Differential phosphoproteomics of fibroblast growth factor signaling: identification of Src family kinase-mediated phosphorylation events - PubMed (original) (raw)

. 2010 May 7;9(5):2317-28.

doi: 10.1021/pr9010475.

Affiliations

Free PMC article

Differential phosphoproteomics of fibroblast growth factor signaling: identification of Src family kinase-mediated phosphorylation events

Debbie L Cunningham et al. J Proteome Res. 2010.

Free PMC article

Abstract

Activation of signal transduction by the receptor tyrosine kinase, fibroblast growth factor receptor (FGFR), results in a cascade of protein-protein interactions that rely on the occurrence of specific tyrosine phosphorylation events. One such protein recruited to the activated receptor complex is the nonreceptor tyrosine kinase, Src, which is involved in both initiation and termination of further signaling events. To gain a further understanding of the tyrosine phosphorylation events that occur during FGF signaling, with a specific focus on those that are dependent on Src family kinase (SFK) activity, we have applied SILAC combined with chemical inhibition of SFK activity to search for phosphorylation events that are dependent on SFK activity in FGF stimulated cells. In addition, we used a more targeted approach to carry out high coverage phosphopeptide mapping of one Src substrate protein, the multifunctional adaptor Dok1, and to identify SFK-dependent Dok1 binding partners. From these analyses we identify 80 SFK-dependent phosphorylation events on 40 proteins. We further identify 18 SFK-dependent Dok1 interactions and 9 SFK-dependent Dok1 phosphorylation sites, 6 of which had not previously been known to be SFK-dependent.

PubMed Disclaimer

Figures

Figure 1

Figure 1

Profiles of phosphorylation events in the presence of FGF2. NIH 3T3s were stimulated with 20 ng/mL FGF2 for varying lengths of time. Western blot analysis was carried out on whole cell lysates using antibodies against indicated proteins and phospho-proteins.

Figure 2

Figure 2

Representative mass spectra for identification and site localization of tyrosine phosphorylation. (a) Afadin is phosphorylated at pY203. (b) Catenin delta-1 is phosphorylated at pY865. Peptides are labeled with 13C(6),15N(4) Arg and 13C(6) Lys. pY indicates phosphotyrosine.

Figure 3

Figure 3

Partial co-elution of isobaric phosphopeptides. The Dok1 peptide LPSPPGPQELLDSPPALYAEPLDSLR is present in three different singly phosphorylated forms, with phosphorylation occurring at Ser281, Ser291, and Tyr296. The selected-ion chromatogram for the [M + 3H]3+ light precursor is shown. Comparison of the heavy/light ratios for the three versions indicates that only Tyr296 is Src-dependent.

Similar articles

Cited by

References

    1. Itoh N.; Ornitz D. M. Functional evolutionary history of the mouse Fgf gene family. Dev. Dyn. 2008, 237 (1), 18–27. - PubMed
    1. Beenken A.; Mohammadi M. The FGF family: biology, pathophysiology and therapy. Nat. Rev. Drug Discovery 2009, 8 (3), 235–53. - PMC - PubMed
    1. Ornitz D. M.; Itoh N. Fibroblast growth factors. Genome Biol. 2001, 2 (3), 3005.1–−3005.12. - PMC - PubMed
    1. Grose R.; Dickson C. Fibroblast growth factor signaling in tumorigenesis. Cytokine Growth Factor Rev. 2005, 16 (2), 179–86. - PubMed
    1. Katoh M. Cancer genomics and genetics of FGFR2 (Review). Int. J. Oncol. 2008, 33 (2), 233–7. - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources