Mapping protein phosphorylation in zebrafish development Reversible protein phosphorylation plays a key role in signaling processes that are vital for a cell and organism. It provides a rapid switch for protein activity as it often changes the conformation and function of a protein in the cell. Deregulation of kinase or phosphatase activity may lead to a disturbed balance and can result in inaccurate signaling. As a consequence protein phosphorylation and the elucidation of signaling pathways have received considerable attention over the past years. To date, various approaches have been successfully applied to in vitro cell systems; however in vivo data become essential to relate to physiological processes. Therefore we have explored the possibilities to use zebrafish development as a model system to study in vivo phosphorylation using mass spectrometry. Tyrosine phosphorylation of proteins at two different stages in zebrafish embryonic development was studied by immuno-affinity purification and multi-dimensional LC-MS. Tyrosine kinases including members of the Src family of kinases, Eph receptors and Focal adhesion kinase as well as adaptor proteins such as Paxillin, Crk and p130Cas were identified. Many differences in expression or phosphorylation state were observed for these two embryonic stages. In addition, multiplex in vitro kinase assays were performed, which confirmed most of the in vivo observations. The in vitro kinase chip technology was also used to determine protein tyrosine kinase (PTK) activity profiles from morpholino mediated knock down zebrafish embryo lysates. Morpholino knock down of the kinases Fyn and Yes, induced characteristic phenotypes in 1 day old zebrafish embryos and the lysates induced distinct changes in the PTK activity profiles compared to wild-type lysates. Previously it was shown that Wnt11 knock down phenocopied Fyn and Yes knock down. Interestingly, Wnt11 knock down induced similar changes in the PTK activity profile as Fyn/Yes knock down. An on-line TiO2 based LC MS/MS was used to enrich for all phosphorylated peptides in 1 day old zebrafish embryo samples. We identified more than 1000 phosphorylation sites in 60 embryos. The phosphopeptide dataset was searched for known kinase motifs, using Scansite. This revealed predominant Cdk5 kinase, p38MAPK, PKA and casein kinase 2 substrates in the dataset, indicating high kinase activity. Comparison with human datasets revealed surprising conservation of phosphorylation sites, suggesting specialized function. A quantitative proteomics approach, using stable isotope di-methyl labeling was used to compare protein phosphorylation between wild type and Fyn/Yes knockdown embryos that display convergence and extension cell movement defects. We detected differential phosphorylation of known regulators of gastrulation cell movements, including Gravin, PDLIM5 and small GTPase regulators. In this thesis in vivo phosphorylation was studied at different stages and under different experimental conditions in developing zebrafish embryos. The results established zebrafish as a good model to study in vivo phosphorylation and reveal that surprising homology exists with human phosphoproteins. Taken together this research emphasizes the strength of mass spectrometry based phosphoproteomics in the elucidation of endogenous signaling pathways via the large scale identification of phosphoproteins and phosphorylation sites and the obtained results contribute to a better understanding of biological pathways and critical cellular processes
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