Abstract
Stimulated by its physiological ligand, hepatocyte growth factor, the transmembrane receptor tyrosine kinase Met activates a signaling machinery that leads to mitogenic, motogenic, and morphogenic responses. Remarkably, the food-borne human pathogen Listeria monocytogenes also promotes autophosphorylation of Met through its virulence factor internalin B (InlB) and subsequently exploits Met signaling to induce phagocytosis into a broad range of host cells. Although the interaction between InlB and Met has been studied in detail, the signaling specificity of components involved in InlB-triggered cellular responses remains poorly characterized. The analysis of regulated phosphorylation events on protein kinases is therefore of particular relevance, although this could not as yet be characterized systematically by proteomics. Here, we implemented a new pyridopyrimidine-based strategy that enabled the efficient capture of a considerable subset of the human kinome in a robust one-step affinity chromatographic procedure. Additionally, and to gain functional insights into the InlB/Met-induced bacterial invasion process, a quantitative survey of the phosphorylation pattern of these protein kinases was accomplished. In total, the experimental design of this study comprises affinity chromatographic procedures for the systematic enrichment of kinases, as well as phosphopeptides; the quantification of all peptides based on the iTRAQ reporter system; and a rational statistical strategy to evaluate the quality of phosphosite regulations. With this improved chemical proteomics strategy, we determined and relatively quantified 143 phosphorylation sites detected on 94 human protein kinases. Interestingly, InlB-mediated signaling shows striking similarities compared with the natural ligand hepatocyte growth factor that was intensively studied in the past. In addition, this systematic approach suggests a new subset of protein kinases including Nek9, which are differentially phosphorylated after short time (4-min) treatment of cells with the Met-activating InlB(321). Thus, this quantitative phosphokinome study suggests a general, hypothesis-free concept for the detection of dynamically regulated protein kinases as novel signaling components involved in host-pathogen interactions.
Highlights
Stimulated by its physiological ligand, hepatocyte growth factor, the transmembrane receptor tyrosine kinase Met activates a signaling machinery that leads to mitogenic, motogenic, and morphogenic responses
L. monocytogenes utilizes two different molecular routes to invade nonprofessional 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]
InlB comprising amino acids –321 (InlB321) expression was performed according to Schubert et al [27], and its capacity to stimulate the Met receptor and its known downstream components was investigated by Western blot analyses
Summary
InlB, internalin B; HGF, hepatocyte growth factor; PI3K, phosphatidylinositol-4,5-bisphosphate 3-kinase; Erk, extracellular signal-regulated kinase (Erk ϭ MK03, Erk ϭ MK01); GSK3, glycogen synthase kinase 3; Mek, dual specificity mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (Mek ϭ MP2K1, Mek ϭ MP2K2); PTM, posttranslational modification; iTRAQ, isobaric tag for relative and absolute quantification; SILAC, stable isotope labeling by amino acids in cell culture; RF, regulation factor; SMAC, small molecule affinity chromatography; GK, gatekeeper; RP, reversed phase; MAPK, mitogenactivated protein kinase; DMF, N,N-dimethylformamide; EDC-HCl, N-(3-dimethylaminopropyl)-NЈ-ethylcarbodiimide hydrochloride; DMEM, Dulbecco’s modified Eagle’s medium; HRP, horseradish peroxidase; SPE, solid phase extraction; SCX, strong cation exchange; UPLC, ultraperformance LC; FP, false positive; TP, true positive; p, phospho-; MARK, microtubule-associated protein/microtubule affinity-regulating kinase; AMPK, AMP-activated protein kinase; MT, microtubule. 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. 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. We have recently established a validated statistical strategy for the quality control of quantitative MS methods used in this study [26] 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. This study suggests novel candidates such as Nek involved in signal networks exploited in the process of listerial invasion
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