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Application of adenosine triphosphate affinity probe and scheduled multiple-reaction monitoring analysis for profiling global kinome in human cells in response to arsenite treatment.

Guo L, Xiao Y, Wang Y - Anal. Chem. (2014)

Bottom Line: Several kinases involved in cell cycle progression, including cyclin-dependent kinases (CDK1 and CDK4) and Aurora kinases A, B, and C, were found to be hyperactivated, and the altered expression of CDK1 was further validated by Western analysis.In addition, treatment with a CDK inhibitor, flavopiridol, partially restored the arsenite-induced growth inhibition of human skin fibroblast cells.Thus, sodium arsenite may confer its cytotoxic effect partly through the aberrant activation of CDKs and the resultant perturbation of cell cycle progression.

View Article: PubMed Central - PubMed

Affiliation: Environmental Toxicology Graduate Program and ‡Department of Chemistry, University of California , Riverside, California 92521-0403, United States.

ABSTRACT
Phosphorylation of cellular components catalyzed by kinases plays important roles in cell signaling and proliferation. Quantitative assessment of perturbation in global kinome may provide crucial knowledge for elucidating the mechanisms underlying the cytotoxic effects of environmental toxicants. Here, we utilized an adenosine triphosphate (ATP) affinity probe coupled with stable isotope labeling by amino acids in cell culture (SILAC) to assess quantitatively the arsenite-induced alteration of global kinome in human cells. We constructed a SILAC-compatible kinome library for scheduled multiple-reaction monitoring (MRM) analysis and adopted on-the-fly recalibration of retention time shift, which provided better throughput of the analytical method and enabled the simultaneous quantification of the expression of ∼300 kinases in two LC-MRM runs. With this improved analytical method, we conducted an in-depth quantitative analysis of the perturbation of kinome of GM00637 human skin fibroblast cells induced by arsenite exposure. Several kinases involved in cell cycle progression, including cyclin-dependent kinases (CDK1 and CDK4) and Aurora kinases A, B, and C, were found to be hyperactivated, and the altered expression of CDK1 was further validated by Western analysis. In addition, treatment with a CDK inhibitor, flavopiridol, partially restored the arsenite-induced growth inhibition of human skin fibroblast cells. Thus, sodium arsenite may confer its cytotoxic effect partly through the aberrant activation of CDKs and the resultant perturbation of cell cycle progression. Together, we developed a high-throughput, SILAC-compatible, and MRM-based kinome profiling method and demonstrated that the method is powerful in deciphering the molecular modes of action of a widespread environmental toxicant. The method should be generally applicable for uncovering the cellular pathways triggered by other extracellular stimuli.

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Global kinome profilingfor GM00637 human skin fibroblasts aftertreatment with 5 μM sodium arsenite for 24 h. Blue and red barsdenote kinases that are down- and up-regulated after sodium arseniteexposure, respectively.
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fig4: Global kinome profilingfor GM00637 human skin fibroblasts aftertreatment with 5 μM sodium arsenite for 24 h. Blue and red barsdenote kinases that are down- and up-regulated after sodium arseniteexposure, respectively.

Mentions: GM00637 human skin fibroblast cells weretreated with 5 μM sodium arsenite [iAs(III)] for 24 h. The subsequentATP affinity probe labeling and sample preparation were as describedin Figure 1B. The resulting enriched kinasepeptides were analyzed by two scheduled MRM runs with a 6 min retentiontime window and on-the-fly retention time correction strategy. Intotal, we quantified 234 kinases from one forward and two reverseSILAC experiments (Table S3, Supporting Information). In addition, the excellent correlation between the measured retentiontime with iRT demonstrated the effectiveness of this strategy (Figure 3C). Depicted in Figure 4 isthe histogram of kinome quantification results, which revealed thatkinases from all kinase groups except the PKL group were quantified.Although the expression levels and ATP binding affinities of the majorityof the kinases in the kinome remained unchanged toward iAs(III) treatment,8 and 9 kinases were significantly down- and up-regulated, respectively(Figures 4 and S1 and S2 and Table S2, Supporting Information).


