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Differential Phosphoproteomics of Fibroblast Growth Factor Signaling: Identification of Src Family Kinase-Mediated Phosphorylation Events

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      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.

      Abstract

      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. Also, we used a targeted approach to carry out high coverage phosphopeptide mapping of one Src substrate protein, Dok1, and to identify SFK-dependent Dok1 binding partners.

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      MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification.

      Efficient analysis of very large amounts of raw data for peptide identification and protein quantification is a principal challenge in mass spectrometry (MS)-based proteomics. Here we describe MaxQuant, an integrated suite of algorithms specifically developed for high-resolution, quantitative MS data. Using correlation analysis and graph theory, MaxQuant detects peaks, isotope clusters and stable amino acid isotope-labeled (SILAC) peptide pairs as three-dimensional objects in m/z, elution time and signal intensity space. By integrating multiple mass measurements and correcting for linear and nonlinear mass offsets, we achieve mass accuracy in the p.p.b. range, a sixfold increase over standard techniques. We increase the proportion of identified fragmentation spectra to 73% for SILAC peptide pairs via unambiguous assignment of isotope and missed-cleavage state and individual mass precision. MaxQuant automatically quantifies several hundred thousand peptides per SILAC-proteome experiment and allows statistically robust identification and quantification of >4,000 proteins in mammalian cell lysates.
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        Global, in vivo, and site-specific phosphorylation dynamics in signaling networks.

        Cell signaling mechanisms often transmit information via posttranslational protein modifications, most importantly reversible protein phosphorylation. Here we develop and apply a general mass spectrometric technology for identification and quantitation of phosphorylation sites as a function of stimulus, time, and subcellular location. We have detected 6,600 phosphorylation sites on 2,244 proteins and have determined their temporal dynamics after stimulating HeLa cells with epidermal growth factor (EGF) and recorded them in the Phosida database. Fourteen percent of phosphorylation sites are modulated at least 2-fold by EGF, and these were classified by their temporal profiles. Surprisingly, a majority of proteins contain multiple phosphorylation sites showing different kinetics, suggesting that they serve as platforms for integrating signals. In addition to protein kinase cascades, the targets of reversible phosphorylation include ubiquitin ligases, guanine nucleotide exchange factors, and at least 46 different transcriptional regulators. The dynamic phosphoproteome provides a missing link in a global, integrative view of cellular regulation.
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          Cellular signaling by fibroblast growth factor receptors.

          The 22 members of the fibroblast growth factor (FGF) family of growth factors mediate their cellular responses by binding to and activating the different isoforms encoded by the four receptor tyrosine kinases (RTKs) designated FGFR1, FGFR2, FGFR3 and FGFR4. Unlike other growth factors, FGFs act in concert with heparin or heparan sulfate proteoglycan (HSPG) to activate FGFRs and to induce the pleiotropic responses that lead to the variety of cellular responses induced by this large family of growth factors. A variety of human skeletal dysplasias have been linked to specific point mutations in FGFR1, FGFR2 and FGFR3 leading to severe impairment in cranial, digital and skeletal development. Gain of function mutations in FGFRs were also identified in a variety of human cancers such as myeloproliferative syndromes, lymphomas, prostate and breast cancers as well as other malignant diseases. The binding of FGF and HSPG to the extracellular ligand domain of FGFR induces receptor dimerization, activation and autophosphorylation of multiple tyrosine residues in the cytoplasmic domain of the receptor molecule. A variety of signaling proteins are phosphorylated in response to FGF stimulation including Shc, phospholipase-Cgamma, STAT1, Gab1 and FRS2alpha leading to stimulation of intracellular signaling pathways that control cell proliferation, cell differentiation, cell migration, cell survival and cell shape. The docking proteins FRS2alpha and FRS2beta are major mediators of the Ras/MAPK and PI-3 kinase/Akt signaling pathways as well as negative feedback mechanisms that fine-tune the signal that is initiated at the cell surface following FGFR stimulation.
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            Author and article information

            Affiliations
            School of Biosciences, College of Life and Environmental Sciences, University of Birmingham, Edgbaston, Birmingham, B15 2TT, United Kingdom
            Author notes
            [* ] To whom correspondence should be addressed. Prof. John K. Heath, School of Biosciences, College of Life and Environmental Sciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, U.K. Telephone: +44 (0)121 414 7533. Fax: +44 (0)121 414 5925. Email: J.K.Heath@ 123456bham.ac.uk .
            [†]

            These authors contributed equally to this work.

            [‡]

            Cancer Research UK Growth Factor Signalling Group.

            [§]

            Current address: Department of Chemistry, University of Illinois at Urbana−Champaign, Urbana, IL 61801.

            Journal
            J Proteome Res
            pr
            jprobs
            Journal of Proteome Research
            American Chemical Society
            1535-3893
            1535-3907
            12 March 2010
            07 May 2010
            : 9
            : 5
            : 2317-2328
            2950672
            20225815
            10.1021/pr9010475
            Copyright © 2010 American Chemical Society

            This is an open-access article distributed under the ACS AuthorChoice Terms & Conditions. Any use of this article, must conform to the terms of that license which are available at http://pubs.acs.org.

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            pr9010475
            pr-2009-010475

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