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Molecular & Cellular Proteomics 6:2088-2099, 2007.
© 2007 by The American Society for Biochemistry and Molecular Biology, Inc.

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Research

Endogenous Phosphotyrosine Signaling in Zebrafish Embryos ^*,S

Simone Lemeer^,, Rob Ruijtenbeek^¶, Martijn W. H. Pinkse, Chris Jopling, Albert J. R. Heck, Jeroen den Hertog^,|| and Monique Slijper^,**

From the Department of Biomolecular Mass Spectrometry, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Sorbonnelaan 16, 3584 CA Utrecht, The Netherlands, Hubrecht Institute, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands, and ^¶ PamGene International B. V., Nieuwstraat 30, 5211 BJ 's Hertogenbosch, The Netherlands

In the developing embryo, cell growth, differentiation, and migration are strictly regulated by complex signaling pathways. One of the most important cell signaling mechanisms is protein phosphorylation on tyrosine residues, which is tightly controlled by protein-tyrosine kinases and protein-tyrosine phosphatases. Here we investigated endogenous phosphotyrosine signaling in developing zebrafish embryos. Tyrosine phosphorylated proteins were immunoaffinity-purified from zebrafish embryos at 3 and 5 days postfertilization and identified by multidimensional LC-MS. Among the identified proteins were tyrosine kinases, including Src family kinases, Eph receptor kinases, and focal adhesion kinases, as well as the adaptor proteins paxillin, p130Cas, and Crk. We identified several known and some unknown in vivo tyrosine phosphorylation sites in these proteins. Whereas most immunoaffinity-purified proteins were detected at both developmental stages, significant differences in abundance and/or phosphorylation state were also observed. In addition, multiplex in vitro kinase assays were performed by incubating a microarray of peptide substrates with the lysates of the two developmental stages. Many of the in vivo observations were confirmed by this on-chip in vitro kinase assay. Our experiments are the first to show that global tyrosine phosphorylation-mediated signaling can be studied at endogenous levels in complex multicellular organisms.

^** To whom correspondence may be addressed. Tel.: 31-30-2533789; Fax: 31-30-2518219; E-mail: m.slijper{at}uu.nl

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