Application of adenosine triphosphate affinity probe and scheduled multiple-reaction monitoring analysis for profiling global kinome in human cells in response to arsenite treatment.

Guo L, Xiao Y, Wang Y - Anal. Chem. (2014)

Global kinome profilingfor GM00637 human skin fibroblasts aftertreatment with 5 μM sodium arsenite for 24 h. Blue and red barsdenote kinases that are down- and up-regulated after sodium arseniteexposure, respectively.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4222629&req=5

fig4: Global kinome profilingfor GM00637 human skin fibroblasts aftertreatment with 5 μM sodium arsenite for 24 h. Blue and red barsdenote kinases that are down- and up-regulated after sodium arseniteexposure, respectively.
Mentions: GM00637 human skin fibroblast cells weretreated with 5 μM sodium arsenite [iAs(III)] for 24 h. The subsequentATP affinity probe labeling and sample preparation were as describedin Figure 1B. The resulting enriched kinasepeptides were analyzed by two scheduled MRM runs with a 6 min retentiontime window and on-the-fly retention time correction strategy. Intotal, we quantified 234 kinases from one forward and two reverseSILAC experiments (Table S3, Supporting Information). In addition, the excellent correlation between the measured retentiontime with iRT demonstrated the effectiveness of this strategy (Figure 3C). Depicted in Figure 4 isthe histogram of kinome quantification results, which revealed thatkinases from all kinase groups except the PKL group were quantified.Although the expression levels and ATP binding affinities of the majorityof the kinases in the kinome remained unchanged toward iAs(III) treatment,8 and 9 kinases were significantly down- and up-regulated, respectively(Figures 4 and S1 and S2 and Table S2, Supporting Information).

Bottom Line: Several kinases involved in cell cycle progression, including cyclin-dependent kinases (CDK1 and CDK4) and Aurora kinases A, B, and C, were found to be hyperactivated, and the altered expression of CDK1 was further validated by Western analysis.In addition, treatment with a CDK inhibitor, flavopiridol, partially restored the arsenite-induced growth inhibition of human skin fibroblast cells.Thus, sodium arsenite may confer its cytotoxic effect partly through the aberrant activation of CDKs and the resultant perturbation of cell cycle progression.

View Article: PubMed Central - PubMed

Affiliation: Environmental Toxicology Graduate Program and ‡Department of Chemistry, University of California , Riverside, California 92521-0403, United States.

ABSTRACT
Phosphorylation of cellular components catalyzed by kinases plays important roles in cell signaling and proliferation. Quantitative assessment of perturbation in global kinome may provide crucial knowledge for elucidating the mechanisms underlying the cytotoxic effects of environmental toxicants. Here, we utilized an adenosine triphosphate (ATP) affinity probe coupled with stable isotope labeling by amino acids in cell culture (SILAC) to assess quantitatively the arsenite-induced alteration of global kinome in human cells. We constructed a SILAC-compatible kinome library for scheduled multiple-reaction monitoring (MRM) analysis and adopted on-the-fly recalibration of retention time shift, which provided better throughput of the analytical method and enabled the simultaneous quantification of the expression of ∼300 kinases in two LC-MRM runs. With this improved analytical method, we conducted an in-depth quantitative analysis of the perturbation of kinome of GM00637 human skin fibroblast cells induced by arsenite exposure. Several kinases involved in cell cycle progression, including cyclin-dependent kinases (CDK1 and CDK4) and Aurora kinases A, B, and C, were found to be hyperactivated, and the altered expression of CDK1 was further validated by Western analysis. In addition, treatment with a CDK inhibitor, flavopiridol, partially restored the arsenite-induced growth inhibition of human skin fibroblast cells. Thus, sodium arsenite may confer its cytotoxic effect partly through the aberrant activation of CDKs and the resultant perturbation of cell cycle progression. Together, we developed a high-throughput, SILAC-compatible, and MRM-based kinome profiling method and demonstrated that the method is powerful in deciphering the molecular modes of action of a widespread environmental toxicant. The method should be generally applicable for uncovering the cellular pathways triggered by other extracellular stimuli.

Show MeSH
Related in: MedlinePlus