Endogenous Phosphotyrosine Signaling in Zebrafish Embryos

Abstract

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. Molecular & Cellular Proteomics 6:2088 -2099, 2007.Embryonic development is tightly regulated, and numerous developmental processes like cell growth, differentiation, and migration are controlled by phosphotyrosine signaling, which is mediated by protein-tyrosine kinases and protein-tyrosine phosphatases. Improved knowledge about the identity of tyrosine phosphorylated proteins, their interacting proteins, and their involvement in signaling will improve our understanding of biological pathways and critical cellular processes. A first step toward elucidation of the critical signaling pathways is the identification of tyrosine phosphorylated proteins in the developing embryo.In recent years the availability of selective antibodies (1-4), targeted at tyrosine phosphorylated proteins, facilitated more global purification of these proteins and their binding partners, allowing identification by very sensitive LC-MS-based analysis of the proteins tryptic digests (1, 4, 5). Such approaches have been used for instance to study elegantly the temporal, global response to receptor stimulation. However, all of these data have been obtained from well defined cell systems under stimulated conditions. Although these data contribute well to our understanding of complex tyrosine phosphorylation-mediated signaling pathways, it is still unclear how they relate to in vivo processes. Therefore, in vivo studies are essential (6). Here we explored whether the above described technologies may be extended to perform a global analysis of in vivo signaling processes involved in zebrafish development. Zebrafish is n...

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