Fibroblast Growth Factor Receptor-1-mediated Endothelial Cell Proliferation Is Dependent on the Src Homology (SH) 2/SH3 Domain-containing Adaptor Protein Crk*
- From the Department of Medical Biochemistry and Microbiology, Uppsala University Biomedical Center, Box 575, S-751 23 Uppsala, Sweden
Abstract
Stimulation of fibroblast growth factor receptor-1 (FGFR-1) expressed on endothelial cells leads to cellular migration and proliferation. We have examined the role of the Src homology (SH) 2/SH3 domain-containing adaptor protein Crk in these processes. Transient tyrosine phosphorylation of Crk in fibroblast growth factor-2-stimulated endothelial cells was dependent on the juxtamembrane tyrosine residue 463 in FGFR-1, and a Crk SH2 domain precipitated FGFR-1 via phosphorylated Tyr-463, indicating direct complex formation between Crk and FGFR-1. Furthermore, Crk SH2 and SH3 domains formed ligand-independent complexes with Shc, C3G, and the Crk-associated substrate (Cas). Tyrosine phosphorylation of C3G and Cas increased as a consequence of growth factor treatment. We examined the role of Crk in FGFR-1-mediated cellular responses by use of cells expressing chimeric platelet-derived growth factor receptor-α/FGFR-1 (αR/FR) wild type and mutant Y463F receptors. The kinase activity of αR/FR Y463F was intact, but both Crk and the adaptor FRS-2 were no longer tyrosine-phosphorylated in the mutant cells. Both wild type and mutant receptor cells migrated efficiently, whereas cells expressing the mutant αR/FR Y463F failed to proliferate and Erk2 and Jun kinase activities were suppressed in these cells. In wild type αR/FR cells transiently expressing an SH2 domain mutant of Crk, Erk and Jun kinase activities as well as DNA synthesis were attenuated. Our data indicate that Crk participates in signaling complexes downstream of FGFR-1, which propagate mitogenic signals.
Footnotes
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↵* This work was supported by Swedish Cancer Foundation Project 3820-B97-02XBB.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵‡ Present address: Dept. of Medical Sciences, University Hospital, S-75185, Uppsala, Sweden.
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↵§ To whom all correspondence should be addressed. Tel.: 46-18-471-43-63; Fax: 46-18-471-49-75; E-mail: lena.welsh@medkem.uu.se.
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↵2 Klint, P., Kanda, S., Kloog, Y., and Claesson-Welsh, L. (1999) Oncogene 18,3354–3364.
- Abbreviations:
- FGF
-
fibroblast growth factor
- FGFR
-
fibroblast growth factor receptor
- GST
-
glutathione S-transferase
- HA
-
hemagglutinin
- wt
-
wild type
- MAPK
-
mitogen-activated protein kinase
- PBS
-
phosphate-buffered saline
- PAGE
-
polyacrylamide gel electrophoresis
- PLCγ
-
phospholipase Cγ
- DTT
-
dithiothreitol
- PDGF
-
platelet-derived growth factor
- PDGFR
-
platelet-derived growth factor receptor
- BSA
-
bovine serum albumin
- RIPA
-
radioimmune precipitation buffer
- MBP
-
myelin basic protein
- BCE
-
bovine adrenal cortex capillary endothelial
- PAE
-
porcine aortic endothelial
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- Received September 9, 1998.
- Revision received June 2, 1999.
- The American Society for Biochemistry and Molecular Biology, Inc.
