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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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Sodium arsenite-induced elevated expressionof cyclin-dependentkinase 1 (CDK1) (A, B) and arsenite-induced growth inhibition of cellscan be rescued by a CDK inhibitor, flavopiridol (C). (A) Quantitativeresults by LC-MRM analysis for peptide DLK*PQNLLIDDK (“K*”designates the desthiobiotin-labeled lysine) from CDK1. Shown arethe extracted-ion chromatograms for three transitions monitored forthe peptide with light (red) and heavy (blue) labels in forward (left)and reverse (right) SILAC experiments. (B) Western blot analysis revealedthe elevated expression of CDK1 upon arsenite treatment. (C) The MTTassay showed that flavopiridol could significantly restore the cellproliferation rate after sodium arsenite treatment. “∗”, p < 0.05; “∗∗”, p < 0.01; “∗∗∗”, p < 0.001. The p values were calculated by usingan unpaired two-tailed t test. The values representthe mean of results obtained from three independent experiments.
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fig5: Sodium arsenite-induced elevated expressionof cyclin-dependentkinase 1 (CDK1) (A, B) and arsenite-induced growth inhibition of cellscan be rescued by a CDK inhibitor, flavopiridol (C). (A) Quantitativeresults by LC-MRM analysis for peptide DLK*PQNLLIDDK (“K*”designates the desthiobiotin-labeled lysine) from CDK1. Shown arethe extracted-ion chromatograms for three transitions monitored forthe peptide with light (red) and heavy (blue) labels in forward (left)and reverse (right) SILAC experiments. (B) Western blot analysis revealedthe elevated expression of CDK1 upon arsenite treatment. (C) The MTTassay showed that flavopiridol could significantly restore the cellproliferation rate after sodium arsenite treatment. “∗”, p < 0.05; “∗∗”, p < 0.01; “∗∗∗”, p < 0.001. The p values were calculated by usingan unpaired two-tailed t test. The values representthe mean of results obtained from three independent experiments.

Mentions: In the current study, we observed theoverexpression of multiple protein kinases associated with cell cycleprogression. For instance, we observed an elevated level of cyclin-dependentkinase 1 (CDK1) in both forward and reverse SILAC experiments (ratioof 2.07, Figures 5A and S2, Supporting Information). Along this line, our Western blotanalysis independently confirmed the increased expression of CDK1,as illustrated in Figure 5B. In agreement withour observation, cDNA microarray analysis also revealed the augmentedexpression of CDK1 in sodium arsenite-treated HFW human fibroblastcells.28


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)

Sodium arsenite-induced elevated expressionof cyclin-dependentkinase 1 (CDK1) (A, B) and arsenite-induced growth inhibition of cellscan be rescued by a CDK inhibitor, flavopiridol (C). (A) Quantitativeresults by LC-MRM analysis for peptide DLK*PQNLLIDDK (“K*”designates the desthiobiotin-labeled lysine) from CDK1. Shown arethe extracted-ion chromatograms for three transitions monitored forthe peptide with light (red) and heavy (blue) labels in forward (left)and reverse (right) SILAC experiments. (B) Western blot analysis revealedthe elevated expression of CDK1 upon arsenite treatment. (C) The MTTassay showed that flavopiridol could significantly restore the cellproliferation rate after sodium arsenite treatment. “∗”, p < 0.05; “∗∗”, p < 0.01; “∗∗∗”, p < 0.001. The p values were calculated by usingan unpaired two-tailed t test. The values representthe mean of results obtained from three independent experiments.
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Related In: Results  -  Collection

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

fig5: Sodium arsenite-induced elevated expressionof cyclin-dependentkinase 1 (CDK1) (A, B) and arsenite-induced growth inhibition of cellscan be rescued by a CDK inhibitor, flavopiridol (C). (A) Quantitativeresults by LC-MRM analysis for peptide DLK*PQNLLIDDK (“K*”designates the desthiobiotin-labeled lysine) from CDK1. Shown arethe extracted-ion chromatograms for three transitions monitored forthe peptide with light (red) and heavy (blue) labels in forward (left)and reverse (right) SILAC experiments. (B) Western blot analysis revealedthe elevated expression of CDK1 upon arsenite treatment. (C) The MTTassay showed that flavopiridol could significantly restore the cellproliferation rate after sodium arsenite treatment. “∗”, p < 0.05; “∗∗”, p < 0.01; “∗∗∗”, p < 0.001. The p values were calculated by usingan unpaired two-tailed t test. The values representthe mean of results obtained from three independent experiments.
Mentions: In the current study, we observed theoverexpression of multiple protein kinases associated with cell cycleprogression. For instance, we observed an elevated level of cyclin-dependentkinase 1 (CDK1) in both forward and reverse SILAC experiments (ratioof 2.07, Figures 5A and S2, Supporting Information). Along this line, our Western blotanalysis independently confirmed the increased expression of CDK1,as illustrated in Figure 5B. In agreement withour observation, cDNA microarray analysis also revealed the augmentedexpression of CDK1 in sodium arsenite-treated HFW human fibroblastcells.28

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