Involvement of Src tyrosine kinases (SFKs) and of focal adhesion kinase (FAK) in the injurious mechanism in rat primary neuronal cultures exposed to chemical ischemia.
Src family of kinases (SFKs) and focal adhesion kinase (FAK) are two important cellular signaling components known to act cooperatively in the transduction of death and survival signals. We investigated the involvement of these proteins in the mechanism of the injurious response in rat primary neuronal cultures exposed to an insult composed of chemical ischemia (poisoning with iodoacetic acid; 100 muM, for 150 min) followed by 1 h of incubation in the regular medium, an insult shown before to be associated with generation of reactive oxygen species and with the depletion of adenosine triphisphate. The exposure of the neuronal cultures to the insult resulted in cell injury, assessed by the increased release of cytoplasmic lactate dehydrogenase (LDH) into the culture media, which could be attenuated markedly by the presence of the antioxidant LY 231617. The insult resulted in the decreased level of phosphorylation of the SFKs members Src, Fyn, and Yes at the Src Y416-equivalent activation sites and of the FAK Y397 activation site, degradation of FAK to a p85 fragment, and disassembling of the FAK-SFKs complexes. The inhibition of SFKs was found to be responsible for part of the insult-induced cell damage manifested in increased LDH release. Pervanadate, an inhibitor of the phosphotyrosine phosphatases (PTPs), abrogated the inactivation of SFKs and attenuated cell injury, indicating that insult-induced activation of PTPs is involved in SFKs inhibition and the ensued damage. The inhibition of SFKs and FAK is probably the cause of the disassembling of SFKs-FAK complexes, a process known to be associated with apoptosis.
Shani
Vered
V
Department of Human Molecular Genetics and Biochemistry, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, 69978, Israel.
Bromberg
Yael
Y
Sperling
Oded
O
Zoref-Shani
Esther
E
D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology; D002545:Brain Ischemia / Q000378:metabolism* / Q000473:pathology*; D002478:Cells, Cultured; D004791:Enzyme Inhibitors / Q000633:toxicity; D051417:Focal Adhesion Kinase 1 / Q000378:metabolism*; D019807:Iodoacetic Acid / Q000633:toxicity; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D009474:Neurons / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D010766:Phosphorylation / Q000187:drug effects / Q000502:physiology; D051076:Proto-Oncogene Proteins c-fyn / Q000378:metabolism; D051575:Proto-Oncogene Proteins c-yes / Q000378:metabolism; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000378:metabolism; D051381:Rats; D017382:Reactive Oxygen Species / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D014638:Vanadates / Q000494:pharmacology; D019061:src-Family Kinases / Q000378:metabolism*
D004791:Enzyme Inhibitors; D017382:Reactive Oxygen Species; C076715:pervanadate; D014638:Vanadates; D007770:L-Lactate Dehydrogenase; D051417:Focal Adhesion Kinase 1; C494961:Fyn protein, rat; D051076:Proto-Oncogene Proteins c-fyn; D051575:Proto-Oncogene Proteins c-yes; D016392:Proto-Oncogene Proteins pp60(c-src); C496052:Ptk2 protein, rat; C490726:Yes1 protein, rat; D019061:src-Family Kinases; D019807:Iodoacetic Acid