Quantitative Phosphokinome Analysis of the Met Pathway Activated by the Invasin Internalin B from Listeria monocytogenes

The human food-borne pathogen Listeria monocytogenes has evolved mechanisms to cross the intestinal, placental, and blood-brain barriers with severe consequences for pregnant women, newborns, and immunocompromised individuals. As a facultative intracellular pathogen, L. monocytogenes invades host cells within minutes, thus escaping the humoral arm of adaptive immunity. In this protective host niche, the organism replicates and spreads from cell to cell through the formation of so-called membrane protrusions. L. monocytogenes utilizes two different molecular routes to invade non-professional phagocytotic cells. (i) Internalin A binds to the cell adhesion molecule E-cadherin, resulting in the initial penetration of intestinal tissue (1, 2). (ii) In contrast, internalin B (InlB)1 contributes to the systemic infection of the host, promoting the invasion of a broader range of cell types including hepatocytes (3) and endothelial cells (4). A basic GW motif at the C terminus mediates the attachment of InlB to the bacterial cell wall, but the non-covalent nature of this interaction also allows the partial release of InlB into the environment (5, 6). GW domains of soluble InlB interact with glycosaminoglycans (7) and the complement receptor qC1q-R (8) on the host cell surface, although these interactions seem to be dispensable for the process of listerial invasion. In contrast, the N-terminal region of InlB comprising the cap, leucine-rich repeat, and inter-repeat domains (termed InlB321) constitutes structural features that stimulate the bacterial ingestion into the host cell cytosol. The horseshoe-like shape of InlB321 allows binding to and activation of the transmembrane tyrosine kinase Met, which is also the receptor for the host growth factor, hepatocyte growth factor (HGF). Although InlB binds to a different region of Met compared with HGF, it exploits the Met signaling capabilities, ultimately leading to actin cytoskeleton rearrangements, membrane engulfment, and uptake of the pathogen. InlB induces a rapid autophosphorylation in the kinase domain of Met (9) followed by recruitment of specific adapter molecules initiating signal transduction via prominent downstream components such as PI3K and the Raf-Erk pathway (10). Moreover, immobilized InlB321 is sufficient to induce the efficient uptake of latex beads into the host cell (11, 12). Recently, the structure of the InlB321-Met complex was solved at the atomic level, unambiguously demonstrating that InlB321 is mandatory but also sufficient to activate Met signaling (13). Numerous molecular studies of signaling components have been reported, and a complex protein network downstream of Met has been compiled (14). However, the molecular interactions defined so far are still insufficient to derive the InlB-induced signal transduction pathway resulting in uptake of Listeria. As a basic signaling principle, protein kinase-catalyzed phosphorylation regulates virtually every function of substrate proteins, i.e. protein-protein interactions, localization, activity, and stability. With more than 500 members, the superfamily of protein kinases is among the largest protein families encoded by the human genome (15). The functional mechanisms regulated by kinase-mediated phosphorylations on substrate proteins are also involved in the activity control of the kinases themselves. Studying these modifications directly at the kinase level enables classification of their activated states, and their systematic investigation by proteomics has already been used to detect and correlate kinases with potential functions in cell cycle control and cancer biology (16, 17). A detailed knowledge of InlB/Met-affected phosphorylation sites of proteins from the kinase superfamily would contribute to a better understanding of the listerial invasion strategy in addition to complementing our knowledge of the Met signaling pathway. Phosphorylation sites can be detected during the process of automatic peptide sequencing in well established bottom-up proteome approaches. However, the substoichiometric nature and poor ionization properties of phosphopeptides usually require purification strategies such as IMAC to optimize analysis by mass spectrometry (18). Furthermore, the complexity of the total phosphoproteome requires the pre-enrichment of protein kinases as a prerequisite for characterization of the low abundance family members. We and others have demonstrated that the highly conserved ATP-binding region of protein kinases offers possibilities for their systematic purification based on immobilized ATP-competitive small molecule inhibitors with broad kinase selectivity. In combination with phosphopeptide enrichment, this strategy has proven to be highly appropriate for a comprehensive LC-MS/MS-based phosphorylation site analysis of these key signaling components (17, 19, 20). To characterize the role of protein kinases as key regulatory elements in signaling pathways, the acquisition of quantitative peptide data of both the phosphorylated and unmodified proteins is required. Powerful isotopic labeling approaches such as SILAC (21) and iTRAQTM (22) have been devised and successfully applied to dissect cell and signaling states mainly at the substrate protein level (23, 24), but they are also beginning to support the in-depth characterization of the human kinome (17, 20, 25). Because the detection of individually regulated phosphopeptides has to cope with the so-called “one-hit wonder” problem in proteomics, the interpretation of single peptide regulation requires that particular attention must be paid to the process of statistical raw data evaluation. We have recently established a validated statistical strategy for the quality control of quantitative MS methods used in this study (26). In total, this bioinformatics work flow normalizes unequal sample amounts, corrects isotopic impurities of iTRAQ labeling reagents, and importantly can calculate the reliability of regulatory data based on the actual signal-to-noise properties of the mass spectrometer used. The synthesis of an optimized affinity resin as a base for a robust single step capture of protein kinases was the starting point in this study and allowed the systematic analyses of this enzyme class in human epithelial cells. In the following, we explain the biochemical strategy established for the quantitative characterization of phosphorylation events at these kinases in the context of infection. The dissection of one representative data set shows the potential of the selected strategy but also underscores the necessity of our statistical approach for evaluating the regulatory information based on iTRAQ reporter ions. Finally, we apply the total approach to analyze protein kinases systematically in the Met receptor kinase pathway exploited by the invasin InlB from L. monocytogenes. The majority of unambiguously regulated phosphorylation events are in accordance with our existing knowledge about the HGF/Met pathway. Furthermore, this study suggests novel candidates such as Nek9 involved in signal networks exploited in the process of listerial invasion.