Mercury Alters B-Cell Protein Phosphorylation Profiles

Caruthers, Nicholas J.; Stemmer, Paul M.; Shin, Namhee; Dombkowski, Alan A.; Caruso, Joseph A.; Gill, Randall F.; Rosenspire, Allen J.

doi:10.1021/pr400657k

Cited by 10 publications

(8 citation statements)

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“…WEHI-231 cells are a well characterized mouse B cell line that exhibits many of the characteristics of immature B cells (Jakway et al , 1986). Our initial experiments utilized qualitative mass spectroscopic techniques to delineate global effects of mercury exposures on the WEHI-231 phosphoproteome, so as to identify potential protein targets of Hg 2+ (Caruthers et al , 2013). In the present study, bioinformatic data analysis algorithms identified the Src family protein phosphokinase Lyn as the “key node” and the B cell receptor signaling pathway as the primary pathway modulated by Hg 2+ .…”

Section: Introductionmentioning

confidence: 99%

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

Caruso¹,

Stemmer²,

Dombkowski³

et al. 2014

Toxicology and Applied Pharmacology

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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 low 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.

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Section: Introductionmentioning

confidence: 99%

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

Caruso¹,

Stemmer²,

Dombkowski³

et al. 2014

Toxicology and Applied Pharmacology

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“…For instance, the Y342/Y346 cluster is phosphorylated by LYN leading to binding of Vav-1 and PLC-γ with an affinity that is dependent upon whether the sites are individually or dually phosphorylated [36–38]. Previously we have shown that 5 μM Hg does not affect PLC-γ phosphorylation [31], and its not until a very high concentration (250 μM) that we observe an increase [39]. However, low doses of Hg can affect the temporal dynamics of BCR signalosome formation and may alter signaling by outcomes that are yet to be determined.…”

Section: Discussionmentioning

confidence: 92%

Mercury alters endogenous phosphorylation profiles of SYK in murine B cells

et al. 2017

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BackgroundEpidemiological evidence and animal models suggest that exposure to low and non-neurotoxic concentrations of mercury may contribute to idiosyncratic autoimmune disease. Since defects in function and signaling in B cells are often associated with autoimmunity, we investigated whether mercury exposure might alter B cell responsiveness to self-antigens by interfering with B cell receptor (BCR) signal transduction. In this study we determined the effects of mercury on the protein tyrosine kinase SYK, a critical protein involved in regulation of the BCR signaling pathway.MethodsPhosphorylation sites of murine SYK were mapped before and after treatment of WEHI cell cultures with mercury, or with anti-IgM antibody (positive control) or pervanadate (a potent phosphatase inhibitor). Phosphopeptides were enriched by either titanium dioxide chromatography or anti-phosphotyrosine immunoaffinity, and analyzed by liquid chromatography-mass spectrometry. Select SYK phosphosite cluster regions were profiled for responsiveness to treatments using multiple reaction monitoring (MRM) methodology.ResultsA total of 23 phosphosites were identified with high probability in endogenous SYK, including 19 tyrosine and 4 serine residues. For 10 of these sites phosphorylation levels were increased following BCR activation. Using MRM to profile changes in phosphorylation status we found that 4 cluster regions, encompassing 8 phosphosites, were activated by mercury and differentially responsive to all 3 treatments. Phosphorylation of tyrosine-342 and -346 residues were most sensitive to mercury exposure. This cluster is known to propagate normal BCR signal transduction by recruiting adaptor proteins such as PLC-γ and Vav-1 to SYK during formation of the BCR signalosome.ConclusionsOur data shows that mercury alters the phosphorylation status of SYK on tyrosine sites known to have a role in promoting BCR signals. Considering the importance of SYK in the BCR signaling pathway, these data suggest that mercury can alter BCR signaling in B cells, which might affect B cell responsiveness to self-antigen and have implications with respect to autoimmunity and autoimmune disease.Electronic supplementary materialThe online version of this article (doi:10.1186/s12865-017-0221-0) contains supplementary material, which is available to authorized users.

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“…An additional sample treated with 25 µM pervanadate for 15 min was also prepared to improve our ability to detect phosphotyrosine (pTyr). Pervanadate treatment can result in an accumulation of pTyr to up to 20% of phosphorylated residues (Caruthers et al, 2014). We reasoned that the inclusion of a pTyr enriched sample would improve our ability to detect pTyr residues in data-dependent LC-MS3 analysis.…”

Section: Methodsmentioning

confidence: 99%

“…The expanding capabilities of mass spectrometry based phosphoproteomics in terms of depth of coverage and sample multiplexing (Erickson et al, 2015; Sharma et al, 2014), are making it a powerful systems-level approach for mechanism of action studies. It has already been applied to mechanism of action determination for inorganic mercury (Caruso et al, 2014; Caruthers et al, 2014), deoxyvinylinol (Pan et al, 2013), ammonia (Harder et al, 2014), and to identify the target profiles of kinase inhibitors (Li et al, 2010; Pan et al, 2009). It has emerged from these studies and from other investigations of compounds with known mechanisms (Pines et al, 2011) that the phosphoproteome can provide a more precise determination of mechanism of action than transcriptome or total proteome analysis.…”

Section: Introductionmentioning

confidence: 99%

Phosphoproteome and transcription factor activity profiling identify actions of the anti-inflammatory agent UTL-5g in LPS stimulated RAW 264.7 cells including disrupting actin remodeling and STAT-3 activation

Carruthers

Stemmer

Chen

et al. 2017

European Journal of Pharmacology

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UTL-5g is a novel small-molecule TNF-alpha modulator. It reduces cisplatin-induced side effects by protecting kidney, liver, and platelets, thereby increasing tolerance for cisplatin. UTL-5g also reduces radiation-induced acute liver toxicity. The mechanism of action for UTL-5g is not clear at the present time. A phosphoproteomic analysis to a depth of 4943 phosphopeptides and a luminescence-based transcription factor activity assay were used to provide complementary analyses of signaling events that were disrupted by UTL-5g in RAW 264.7 cells. Transcriptional activity downstream of the interferon gamma, IL-6, type 1 Interferon, TGF-β, PKC/Ca2+ and the glucocorticoid receptor pathways were disrupted by UTL-5g. Phosphoproteomic analysis indicated that hyperphosphorylation of proteins involved in actin remodeling was suppressed by UTL-5g (gene set analysis, FDR < 1%) as was phosphorylation of Stat3, consistent with the IL-6 results in the transcription factor assay. Neither analysis indicated that LPS-induced activation of the NF-κB, cAMP/PKA and JNK signaling pathways were affected by UTL-5g. This global characterization of UTL-5g activity in a macrophage cell line discovered that it disrupts selected aspects of LPS signaling including Stat3 activation and actin remodeling providing new insight on how UTL-5g acts to reduce cisplatin-induced side effects.

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Mercury Alters B-Cell Protein Phosphorylation Profiles

Cited by 10 publications

References 53 publications

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

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

Mercury alters endogenous phosphorylation profiles of SYK in murine B cells

Phosphoproteome and transcription factor activity profiling identify actions of the anti-inflammatory agent UTL-5g in LPS stimulated RAW 264.7 cells including disrupting actin remodeling and STAT-3 activation

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