QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online September 29, 2004
doi: 10.1242/10.1242/jcs.01397

This Article Figures Only Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Biskup, C. Articles by Böhmer, F.-D. Search for Related Content PubMed PubMed Citation Articles by Biskup, C. Articles by Böhmer, F.-D.

Visualization of SHP-1-target interaction

Christoph Biskup^1,*, Annette Böhmer^2,*, Rico Pusch^2,*, Laimonas Kelbauskas¹, Alexander Gorshokov³, Irina Majoul³, Jörg Lindenau⁴, Klaus Benndorf¹ and Frank-D. Böhmer^2,

¹ Institute of Physiology II, Medical Faculty, Friedrich Schiller University, Drackendorfer Str. 1, 07747 Jena, Germany
² Research Unit Molecular Cell Biology, Medical Faculty, Friedrich Schiller University, Drackendorfer Str. 1, 07747 Jena, Germany
³ Max-Planck Institute of Biophysical Chemistry, Am Fassberg 11, 37077 Göttingen, Germany
⁴ Application Laboratory, Advanced Imaging Microscopy, Carl-Zeiss GmbH, Carl-Zeiss-Promenade 10, 07745 Jena, Germany

Author for correspondence (e-mail: i5frbo{at}rz.uni-jena.de)

Accepted 6 July 2004

Signaling of receptor tyrosine kinases (RTKs) is regulated by protein-tyrosine phosphatases (PTPs). We previously discovered the efficient downregulation of Ros RTK signaling by the SH2 domain PTP SHP-1, which involves a direct interaction of both molecules. Here, we studied the mechanism of this interaction in detail. Phosphopeptides representing the SHP-1 candidate binding sites in the Ros cytoplasmic domain, pY2267 and pY2327, display high affinity binding to the SHP-1 N-terminal SH2 domain (K_d=217 nM and 171 nM, respectively). Y2327 is, however, a poor substrate of Ros kinase and, therefore, contributes little to SHP-1 binding in vitro. To explore the mechanism of association in intact cells, functional fluorescent fusion proteins of Ros and SHP-1 were generated. Complexes of both molecules could be detected by Förster resonance energy transfer (FRET) in intact HEK293 and COS7 cells. As expected, the association required the functional SHP-1 N-terminal SH2 domain. Unexpectedly, pY2267 and pY2327 both contributed to the association. Mutation of Y2327 reduced constitutive association in COS7 cells. Ligand-dependent association was abrogated upon mutation of Y2267 but remained intact when Y2327 was mutated. A phosphopeptide representing the binding site pY2267 was a poor substrate for SHP-1, whereas Ros activation loop phosphotyrosines were effectively dephosphorylated. We propose a model for SHP-1-Ros interaction in which ligand-stimulated phosphorylation of Ros Y2267 by Ros, phosphorylation of Y2327 by a heterologous kinase, and inactivation of Ros by SHP-1-mediated dephosphorylation play a role in the regulation of complex stability.

Key words: Protein-tyrosine phosphatase, SHP-1, Receptor tyrosine kinase, Ros, FRET

This article has been cited by other articles:

				L. Karagyozov, R. Godfrey, S.-A. Bohmer, A. Petermann, S. Holters, A. Ostman, and F.-D. Bohmer The structure of the 5'-end of the protein-tyrosine phosphatase PTPRJ mRNA reveals a novel mechanism for translation attenuation Nucleic Acids Res., August 1, 2008; 36(13): 4443 - 4453. [Abstract] [Full Text] [PDF]