Adaptor Protein Crk Is Required for Ephrin-B1-induced Membrane Ruffling and Focal Complex Assembly of Human Aortic Endothelial Cells
Open Access
- 1 December 2002
- journal article
- Published by American Society for Cell Biology (ASCB) in Molecular Biology of the Cell
- Vol. 13 (12), 4231-4242
- https://doi.org/10.1091/mbc.e02-04-0181
Abstract
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 p130Cas, 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.Keywords
This publication has 58 references indexed in Scilit:
- Focal Adhesions Require Catalytic Activity of Src Family Kinases To Mediate Integrin-Matrix AdhesionMolecular and Cellular Biology, 2002
- Downregulation of the Ras–Mitogen-Activated Protein Kinase Pathway by the EphB2 Receptor Tyrosine Kinase Is Required for Ephrin-Induced Neurite RetractionMolecular and Cellular Biology, 2001
- Chat, a Cas/HEF1-associated Adaptor Protein That Integrates Multiple Signaling PathwaysPublished by Elsevier BV ,2000
- Replacing two conserved tyrosines of the EphB2 receptor with glutamic acid prevents binding of SH2 domains without abrogating kinase activity and biological responsesOncogene, 2000
- Tyrosine-614, the major autophosphorylation site of the receptor tyrosine kinase HEK2, functions as multi-docking site for SH2-domain mediated interactionsOncogene, 1998
- CAS/Crk Coupling Serves as a “Molecular Switch” for Induction of Cell MigrationThe Journal of cell biology, 1998
- Mechanisms of angiogenesisNature, 1997
- Role of Crk Oncogene Product in Physiologic SignalingCritical Reviews™ in Oncogenesis, 1997
- Cell Migration: A Physically Integrated Molecular ProcessCell, 1996
- Efficient selection for high-expression transfectants with a novel eukaryotic vectorGene, 1991