Ischemia induced focal adhesion kinase cleavage in rat lung.
Focal adhesion kinase (FAK), a 125 kDa protein, is located at the focal adhesion sites that stabilize endothelial cells on the matrix. Ischemic stimuli cause FAK cleavage and FAK depletion from focal adhesion sites. To test this hypothesis under ischemic conditions, we established an isolated rat lung that we ventilated at 40 inflations/min at inspiratory and expiratory pressures of 10 and 3 cm H2O, respectively. After 5 min of blood perfusion at pulmonary arterial and left atrial pressures of 13 and 3 cm H2O respectively, we either stopped (ischemic group, n=10) or continued (perfused group, n=10) perfusion for 2 hr. We then recovered endothelial cells from each lung, using our previously reported magnetic cell separation method. The endothelial lysates were separated into cytosol and membrane fractions, then immunoprecipitated and blotted with anti-FAK, and anti-phospho-FAK antibody. In ischemic as compared to perfused lungs, immunoblotting with anti-FAK antibody revealed a greater prominence of bands at 125 kDa as well as at cleavage products 80, 70 and 44 kDa in the cytosol fraction. Immuno-blotting with anti-phosphotyrosine antibody revealed decreased enhancement of bands at 80, 70 and 44 kDa, also at 125 kDa in the cytosol fraction. We propose that during lung ischemia, FAK is dephosphorylated, cleaved and translocated to the cytosol. This first evidence of the involvement of FAK in lung ischemia suggests that destabilization of focal adhesions may underlie endothelial barrier deterioration, contributing to increased vascular permeability in ischemic lung injury.
Yang
Stephen
S
Columbia University, New York, NY 10032, USA. xy43@columbia.edu
Yip
Rowena
R
Polena
Sotir
S
Gricius
Jonas
J
Desai
Kartik J
KJ
Sharma
Mohan
M
Ruby
Candice
C
Gintautas
Jonas
J
Jerome
Heidi
H
D000107:Acetylation; D000818:Animals; D015153:Blotting, Western; D042783:Endothelial Cells / Q000201:enzymology; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; D007074:Immunoglobulin G; D047468:Immunoprecipitation; D066298:In Vitro Techniques; D007511:Ischemia / Q000201:enzymology* / Q000473:pathology; D008077:Lipoproteins, LDL / Q000378:metabolism; D008168:Lung / Q000201:enzymology* / Q000473:pathology; D008856:Microscopy, Fluorescence; D010477:Perfusion; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011652:Pulmonary Circulation / Q000502:physiology*; D051381:Rats; D017207:Rats, Sprague-Dawley
D007074:Immunoglobulin G; D008077:Lipoproteins, LDL; D011505:Protein-Tyrosine Kinases; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; C496052:Ptk2 protein, rat