Phosphoproteom Analysis of human Kinases: Characterisation of the c-Met Pathway after Activation with InlB from Listeria monocytogenes

Das human-pathogene Bakterium Listeria monocytogenes induziert seine Phagozytose in eine Vielzahl unterschiedlicher Zelltypen. Die Aktivierung der Rezeptor-Tyrosinkinase c-Met durch den listeriellen Virulenzfaktor Internalin B (InlB) resultiert in einer Reißverschluss-artigen Aufnahme. Die Phosphorylierungs-abhängige Signalübertragung im c-Met-Signalnetzwerk wird durch Kinasen wie Src und Akt reguliert. Mit über 500 Mitgliedern zählen Proteinkinasen zu einer der größten Proteinfamilien im Menschen. Deren Aktivitätsstatus wird selbst über Phosphorylierungen an Serin-, Threonin- und Tyrosinseitenketten reguliert. Ein detailliertes Wissen über den Phosphorylierungs-Status InlB-beeinflusster Kinasen würde unser Verständnis über die zellulären Konsequenzen der listeriellen Wirt-Pathogen-Interaktion deutlich verbessern. Die vergleichende Analyse von Phosphorylierungs-Stellen an niedrig-exprimierten Proteinen wie Kinasen ist immer noch eine Herausforderung und benötigt neuartige Ansätze. Die vorliegende Studie demonstriert zum ersten Mal die erfolgreiche Kombination einer Kinase-spezifischen Affinitäts-Chromatographie mit einer IMAC-basierten Phosphopeptid-Anreicherung und der iTRAQ™-Quantifizierung für die Identifizierung InlB-beeinflusster Proteinkinasen im c-Met-Signalweg. Durch diese Strategie konnten 166 Phosphorylierungs-Stellen in 106 humanen Kinasen exakt bestimmt werden. Die vergleichende Quantifizierung jeder einzelnen Phosphorylierungs-Stelle führte zur Identifizierung von vier aus dem physiologischen HGF/SF-stimulierten c-Met-Signalweg bekannten Proteinkinasen. Darüber hinaus konnten neun Proteinkinasen analysiert werden, welche bislang nicht zum c-Met-Signalweg zugeordnet wurden. Die vorliegende Studie bietet somit eine innovative Kombination von Technologien an, welche neue Einblicke in die c-Met-Signalkaskade schafft und zukünftig die detaillierte Charakterisierung pathologisch relevanter Signalwege erleichtern wird.The human pathogen Listeria monocytogenes exploits the c-Met signaling network to induce its phagocytosis into a broad range of host cells. Activation of c-Met is mediated by the listerial virulence factor Internalin B (InlB) and results in a zipper-like process of invasion. Phosphorylation dependend signal transduction downstream of c-Met is regulated by kinases like Src or Akt. Consisting of about 500 members, protein kinases belong to one of the largest protein families encoded by the human genome. Their activity states are mainly determined by specific phosphorylation on serine, threonine or tyrosine residues. A detailed knowledge about the phosphorylation state of InlB affected kinases subsequent to c-Met activation would significantly complement our existing knowledge of the cellular consequences induced by listerial host pathogen interaction. Comparative mapping of phosphorylation sites directly derived from low abundant signaling proteins such as kinases is still a challenge and requires sophisticated purification and quantitation technologies. This study demonstrates for the first time the successful combination of kinase-directed affinity chromatography with IMAC phosphopeptide purification and iTRAQ™ quantitation to investigate putative new kinases involved in InlB activated c-Met signaling cascade. With this strategy access to 166 phosphorylation sites derived from 106 human kinases was achieved. iTRAQTM quantitation of phosphorylation sites revealed InlB induced posttranslational modifications of kinases already known from HGF/SF studies. Moreover, nine additional kinases have been identified for the first time as InlB effector proteins. A comparable approach using the physiological ligand HGF/SF revealed common and different kinases downstream of c-Met. The present study offers an outstanding combination of technologies for the characterisation of pathological relevant signaling networks and provides new insides in the c-Met pathway

First insight into the kinome of human regulatory T cells.

