Quantitative phosphokinome analysis of the Met pathway activated by the invasin internalin B from Listeria monocytogenes.
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.
Reinl
Tobias
T
Department of Cell Biology, Helmholtz Centre for Infection Research, 38124 Braunschweig, Germany.
Nimtz
Manfred
M
Hundertmark
Claudia
C
Johl
Thorsten
T
Kéri
György
G
Wehland
Jürgen
J
Daub
Henrik
H
Jänsch
Lothar
L
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000378:metabolism*; D002846:Chromatography, Affinity; D002853:Chromatography, Liquid; D016000:Cluster Analysis; D006367:HeLa Cells; D054884:Host-Pathogen Interactions; D006801:Humans; D007553:Isotope Labeling; D008024:Ligands; D008089:Listeria monocytogenes / Q000187:drug effects / Q000378:metabolism*; D013058:Mass Spectrometry; D008565:Membrane Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D040901:Proteomics / Q000379:methods*; D019859:Proto-Oncogene Proteins c-met / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects
D001426:Bacterial Proteins; D008024:Ligands; D008565:Membrane Proteins; D010455:Peptides; D010750:Phosphoproteins; D047428:Protein Kinase Inhibitors; C095768:inlB protein, Listeria monocytogenes; D019859:Proto-Oncogene Proteins c-met