Dissecting the Roles of Tyrosines 490 and 785 of TrkA Protein in the Induction of Downstream Protein Phosphorylation Using Chimeric Receptors

Abstract

Receptor tyrosine kinases generally act by forming phosphotyrosine-docking sites on their own endodomains that propagate signals through cascades of post-translational modifications driven by the binding of adaptor/effector proteins. The pathways that are stimulated in any given receptor tyrosine kinase are a function of the initial docking sites that are activated and the availability of downstream participants. In the case of the Trk receptors, which are activated by nerve growth factor, there are only two established phosphotyrosine-docking sites (Tyr-490 and Tyr-785 on TrkA) that are known to be directly involved in signal transduction. Taking advantage of this limited repertoire of docking sites and the availability of PC12 cell lines stably transfected with chimeric receptors composed of the extracellular domain of the PDGF receptor and the transmembrane and intracellular domains of TrkA, the downstream TrkA-induced phosphoproteome was assessed for the “native” receptor and mutants lacking Tyr-490 or both Tyr-490 and Tyr-785. Basal phosphorylation levels were compared with those formed after 20 min of stimulation with PDGF. Several thousand phosphopeptides were identified after TiO2 enrichment, and many were up- or down-regulated by receptor activation. The modified proteins in the native sample contained many of the well established participants in TrkA signaling. The results from the mutant receptors allowed grouping of these downstream targets by their dependence on the two characterized docking site(s). A clear subset that was not dependent on either Tyr-490 or Tyr-785 emerged, providing direct evidence that there are other sites on TrkA that are involved in downstream signaling.

The Inorganic Side of NGF: Copper(II) and Zinc(II) Affect the NGF Mimicking Signaling of the N-Terminus Peptides Encompassing the Recognition Domain of TrkA Receptor

“…The typical TrkA engagement by NGF (Biarc et al, 2013) was also reproduced using the NGF-mimetic peptides NGF(1–14) and AcNGF(1–14), which were able to phosphorylate Tyr-490, thus triggering an intracellular signaling with strength similar to that elicited after NGF stimulation (Figure 5). This, in turn, was followed by Akt phosphorylation (Ser-473) (Figure 6), and the downstream ERK1/2 phosphorylation that paralleled the pattern observed for Akt phosphorylation (Figure 7).…”

NGF and ProNGF: Regulation of neuronal and neoplastic responses through receptor signaling

“…Further analyses of these samples underscored the central role of Y490 in activating the Erks and effecting changes in transcription while Y785 (which is known to activate PLCγ) is more involved in cell cycle/mitotic control. Interestingly these studies also established that there was still signaling by the double mutant, indicating at least one additional docking site (perhaps involving the activation loop tyrosines) that was strongly manifested in CK2 regulation (Biarc et al ., 2013). …”

“…988 phosphopeptides (gray part) were identified in all four conditions. Adapted from (Biarc et al ., 2013). …”

“…Plotted is the enrichment factor (how frequently phosphorylation in a particular motif was observed in comparison to the motif’s frequency in all rat proteins) in up-regulated phosphopeptides upon stimulation of the TrkA chimera in PC12 cells (orange bars) and EGFR in Hela cells (Olsen et al ., 2006) (green bars). Adapted from (Biarc et al ., 2013). …”

Biogenesis and Function of the NGF/TrkA Signaling Endosome

“…Phosphorylation in the activation loop of the kinase domain on Y670, Y674, and Y675 enhances the kinase activity (Cunningham and Greene, 1998; Stephens et al, 1994) (Figure 2). Outside the kinase domain, phosphorylation of Y490 and Y785 is most extensively characterized, but other tyrosine residues may also contribute to TrkA signaling (Biarc et al, 2013; Inagaki et al, 1995). Phosphorylated Y490 recruits adaptors Shc or Frs2 for activation of the MAPK and PI3K pathways, while phosphorylated Y785 recruits PLC-γ1 (Obermeier et al, 1993; Stephens et al, 1994) (Figure 2).…”