Fluid shear stress activates proline-rich tyrosine kinase via reactive oxygen species-dependent pathway.
OBJECTIVE
Fluid shear stress (flow) modulates endothelial cell (EC) function via specific signal transduction events. Previously, we showed that flow-mediated tyrosine phosphorylation of p130 Crk-associated substrate (Cas) required calcium-dependent c-Src activation. Because flow increases reactive oxygen species (ROS) production in ECs and because H(2)O(2) increases tyrosine phosphorylation of proline-rich tyrosine kinase (PYK2), we hypothesized that flow may activate PYK2 via ROS.


RESULTS
Exposure of bovine aortic ECs to flow stimulated PYK2 phosphorylation rapidly, with a peak at 2 minutes. The activation of PYK2 and phosphorylation of Cas induced by flow were inhibited by pretreatment with the antioxidant N-acetylcysteine. Flow-induced PYK2 phosphorylation was inhibited by BAPTA-AM, an intracellular calcium chelator. Bovine aortic ECs transfected with kinase-inactive PYK2 showed attenuated flow-stimulated Cas tyrosine phosphorylation. Although flow-induced Cas phosphorylation was inhibited by kinase-inactive Src, PYK2 activation induced by flow was not inhibited by overexpression of kinase-inactive Src.


CONCLUSIONS
These results show a redox-sensitive pathway for flow-mediated activation of nonreceptor tyrosine kinase activity that requires ROS and intracellular calcium, but not Src kinase.
Tai
Lung-Kuo
LK
Center for Cardiovascular Research, University of Rochester, Rochester, NY 14642, USA.
Okuda
Masanori
M
Abe
Jun-ichi
J
Yan
Chen
C
Berk
Bradford C
BC
D000111:Acetylcysteine / Q000494:pharmacology; D000818:Animals; D001011:Aorta / Q000166:cytology / Q000187:drug effects / Q000201:enzymology; D002118:Calcium / Q000502:physiology; D002417:Cattle; D002478:Cells, Cultured; D015638:Cytochalasin D / Q000494:pharmacology; D003598:Cytoskeletal Proteins / Q000037:antagonists & inhibitors; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects / Q000201:enzymology; D004789:Enzyme Activation / Q000187:drug effects / Q000502:physiology; D051418:Focal Adhesion Kinase 2; D006861:Hydrogen Peroxide / Q000037:antagonists & inhibitors / Q000494:pharmacology; D010084:Oxidation-Reduction; D010766:Phosphorylation / Q000187:drug effects; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000172:deficiency / Q000378:metabolism* / Q000502:physiology; D011518:Proto-Oncogene Proteins / Q000378:metabolism / Q000502:physiology; D050721:Proto-Oncogene Proteins c-cbl; D017382:Reactive Oxygen Species / Q000378:metabolism*; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D012212:Rheology; D013314:Stress, Mechanical; D013379:Substrate Specificity / Q000502:physiology; D013997:Time Factors; D010738:Type C Phospholipases / Q000502:physiology; D044767:Ubiquitin-Protein Ligases; D019061:src-Family Kinases / Q000502:physiology
D003598:Cytoskeletal Proteins; D011518:Proto-Oncogene Proteins; D017382:Reactive Oxygen Species; D011994:Recombinant Proteins; D015638:Cytochalasin D; D006861:Hydrogen Peroxide; D050721:Proto-Oncogene Proteins c-cbl; D044767:Ubiquitin-Protein Ligases; D011505:Protein-Tyrosine Kinases; D051418:Focal Adhesion Kinase 2; D019061:src-Family Kinases; D010738:Type C Phospholipases; D002118:Calcium; D000111:Acetylcysteine