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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 affects Ros signaling in vivo. mev/mev 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.
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Figure 3: SHP-1 affects Ros signaling in vivo. mev/mev 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.

Mentions: The overlapping expression of SHP-1 and Ros prompted us to test whether impairment of SHP-1 activity in mev/mev mice might affect Ros signaling. We analyzed tyrosine phosphorylation of immunoprecipitated Ros from the epididymis of mev/mevmice. Ros from mev/mevmice displayed an elevated tyrosine phosphorylation compared to Ros from heterozygous animals (Fig. 3), indicating that SHP-1 is capable of downregulating Ros signaling in the epithelium of the epididymis in vivo. Intraperitoneal injection of peroxovanadate rapidly triggers tyrosine phosphorylation in multiple murine tissues (Ruff et al. 1997). After treatment with peroxovanadate, a dramatic increase in overall tyrosyl phosphorylation was detected in epididymal extracts from mev/mevmice, which was less pronounced in heterozygous mice (Fig. 3). In particular, phosphorylation of Ros was strongly elevated in peroxovanadate-treated homozygous, but not in heterozygous mevmice. Peroxovanadate is an effective inhibitor of many PTPs, but a relatively weak inhibitor of SHP-1 (Wetzker, M., and F.D. Böhmer, unpublished data). We assume that partial inhibition of SHP-1 by the pervanadate treatment eliminates the residual activity of SHP-1 in the mev/mevmice, but leaves substantial activity in the heterozygous mice intact, resulting in a further enhancement of the difference in Ros phosphorylation level.


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 affects Ros signaling in vivo. mev/mev 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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199605&req=5

Figure 3: SHP-1 affects Ros signaling in vivo. mev/mev 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.
Mentions: The overlapping expression of SHP-1 and Ros prompted us to test whether impairment of SHP-1 activity in mev/mev mice might affect Ros signaling. We analyzed tyrosine phosphorylation of immunoprecipitated Ros from the epididymis of mev/mevmice. Ros from mev/mevmice displayed an elevated tyrosine phosphorylation compared to Ros from heterozygous animals (Fig. 3), indicating that SHP-1 is capable of downregulating Ros signaling in the epithelium of the epididymis in vivo. Intraperitoneal injection of peroxovanadate rapidly triggers tyrosine phosphorylation in multiple murine tissues (Ruff et al. 1997). After treatment with peroxovanadate, a dramatic increase in overall tyrosyl phosphorylation was detected in epididymal extracts from mev/mevmice, which was less pronounced in heterozygous mice (Fig. 3). In particular, phosphorylation of Ros was strongly elevated in peroxovanadate-treated homozygous, but not in heterozygous mevmice. Peroxovanadate is an effective inhibitor of many PTPs, but a relatively weak inhibitor of SHP-1 (Wetzker, M., and F.D. Böhmer, unpublished data). We assume that partial inhibition of SHP-1 by the pervanadate treatment eliminates the residual activity of SHP-1 in the mev/mevmice, but leaves substantial activity in the heterozygous mice intact, resulting in a further enhancement of the difference in Ros phosphorylation level.

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