Journal
AMERICAN JOURNAL OF PATHOLOGY
Volume 183, Issue 6, Pages 1841-1852Publisher
ELSEVIER SCIENCE INC
DOI: 10.1016/j.ajpath.2013.08.015
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Funding
- NIH [EY06000, EY06000S, S10 RR017967, P41 RR10888/GM104603, S10 RR15942, S10 RR020946]
- New England Corneal Transplant Fund
- NTH-National Heart, Lung, and Blood Institute [HHSN268201000031C]
- Massachusetts Lions Eye Research Fund Ophthalmology Departmental grant
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Protein phosphorylation is a dynamic post-translational modification. Mass spectrometry-based quantitation was performed to determine the phosphoproteome profile of epithelial cells in response to injury, nucleotide, or epidermal growth factor. Phosphotyrosine enrichment used immunoprecipitation and immobilized metal affinity chromatography. Nucleotides released after scratch wounding activate purinergic receptors, leading to a distinct phosphorylation profile on epidermal growth factor receptor (EGFR) compared with its natural Ligand. ATP induced a 2- to 15-fold phosphorylation increase over control on EGFR Y974, Y1086, and Y1148, with minimal phosphorylation intensity on EGFRY1173 compared with the Level measured in response to epidermal growth factor. Differential phosphorylation induced by epidermal growth factor or ATP was site specific on Src, Shc, phospholipase C gamma, protein kinase C, focal adhesion kinase, paxillin, and mitogen-activated protein kinases 1, 12, and 13. After wounding, the P2Y(2) receptor mRNA expression increased, and after knockdown, migration and Ca2+ mobilization were impaired. To examine phosphorylation mediated by P2Y(2), cells were cultured in media containing stable isotope-labeled amino acids, the receptor was knocked down, and the cells were stimulated. Mass spectrometry based comparison of the phosphorylation profiles of control versus transfected cells revealed a 50-fold decrease in phosphorylation of EGFRY974 and 1086, with no decrease in Y1173 phosphorylation. A similarfold decrease in SrcY421 and Y446 and paxillin Y118 was detected, indicating the far-reaching importance of the P2Y(2) receptor in mediating migration.
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