Nikolaus Schiering

Novartis, Bâle, Basel-City, Switzerland

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Publications (3)18.05 Total impact

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    ABSTRACT: Point mutations emerge as one of the rate-limiting steps in tumor response to small molecule inhibitors of protein kinases. Here we characterized the response of the MET mutated variants, V1110I, V1238I, V1206L and H1112L to the small molecule SU11274. Our results reveal a distinct inhibition pattern of the four mutations with IC(50) values for autophosphorylation inhibition ranging between 0.15 and 1.5muM. Differences were further seen on the ability of SU11274 to inhibit phosphorylation of downstream MET transducers such as AKT, ERK, PLCgamma and STAT3 and a variety of MET-dependent biological endpoints. In all the assays, H1112L was the most sensitive to SU11274, while V1206L was less affected under the used concentration range. The differences in responses to SU11274 are discussed based on a structural model of the MET kinase domain.
    Cancer letters 09/2009; 289(2):228-36. · 4.86 Impact Factor
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    ABSTRACT: Met receptor tyrosine kinase plays a crucial role in the regulation of a large number of cellular processes and, when deregulated by overexpression or mutations, leads to tumor growth and invasion. The Y1235D mutation identified in metastases was shown to induce constitutive activation and a motile-invasive phenotype on transduced carcinoma cells. Wild-type Met activation requires phosphorylation of both Y1234 and Y1235 in the activation loop. We mapped the major phosphorylation sites in the kinase domain of a recombinant Met protein and identified the known residues Y1234 and Y1235 as well as a new phosphorylation site at Y1194 in the hinge region. Combining activating and silencing mutations at these sites, we characterized in depth the mechanism of activation of wild-type and mutant Met proteins. We found that the phosphotyrosine mimetic mutation Y1235D is sufficient to confer constitutive kinase activity, which is not influenced by phosphorylation at Y1234. However, the specific activity of this mutant was lower than that observed for fully activated wild-type Met and induced less phosphorylation of Y1349 in the signaling site, indicating that this mutation cannot entirely compensate for a phosphorylated tyrosine at this position. The Y1194F silencing mutation yielded an enzyme that could be activated to a similar extent as the wild type but with significantly slower activation kinetics, underlying the importance of this residue, which is conserved among different tyrosine kinase receptors. Finally, we observed different interactions of wild-type and mutant Met with the inhibitor K252a that may have therapeutic implications for the selective inhibition of this kinase.
    Biochemistry 12/2005; 44(43):14110-9. · 3.38 Impact Factor
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    ABSTRACT: The protooncogene c-met codes for the hepatocyte growth factor receptor tyrosine kinase. Binding of its ligand, hepatocyte growth factor/scatter factor, stimulates receptor autophosphorylation, which leads to pleiotropic downstream signaling events in epithelial cells, including cell growth, motility, and invasion. These events are mediated by interaction of cytoplasmic effectors, generally through Src homology 2 (SH2) domains, with two phosphotyrosine-containing sequence motifs in the unique C-terminal tail of c-Met (supersite). There is a strong link between aberrant c-Met activity and oncogenesis, which makes this kinase an important cancer drug target. The furanosylated indolocarbazole K-252a belongs to a family of microbial alkaloids that also includes staurosporine. It was recently shown to be a potent inhibitor of c-Met. Here we report the crystal structures of an unphosphorylated c-Met kinase domain harboring a human cancer mutation and its complex with K-252a at 1.8-A resolution. The structure follows the well established architecture of protein kinases. It adopts a unique, inhibitory conformation of the activation loop, a catalytically noncompetent orientation of helix alphaC, and reveals the complete C-terminal docking site. The first SH2-binding motif (1349YVHV) adopts an extended conformation, whereas the second motif (1356YVNV), a binding site for Grb2-SH2, folds as a type II Beta-turn. The intermediate portion of the supersite (1353NATY) assumes a type I Beta-turn conformation as in an Shc-phosphotyrosine binding domain peptide complex. K-252a is bound in the adenosine pocket with an analogous binding mode to those observed in previously reported structures of protein kinases in complex with staurosporine.
    Proceedings of the National Academy of Sciences 11/2003; 100(22):12654-9. · 9.81 Impact Factor