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Negative regulation of Ros receptor tyrosine kinase signaling. An epithelial function of the SH2 domain protein tyrosine phosphatase SHP-1.

Keilhack H, Müller M, Böhmer SA, Frank C, Weidner KM, Birchmeier W, Ligensa T, Berndt A, Kosmehl H, Günther B, Müller T, Birchmeier C, Böhmer FD - J. Cell Biol. (2001)

Bottom Line: Strong binding of SHP-1 to Ros is selective compared to six other receptor tyrosine kinases.Overexpression of SHP-1 results in Ros dephosphorylation and effectively downregulates Ros-dependent proliferation and transformation.We propose that SHP-1 is an important downstream regulator of Ros signaling.

View Article: PubMed Central - PubMed

Affiliation: Research Unit, Molecular Cell Biology, D-07747 Jena, Germany.

ABSTRACT
Male "viable motheaten" (me(v)) mice, with a naturally occurring mutation in the gene of the SH2 domain protein tyrosine phosphatase SHP-1, are sterile. Known defects in sperm maturation in these mice correlate with an impaired differentiation of the epididymis, which has similarities to the phenotype of mice with a targeted inactivation of the Ros receptor tyrosine kinase. Ros and SHP-1 are coexpressed in epididymal epithelium, and elevated phosphorylation of Ros in the epididymis of me(v) mice suggests that Ros signaling is under control of SHP-1 in vivo. Phosphorylated Ros strongly and directly associates with SHP-1 in yeast two-hybrid, glutathione S-transferase pull-down, and coimmunoprecipitation experiments. Strong binding of SHP-1 to Ros is selective compared to six other receptor tyrosine kinases. The interaction is mediated by the SHP-1 NH(2)-terminal SH2 domain and Ros phosphotyrosine 2267. Overexpression of SHP-1 results in Ros dephosphorylation and effectively downregulates Ros-dependent proliferation and transformation. We propose that SHP-1 is an important downstream regulator of Ros signaling.

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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.
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Figure 6: 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.

Mentions: Proliferation of stably TrkA-Ros–expressing NIH3T3 fibroblasts can be stimulated with NGFβ; upon prolonged NGFβ stimulation, the cells assume a transformed phenotype (Riethmacher et al. 1994). We established derivatives of TrkA-Ros–expressing NIH3T3 cells, which express SHP-1 in a tetracycline-regulated manner. Cell clones containing the inducible SHP-1 expression vector were isolated and tested for the effect of SHP-1 on the TrkA-Ros phosphorylation. To induce or suppress SHP-1 expression, cells were cultured in the absence or presence of ATc. After stimulation with different NGFβ concentrations (Fig. 6 A), phosphorylation of TrkA-Ros was monitored. SHP-1 expression led to a reduction of the NGFβ-stimulated tyrosine phosphorylation of TrkA-Ros. Thus, as in HEK293 cells, TrkA-Ros is an efficient substrate for SHP-1 in NIH3T3 cells.


Negative regulation of Ros receptor tyrosine kinase signaling. An epithelial function of the SH2 domain protein tyrosine phosphatase SHP-1.

Keilhack H, Müller M, Böhmer SA, Frank C, Weidner KM, Birchmeier W, Ligensa T, Berndt A, Kosmehl H, Günther B, Müller T, Birchmeier C, Böhmer FD - J. Cell Biol. (2001)

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.
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Related In: Results  -  Collection

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Figure 6: 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.
Mentions: Proliferation of stably TrkA-Ros–expressing NIH3T3 fibroblasts can be stimulated with NGFβ; upon prolonged NGFβ stimulation, the cells assume a transformed phenotype (Riethmacher et al. 1994). We established derivatives of TrkA-Ros–expressing NIH3T3 cells, which express SHP-1 in a tetracycline-regulated manner. Cell clones containing the inducible SHP-1 expression vector were isolated and tested for the effect of SHP-1 on the TrkA-Ros phosphorylation. To induce or suppress SHP-1 expression, cells were cultured in the absence or presence of ATc. After stimulation with different NGFβ concentrations (Fig. 6 A), phosphorylation of TrkA-Ros was monitored. SHP-1 expression led to a reduction of the NGFβ-stimulated tyrosine phosphorylation of TrkA-Ros. Thus, as in HEK293 cells, TrkA-Ros is an efficient substrate for SHP-1 in NIH3T3 cells.

Bottom Line: Strong binding of SHP-1 to Ros is selective compared to six other receptor tyrosine kinases.Overexpression of SHP-1 results in Ros dephosphorylation and effectively downregulates Ros-dependent proliferation and transformation.We propose that SHP-1 is an important downstream regulator of Ros signaling.

View Article: PubMed Central - PubMed

Affiliation: Research Unit, Molecular Cell Biology, D-07747 Jena, Germany.

ABSTRACT
Male "viable motheaten" (me(v)) mice, with a naturally occurring mutation in the gene of the SH2 domain protein tyrosine phosphatase SHP-1, are sterile. Known defects in sperm maturation in these mice correlate with an impaired differentiation of the epididymis, which has similarities to the phenotype of mice with a targeted inactivation of the Ros receptor tyrosine kinase. Ros and SHP-1 are coexpressed in epididymal epithelium, and elevated phosphorylation of Ros in the epididymis of me(v) mice suggests that Ros signaling is under control of SHP-1 in vivo. Phosphorylated Ros strongly and directly associates with SHP-1 in yeast two-hybrid, glutathione S-transferase pull-down, and coimmunoprecipitation experiments. Strong binding of SHP-1 to Ros is selective compared to six other receptor tyrosine kinases. The interaction is mediated by the SHP-1 NH(2)-terminal SH2 domain and Ros phosphotyrosine 2267. Overexpression of SHP-1 results in Ros dephosphorylation and effectively downregulates Ros-dependent proliferation and transformation. We propose that SHP-1 is an important downstream regulator of Ros signaling.

Show MeSH
Related in: MedlinePlus