Biochem. J. (2008) 416
(189–199) (Printed in Great Britain)
Indirect recruitment of the signalling adaptor Shc to the fibroblast growth factor receptor 2 (FGFR2)
Annika C. SCHÜLLER*, Zamal AHMED*, James A. LEVITT†, Kin M. SUEN*, Klaus SUHLING† and John E. LADBURY*1
*Department of Biochemistry and Molecular Biology, Darwin Building, Gower Street, London WC1E 6BT, U.K., and †Department of Physics, King's College London, The Strand, London, WC2R 2LS, U.K.
The adaptor protein Shc (Src homology and collagen-containing protein) plays an important role in the activation of signalling pathways downstream of RTKs (receptor tyrosine kinases) regulating diverse cellular functions, such as differentiation, adhesion, migration and mitogenesis. Despite being phosphorylated downstream of members of the FGFR (fibroblast growth factor receptor) family, a direct interaction of Shc with this receptor family has not been described to date. Various studies have suggested potential binding sites for the Shc PTB domain (phosphotyrosine-binding domain) and/or the SH2 (Src homology 2) domain on FGFR1, but no interaction of full-length Shc with these sites has been reported in vivo. In the present study, we investigated the importance of the SH2 domain and the PTB domain in recruitment of Shc to FGFR2(IIIc) to characterize the interaction of these two proteins. Confocal microscopy revealed extensive co-localization of Shc with FGFR2. The PTB domain was identified as the critical component of Shc which mediates membrane localization. Results from FLIM (fluorescence lifetime imaging microscopy) revealed that the interaction between Shc and FGFR2 is indirect, suggesting that the adaptor protein forms part of a signalling complex containing the receptor. We identified the non-RTK Src as a protein which potentially mediates the formation of such a ternary complex. Although an interaction between Src and Shc has been described previously, in the present study we implicate the Shc SH2 domain as a novel mediator of this association. The recruitment of Shc to FGFR2 via an indirect mechanism provides new insight into the regulation of protein assembly and activation of various signalling pathways downstream of this RTK.
Key words: fibroblast growth factor receptor 2 (FGFR2), fluorescence lifetime imaging microscopy (FLIM), Förster resonance energy transfer (FRET), Src, Src homology and collagen containing protein (Shc), tyrosine kinase.
Abbreviations used: CH1 domain, collagen homology 1 domain; CrkII, CT10 sarcoma oncogene cellular homologue II; DMEM, Dulbecco's modified Eagle's medium; EGF, epidermal growth factor; EGFR, EGF receptor; ERK, extracellular-signal-regulated kinase; FGF, fibroblast growth factor; FGFR, FGF receptor; FLIM, fluorescence lifetime imaging microscopy; FRET, Förster resonance energy transfer; FRS2, FGFR substrate 2; GFP, green fluorescent protein; Grb, growth-factor-receptor-bound protein; GST, glutathione transferase; HEK-293T cell, human embryonic kidney cell expressing the large T-antigen of simian virus 40; MAPK, mitogen-activated protein kinase; PDGFR, platelet-derived growth factor receptor; PLCγ, phospholipase Cγ; PTB domain, phosphotyrosine-binding domain; RFP, red fluorescent protein; mRFP, monomeric RFP; RTK, receptor tyrosine kinase; SH2, Src homology 2; Shc, Src homology and collagen-containing protein; p46 Shc, 46 kDa Shc; p52 Shc, 52 kDa Shc; p66 Shc, 66 kDa Shc; Shc3F, Shc Y239F/Y240F/Y317F; Shc ΔPTB, Shc lacking the PTB domain; Shc Triple, Shc R112Q/K116A/K139A; Sos, Son of sevenless; TrkA, tyrosine kinase neurotrophin receptor A.
1To whom correspondence should be addressed (email j.ladbury@biochem.ucl.ac.uk).
Received 1 May 2008/24 September 2008; accepted 7 October 2008
Published as BJ Immediate Publication 7 October 2008, doi:10.1042/BJ20080887
© The Authors Journal compilation © 2008 Biochemical Society
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