Ionizing radiation modules of the expression and tyrosine phosphorylation of the focal adhesion-associated proteins focal adhesion kinase (FAK) and its substrates p130cas and paxillin in A549 human lung carcinoma cells in vitro.
OBJECTIVE
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.


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.
Beinke
C
C
Institute of Radiobiology, German Armed Forces, Neuherbergstrasse 11, D-80937 Munich, Germany. cbeinke@gmx.net
Van Beuningen
D
D
Cordes
N
N
D045744:Cell Line, Tumor / Q000378:metabolism / Q000528:radiation effects; D050719:Crk-Associated Substrate Protein; D003598:Cytoskeletal Proteins / Q000378:metabolism*; D004307:Dose-Response Relationship, Radiation; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; D015971:Gene Expression Regulation, Enzymologic / Q000528:radiation effects; D006801:Humans; D008175:Lung Neoplasms / Q000378:metabolism* / Q000473:pathology; D051419:Paxillin; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation / Q000528:radiation effects; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011506:Proteins; D011829:Radiation Dosage; D011839:Radiation, Ionizing; D050717:Retinoblastoma-Like Protein p130; D014443:Tyrosine / Q000378:metabolism*; D014965:X-Rays
C493841:BCAR1 protein, human; D050719:Crk-Associated Substrate Protein; D003598:Cytoskeletal Proteins; C496061:PXN protein, human; D051419:Paxillin; D010750:Phosphoproteins; D011506:Proteins; D050717:Retinoblastoma-Like Protein p130; D014443:Tyrosine; D011505:Protein-Tyrosine Kinases; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; C496050:PTK2 protein, human