Molecular mechanisms of the PRL phosphatases.
Rios, P., Li, X. & Köhn, M.
FEBS J. 2013 Jan;280(2):505-24. doi: 10.1111/j.1742-4658.2012.08565.x. Epub 2012Apr 10.
The phosphatases of regenerating liver (PRLs) are an intriguing family of dual specificity phosphatases due to their oncogenicity. The three members are small, single domain enzymes. We provide an overview of the phosphatases of regenerating liver, compare them to related phosphatases, and review recent reports about each phosphatase. Finally, we discuss similarities and differences between the phosphatases of regenerating liver, focusing on their molecular mechanisms and signalling pathways.
Development of a peptide that selectively activates protein phosphatase-1 in living cells.
Chatterjee, J., Beullens, M., Sukackaite, R., Qian, J., Lesage, B., Hart, D.J., Bollen, M. & Köhn, M.
Angew Chem Int Ed Engl. 2012 Oct 1;51(40):10054-9. doi: 10.1002/anie.201204308.Epub 2012 Sep 7. Europe PMC
Development of a solid phase synthesis strategy for soluble phosphoinositide analogues.
Bru, M., Kotkar, S.P., Kar, N. & Köhn, M.
Chem. Sci., 2012, 3, 1893-1902, doi: 10.1039/C2SC01061E.
Phosphatidyl inositol phosphates (PIPn) exist in nature with a variety of phosphorylation patterns on the inositol head group. These compounds are vital signaling molecules that regulate numerous key cellular processes. Different enzymes such as kinases and phosphatases modify the phosphorylation patterns. There are only a few structure activity relationship (SAR) studies with PIPn analogues to gather information for the development of PIPn analogue-based inhibitors, because chemical modifications of the inositol head group are very challenging and laborious due to multiple synthetic steps that often require tedious purifications. To simplify such studies, we describe here the development of the first solid phase organic synthesis (SPOS) strategy for PIPn analogues and derivatives providing the basis for the modification of the inositol head group in a combinatorial fashion. Our strategy builds for the first time on only four inositol building blocks to address the seven phosphorylation patterns, and this is enabled by a novel selective benzylidene acetal ring opening on a solid support, which is not possible in solution so far. The first structure?activity-relationship studies using PI(4,5)P2 analogues and derivatives with lipid phosphatases are presented.
The Metastasis-Promoting Phosphatase PRL-3 Shows Activity toward Phosphoinositides.
McParland, V., Varsano, G., Li, X., Thornton, J., Baby, J., Aravind, A., Meyer, C., Pavic, K., Rios, P. & Köhn, M.
Biochemistry. 2011 Sep 6;50(35):7579-7590. 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.
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