N-terminal cleavage fragment of focal adhesion kinase is required to activate the survival signalling pathway in cultured myoblasts under oxidative stress.
We have previously shown that the cultured L6 myoblasts are susceptible to menadione-induced oxidative stress. Damaged cells were detached from the culture dishes. In the present study, we focused on focal adhesion kinase (FAK), which plays pivotal roles in maintaining focal adhesion function and cell survival. FAK, normally localized at the focal adhesion regions of the myoblasts, was not observed at the regions under oxidative stress induced by menadione and H(2) O(2) . Two cleavage products, 80-kDa N-terminal FAK and 35-kDa C-terminal FAK fragments, as well as full-length FAK (125 kDa) were detected in myoblasts cultured under normal conditions by western blotting with anti-N-terminal FAK or anti-C-terminal FAK sera. Of interest was the finding that the cleavage products of FAK (but not full-length FAK) disappeared under oxidative stress. The cleavage of full-length FAK to N-terminal FAK and C-terminal FAK was inhibited by calpeptin, a specific calpain inhibitor. In addition, pre-incubation of cells with calpeptin resulted in a sharp decrease in survival signals, such as Akt phosphorylation and the ratio of Bcl-2/Bax, under stress conditions. By contrast, not only relative viability, but also Akt phosphorylation and the ratio of Bcl-2/Bax was significantly improved when cells were transfected with a DNA construct of N-terminal FAK-Myc. These results suggest that the N-terminal FAK positively regulates survival signalling in early phases of oxidative stress in the cultured myoblasts.
Department of Biological Science, College of Natural Sciences, Ajou University, Suwon, KoreaRadiation Medicine Branch, National Cancer Center, Goyang, Korea.
Jeong A
JA
Lim
Department of Biological Science, College of Natural Sciences, Ajou University, Suwon, KoreaRadiation Medicine Branch, National Cancer Center, Goyang, Korea.
Sung Ho
SH
Hwang
Department of Biological Science, College of Natural Sciences, Ajou University, Suwon, KoreaRadiation Medicine Branch, National Cancer Center, Goyang, Korea.
Min Jeong
MJ
Kim
Department of Biological Science, College of Natural Sciences, Ajou University, Suwon, KoreaRadiation Medicine Branch, National Cancer Center, Goyang, Korea.
Sang Soo
SS
Kim
Department of Biological Science, College of Natural Sciences, Ajou University, Suwon, KoreaRadiation Medicine Branch, National Cancer Center, Goyang, Korea.
Hye Sun
HS
Kim
D000818:Animals; D017209:Apoptosis; D015153:Blotting, Western; D002154:Calpain / Q000378:metabolism; D002448:Cell Adhesion; D002470:Cell Survival; D002478:Cells, Cultured; D004151:Dipeptides / Q000378:metabolism; D051416:Focal Adhesion Protein-Tyrosine Kinases / Q000378:metabolism*; D051379:Mice; D032446:Myoblasts / Q000166:cytology* / Q000378:metabolism*; D018384:Oxidative Stress; D010766:Phosphorylation; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism*; D051381:Rats; D015398:Signal Transduction
D004151:Dipeptides; C071482:calpeptin; D051416:Focal Adhesion Protein-Tyrosine Kinases; D051057:Proto-Oncogene Proteins c-akt; D002154:Calpain