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J Biol Chem. 2013 Jun 7;288(23):16606-18. doi: 10.1074/jbc.M113.475285. Epub 2013 Apr 15.

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

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  • 1Department of Pharmaceutical Chemistry, University of California, San Francisco, California 94158, USA.

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

PMID:
23589303
PMCID:
PMC3675596
DOI:
10.1074/jbc.M113.475285
[PubMed - indexed for MEDLINE]
Free PMC Article
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