title: Effective dephosphorylation of Src substrates by SHP-1.
abstract: 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, DDLDpY(296)GMMSD, 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.
authors.affiliation: Institute of Molecular Cell Biology, Medical Faculty, Faculty of Biology and Pharmacy, Friedrich Schiller University, D-07747 Jena, Germany.
authors.forename: Carsten
authors.initials: C
authors.lastname: Frank
authors.forename: Carmen
authors.initials: C
authors.lastname: Burkhardt
authors.forename: Diana
authors.initials: D
authors.lastname: Imhof
authors.forename: Jens
authors.initials: J
authors.lastname: Ringel
authors.forename: Olaf
authors.initials: O
authors.lastname: Zschörnig
authors.forename: Karin
authors.initials: K
authors.lastname: Wieligmann
authors.forename: Martin
authors.initials: M
authors.lastname: Zacharias
authors.forename: Frank-D
authors.initials: FD
authors.lastname: Böhmer
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D017931:DNA Primers; D004789:Enzyme Activation; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010766:Phosphorylation; D053824:Protein Tyrosine Phosphatase, Non-Receptor Type 6; D017027:Protein Tyrosine Phosphatases / Q000378:metabolism*; D013379:Substrate Specificity; D019061:src-Family Kinases / Q000378:metabolism*
chemical_list: D017931:DNA Primers; D047908:Intracellular Signaling Peptides and Proteins; D019061:src-Family Kinases; C509249:PTPN6 protein, human; D053824:Protein Tyrosine Phosphatase, Non-Receptor Type 6; D017027:Protein Tyrosine Phosphatases