Shc and a Novel 89-kDa Component Couple to the Grb2-Sos Complex in Fibroblast Growth Factor-2-stimulated Cells

doi:10.1074/jbc.270.40.23337

Shc and a Novel 89-kDa Component Couple to the Grb2-Sos Complex in Fibroblast Growth Factor-2-stimulated Cells (*)

From the Ludwig Institute for Cancer Research, Biomedical Center, Box 595, S-751 24 Uppsala, Sweden

^§ To whom correspondence should be addressed. Tel.: 46-18-174146; Fax: 46-18-506867.

Abstract

A major pathway for mitogenicity is gated via the small GTP-binding protein Ras. Receptor tyrosine kinases couple to Ras through the Src homology 2 (SH2) domain protein Grb2. The activated fibroblast growth factor receptor-1 (FGFR-1) expressed in L6 myoblasts did not bind Grb2 directly, but indirectly, through the small adaptor protein Shc, which was tyrosine-phosphorylated in response to fibroblast growth factor-2 (FGF-2) stimulation. A FGFR-1 mutant in which Tyr, a known autophosphorylation site, was changed to Phe, mediated less efficient tyrosine phosphorylation of Shc. FGF-2 stimulation of mutant FGFR-1-expressing cells still allowed formation of complexes containing Shc, Grb2, and the nucleotide exchange factor Sos and mediation of a mitogenic signal. Another pool of Grb2 was found in complex with a tyrosine-phosphorylated 89-kDa component after FGF-2 stimulation. Stimulation with other growth factors did not lead to tyrosine phosphorylation of p89. As shown by “far-Western” analysis, p89 bound directly to the Grb2 SH2 domain, and this interaction was inhibited by a peptide containing the Y(P)-X-N motif. Tyrosine-phosphorylated p89 was found exclusively in the membrane fraction, indicating its role in bringing Grb2, as well as Sos, to the plasma membrane. These data support the concept of growth factor-specific coupling of Grb2 to the Ras pathway.

Footnotes

↵* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

FGF

fibroblast growth factor

FGFR

fibroblast growth factor receptor

EGF

epidermal growth factor

EGFR

epidermal growth factor receptor

Grb2

growth factor receptor bound protein 2

GST

glutathione S-transferase

MAPK

mitogen-activated protein kinase

MEK

MAP kinase (or ERK) kinase

PBS

phosphate-buffered saline

PDGF

platelet derived growth factor

PLC-

phospholipase C-

PAGE

polyacrylamide gel electrophoresis

SH2

Src homology 2

Tyr(P)

phosphotyrosine

Shc

Src homologous and collagen

Sos

son of sevenless.
↵² S. Kanda, E. Landgren, M. Ljungström, and L. Claesson-Welsh, submitted for publication.
- Received March 16, 1995.
- Revision received June 13, 1995.