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Molecular and Cellular Biology, July 2000, p. 4791-4805, Vol. 20, No. 13
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Phosphorylation of Tyrosine Residues in the Kinase Domain and Juxtamembrane Region Regulates the Biological and Catalytic Activities of Eph Receptors

Kathleen L. Binns,1,2 Paul P. Taylor,1 Frank Sicheri,1,2 Tony Pawson,1,2,* and Sacha J. Holland1,dagger

Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario M5G 1X5,1 and Department of Molecular and Medical Genetics, University of Toronto, Toronto, Ontario M5G 1A8,2 Canada

Received 15 November 1999/Returned for modification 5 January 2000/Accepted 28 February 2000

Members of the Eph family of receptor tyrosine kinases exhibit a striking degree of amino acid homology, particularly notable in the kinase and membrane-proximal regions. A mutagenesis approach was taken to address the functions of specific conserved tyrosine residues within these catalytic and juxtamembrane domains. Ligand stimulation of wild-type EphB2 in neuronal NG108-15 cells resulted in an upregulation of catalytic activity and an increase in cellular tyrosine phosphorylation, accompanied by a retraction of neuritic processes. Tyrosine-to-phenylalanine substitutions within the conserved juxtamembrane motif abolished these responses. The mechanistic basis for these observations was examined using the highly related EphA4 receptor in a continuous coupled kinase assay. Tandem mass spectrometry experiments confirmed autophosphorylation of the two juxtamembrane tyrosine residues and also identified a tyrosine within the kinase domain activation segment as a phosphorylation site. Kinetic analysis revealed a decreased affinity for peptide substrate upon substitution of activation segment or juxtamembrane tyrosines. Together, our data suggest that the catalytic and therefore biological activities of Eph receptors are controlled by a two-component inhibitory mechanism, which is released by phosphorylation of the juxtamembrane and activation segment tyrosine residues.

* Corresponding author. Mailing address: Samuel Lunenfeld Research Institute, Mt. Sinai Hospital, 600 University Ave., Toronto, Ontario, Canada M5G 1X5. Phone: (416) 586-8262. Fax: (416) 586-8869. E-mail: pawson{at}mshri.on.ca.

dagger Present address: Rigel Inc., South San Francisco, CA 94080.

Molecular and Cellular Biology, July 2000, p. 4791-4805, Vol. 20, No. 13
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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