Phosphatase and Pseudo-Phosphatase Functions of Phosphatase of Regenerating Liver 3 (PRL-3) Are Insensitive to Divalent Metals In Vitro

[Image: see text] Phosphatase of regenerating liver 3 (PRL-3) is associated with cancer metastasis and has been shown to interact with the cyclin and CBS domain divalent metal cation transport mediator (CNNM) family of proteins to regulate the intracellular concentration of magnesium and other divalent metals. Despite PRL-3’s importance in cancer, factors that regulate PRL-3’s phosphatase activity and its interactions with CNNM proteins remain unknown. Here, we show that divalent metal ions, including magnesium, calcium, and manganese, have no impact on PRL-3’s structure, stability, phosphatase activity, or CNNM binding capacity, indicating that PRL-3 does not act as a metal sensor, despite its interaction with CNNM metal transporters. In vitro approaches found that PRL-3 is a broad but not indiscriminate phosphatase, with activity toward di- and tri-nucleotides, phosphoinositols, and NADPH but not other common metabolites. Although calcium, magnesium, manganese, and zinc-binding sites were predicted near the PRL-3 active site, these divalent metals did not specifically alter PRL-3’s phosphatase activity toward a generic substrate, its transition from an inactive phospho-cysteine intermediate state, or its direct binding with the CBS domain of CNNM. PRL-3’s insensitivity to metal cations negates the possibility of its role as an intracellular metal content sensor for regulating CNNM activity. Further investigation is warranted to define the regulatory mechanisms governing PRL-3’s phosphatase activity and CNNM interactions, as these findings could hold potential therapeutic implications in cancer treatment.