Prostate tumor overexpressed 1 (PTOV1) is highly expressed in several forms of cancer. High expression of PTOV1 is associated with tumor aggressiveness in several tumor types, including ovarian and breast cancer. Currently, PTOV1 is known to act both as a translational and transcriptional regulator aiding in the expression of prosurvival genes. Although PTOV1 is known to pass in and out of the nucleus in a cell cycle-dependent manner, the regulation of PTOV1 activity is not well understood and here we identify 14-3-3 as a PTOV1 interactor and show that high levels of 14-3-3 expression, like PTOV1, correlate with prostate cancer progression. Further, we identify SGK2-mediated phosphorylation at S36 of PTOV1 that is required for 14-3-3 binding. Disruption of the PTOV1-14-3-3 interaction results in an accumulation of PTOV1 in the nucleus and a proteasome-dependent reduction in PTOV1 protein levels, which requires ubiquitination at K114 of PTOV1. We also observed HUWE1 as a PTOV1-interacting partner responsible for the degradation of PTOV1 through the proteasome. We show that loss of 14-3-3 binding leads to an increase in PTOV1-HUWE1 binding, suggesting that 14-3-3 stabilizes PTOV1 protein by sequestering PTOV1 in the cytosol and inhibiting its interaction with HUWE1. Finally, our data suggest that stabilization of the 14-3-3-bound form of PTOV1 promotes PTOV1-mediated expression of cJun. Together, these data support a model that explains how 14-3-3 and HUWE1 regulate the PTOV1 stability, localization, and function within the cell.

14-3-3 proteins are among a family of phospho-binding proteins that are known to regulate many essential cellular mechanisms. By binding to sites of phosphorylation, 14-3-3s are integrated into multiple signaling pathways that govern critical processes, such as apoptosis, cell cycle progression, autophagy, glucose metabolism, and cell motility. These processes are crucial for tumorigenesis and 14-3-3 proteins are known to play a central role in facilitating cancer progression. In this study, my colleagues and I discover two novel 14-3-3 interacting proteins, ATG9A and PTOV1, that are both vital to essential cellular functions and describe various mechanisms that these two proteins regulate. ATG9A is a multi-pass transmembrane lipid scramblase that is found primarily as a homotrimer in the ER or small ATG9A vesicles. It is essential in the cellular recycling process called autophagy and is believed to act at the earliest stages of autophagy by providing the seed for the growth of the double membrane vesicle called an autophagosome. Previous work in our lab demonstrated that upon hypoxic stress, AMPK, the master nutrient-sensing kinase, phosphorylates S761 on the C-terminus of ATG9A. This triggers the binding of 14-3-3ζ to contribute to ATG9A function in hypoxia induced autophagy. Despite this revelation, the exact function of ATG9A is still poorly understood, especially in unstimulated conditions where autophagy functions at a basal level and AMPK is inactive. In this study, we sought to understand ATG9A function more broadly by identifying novel interactors of ATG9A and the role ATG9A plays in basal autophagy. To do this, we employed BioID mass spectrometry and various biochemical approaches to identify LRBA as a bona fide ATG9A interactor and autophagy regulator. Furthermore, using deuterium labeling and quantitative whole proteome mass spectrometry, and various other biochemical techniques, we show that ATG9A regulates the basal degradation of p62 and is recruited to sites of basal autophagy by active poly-ubiquitination to initiate basal autophagy. PTOV1 is an oncogenic protein that is poorly understood. Our current understanding of PTOV1 is limited to a few studies, which demonstrate that PTOV1 is highly expressed in primary prostate tumor samples and is correlated with metastasis, drug resistance, and poor clinical outcomes. In this study, we identify a mechanism by which SGK2, a poorly understood kinase, phosphorylates PTOV1 at S36 to trigger 14-3-3 binding at that site to increase PTOV1 stability in the cytosol and increase c-Jun expression. Upon SGK2 inhibition, 14-3-3 releases PTOV1 and PTOV1 is shuttled into the nucleus where HUWE1, an E3 ubiquitin ligase, ubiquitinates PTOV1 and initiates PTOV1 degradation by the proteasome. This is the first detailed mechanism of regulation identified for the poorly understood oncogene, PTOV1, and sheds light on potential therapeutic targets for cancer treatments.