，并将证明PAC1在肿瘤发生中的重要性。成功实现我们的特定目标将为p53， p73和E2F-1如何协同调节双特异性磷酸酶提供强有力的证据，从而使MAP激酶失活，MAP激酶是细胞生长和增殖的信号转导途径。这项资助可能会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.