European Molecular Biology Laboratory, Genome Biology Unit, Meyerhofstrasse 1, 69117 Heidelberg, Germany.
Biochemistry. 2011 Sep 6;50(35):7579-90. doi: 10.1021/bi201095z. Epub 2011 Aug 11.
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.
磷酸酶肝再生 3（PRL-3）被认为是几种癌症的生物标志物和治疗靶点。它在癌症转移中具有明确的因果作用。然而，人们对其天然底物、途径和生物学功能知之甚少，迄今为止仅提出了少数几种蛋白质底物。为了提高我们对 PRL-3 活性的底物特异性和分子决定因素的理解，对野生型（WT）蛋白、两个假定的催化失活突变体 D72A 和 C104S 以及报道的高活性突变体 A111S 进行了体外底物特异性和对磷酸肽和磷酸肌醇（PIPs）的活性、其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的测试。我们发现 WT PRL-3 不会在体外使测试的磷酸肽去磷酸化。然而，通过两种互补的生化测定表明，PRL-3 对磷酸肌醇 PI(4,5)P(2)具有活性。分子对接研究证实的实验结果表明，PRL-3 是一种磷脂酰肌醇 5-磷酸酶。C104S 变体不仅催化失活，而且结构不稳定，无法促进细胞迁移，而 WT PRL-3 则促进细胞迁移。D72A 突变体结构稳定，不使非天然底物 3-O-甲基荧光素磷酸酯（OMFP）去磷酸化。然而，我们观察到 D72A 对 PI(4,5)P(2)仍有残留的体外活性，并且与此一致，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，对非天然 OMFP 底物的高活性在磷酸肌醇的去磷酸化测定中并不明显：突变体对 PIPs 完全没有活性。我们观察到这种变体的结构明显不稳定。该突变体的细胞表型与催化失活的 C104S 突变体相同。这些结果为 PRL 家族中缺乏 PTP 催化基序中的保守丝氨酸提供了可能的解释。WT PRL-3 和突变体的 PI(4,5)P(2)去磷酸化活性与观察到的表型之间的相关性表明，PRL-3 对 PI(4,5)P(2)的去磷酸化与其在细胞迁移中的作用之间存在联系。