Regulatory T cells (Tregs) are essential for controlling peripheral tolerance by the active suppression of various immune cells including conventional T effector cells (Teffs). Downstream of the T cell receptor (TCR), more than 500 protein kinases encoded by the human genome have to be considered in signaling cascades regulating the activation of Tregs and Teffs, respectively. Following TCR engagement, Tregs posses a number of unique attributes, such as constitutive expression of Foxp3, hyporesponsiveness and poor cytokine production. Furthermore, recent studies showed that altered regulation of protein kinases is important for Treg function. These data indicate that signaling pathways in Tregs are distinctly organized and alterations at the level of protein kinases contribute to the unique Treg phenotype. However, kinase-based signaling networks in Tregs are poorly understood and necessitate further systematic characterization. In this study, we analyzed the differential expression of kinases in Tregs and Teffs by using a kinase-selective proteome strategy. In total, we revealed quantitative information on 185 kinases expressed in the human CD4(+) T cell subsets. The majority of kinases was equally abundant in both T cell subsets, but 11 kinases were differentially expressed in Tregs. Most strikingly, Tregs showed an altered expression of cell cycle kinases including CDK6. Quantitative proteomics generates first comparative insight into the kinase complements of the CD4(+) Teff and Treg subset. Treg-specific expression pattern of 11 protein kinases substantiate the current opinion that TCR-mediated signaling cascades are altered in Tregs and further suggests that Tregs exhibit significant specificities in cell-cycle control and progression

High-resolution mass spectrometric analysis of the secretome from mouse lung endothelial progenitor cells.

Recently, we isolated and characterized resident endothelial progenitor cells from the lungs of adult mice. These cells have a high proliferation potential, are not transformed and can differentiate into blood- and lymph-vascular endothelial cells under in vitro and in vivo conditions. Here we studied the secretome of these cells by nanoflow liquid chromatographic mass spectrometry (LC-MS). For analysis, 3-day conditioned serum-free media were used. We found 133 proteins belonging to the categories of membrane-bound or secreted proteins. Thereby, several of the membrane-bound proteins also existed as released variants. Thirty-five proteins from this group are well known as endothelial cell- or angiogenesis-related proteins. The MS analysis of the secretome was supplemented and confirmed by fluorescence activated cell sorting analyses, ELISA measurements and immunocytological studies of selected proteins. The secretome data presented in this study provides a platform for the in-depth analysis of endothelial progenitor cells and characterizes potential cellular markers and signaling components in hem- and lymphangiogenesis

Kinase-selective proteomics provided comprehensive insight into the kinase complement of human Tregs and Teffs.

<p>(<b>A</b>) Enrichment of kinases. Percentage of kinases and non-kinases (others: proteins without kinase activity) after VI16743/Purvalanol-B-mediated affinity purification is shown. (<b>B</b>) Quality of kinase identification. Mean Mascot Mowse score of kinases <i>vs</i>. non-kinases (others). The Mascot score is reflecting the probability of the match between the mass spectrometry spectra/data and the protein sequence given in the database. Data shown in A and B are representative for four independently performed LC-MS/MS experiments. (<b>C</b>) Kinase complement of human Tregs and Teffs. In total, 185 kinases were identified. Kinases from nearly all groups of the human kinome were detected: AGC, PKA/PKG/PKC-family kinases; CAMK, calcium/calmodulin-dependent kinases; CK1, casein kinases; CMGC, CDK/MAPK/GSK3/CLK-family kinases; RCG, receptor guanylate cyclases; STE, sterile homologue kinases; TK, tyrosine kinases; TKL, tyrosine kinase-like kinases; atypical protein kinases; Other, kinases belonging to non of the mentioned groups; NPK, non-protein-kinases (kinases having non protein substrates). The kinase dendrogram was adapted with permission from Cell Signaling Technology, Inc. (<a href="http://www.cellsignal.com" target="_blank">www.cellsignal.com</a>).</p

Experimental strategy for the comparative analysis of kinase-dependent signaling networks in Tregs and Teffs.

