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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 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.
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Figure 5: 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.

Mentions: 293 cells were transfected with expression constructs for TrkA-Ros or TrkA-Ros mutants, SHP-1, SHP-1 CS, or empty vector using calcium phosphate coprecipitation (Lammers et al. 1993). For coimmunoprecipitation, 10 μg pRK5RS SHP-1 or SHP-1 C455S DNA was cotransfected with 10 μg pcDNA3 TrkA-Ros DNA per 10-cm dish. For assessment of dephosphorylation, 0.5 μg pcDNA3 TrkA-Ros was cotransfected with 3.5 μg pRK5RS SHP-1, pcDNA3 SHP-1 C455S, or empty vector in 6-well plates. For titration of SHP-1 efficacy, variable amounts of pRK5RS SHP-1 were used (see the legend to Fig. 5 E). After transfection, the cells were stimulated with 50 ng/ml NGFβ or vehicle for 10 min and lysed in 700 (10-cm dish) or 200 μl (6-well plate) lysis buffer (20 mM Hepes, pH 7.4, 150 mM NaCl, 2 mM EDTA, 2 mM EGTA, 20 μM zinc acetate, 50 mM NaF, 10 mM NaPP, 1 mM Na3VO4, 1% Triton X-100, 1 mM PMSF, 1 μg/ml pepstatin A, 2 μg/ml aprotinin, 10 μg/ml leupeptin). The lysates were clarified by centrifugation at 25,000 g and 4°C for 20 min. For coimmunoprecipitation, 10 μl anti-Ros antiserum or 1 μg monoclonal anti–SHP-1 antibody were used. Immunoprecipitation was carried out as described, and associated proteins were analyzed by SDS-PAGE and immunoblotting. For dephosphorylation assays, lysate aliquots were resolved by SDS-PAGE, and the tyrosyl phosphorylation or activation of endogenous Erk was visualized by immunoblotting.


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 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.
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Related In: Results  -  Collection

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Figure 5: 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.
Mentions: 293 cells were transfected with expression constructs for TrkA-Ros or TrkA-Ros mutants, SHP-1, SHP-1 CS, or empty vector using calcium phosphate coprecipitation (Lammers et al. 1993). For coimmunoprecipitation, 10 μg pRK5RS SHP-1 or SHP-1 C455S DNA was cotransfected with 10 μg pcDNA3 TrkA-Ros DNA per 10-cm dish. For assessment of dephosphorylation, 0.5 μg pcDNA3 TrkA-Ros was cotransfected with 3.5 μg pRK5RS SHP-1, pcDNA3 SHP-1 C455S, or empty vector in 6-well plates. For titration of SHP-1 efficacy, variable amounts of pRK5RS SHP-1 were used (see the legend to Fig. 5 E). After transfection, the cells were stimulated with 50 ng/ml NGFβ or vehicle for 10 min and lysed in 700 (10-cm dish) or 200 μl (6-well plate) lysis buffer (20 mM Hepes, pH 7.4, 150 mM NaCl, 2 mM EDTA, 2 mM EGTA, 20 μM zinc acetate, 50 mM NaF, 10 mM NaPP, 1 mM Na3VO4, 1% Triton X-100, 1 mM PMSF, 1 μg/ml pepstatin A, 2 μg/ml aprotinin, 10 μg/ml leupeptin). The lysates were clarified by centrifugation at 25,000 g and 4°C for 20 min. For coimmunoprecipitation, 10 μl anti-Ros antiserum or 1 μg monoclonal anti–SHP-1 antibody were used. Immunoprecipitation was carried out as described, and associated proteins were analyzed by SDS-PAGE and immunoblotting. For dephosphorylation assays, lysate aliquots were resolved by SDS-PAGE, and the tyrosyl phosphorylation or activation of endogenous Erk was visualized by immunoblotting.

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