Quantitative profiling of spreading-coupled protein tyrosine phosphorylation in migratory cells.

Yajun Xie,Xiahe Huang,Jinlong Wang,Xiaofei Liu,Kehui Liu,Yingchun Wang,Yuanya Zhang,Xiaorong Wang

doi:10.1038/srep31811

Abstract

Protein tyrosine phosphorylation is an important mechanism that regulates cytoskeleton reorganization and cell spreading of migratory cells. A number of cytoskeletal proteins are known to be tyrosine phosphorylated (pY) in different cellular processes. However, the profile of pY proteins during different stages of cell spreading has not been available. Using immunoafffinity enrichment of pY proteins coupled with label free quantitative proteomics, we quantitatively identified 447 pY proteins in the migratory ECV-304 cells at the early spreading (adhesion) and the active spreading stages. We found that pY levels of the majority of the quantified proteins were significantly increased in the active spreading stage compared with the early spreading stage, suggesting that active cell spreading is concomitant with extra tyrosine phosphorylation. The major categories of proteins impacted by tyrosine phosphorylation are involved in cytoskeleton and focal adhesion regulation, protein translation and degradation. Our findings, for the first time, dissect the cell spreading-specific pY signals from the adhesion induced pY signals, and provide a valuable resource for the future mechanistic research regarding the regulation of cell spreading.

Highlights

Documented, at least at a global level
To determine the time points representing the early and the active spreading stages, we performed the spreading assay using ECV-304, a cell line derived from human umbilical vein endothelial cells and has been used as a model for angiogenesis study[14]
Many proteins involved in the two processes including FAK, Paxillin, Cortactin, Profilin, Cofilin, and Arp2/3 components are known to be affected functionally by tyrosine phosphorylation[3,6,7,19,20]

Summary

Introduction

Documented, at least at a global level. Lacking of this information has seriously impeded the paces towards the understanding of the mechanism regulating cell adhesion and spreading. Using ECV-304 cell line, we determined the time points when suspended cells reach the states of fully attached and active spreading on fibronectin-coated dishes, and quantitatively compared the pY proteomes of the cells in the two states. We found that overall more proteins have increased tyrosine phosphorylation in active spreading cells than in fully attached cells. We bioinformatically analyzed the functional significance of the pY proteins differentially phosphorylated in the two stages. Our results should serve as a useful resource for the future mechanistic studies of the regulation of cell adhesion and spreading

Methods

Results

Conclusion