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Toxicol Appl Pharmacol. 2014 Apr 1;276(1):47-54. doi: 10.1016/j.taap.2014.01.002. Epub 2014 Jan 14.

A systems toxicology approach identifies Lyn as a key signaling phosphoprotein modulated by mercury in a B lymphocyte cell model.

Author information

1
Institute of Environmental Health Sciences, Wayne State University, Detroit, MI, USA.
2
Department of Pediatrics, Wayne State University, Detroit, MI, USA.
3
Department of Immunology and Microbiology, Wayne State University, Detroit, MI, USA.
4
Department of Immunology and Microbiology, Wayne State University, Detroit, MI, USA. Electronic address: arosenspire@wayne.edu.

Abstract

Network and protein-protein interaction analyses of proteins undergoing Hg²⁺-induced phosphorylation and dephosphorylation in Hg²⁺-intoxicated mouse WEHI-231 B cells identified Lyn as the most interconnected node. Lyn is a Src family protein tyrosine kinase known to be intimately involved in the B cell receptor (BCR) signaling pathway. Under normal signaling conditions the tyrosine kinase activity of Lyn is controlled by phosphorylation, primarily of two well known canonical regulatory tyrosine sites, Y-397 and Y-508. However, Lyn has several tyrosine residues that have not yet been determined to play a major role under normal signaling conditions, but are potentially important sites for phosphorylation following mercury exposure. In order to determine how Hg²⁺ exposure modulates the phosphorylation of additional residues in Lyn, a targeted MS assay was developed. Initial mass spectrometric surveys of purified Lyn identified 7 phosphorylated tyrosine residues. A quantitative assay was developed from these results using the multiple reaction monitoring (MRM) strategy. WEHI-231 cells were treated with Hg²⁺, pervanadate (a phosphatase inhibitor), or anti-Ig antibody (to stimulate the BCR). Results from these studies showed that the phosphoproteomic profile of Lyn after exposure of the WEHI-231 cells to a low concentration of Hg²⁺ closely resembled that of anti-Ig antibody stimulation, whereas exposure to higher concentrations of Hg²⁺ led to increases in the phosphorylation of Y-193/Y-194, Y-501 and Y-508 residues. These data indicate that mercury can disrupt a key regulatory signal transduction pathway in B cells and point to phospho-Lyn as a potential biomarker for mercury exposure.

KEYWORDS:

Autoimmune disease; B cell; Lyn; Mercury; Multiple reaction monitoring; Phosphoproteomics

PMID:
24440445
PMCID:
PMC4005802
DOI:
10.1016/j.taap.2014.01.002
[Indexed for MEDLINE]
Free PMC Article
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