p53-induced FBXL20 suppresses the PP2A regulatory subunit PR55alpha

Background :Protein phosphatase 2A (PP2A) is a major serine/threonine phosphatase that regulates cell survival and proliferation. Each PP2A holoenzyme consists of a scaffolding subunit, a catalytic subunit, and a regulatory subunit, with the regulatory subunit determining substrate specificity and cellular localization. Work from our group and others has shown that the PR55alpha regulatory subunit promotes oncogenic signaling through YAP, c-Myc, beta-catenin, and ERK pathways. Despite its established role in supporting tumorigenesis and progression, the upstream mechanisms controlling PR55alpha expression and activity remain poorly understood. Pancreatic cancer (PC), one of the most lethal human cancers, is characterized by frequent loss of p53 function, with mutations occurring in more than 75% of cases. We recently discovered that p53 induces the F-box protein FBXL20, which in turn reduces PR55alpha stability and expression. This finding reveals a previously unrecognized mechanism whereby p53 restrains PR55alpha through SCF-mediated ubiquitination and proteasomal degradation. Defining this regulatory interaction is essential for understanding how p53 loss enhances PR55alpha-driven oncogenic signaling in PC. Methods :To identify the FBXL20 interaction site on PR55alpha, we generated Myc-tagged PR55alpha truncation constructs and performed co-immunoprecipitation assays. Additional immunoprecipitations are underway to determine whether FBXL20 engages PR55alpha within an assembled SCF complex by assessing SKP1 and CUL1-4 association. To evaluate functional consequences, we are generating PR55alpha point mutations that disrupt FBXL20 binding and will examine their biological effects on PR55alpha stability and downstream signaling pathways. Results :Mapping studies indicate that FBXL20 binds to a region between WD40 domains 5-6 of PR55alpha, potentially involving an alpha-helical linker. FBXL20 also associates with the heterotrimeric PR55alpha-PP2A complex, suggesting that PR55alpha engagement with PP2A does not block its recognition by the SCF ligase. Ongoing experiments are testing whether core SCF components co-precipitate with PR55alpha, which will determine whether a functional ubiquitin ligase complex forms. Parallel studies using PR55alpha point-mutation constructs are examining how loss of p53/FBXL20 interaction alters PR55alpha stability and downstream signaling. Conclusions :Our findings establish a novel mechanism by which p53 constrains PR55alpha abundance through FBXL20-mediated ubiquitination and proteasomal degradation. Identifying the FBXL20 recognition site on PR55alpha provides a foundation for defining the phosphodegron required for its turnover and significance on PR55alpha stability and oncogenic activity. Understanding this regulatory pathway offers insight into how p53 loss through mutations enables unchecked PR55alpha/PP2A-driven oncogenic signaling in PC and other malignancies and may uncover new PR55alpha-based therapeutic strategies.