The Metastasis-Promoting Phosphatase PRL-3 Shows Activity toward Phosphoinositides

Abstract

Phosphatase of regenerating liver 3 (PRL-3) is suggested as a biomarker and therapeutic target in several cancers. It has a well-established causative role in cancer metastasis. However, little is known about its natural substrates, pathways, and biological functions, and only a few protein substrates have been suggested so far. To improve our understanding of the substrate specificity and molecular determinants of PRL-3 activity, the wild-type (WT) protein, two supposedly catalytically inactive mutants D72A and C104S, and the reported hyperactive mutant A111S were tested in vitro for substrate specificity and activity toward phosphopeptides and phosphoinositides (PIPs), their structural stability, and their ability to promote cell migration using stable HEK293 cell lines. We discovered that WT PRL-3 does not dephosphorylate the tested phosphopeptides in vitro. However, as shown by two complementary biochemical assays, PRL-3 is active toward the phosphoinositide PI(4,5)P(2). Our experimental results substantiated by molecular docking studies suggest that PRL-3 is a phosphatidylinositol 5-phosphatase. The C104S variant was shown to be not only catalytically inactive but also structurally destabilized and unable to promote cell migration, whereas WT PRL-3 promotes cell migration. The D72A mutant is structurally stable and does not dephosphorylate the unnatural substrate 3-O-methylfluorescein phosphate (OMFP). However, we observed residual in vitro activity of D72A against PI(4,5)P(2), and in accordance with this, it exhibits the same cellular phenotype as WT PRL-3. Our analysis of the A111S variant shows that the hyperactivity toward the unnatural OMFP substrate is not apparent in dephosphorylation assays with phosphoinositides: the mutant is completely inactive against PIPs. We observed significant structural destabilization of this variant. The cellular phenotype of this mutant equals that of the catalytically inactive C104S mutant. These results provide a possible explanation for the absence of the conserved Ser of the PTP catalytic motif in the PRL family. The correlation of the phosphatase activity toward PI(4,5)P(2) with the observed phenotypes for WT PRL-3 and the mutants suggests a link between the PI(4,5)P(2) dephosphorylation by PRL-3 and its role in cell migration.

PRL-3 disrupts epithelial architecture by altering the post-mitotic midbody position

“…Two of the suggested PRL-3 substrates, phosphatidylinositol 4,5 bisphosphate [PI(4,5)P 2 ] (McParland et al, 2011) and ezrin (Forte et al, 2008), are particularly interesting with respect to their potential involvement in the mechanism by which PRL-3 could cause this phenotype because they are known determinants of cellular polarity (Martin-Belmonte et al, 2007), marking the apical membrane. In MDCK parental and PRL-3-overexpressing cysts, ezrin was present in the apical membrane and partially colocalized with overexpressed GFP–PRL-3-WT and GFP–PRL-3-C104S (Fig.…”

“…The PRL-3-C104S mutant was generated by using site-directed mutagenesis (McParland et al, 2011). YFP–MKLP1 was a gift from Dr Rainer Pepperkok (EMBL, Heidelberg, Germany).…”

Phosphatase of regenerating liver: a novel target for cancer therapy

“…Several independent studies also show that PRL effects some additional pathways including upregulation of KCNN4 potassium channels (79), activity toward phosphoinositides (80), induction of micro RNAs (miR) 21, 17, 19a (81), and interaction with miR-495 and miR551a (82). One study proposes a novel role of PRL-3 downstream of an internal tandem duplication mutant of fms-like tyrosine kinase 3 (FLT3-ITD) present in approximately 25% of AML patients (43).…”

Phosphatase of regenerating liver-3 inhibits invasiveness and proliferation in non-small cell lung cancer by regulating the epithelial-mesenchymal transition

“…In addition, PRL-3 with C104S mutation in the catalytic site loses biological function [32], which could reveal the effect of catalytic functionality on cell invasion. Our data showed that wild-type PRL-3 inhibits cell invasion in several lung cancer cell lines, but the C104S and C170S variants enhance invasiveness.…”