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EX VIVIO DETECTION OF KINASE AND PHOSPHATASE ACTIVITIES IN HUMAN BRONCHIAL BIOPSIES
Citation:
Samet, J M., T. Tal, A J. Ghio, Huang, YuhChin T, L. A. Dailey, AND R Silbajoris. EX VIVIO DETECTION OF KINASE AND PHOSPHATASE ACTIVITIES IN HUMAN BRONCHIAL BIOPSIES. Presented at American Thoracic Society Annual Conference, San Diego, CA, March 20 - 25, 2005.
Description:
Protein phosphorylation is a posttranslational modification involved in every aspect cellular function. Levels of protein phosphotyrosine, phosphoserine and phosphothreonine are regulated by the opposing activities of kinases and phosphatases, the expression of which can be altered by environmental stimuli, including culture conditions. To test the relevance of cultured human airway epithelial cells (HAEC) as an in vitro model of the human bronchial epithelium in vivo, we compared the profile of kinase and phosphatase activities in HAEC and fresh bronchial biopsies. Endobronchial biopsies were obtained from the right mainstem bronchus of normal volunteers undergoing fiberotic bronchoscopy following an IRB-approved protocol. Bronchial tissue was frozen immediately or dispersed and the HAEC cultured for 14-21 days in a growth-factor supplemented medium. Protein extracts prepared from HAEC and fresh biopsies were subjected to in-gel kinase and in-gel phosphatase activity assays using myelin basic protein and 32P-polyglutyr as a substrate, respectively. The activities of over 12 individual tyrosine and Ser/Thr kinases ranging in molecular weight 20-200 kD with varying levels of activity could be discerned by in-gel kinase assay of HAEC and biopsy tissue. Similarly, at least 20 individual active tyrosine phosphatases between 30 and 180 kD were detected in the HAEC and biopsy samples by the in-gel phosphatase assay. Kinase and phosphatase activity profiles in the HAEC closely matched those of the biopsy tissues. These findings show that HAEC retain their pattern of kinase and phosphatase activity when cultured, suggesting that HAEC are a relevant model of the human bronchial epithelium for the study of signaling processes. THIS ABSTRACT OF A PROPOSED PRESENTATION DOES NOT NECESSARILY REFLECT