Adaptor protein Crk is required for ephrin-B1-induced membrane ruffling and focal complex assembly of human aortic endothelial cells.
Endothelial cell migration is an essential step in vasculogenesis and angiogenesis, in which receptor tyrosine kinases play a pivotal role. We investigated the mechanism by which ephrin-B1 promotes membrane ruffling in human aortic endothelial cells, because membrane ruffling heralds cell body migration. We especially focused on the role of Crk adaptor protein in EphB-mediated signaling. Using DsRed-tagged Crk and a fluorescent time-lapse microscope, we showed that Crk was recruited to the nascent focal complex after ephrin-B1 stimulation. Furthermore, we found that p130(Cas), but not paxillin, recruited Crk to the nascent focal complex. The necessity of Crk in ephrin-B1-induced membrane ruffling was shown both by the overexpression of dominant negative Crk mutants and by the depletion of Crk by using RNA interference. Then, we examined the role of two major downstream molecules of Crk, Rac1 and Rap1. The dominant negative mutant of Rac1 completely inhibited ephrin-B1-induced membrane ruffling and focal complex assembly. In contrast, rap1GAPII, a negative regulator of Rap1, did not inhibit ephrin-B1-induced membrane ruffling. However, in rap1GAPII-expressing cells, ephrin-B1 did not induce membrane spreading, probably due to instability of the focal complex. These results indicated that Crk plays a critical role in Rac1-induced membrane ruffling and Rap1-mediated nascent focal complex stabilization contributing to ephrin-B1-induced human aortic endothelial cells migration.
Nagashima
Ken-Ichiro
K
Department of Structural Analysis, National Cardiovascular Center Research Institute, Suita, Osaka 565-8565, Japan.
Endo
Akira
A
Ogita
Hisakazu
H
Kawana
Akiko
A
Yamagishi
Akiko
A
Kitabatake
Akira
A
Matsuda
Michiyuki
M
Mochizuki
Naoki
N
D001011:Aorta / Q000166:cytology*; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D002465:Cell Movement; D002478:Cells, Cultured; D050719:Crk-Associated Substrate Protein; D004730:Endothelium, Vascular / Q000166:cytology*; D036387:Ephrin-B1 / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D015151:Immunoblotting; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D010750:Phosphoproteins / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D017434:Protein Structure, Tertiary; D011506:Proteins; D011518:Proto-Oncogene Proteins / Q000502:physiology*; D050737:Proto-Oncogene Proteins c-crk; D034622:RNA Interference; D011994:Recombinant Proteins / Q000378:metabolism; D050717:Retinoblastoma-Like Protein p130; D013997:Time Factors; D014162:Transfection
C493841:BCAR1 protein, human; D050719:Crk-Associated Substrate Protein; D036387:Ephrin-B1; D010750:Phosphoproteins; D011506:Proteins; D011518:Proto-Oncogene Proteins; D050737:Proto-Oncogene Proteins c-crk; D011994:Recombinant Proteins; D050717:Retinoblastoma-Like Protein p130