Investigation of the Oncogenic Properties of Phosphatase of Regenerating Liver 3

Abstract

Phosphatase regenerating liver (PRL) is an enzymatic phosphatase whose oncogenic properties warrant its investigation as a therapeutic drug target. Specifically, it is well understood that PRL3 overexpression can be directly linked to cancer, as the phosphatase can activate PI3K/AKT pathways and downregulate PTEN levels, promoting metastasis and epithelial-mesenchymal transition (EMT). The purpose of this project was to further investigate mutated PRL3 proteins in order to assess whether mutations, rather than overexpression, of PRL3 can lead to cancer. Furthermore, whether these mutations specifically caused a gain-of-function or loss-of-function in PRL3 activity was examined. This study began by investigating common point mutations associated with PRL3 using the cBioPortal for Cancer Genomics database, through which R138 was identified as the most commonly mutated site. The four most frequent PRL3 mutations were created using QuikChange® site-directed mutagenesis. After sequencing the DNA to confirm mutagenesis, the recombinant proteins were purified to investigate their biochemical properties in vitro, such as their trimerization ability and phosphatase activity. Results indicated the most common mutation, R138C, had similar trimerization ability compared to wild-type PRL3, however, the other three mutants had lower trimerization ability. Interestingly, all four oncogenic mutants showed decreased kinetic activity and lower binding affinity for the pNPP substrate. Although these results imply that these oncogenic mutations are indeed loss-of-function, further in vivo experimentation should be conducted to confirm the applicability of these results in transfected human cell lines.