C Beinke1, D Van Beuningen, N Cordes. 1. Institute of Radiobiology, German Armed Forces, Neuherbergstrasse 11, D-80937 Munich, Germany. cbeinke@gmx.net
Abstract
PURPOSE: Focal adhesion kinase (FAK) is involved in the regulation of many cellular processes, including cell survival and death, proliferation and migration. The same endpoints are influenced by ionizing radiation (IR). Therefore, study was performed to determine the effect of IR on the expression and phosphorylation of FAK and two of its substrates, p130cas and paxillin, in vitro. MATERIALS AND METHODS: Exponentially growing A549 lung carcinoma cells were exposed to 6 Gy X-rays. Protein expression and the extent of tyrosine phosphorylation were investigated by immunoprecipitation experiments and Western blotting analysis using specific or unspecific phosphotyrosine antibodies. Immunofluorescence staining in combination with confocal laser scanning microscopy was done to localize the proteins within the cell. RESULTS: Tyrosine phosphorylation, of Mr 110 000 150 000 and 65 000-75 000 protein bands, was induced within 30 min after exposure to IR. Three of these proteins were identified as FAK, p130cas and paxillin. IR induced phosphorylation of FAK (tyr397 and tyr925) but did not change FAK expression. Additionally, IR induced phosphorylation of paxillin (tyr31 and tyr181) within 30 min and an up-regulation of paxillin expression 2-6 h after exposure. Furthermore, a higher amount of phosphorylated p130cas was found in irradiated cells. Immunofluorescence staining demonstrated that in A549 cells, all three proteins colocalize at sites of focal adhesions at the cytoplasmic face of the cell membrane and to lamellopodia. CONCLUSIONS: The data indicate that these focal adhesion-associated proteins are modulated by IR and thus are likely to play a role in the cellular response to IR. These proteins might represent attractive targets to modulate FAK-initiated signalling pathways, which may be involved in improved radioresistance and, furthermore, in important pathological phenomena such as tumour growth and metastatic phenotypes.
PURPOSE:Focal adhesion kinase (FAK) is involved in the regulation of many cellular processes, including cell survival and death, proliferation and migration. The same endpoints are influenced by ionizing radiation (IR). Therefore, study was performed to determine the effect of IR on the expression and phosphorylation of FAK and two of its substrates, p130cas and paxillin, in vitro. MATERIALS AND METHODS: Exponentially growing A549 lung carcinoma cells were exposed to 6 Gy X-rays. Protein expression and the extent of tyrosine phosphorylation were investigated by immunoprecipitation experiments and Western blotting analysis using specific or unspecific phosphotyrosine antibodies. Immunofluorescence staining in combination with confocal laser scanning microscopy was done to localize the proteins within the cell. RESULTS:Tyrosine phosphorylation, of Mr 110 000 150 000 and 65 000-75 000 protein bands, was induced within 30 min after exposure to IR. Three of these proteins were identified as FAK, p130cas and paxillin. IR induced phosphorylation of FAK (tyr397 and tyr925) but did not change FAK expression. Additionally, IR induced phosphorylation of paxillin (tyr31 and tyr181) within 30 min and an up-regulation of paxillin expression 2-6 h after exposure. Furthermore, a higher amount of phosphorylated p130cas was found in irradiated cells. Immunofluorescence staining demonstrated that in A549 cells, all three proteins colocalize at sites of focal adhesions at the cytoplasmic face of the cell membrane and to lamellopodia. CONCLUSIONS: The data indicate that these focal adhesion-associated proteins are modulated by IR and thus are likely to play a role in the cellular response to IR. These proteins might represent attractive targets to modulate FAK-initiated signalling pathways, which may be involved in improved radioresistance and, furthermore, in important pathological phenomena such as tumour growth and metastatic phenotypes.
Authors: Jordan J Toutounchian; Jena J Steinle; Patrudu S Makena; Christopher M Waters; Matthew W Wilson; Barrett G Haik; Duane D Miller; Charles R Yates Journal: PLoS One Date: 2014-06-23 Impact factor: 3.240