Examining Phosphatases Through Immunofluorescent Microscopy.

Immunofluorescent microscopy enables the examination of cellular expression and localization of proteins. Cellular localization can often impact protein function, as certain molecular interactions occur in specific cellular compartments. Here we describe in detail the processes necessary for identifying phosphatases in the cell through immunofluorescent microscopy. Identification of phosphatase expression and localization could lead to the discovery of protein function in disease states along with potential substrates and binding partners.

Development and characterization of nanobodies that specifically target the oncogenic Phosphatase of Regenerating Liver-3 (PRL-3) and impact its interaction with a known binding partner, CNNM3.

Phosphatase of Regenerating Liver-3 (PRL-3) is associated with cancer progression and metastasis. The mechanisms that drive PRL-3's oncogenic functions are not well understood, partly due to a lack of research tools available to study this protein. We have begun to address these issues by developing alpaca-derived single domain antibodies, or nanobodies, targeting PRL-3 with a KD of 30-300 nM and no activity towards highly homologous family members PRL-1 and PRL-2. We found that longer and charged N-terminal tags on PRL-3, such as GFP and FLAG, changed PRL-3 localization compared to untagged protein, indicating that the nanobodies may provide new insights into PRL-3 trafficking and function. The nanobodies perform equally, if not better, than commercially available antibodies in immunofluorescence and immunoprecipitation. Finally, hydrogen-deuterium exchange mass spectrometry (HDX-MS) showed that the nanobodies bind partially within the PRL-3 active site and can interfere with PRL-3 phosphatase activity. Co-immunoprecipitation with a known PRL-3 active site binding partner, the CBS domain of metal transporter CNNM3, showed that the nanobodies reduced the amount of PRL-3:CBS inter-action. The potential of blocking this interaction is highly relevant in cancer, as multiple research groups have shown that PRL-3 binding to CNNM proteins is sufficient to promote metastatic growth in mouse models. The anti-PRL-3 nanobodies represent an important expansion of the research tools available to study PRL-3 function and can be used to define the role of PRL-3 in cancer progression.

PRL-3 promotes a positive feedback loop between STAT1/2-induced gene expression and glycolysis in multiple myeloma.

Over 34 000 patients are diagnosed yearly with multiple myeloma (MM), which remains a fatal malignancy. Expression of the phosphatase PRL-3 is associated with poor prognosis in MM patients, and Vandsemb et al. have demonstrated that PRL-3 contributes to enhanced MM cell fitness through activation of a glycolysis-associated feedback loop. PRL-3 resulted in increased expression of signal transducer and activator of transcription 1 (STAT1) and 2 (STAT2) and increased glycolysis. Increased glucose metabolism in turn activated STAT1/2 and interferon 1-related genes. This discovery advances the MM field by providing a new potential treatment avenue. Comment on: https://doi.org/10.1111/febs.16058.

A screen of FDA-approved drugs identifies inhibitors of protein tyrosine phosphatase 4A3 (PTP4A3 or PRL-3).

Protein tyrosine phosphatase 4A3 (PTP4A3 or PRL-3) is highly expressed in a variety of cancers, where it promotes tumor cell migration and metastasis leading to poor prognosis. Despite its clinical significance, small molecule inhibitors of PRL-3 are lacking. Here, we screened 1443 FDA-approved drugs for their ability to inhibit the activity of the PRL phosphatase family. We identified five specific inhibitors for PRL-3 as well as one selective inhibitor of PRL-2. Additionally, we found nine drugs that broadly and significantly suppressed PRL activity. Two of these broad-spectrum PRL inhibitors, Salirasib and Candesartan, blocked PRL-3-induced migration in human embryonic kidney cells with no impact on cell viability. Both drugs prevented migration of human colorectal cancer cells in a PRL-3 dependent manner and were selective towards PRLs over other phosphatases. In silico modeling revealed that Salirasib binds a putative allosteric site near the WPD loop of PRL-3, while Candesartan binds a potentially novel targetable site adjacent to the CX5R motif. Inhibitor binding at either of these sites is predicted to trap PRL-3 in a closed conformation, preventing substrate binding and inhibiting function.