PAC1 in Signaling Apoptosis and Tumor Suppression

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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.