Adaptor Protein Crk Is Required for Ephrin-B1-induced Membrane Ruffling and Focal Complex Assembly of Human Aortic Endothelial Cells

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.

A Novel Dynamin-associating Molecule, Formin-binding Protein 17, Induces Tubular Membrane Invaginations and Participates in Endocytosis

“…Actin was visualized with rhodamine-conjugated phalloidin. Fluorescent images of EGFP and of Alexa 546 or rhodamine were obtained with an Olympus BX50EI confocal microscope controlled by Fluoview (Olympus, Tokyo) as described previously (21). Time-lapse fluorescence imaging was performed as described previously (21).…”

“…Fluorescent images of EGFP and of Alexa 546 or rhodamine were obtained with an Olympus BX50EI confocal microscope controlled by Fluoview (Olympus, Tokyo) as described previously (21). Time-lapse fluorescence imaging was performed as described previously (21). Briefly, COS-1 cells expressing EGFP-FBP17 were cultured on a collagen-coated glass-bottom dish in DMEM/nutrient mixture F-12 (Invitrogen) supplemented with 10% fetal bovine serum, 2 mM L-glutamine, and 10 mM HEPES without phenol red.…”

Signaling adaptor protein Crk is indispensable for malignant feature of glioblastoma cell line KMG4

“…All cells used were maintained in Dulbecco's modified eagle medium (Seikagaku Co., Tokyo, Japan), supplemented with 10% fetal calf serum. For establishment of Crk knockdown glioma cells, the pSUPER-vector [19] expressing small interference RNA for human Crk [20] was transfected into the KMG4 cells 6 by means of Fugene 6 (Roche, Indianapolis, IN, USA). Cells were cultured in the presence of 500 ng/ml puromycin (Sigma) and drug-resistant clones were isolated.…”

Adaptor Molecule Crk Is Required for Sustained Phosphorylation of Grb2-Associated Binder 1 and Hepatocyte Growth Factor–Induced Cell Motility of Human Synovial Sarcoma Cell Lines

“…The plasmid containing small interfering RNAs for Crk (CrkI) was described previously (63). The CrkI corresponding to the bases 277 to 296 of human c-CrkII encoding sequences as 5 ¶-GAGTTTGATTCATTGCCTG-3 ¶ and 5 ¶-CAGGCAATGAATCAAACTC-3 ¶ were subcloned into SalI and KpnI sites of the pSUPER (suppression of endogenous RNA) vector (64) and transfected into synovial sarcoma cell lines by Fugene 6 transfection reagent (Roche, Indianapolis, IN).…”