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J. Biol. Chem., Vol. 279, Issue 12, 11375-11383, March 19, 2004

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Effective Dephosphorylation of Src Substrates by SHP-1^*

Carsten Frank, Carmen Burkhardt¶, Diana Imhof||, Jens Ringel, Olaf Zschörnig**, Karin Wieligmann, Martin Zacharias, and Frank-D. Böhmer¶¶

From the Institute of Molecular Cell Biology, Medical Faculty, ^||Institute of Biochemistry and Biophysics, Faculty of Biology and Pharmacy, Friedrich Schiller University, D-07747 Jena, Germany, the ^**Institute for Medical Physics and Biophysics, University of Leipzig, D-04103 Leipzig, Germany, and the Institute of Molecular Biotechnology, D-07745 Jena, Germany

The protein-tyrosine phosphatase SHP-1 is a negative regulator of multiple signal transduction pathways. We observed that SHP-1 effectively antagonized Src-dependent phosphorylations in HEK293 cells. This occurred by dephosphorylation of Src substrates, because Src activity was unaffected in the presence of SHP-1. One reason for efficient dephosphorylation was activation of SHP-1 by Src. Recombinant SHP-1 had elevated activity subsequent to phosphorylation by Src in vitro, and SHP-1 variants with mutated phosphorylation sites in the C terminus, SHP-1 Y538F, and SHP-1 Y538F,Y566F were less active toward Src-generated phosphoproteins in intact cells. A second reason for efficient dephosphorylation is the substrate selectivity of SHP-1. Pull-down experiments with different GST-SHP-1 fusion proteins revealed efficient interaction of Src-generated phosphoproteins with the SHP-1 catalytic domain rather than with the SH2 domains. Phosphopeptides that correspond to good Src substrates were efficiently dephosphorylated by SHP-1 in vitro. Phosphorylated "optimal Src substrate" AEEEIpYGEFEA (where pY is phosphotyrosine) and a phosphopeptide corresponding to a recently identified Src phosphorylation site in p120 catenin, DDLDpY²⁹⁶GMMSD, were excellent SHP-1 substrates. Docking of these phosphopeptides into the catalytic domain of SHP-1 by molecular modeling was consistent with the biochemical data and explains the efficient interaction. Acidic residues N-terminal of the phosphotyrosine seem to be of major importance for efficient substrate interaction. Residues C-terminal of the phosphotyrosine probably contribute to the substrate selectivity of SHP-1. We propose that activation of SHP-1 by Src and complementary substrate specificities of SHP-1 and Src may lead to very transient Src signals in the presence of SHP-1.

Received for publication, August 18, 2003 , and in revised form, December 15, 2003.

^* This work was supported by Deutsche Forschungsgemeinschaft Grant SFB 604 (to F. D. B. and M. Z.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Current address: Division of Immunology, Allergy and Infectious Diseases, Dept. of Dermatology at Vienna International Research Cooperation Center, Bio-Molecular Therapeutics, University of Vienna Medical School, Brunnerstr. 59, A-1235 Vienna, Austria.

^¶ Current address: ALTANA Pharma AG, Byk-Gulden-Strasse 2, 78467 Konstanz, Germany.

Current address: International University Bremen, School of Engineering and Science, P. O. Box 750 561, 28725 Bremen, Germany.

^¶¶ To whom correspondence should be addressed: Institute of Molecular Cell Biology, Medical Faculty, Friedrich Schiller University, Drackendorfer Str. 1, D-07747 Jena, Germany. Fax: 49-3641-9325652; E-mail: i5frbo{at}rz.uni-jena.de.

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