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Köhn GroupPublications

Elucidating human phosphatase-substrate networks.
Li, X., Wilmanns, M., Thornton, J. & Köhn, M.
Sci Signal. 2013 May 14;6(275):rs10. doi: 10.1126/scisignal.2003203.
Phosphatases are crucially involved in cellular processes by dephosphorylating cellular components. We describe a structure-based classification scheme for all active human phosphatases that reveals previously unrecognized relationships between them. By collating protein and nonprotein substrates and integrating colocalization and coexpression data, we generated a human phosphatase-substrate network. Analysis of the protein sequences surrounding sites of dephosphorylation suggested that common recognition mechanisms may apply to both kinases and a subset of phosphatases. Analysis of three-dimensional substrate recognition by protein phosphatases revealed preferred domains in the substrates. We identified phosphatases with highly specific substrates and those with less specificity by examining the relationship between phosphatases, kinases, and their shared substrates and showed how this analysis can be used to generate testable hypotheses about phosphatase biological function. DEPOD (human DEPhOsphorylation Database, version 1.0, is an online resource with information about active human phosphatases, their substrates, and the pathways in which they function. The database includes links to kinases and chemical modulators of phosphatase activity and contains a sequence similarity search function for identifying related proteins in other species.
Europe PMC

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.
Europe PMC