Abstract
Environmental pollutant exposure is associated with adverse respiratory outcomes. The phosphorylation of enzymes activates or deactivates many cellular processes and is related to the development of lung diseases such as asthma and chronic obstructive pulmonary disease. However, little is known about protein phosphorylation of bronchial epithelial cells in response to airborne particulates. Herein, we screened differentially phosphorylated proteins in TiO 2-treated epithelial cells and validated the change in GSTP1 protein phosphorylation. Two-dimensional electrophoresis was adopted for differential display proteomics of TiO 2-treated BEAS-2B cell lysates. Phosphoproteins were screened using Pro-Q ® Diamond phosphoprotein gel stain and identified by MALDI-TOF/TOF analysis. Immunoprecipitation and immunoblotting were performed for quantitative measurement of GSTP1 phosphorylation in cell lysates. Normalized relative intensities of nine phosphorylated proteins increased after TiO 2 treatment, whereas those of 12 proteins decreased in the BEAS-2B cell lysates. From gene ontology and pathway analysis, proteins involved in signal transduction were commonly identified, followed by cytoskeletal proteins, proteins from oxidation and antioxidation pathways, proteins catalyzing reductions, and those involved in cellular process, transport, and modification. Immunoblotting with anti-GSTP1 antibody demonstrated no change in GSTP1 protein levels in the lysates of BEAS-2B cells after treatment with TiO 2 particles; blotting with anti-phosphoserine and anti-phosphotyrosine antibodies showed dose-dependent decreases in phosphoserine and phosphotyrosine proteins. Stimulation with particulates phosphorylated and dephosphorylated several proteins in epithelial cells, and serine and tyrosine protein phosphorylation of GSTP1 decreased. These data indicate that airborne particles affect the pattern of phosphorylation of proteins involved in defense or apoptosis of respiratory epithelium.
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