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Dissecting the roles of tyrosines 490 and 785 of TrkA protein in the induction of downstream protein phosphorylation using chimeric receptors - PubMed

  • ️Tue Jan 01 2013

Dissecting the roles of tyrosines 490 and 785 of TrkA protein in the induction of downstream protein phosphorylation using chimeric receptors

Jordane Biarc et al. J Biol Chem. 2013.

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.

Keywords: Cell Signaling; Mass Spectrometry (MS); Mutant Receptor; Phosphoproteomics; Phosphotyrosine Receptor; Phosphotyrosine Signaling; Protein Phosphorylation.

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Figures

FIGURE 1.
FIGURE 1.

Chimeric receptor construction. The chimeric PTR receptor consists of the intracellular and transmembrane (TM) domains of the rat TrkA receptor fused to the extracellular region of human PDGF receptor-β. Black and white bars indicate human PDGF receptor sequence and rat TrkA sequence, respectively. The mutant receptors annotated PTR Y490F and PTR Y490F/Y785F contain mutations of tyrosine 490 and 490/785, respectively.

FIGURE 2.
FIGURE 2.

Experimental strategy. PC12 cells stably transfected with chimeric receptors PTR Y490F and PTR Y490F/Y785F were cultivated in an isotopically unlabeled medium containing 200 mg/ml unlabeled proline and stimulated with PDGF-BB (50 ng/ml) for 20 min. As a heavy standard for the relative quantification, PC12-PTRs were cultivated in medium containing heavy lysine and arginine and unlabeled proline and stimulated with PDGF-BB (50 ng/ml) for 20 min. 3 mg of each sample were mixed with 3 mg of the common heavy sample and were then reduced, alkylated, and digested with trypsin. The phosphopeptides were enriched on a TiO2 column. The phosphopeptides and the nonphosphopeptides (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.

FIGURE 3.
FIGURE 3.

Overlap of phosphopeptides identified and the distribution of their changes upon receptor stimulation. A, Venn diagram describing the phosphopeptides identified in PC12 cells (PC12c), PC12 cells stably transfected with chimeric receptor PTR stimulated for 20 min with PDGF-BB (PTRs), PTR Y490F stimulated (PTR Y490F(s)) or PTR Y490F/Y785F stimulated with a peptide false-positive rate of 0.5%. 988 phosphopeptides (gray part) were identified in all four conditions. (Data were produced using Venny.) B, distribution of the log(ratio) of the sample PC12c normalized against the log(ratio) of the peptides from the PTRs sample. Each phosphopeptide with a log(ratio) above 0.25 or below −0.25 represents down-regulated or up-regulated stimulation modification due to stimulation, respectively, and phosphopeptides with a log(ratio) between −0.125 and 0.125 represent peptides that are not regulated.

FIGURE 4.
FIGURE 4.

Phosphorylation patterns in extracted populations. The threshold selected to determine a significant change in phosphorylation was set at a log of ratio of +0.25 and −0.25 for down- and up-regulation, respectively, and the threshold selected to determine an absence of change was set between −0.125 and 0.125. Using these parameters, different populations can be extracted from the phosphopeptides affected by PTR stimulation. Each circle corresponds to a group of phosphopeptides displaying the same responses to each version of the PTR receptor. The numbers in each intersection correspond to the number of phosphopeptides common to both categories. 55 phosphopeptides are affected by the mutation Y490F (still visible with the mutation Y490F/Y785F) (orange border); 77 phosphopeptides are affected by the mutation Y785F only (red border), and 41 phosphopeptides do not show any changes after both mutations (blue border). 24 phosphopeptides show a change after mutation Tyr-490 that seems to be rescued by the mutation of the second tyrosine Tyr-785 (green border).

FIGURE 5.
FIGURE 5.

Regulation of phosphorylation motifs. 16 phosphorylation motifs modified by different kinases (among others), which are represented at the top of the figure, were analyzed by determining their enrichment in the populations of phosphopeptides displaying the same regulation by each version of the PTR receptor: Y490 dependent represents phosphorylations affected by the mutation of the Tyr-490 (yellow bars); Y785 dependent represents phosphorylations affected by the mutation of the Tyr-785 (red bars); other dependent represents phosphorylations not affected by the either mutation of the Tyr-490 and Y785F (blue bars), and Y490 dependent/rescue Y785 represent phosphorylations affected by the mutation of the Tyr-490 and rescued by the mutation on Y785F (green bars). Specificity of each motif has been designated according to the Human Protein Reference Database (54).

FIGURE 6.
FIGURE 6.

Regulated phosphoproteins grouped by molecular function. Selected phosphoproteins regulated after 20 min of stimulation by the chimeric receptor PTR were classified according to their molecular function. Each protein was manually assigned using the Gene Ontology terms found in the DAVID National Institutes of Health database (21). The closed and open symbols indicate up-regulated and down-regulated phosphorylations, respectively. The changes are classified according to their magnitude as follows: more than 2-fold (square), between 1.8- and 2-fold (triangle), and between 1.5- and 1.8-fold (circle). The colored frames indicate the sensitivity of these phosphorylations to the different tyrosines of TrkA: Tyr-490 (orange frame), Tyr-785 (red frame), other (blue frame), or affected by Tyr-490 and rescued by Tyr-785 (green frame). No frame surrounding a protein either means that the phosphoprotein could not be identified in all four conditions or that its behavior to the mutations was not above the significance thresholds selected for grouping. GAPs, GTPase-activating proteins; GEFs, guanine nucleotide exchange factors.

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References

    1. Pawson T. (2002) Regulation and targets of receptor tyrosine kinases. Eur. J. Cancer 38, S3–S10 - PubMed
    1. Kholodenko B. N. (2006) Cell-signalling dynamics in time and space. Nat. Rev. Mol. Cell Biol. 7, 165–176 - PMC - PubMed
    1. Manning G., Whyte D. B., Martinez R., Hunter T., Sudarsanam S. (2002) The protein kinase complement of the human genome. Science 298, 1912–1934 - PubMed
    1. Choudhary C., Olsen J. V., Brandts C., Cox J., Reddy P. N., Böhmer F. D., Gerke V., Schmidt-Arras D. E., Berdel W. E., Müller-Tidow C., Mann M., Serve H. (2009) Mislocalized activation of oncogenic RTKs switches downstream signaling outcomes. Mol. Cell 36, 326–339 - PubMed
    1. Olsen J. V., Blagoev B., Gnad F., Macek B., Kumar C., Mortensen P., Mann M. (2006) Global, in vivo, and site-specific phosphorylation dynamics in signaling networks. Cell 127, 635–648 - PubMed

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