c-Src interacts with and phosphorylates RelA/p65 to promote thrombin-induced ICAM-1 expression in endothelial cells.
The procoagulant thrombin promotes polymorphonuclear leukocyte (PMN) adhesion to endothelial cells by a mechanism involving expression of intercellular adhesion molecule-1 (ICAM-1) via an NF-kappaB-dependent pathway. We now provide evidence that activation of c-Src is crucial in signaling thrombin-induced ICAM-1 expression via tyrosine phosphorylation of RelA/p65. Stimulation of human umbilical vein endothelial cells with thrombin resulted in a time-dependent activation of c-Src, with maximal activation occurring at 30 min after thrombin challenge. Inhibition of c-Src by pharmacological and genetic approaches impaired thrombin-induced NF-kappaB-dependent reporter activity and ICAM-1 expression. Analysis of the NF-kappaB pathway revealed that the effect of c-Src inhibition occurred independently of IkappaBalpha degradation and NF-kappaB DNA binding function and was not associated with exchange of NF-kappaB dimers. Phosphorylation of RelA/p65 at Ser(536), an event mediating the transcriptional activity of DNA-bound RelA/p65, was also insensitive to c-Src inhibition. Interestingly, thrombin induced association of c-Src with RelA/p65, and inhibition of c-Src prevented this response, indicating that this interaction is contingent on activation of c-Src. We also observed that thrombin induced tyrosine phosphorylation of RelA/p65, and this phosphorylation was lost upon inhibition of c-Src, consistent with the requirement of activated c-Src for interaction with RelA/p65. These data implicate an important role of c-Src in phosphorylating RelA/p65 to promote the transcriptional activity of NF-kappaB and thereby ICAM-1 expression in endothelial cells.
Bijli
Kaiser M
KM
Department of Pediatrics, Lung Biology and Disease Program, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642, USA.
Minhajuddin
Mohd
M
Fazal
Fabeha
F
O'Reilly
Michael A
MA
Platanias
Leonidas C
LC
Rahman
Arshad
A
D000081247:CSK Tyrosine-Protein Kinase; D004247:DNA / Q000378:metabolism; D019281:Dimerization; D042783:Endothelial Cells / Q000187:drug effects / Q000201:enzymology*; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D020672:I-kappa B Proteins / Q000378:metabolism; D018799:Intercellular Adhesion Molecule-1 / Q000235:genetics / Q000378:metabolism*; D000072000:NF-KappaB Inhibitor alpha; D010766:Phosphorylation / Q000187:drug effects; D010768:Phosphoserine / Q000378:metabolism; D019000:Phosphotyrosine / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000172:deficiency / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000037:antagonists & inhibitors / Q000172:deficiency / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D013917:Thrombin / Q000494:pharmacology*; D051996:Transcription Factor RelA / Q000378:metabolism*; D019061:src-Family Kinases
D020672:I-kappa B Proteins; C000606204:NFKBIA protein, human; D011518:Proto-Oncogene Proteins; D012333:RNA, Messenger; D051996:Transcription Factor RelA; D018799:Intercellular Adhesion Molecule-1; D000072000:NF-KappaB Inhibitor alpha; D010768:Phosphoserine; D019000:Phosphotyrosine; D004247:DNA; D011505:Protein-Tyrosine Kinases; D000081247:CSK Tyrosine-Protein Kinase; D019061:src-Family Kinases; C000631346:CSK protein, human; D013917:Thrombin