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J. Biol. Chem., Vol. 279, Issue 45, 46438-46447, November 5, 2004

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Tyrosine Phosphoproteomics of Fibroblast Growth Factor Signaling

A ROLE FOR INSULIN RECEPTOR SUBSTRATE-4^*

Anders M. Hinsby¶, Jesper V. Olsen¶||, and Matthias Mann||**

From the Protein Laboratory, Panum Institute 6.1, Blegdamsvej 3C, University of Copenhagen, DK-2200, Denmark and the ^||Center for Experimental BioInformatics, Department of Biochemistry and Molecular Biology, Campusvej 55, University of Southern Denmark, Odense 5230, Denmark

Signal transduction by receptor tyrosine kinases is initiated by recruitment of a variety of signaling proteins to tyrosine-phosphorylated motifs in the activated receptors. Several signaling pathways are thus activated in parallel, the combination of which decides the cellular response. Here, we present a dual strategy for extensive mapping of tyrosine-phosphorylated proteins and probing of signal-dependent protein interactions of a signaling cascade. The approach relies on labeling of cells with "heavy" and "light" isotopic forms of Arg to distinguish two cell populations. First, tyrosine-phosphorylated proteins from stimulated ("heavy"-labeled) and control samples ("normal"-labeled) are isolated and subjected to high sensitivity Fourier transform ion cyclotron resonance mass spectrometry analysis. Next, phosphopeptides corresponding to tyrosine phosphorylation sites identified during the tyrosine phosphoproteomic analysis are used as baits to isolate phosphospecific protein binding partners, which are subsequently identified by mass spectrometry. We used this approach to identify 28 components of the signaling cascade induced by stimulation with the basic fibroblast growth factor. Insulin receptor substrate-4 was identified as a novel candidate in fibroblast growth factor receptor signaling, and we defined phosphorylation-dependent interactions with other components, such as adaptor protein Grb2, of the signaling cascade. Finally, we present evidence for a complex containing insulin receptor substrate-4 and ShcA in signaling by the fibroblast growth factor receptor.

Received for publication, April 23, 2004 , and in revised form, July 30, 2004.

^* Work in the Center for Experimental BioInformatics was supported by a grant by the Danish National Research foundation and by Interaction Proteome, a European Union 6th framework program. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Supported by a Ph.D. fellowship from the Academy of Technical Sciences, Denmark.

^¶ These authors contributed equally to this work.

^** To whom correspondence should be addressed. E-mail: mann{at}bmb.sdu.dk.

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