Function and regulation of cytoplasmic p53

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ABSTRACT While the principal function of tumor suppressor p53 is a nuclear transcription regulator that transcribes a diverse set of genes involved in apoptosis, cell cycle regulation, and numerous other processes, compelling evidences have also unraveled extra-nuclear, transcription-independent activities of p53 in the cytoplasm, where it induces apoptosis and inhibits autophagy. However, significant gaps exist in the knowledge of p53 in the cytoplasm. The lack of knowledge is due at least in part to the fact that approaches investigating cytoplasmic p53 have been primarily in vitro studies in artificial settings. Dissection of the mechanistic aspects of cytoplasmic p53 in an in vivo system is necessary to clarify the biological events by which cytoplasmic p53 exerts its extra-nuclear, transcription-independent functions. Recent genome sequencing analyses of bats discovered many unexpected variances between bats and other mammals. Among them are the bat-specific alterations in the nuclear localization signal (NLS) sequences in p53. The alterations are predicted to hamper bat p53 nuclear import, resulting in increased cytoplasmic accumulation. To explore the significance of cytoplasmic p53, we generated mice expressing a mutant p53 that has an altered NLS sequence to mimic bat p53 NLS. We identified that p53 interacts with lactate dehydrogenase B (LDHB), which plays a critical role in lipid metabolism. We also found that cytoplasmic p53 promotes autophagy through disrupting Beclin 1-Bcl-2/Bcl-xL interaction and activating Beclin 1. IP-mass spectrometry experiments using cytoplasm-sequestered p53 and identified CUL9, a cytoplasmic cullin RING E3 ubiquitin ligase, as a major p53 binding partner. Biochemical experiments revealed that CUL9 catalyzes K63-linked, non-proteolytic polyubiquitination of p53. In this proposal, we test the hypothesis that cytoplasmic p53 exerts tumor suppression and anti-aging functions through inhibiting glycolysis, increasing lipogenesis, and promoting autophagy. Such functions of p53 are regulated by CUL9 catalyzed, K63-linked p53 polyubiquitination.