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首页> 外文期刊>Biochemistry >The Metastasis-Promoting Phosphatase PRL-3 Shows Activity toward Phosphoinositides
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The Metastasis-Promoting Phosphatase PRL-3 Shows Activity toward Phosphoinositides

机译：促进转移的磷酸酶PRL-3表现出对磷酸肌醇的活性

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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)P2. 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)P2, 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)P2 with the observed phenotypes for WT PRL-3 and the mutants suggests a link between the PI(4,5)P2 dephosphorylation by PRL-3 and its role in cell migration.
机译：再生肝3（PRL-3）的磷酸酶被认为是几种癌症的生物标志物和治疗靶标。它在癌症转移中具有公认的致病作用。但是，对其天然底物，途径和生物学功能了解甚少，到目前为止，仅提出了几种蛋白质底物。为了增进我们对PRL-3活性的底物特异性和分子决定因素的了解，在体外测试了野生型（WT）蛋白，两个据称催化失活的突变体D72A和C104S以及报道的高活性突变体A111S的底物特异性和活性。磷酸肽和磷酸肌醇（PIP）的合成，它们的结构稳定性以及使用稳定的HEK293细胞系促进细胞迁移的能力。我们发现WT PRL-3不会在体外使所测试的磷酸肽去磷酸化。但是，如两个互补的生化分析所示，PRL-3对磷酸肌醇PI（4,5）P2具有活性。通过分子对接研究证实的我们的实验结果表明PRL-3是磷脂酰肌醇5-磷酸酶。已显示C104S变体不仅不具有催化活性，而且在结构上不稳定并且不能促进细胞迁移，而WT PRL-3可以促进细胞迁移。 D72A突变体在结构上稳定，不会使非天然底物3-O-甲基荧光素磷酸酯（OMFP）磷酸化。但是，我们观察到D72A对PI（4,5）P2的体外残留活性，因此，它表现出与WT PRL-3相同的细胞表型。我们对A111S变体的分析表明，在用磷酸肌醇进行的去磷酸化测定中，对非天然OMFP底物的过度活性并不明显：该突变体对PIP完全无活性。我们观察到此变体的重大结构不稳定。该突变体的细胞表型等于无催化活性的C104S突变体的表型。这些结果为PRL家族中PTP催化基序的保守Ser不存在提供了可能的解释。观察到的WT PRL-3表型和突变体对PI（4,5）P2的磷酸酶活性的相关性表明PRL-3的PI（4,5）P2去磷酸化与其在细胞迁移中的作用之间存在联系。

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