Time-resolved characterization of the HGF/Met response and the influence of L. monocytogenes InlB on the physiological phosphoproteome

The vertebrate receptor tyrosine kinase Met is bound and activated by its physiological ligand hepatocyte growth factor (HGF). This interaction regulates proliferation, survival, motility, and angiogenesis in cells and plays an important role during embryogenesis and wound healing. However, HGF/Met is often deregulated in cancer which promotes metastasis. Furthermore, the Met receptor is exploited by the facultative intracellular human pathogen Listeria monocytogenes. This bacterium activates the Met receptor through binding with its surface protein internalin B (InlB), a process that induces the uptake of the pathogen into its host cell. Since HGF and InlB bind to Met at different sites and the cellular responses are clearly different, this thesis aimed to compare the signal transduction pathways downstream of the Met receptor induced by both ligands in human cell lines. Quantitative phosphoproteomics was used to characterize the physiologic Met signaling over a time course in the first 20 minutes after stimulation with HGF. About 8000 phosphorylation sites were identified, with 95 belonging to 80 proteins being significantly regulated compared to the non-stimulated control. 40 proteins of them are likely new Met pathway members, because they interact with established components or are involved in cellular responses known for HGF/Met. Two of them were characterized in detail by knockdown and gene expression analysis. The EPHA2 receptor is involved in a feedback loop controlling HGF-induced gene expression. The kinase NEK9 was described in mitosis, but does not affect HGF-induced proliferation. In contrast, it regulates motility in usually non-motile cells. Comparing the HGF/Met phosphoproteome to signaling triggered by pathogenic InlB revealed downstream pathway members activated by both ligands. Importantly however, around 10-15 proteins were HGF- or InlB-specific and could make the difference between tissue regeneration and bacterial invasion.