A systems toxicology approach identifies Lyn as a key signaling phosphoprotein modulated by mercury in a B lymphocyte cell model.
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.
Institute of Environmental Health Sciences, Wayne State University, Detroit, MI, USA.
Joseph A
JA
Caruso
Institute of Environmental Health Sciences, Wayne State University, Detroit, MI, USA.
Paul M
PM
Stemmer
Department of Pediatrics, Wayne State University, Detroit, MI, USA.
Alan
A
Dombkowski
Institute of Environmental Health Sciences, Wayne State University, Detroit, MI, USA.
Nicholas J
NJ
Caruthers
Department of Immunology and Microbiology, Wayne State University, Detroit, MI, USA.
Randall
R
Gill
Department of Immunology and Microbiology, Wayne State University, Detroit, MI, USA. Electronic address: arosenspire@wayne.edu.
Allen J
AJ
Rosenspire
D000818:Animals; D001327:Autoimmune Diseases / Q000139:chemically induced / Q000276:immunology / Q000378:metabolism; D001402:B-Lymphocytes / Q000187:drug effects* / Q000276:immunology / Q000378:metabolism; D015415:Biomarkers / Q000378:metabolism; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D008628:Mercury / Q000633:toxicity*; D008630:Mercury Poisoning / Q000201:enzymology / Q000276:immunology / Q000378:metabolism; D051379:Mice; D009994:Osmolar Concentration; D010446:Peptide Fragments / Q000819:agonists / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D017027:Protein Tyrosine Phosphatases / Q000494:pharmacology; D011947:Receptors, Antigen, B-Cell / Q000819:agonists / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects*; D053719:Tandem Mass Spectrometry; D014443:Tyrosine / Q000378:metabolism; D014638:Vanadates / Q000494:pharmacology; D019061:src-Family Kinases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*
D015415:Biomarkers; D010446:Peptide Fragments; D011947:Receptors, Antigen, B-Cell; C076715:pervanadate; D014638:Vanadates; D014443:Tyrosine; C060902:lyn protein-tyrosine kinase; D019061:src-Family Kinases; D017027:Protein Tyrosine Phosphatases; D008628:Mercury
Autoimmune disease; B cell; Lyn; Mercury; Multiple reaction monitoring; Phosphoproteomics