<p>(<b>A</b>) <i>Ex vivo</i> expansion of human T cell subsets. (<b>B</b>) Protein kinase purification by VI16743/Purvalanol-B-based small molecule affinity chromatography (SMAC). (<b>C</b>) Kinase elution, denaturation and tryptic digestion. (<b>D</b>) Quantitative peptide labeling with iTRAQ™ and sample combination. (<b>E</b>) Separation of complex peptide sample by Strong Cation Exchange Chromatography (SCX). (<b>F</b>) LC-MS/MS analysis of multi-dimensional separated peptide fractions. (<b>G</b>) Mascot database search and peptide/kinase identification. (<b>H</b>) Statistical evaluation of quantitative MS data (iTRAQassist). (<b>I</b>) Visualization of expression data. Regulation factors (RFs) describe the relative kinase expression level in Tregs vs. Teffs (RF = Treg/Teff).</p

iTRAQ-based quantification and statistical evaluation of relative kinase expression in Tregs and Teffs.

<p>iTRAQ-based quantification and statistical analysis of differential kinase expression in Tregs and Teffs is exemplarily shown using the example of cyclin-dependent kinase 6 (CDK6). (<b>A</b>) MS/MS fragmentation spectrum of a tryptic peptide derived from cyclin-dependent kinase 6 (CDK6). Ions used for peptide sequencing and protein identification are indicated in red (b ions series) or blue (y ions series). The resulting peptide sequence (GSSDVDQLGK) is shown in the upper part of the diagram. The box embedded in the graph displays a magnification of the low molecular mass range where the iTRAQ reporter intensities could be observed. The intensity of the iTRAQ reporter 115 is correlating with the peptide abundance in Tregs. The peak at 114 Da represents the amount of the respective peptide in Teffs. (<b>B</b>) Statistical analysis of relative CDK6 peptide abundances. Analyses were performed by iTRAQassist as described previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040896#pone.0040896-Hundertmark1" target="_blank">[40]</a>. Most likely (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040896#pone-0040896-t001" target="_blank">Table 1</a>) and further possible peptide regulations were calculated and depicted as likelihood curves for every peptide. All peptides derived from CDK6 were significantly less abundant in Tregs. All peptide regulations calculated for individual CDK6 peptides were used to determine the overall regulation of the CDK6 protein (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040896#pone-0040896-t001" target="_blank">Table 1</a>). Here CDK6 peptide data are shown obtained from one representative LC-MS/MS experiments.</p

Distribution of iTRAQ-based kinase regulation in Tregs and Teffs.

<p>Here the log2 values of kinase expression ratios in Tregs and Teffs overall conducted LC-MS/MS experiments are shown (log2 RF_kinase = Treg/Teff). The majority of kinases was equally expressed in both T cell subsets.</p

Differential kinase abundances in Tregs and Teffs.

<p>Kinases regarded as expressed differentially in both T cell subsets (RF = Treg/Teff) had to met two criteria: −1.5>RF>1.5 at least in two of the conducted experiments and −1.5>RF_[median] >1.5 (¹median RF over all performed experiments).</p

Kinase signaling network specificities of human Tregs.

<p>Comparative kinome analysis revealed differential kinase expression in Tregs and Teffs. Altered kinase expression in Tregs (RF = Treg/Teff) is indicated by green (increased expression in Tregs) and red boxes (reduced expression in Tregs). Kinases regarded as expressed differentially had to met two criteria: −1.5>RF>1.5 at least in two of the conducted experiments and −1.5>RF_[median] >1.5 (median RF over all performed experiments). The figure provides an overview of possible kinase functions in Tregs. Median RF values and references for indicated kinase functions are given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040896#pone-0040896-t002" target="_blank">Table 2</a>.</p

Phenotype and function of <i>ex vivo</i> expanded Tregs and Teffs.

<p>(<b>A</b>) Allo-reactive CD4⁺CD25^hi Tregs expressed high levels of CD25 (95–99%) and Foxp3 (80–90%). Allo-reactive CD4⁺CD25⁻ Teffs showed low levels of CD25 and Foxp3 expression. (<b>B</b>) T cell proliferation and suppressive capacity of Tregs was accessed by ³H (thymidine)-incorporation on day 3 in triplicates prior each kinome analysis. Expanded Tregs consistently suppressed the proliferation of corresponding Teffs. Data shown are representative for Teffs and Tregs used in this study.</p