Quantitative phosphokinome analysis of the Met pathway activated by the invasin internalin B from Listeria monocytogenes

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.

Fig. 1.

N-terminal variant InlB₃₂₁ is a potent activator of Met signal transduction. A, schematic representation of the Met₇₄₁-InlB₃₂₁ complex according to Niemann et al. (13). Amino acids 36–321 from InlB are necessary for Met binding and activation. B, Western blot analyses of the activation of Met, Akt1, Mek1/2, and Erk1/2 and the inhibition of GSK3A/B by phosphorylation site-specific antibodies reveal a fast InlB₃₂₁-dependent induction of the Met signaling machinery. Within the first minutes after addition, InlB₃₂₁ activates the mitogen-activated protein kinase pathway and the PI3K/Akt1 module (pMet, pAkt, and pErk1/2, n = 3; pMek1/2 and pGSK3A/B, n = 2). IR, inter-repeat; LRR, leucine-rich repeat; TM, transmembrane; JM, juxtamembrane; KD, kinase domain; SEMA, semaphorin. B, B-repeat; PSI, plexin-semaphorin-integrin domain.

Fig. 2.

VI16743 affinity matrix permits comprehensive one-step purification of human protein kinases. A, evaluation of protein distribution and Mascot identification score in VI16743-purified fractions demonstrate the high selectivity of the immobilized inhibitor for protein kinases. Error bars indicate the standard deviation within four independent experiments. B, VI16743 enables purification of protein kinases from nearly all groups of the human kinome. aPK, atypical protein kinases; PK, protein kinases; CAMK, calcium/calmodulin-dependent kinases; TK, tyrosine kinases; TKL, tyrosine kinase-like kinases. STE, sterile homologue kinases; AGC, PKA/PKG/PKC-family kinases; RCG, receptor guanylate cyclases; CMGC, CDK/MAPK/GSK3/CLK family kinases.

Fig. 3.

Experimental strategy for MS/MS-based identification and quantification of ligand-induced phosphorylation events on protein kinases in cellular signaling. InlB₃₂₁-treated and untreated HeLa S3 cells (1.5 × 10⁸ per experiment) were lysed, and the resulting protein extracts were applied in parallel for protein kinase affinity chromatography. Bound proteins were eluted, alkylated, and digested. Peptides of each sample were modified by specific iTRAQ reagents prior to sample combination. Before LC-MS/MS analysis, IMAC was used to separate phosphorylated from non-phosphorylated peptides. The acquired fragmentation spectra were interpreted with the Mascot search algorithm. Quantification analysis was performed with the in-house developed software iTRAQassist (26).

Fig. 4.

Qualitative and quantitative phosphosite analysis at protein kinases. Two MS/MS spectra represent the identification of already known (A) and novel (B) InlB₃₂₁-induced phosphorylation sites. In A, the fragmentation spectrum is derived from a doubly phosphorylated peptide from the protein kinase MK01 (Erk2), indicating MK01 kinase activity. The magnification inset shows the low molecular mass range. Under MS/MS conditions, peptides from the InlB₃₂₁-treated sample release the iTRAQ reporter ion with a mass of 117 Da, whereas the iTRAQ reporter ion with a mass of 115 Da is generated from peptides from the control (CTRL) approach. MK01 phosphorylation is strongly induced in the stimulated cell state. In B, a spectrum is shown from a tryptic peptide from Nek9 carrying a phosphate group at Thr³³³. This modification is significantly increased after treatment of cells with InlB₃₂₁.

Fig. 5.

Cluster analysis of single peptide regulation factors revealed InlB₃₂₁-induced alterations in protein kinases. A, distribution of peptide regulation factors calculated from raw signal intensities (117 Da, InlB321; 115 Da, control) from all MS/MS scans from one Met activation experiment is plotted. The majority of the analyzed 1833 MS/MS scans are not regulated. Phosphopeptide regulations show a non-normal and shifted distribution, indicating their pronounced up-regulation in comparison with non-modified peptides. B, exemplified view of relative peptide regulations of the 15 most robustly identified protein kinases from one typical experiment. All MS/MS scans from A were normalized and iTRAQ by-product-corrected, and RFs from scans corresponding to a unique peptide were accumulated by iTRAQassist. Resulting RFs indicate Met pathway-dependent regulations of specific peptides in this small set of protein kinases after 4 min of InlB₃₂₁ treatment. The two marked peptides from MK01 (Erk2) correspond to the same amino acid sequence in its unmodified and phosphorylated form with oppositely directed RFs, whereas the two highlighted peptides from GSK3B have different sequences. The marked phosphopeptide from Nek9 comprising Thr³³³ and the doubly phosphorylated peptide from MK01 correspond to the raw data shown in Fig. 4.

Fig. 6.

Statistical evaluation of iTRAQ-based peptide regulations. Analyses were done by iTRAQassist as described previously (26). The most likely and possible regulations were calculated based on a work flow-specific noise model and were depicted as likelihood curves for every peptide. Significantly regulated phosphopeptides can be detected after 4-min InlB₃₂₁ treatment of HeLa cells at MK01 (A) and Nek9 (B).

Fig. 7.

Met signal transduction exploited by InlB from L. monocytogenes. InlB₃₂₁-dependent differentially phosphorylated protein kinases identified in the present study are highlighted in blue. L. monocytogenes activates the PI3K and MAPK pathways, both essential for actin cytoskeleton remodeling as a prerequisite for invasion. The identified induction of phosphorylation events on RSK2 and MEK support a negative feedback theory in this signaling module. Dephosphorylation of MARK2 in the kinase domain as one novel finding is suggested to block MARK2 activation, which is probably essential for Listeria invasion. Active MARK2 leads to destabilization of MTs by phosphorylation of microtubule-associated proteins. Earlier studies already demonstrated that destabilization of MTs by nocodazole impaired efficient Listeria uptake by the host cell. The functional contribution of novel candidates such as Nek9 or CK1D to the InlB/Met invasion strategy will be the subject of further studies. Ub, ubiquitin.