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5 June 1997, Volume 14, Number 22, Pages 2619-2631
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Article
Intracellular signaling of the Ufo/Axl receptor tyrosine kinase is mediated mainly by a multi-substrate docking-site
Jürgen Braunger1,2, Lothar Schleithoff2,b, Ansgar S Schulz2, Heidi Kessler2, Reiner Lammers3,c, Axel Ullrich3, Claus R Bartram1,2 and Johannes WG Janssen1,2,a

1Institut für Humangenetik, Ruprecht-Karls-Universität Heidelberg, Im Neuenheimer Feld 328, 69120 Heidelberg, Germany

2Abteilung Kinderheilkunde II, Universität Ulm, Prittwitzstrae 43, 89075 Ulm, Germany

3Max-Planck-Institut für Biochemie, Am Klopferspitz 18A, 82152 Martinsried, Germany

aAuthor for correspondence

bPresent addresses: Abteilung für Angewandte Physiologie, Universität Ulm, Albert-Einstein-Allee 11, 89081 Ulm, Germany

cMedizinische Linik und Poliklinik, Eberhard-Karls-Universität Tübingen, Otfried-Müller-Strae 10, 72076 Tübingen, Germany

The first two authors contributed equally to this work

Abstract

Ufo/Axl belongs to a new family of receptor tyrosine kinases with an extracellular structure similar to that of neural cell adhesion molecules. In order to elucidate intracellular signaling, the cytoplasmic moiety of Ufo/Axl was used to screen an expression library according to the CORT (cloning of receptor targets) method. Three putative Ufo substrates were identified: phospholipase Cbold gamma1 (PLCbold gamma), as well as p85alpha and p85beta subunits of phosphatidylinositol 3'-kinase (PI3-kinase). Subsequently, chimeric EGFR/Ufo receptors consisting of the extracellular domains of the epidermal growth factor receptor (EGFR) and the transmembrane and intracellular moiety of Ufo were engineered. Using different far-Western blot analyses and coimmunoprecipitation assays, receptor binding of PLCbold gamma and p85 proteins as well as GRB2, c-src and lck was examined in vitro and in vivo. Competitive inhibition of substrate binding and mutagenesis experiments with EGFR/Ufo constructs revealed C-terminal tyrosine 821 (EILpYVNMDEG) as a docking site for multiple effectors, namely PLCgamma, p85 proteins, GRB2, c-src and lck. Tyrosine 779 (DGLpYALMSRC) demonstrated an additional, but lower binding affinity for the p85 proteins in vitro. In addition, binding of PLCgamma occurred through tyrosine 866 (AGRpYVLCPST). Moreover, our in vivo data indicate that further direct or indirect binding sites for PLCgamma, GRB2, c-src and lck on the human Ufo receptor may exist.

Keywords

Ufo/Axl; receptor tyrosine kinase; CORT; SH2 domain; multi-substrate docking-site

Received 23 December 1999; revised 28 February 1999; accepted 28 February 1999
5 June 1997, Volume 14, Number 22, Pages 2619-2631
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