SHP-1 is expressed in the epididymal epithelium. A–E show the expression of SHP-1 mRNA in murine epididymis (wild-type animals) by in situ hybridization. (A) Overview in dark-field representation (antisense riboprobe). Bar, 1 mm. (B) SHP-1 mRNA expression in the proximal tubules at higher magnification. (C) Sense riboprobe control. (D and E) Sections from B and C, respectively, shown in differential interference contrast. Bar, 200 μm. (F) Expression of SHP-1 in human epididymis by immunocytochemistry with anti–SHP-1 antibodies. (G) Negative control for F. The incubation with the primary antibody was performed in the presence of excess antigenic peptide. Bar, 50 μm.

The SHP-1 SH2 domains are strong binding partners for Ros in yeast two-hybrid assays. (A) The interaction of various signaling molecules as “prey” (indicated) with the autophosphorylated cytoplasmic domain of Ros as “bait” was tested in yeast colony growth assays. (B) The interaction of the SH2 domains of either SHP-1 or the related PTP SHP-2 with Ros or, for comparison, with the PDGFβ receptor was quantitatively measured with a β-galactosidase reporter gene assay. Colonies were spotted on X-gal–containing plates and color development, as depicted in the top picture, were quantitated by densitometry (graph, mean ± SD, n = 6).

SHP-1 interacts with and dephosphorylates autophosphorylated TrkA-Ros. (A) SHP-1 wild-type (WT) or a catalytically inactive SHP-1 mutant (CS) were coexpressed with TrkA-Ros in HEK293 cells, and the cells were stimulated with NGFβ or vehicle. Cell lysates were used for reciprocal coimmunoprecipitation experiments. (B) HEK293 cell lysates containing autophosphorylated TrkA-Ros were used in GST pull-down assays using different fragments of SHP-1 as GST fusion proteins. CAT, catalytic domain; SH2, SH2 domains; NSH2, isolated NH₂-terminal SH2 domain; CSH2, isolated COOH-terminal SH2 domain; SHP-1, full-length enzyme; SHP-1 Δ41, 41 amino acids deleted at the COOH-terminus; SHP-1 R32K, inactivated NH₂-terminal SH2 domain; SHP-1 R138K, inactivated COOH-terminal SH2 domain. (C) Denatured and partially renatured anti-Ros immunoprecipitates containing wild-type TrkA-Ros or point mutants were used in GST pull-down assays using the isolated SH2 domains of SHP-1 as a GST fusion protein or GST alone (sequential immuno- and affinity precipitation). Expression controls for the different TrkA-Ros variants are shown in the bottom gel. (D) SHP-1 WT and SHP-1 C455S were coexpressed with TrkA-Ros in HEK293 cells, and the cells were stimulated with NGFβ. Lysate aliquots were used to analyze the phosphotyrosine content by SDS-PAGE and immunoblotting. (E) Effect of different amounts of SHP-1 on Ros-dependent tyrosine phosphorylation and Erk activation. TrkA-Ros (wild-type) and TrkA-Ros Y2267F were coexpressed with different amounts of SHP-1 (expression plasmid amounts indicated). Total cell lysates were evaluated for tyrosine phosphorylation and activity of endogenous Erk by immunoblotting with anti-phosphotyrosine (anti-PY) and anti–phospho-Erk (pErk) antibodies. Quantification of Ros tyrosine phosphorylation and phospho-Erk signals is depicted in the graph. Consistent results were obtained in three independent experiments.

Epididymal differentiation is impaired in homozygous me^v mice. Paraffin sections of the epididymis of heterozygous, me^v/me^v, and Ros^−/− mice were stained with hematoxylin/eosin. Depicted sections represent the proximal segment of the epididymal caput at a magnification of 64×. Bars, 30 μm.

SHP-1 affects Ros signaling in vivo. me^v/me^v mice or heterozygous control animals were challenged with peroxovanadate (POV) or mock treated (−). The epididymis was prepared and the tissue was lysed. Lysates were used to analyze the phosphotyrosine content (left gel, anti-PY) by immunoblotting. In addition, lysates were used for immunoprecipitation (IP) of Ros and for the subsequent analysis of Ros phosphorylation, by immunoblotting (right gel). The data are representative of three independent experiments with consistent results.

SHP-1 inhibits Ros-dependent cell growth and transformation. (A) NIH3T3 fibroblasts, stably transfected with TrkA-Ros, were supertransfected with SHP-1 using a tetracycline-inducible expression system (Tet-off system). Cells cultured in the absence or presence of ATc were stimulated with different concentrations of NGFβ. TrkA-Ros was immunoprecipitated and the tyrosine phosphorylation was monitored by immunoblotting. (B) The growth of the cells in the absence or presence of ATc and in the absence or presence of NGFβ was analyzed. The results are represented as fold growth after 8 d and compared to the seeded number of cells (mean of triplicates). (C) To analyze focus formation, cell monolayers were grown in the absence or presence of ATc and in the absence or presence of NGFβ for 12 d. Then, the monolayers were stained with crystal violet dye. In an alternative type of assay, we tested focus formation in the presence of an excess of parental NIH3T3 cells. When 20 transfected cells were mixed with 10⁶ parental cells, the following numbers of foci per dish were observed in a typical experiment: +ATc, +NGFβ: 9; +ATc, −NGFβ: 2; −ATc, −NGFβ: 0; and −ATc, + NGFβ: 0.