Receptor tyrosine kinase signaling mechanisms: Devolving TrkA responses with phosphoproteomics

Abstract

Receptor tyrosine kinases (RTKs) function through protein kinase entities located in the intracellular domain of each protomer. Following activation by ligand binding, they selectively form phosphotyrosine residues by autocatalytic modification. Some of these sites are involved in maintaining the active conformation of the kinase, while others become docking sites for various adaptor/effector/scaffold proteins, which, after complexing with the receptor, then initiate further responses through cascades of post-translational modifications and the generation of lipid second messengers. Although there is substantial overlap in the pathways and activities stimulated by this superfamily, the molecular features of the endodomains of the sub-families and the moieties that they interact with to perpetrate their signals are surprisingly distinct, which may play a significant role in the regulation and responses of the individual RTK types. Some use large scaffold proteins as the basis for most, if not all, of their signal-generating interactions, while others have numerous receptor endodomain phosphotyrosine sites that are quite overlapping in specificity. The members of the Trk family of receptors each have several tyrosine residues that are phosphorylated following stimulation, including those in the kinase activation loop, but there are only two established sites (Y490 and Y785 on TrkA) that are known to be directly involved in signal propagation. Taking advantage of this limited repertoire of docking sites, we have applied phosphoproteomic methods to dissect the signaling responses of both the native protein and derivatives that have had these two sites modified. Interestingly, a clear subset that was not dependent on either docking site was identified. A comparison with a similar set of data for EGFR indicates a considerable degree of similarity in the downstream signaling profile between these two RTKs.

Figure 1

Comparison of six different RTK endodomains, illustrating the diversity in organization and molecular signaling mechanisms. (A) Linear plot showing the location of the tyrosine residues; those that have been shown to be phosphorylated (according to UniprotKB annotation) are highlighted in red. The activation loop region is shaded and annotated by AL. (B) Schematic representation of these six RTKs that emphasizes the differences in the juxtamembrane, kinase and C-terminal extension domains and highlights the major signaling and docking sites.

Figure 2

Experimental strategy for quantifying phosphopeptides. PC12 cells, with or without stably transfected PTR or mutant versions of PTR, were cultivated in an isotopically unlabelled medium containing 200 mg/ml of unlabelled proline and either stimulated or not stimulated with human PDGF-BB (50 ug/mL) for 20 minutes. As a standard for the relative quantification in all comparisons, PC12-PTR were cultivated in medium containing heavy lysine and arginine and unlabelled proline and stimulated with PDGF-BB (50 ug/mL) for 20 minutes was used. Three mg of each sample was mixed with 3 mg of the common heavy sample and were reduced, alkylated and digested with trypsin. The phosphopeptides were enriched on a TiO2 column. The phosphopeptides and the non-phosphopeptides (flow through) were separated on a strong cation exchange column (polysulfoethyl). Samples were analyzed by LC-MS/MS for 90 min on a LTQ-Orbitrap-XL. Adapted from (Biarc et al., 2012)with permission.

Figure 3

Regulated phosphoproteins associated with signal transduction defined by selected molecular function. The closed and open symbols indicate up-regulated and down-regulated phosphorylations, respectively. The changes are classified according to the magnitude of change: more than 2 fold (square), between 1.8 and 2 fold (triangle), and between 1.5 and 1.8 fold (circle). Lipid kinases are indicated in gray. Copied from (Biarc et al., 2012) with permission.

Figure 4

Phosphorylation motifs: Sixteen phosphorylation motifs modified by different kinases that are represented at the top of the figure were analyzed for the regulated phosphopeptides by determining their enrichment (frequency of observation of a motif compared to how often it would be expected at random) in each population. The upper panel represents the enrichment factor (see text for definition) of each motif in the up (dark grey bars) and down (light grey bars) regulated phosphopeptides upon stimulation of PTR. The lower panel shows the same analysis on up-regulated phosphopeptides upon stimulation of PTR (dark grey bars) and EGFR in Hela cells [9](white bars). Copied from (Biarc et al., 2012)with permission.

Figure 5

Summary of activities regulated through different phosphotyrosine docking sites (or combinations) in the TrkA receptor.