Comparative phosphoproteomics of zebrafish Fyn/Yes knockdown embryos (THESIS VERSION)

Abstract

The coordinated movement of cells is indispensable for normal vertebrate gastrulation. Several important players and signaling pathways have been identified in convergence and extension (CE) cell movements during gastrulation, including non-canonical Wnt signaling. Fyn and Yes, members of the Src family of kinases, are key regulators of CE movements as well. Here we investigated CE signaling pathways by comparison of the phosphoproteome of wild type zebrafish embryos with Fyn/Yes knockdown embryos that display specific CE cell movement defects. For quantitation we used differential stable isotope dimethyl labeling of tryptic peptides. Labeled peptides from wild type and Fyn/ Yes knockdown embryos were mixed in a 1:1 ratio and analyzed on an online RP-TiO2-LC-MS/MS. Phosphorylated and non-phosphorylated peptides were quantified and significant changes in protein expression and/or phosphorylation were detected. We found changes in protein phosphorylation in known regulators of gastrulation cell movements, including Gravin, PDLIM5 and Rho guanine nucleotide exchange factors and Rho GTPase activating proteins, whereas the majority of proteins were not affected. Our results indicate that quantitative phosphoproteomics is a useful tool to identify novel signaling pathways in zebrafish development in vivo.