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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 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.
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Figure 2: 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.

Mentions: To assess whether SHP-1 might be relevant for epididymal function, we histologically examined the epididymis of male mev/mev mice and compared them to heterozygous controls. Macroscopically, the epididymis of mev/mev mice was smaller, though the overall shape appeared normal. This size reduction was proportional to the reduced body weight of the mev/mevmice. Histological sections revealed that the tall columnar epithelial cells, which form the tubules of the proximal segment in heterozygous animals, are replaced by considerably flatter epithelial cells in the epithelium of mev/mevmice (Fig. 1). In heterozygous animals, these epithelial cells are clearly structured. The nucleus is located above a clearly defined basal cytoplasmic zone. The cytoplasmic zone above the nucleus is broad and the apical surface is rough. In contrast, the nuclei in epithelial cells of mev/mevare often located closer to the basal membrane, and the apical surfaces appear smooth (Fig. 1). The epithelium in the more distal segments of the epididymis exhibited little differences in mutant and control animals (not shown). Histologically, the epithelium of the proximal segment in mev/mevmice has similarities to that of Ros−/− mice (Fig. 1). We also analyzed the epithelium of other organs. Epithelial cells in the intestine of mev/mev mice were smaller than their counterparts in heterozygous animals, but appeared fully differentiated. No differences between mev/mevand heterozygous animals were detectable in epithelia of stomach and pancreas (not shown). In summary, the epididymal epithelium of mev/mevmice exhibits signs of aberrant differentiation. This phenotype may be related to a loss of function of SHP-1 in the epididymal epithelial cells, since SHP-1 is clearly expressed in these cells (Fig. 2). In situ hybridization with an antisense RNA generated from a 1,184-bp fragment of murine SHP-1 cDNA revealed expression of SHP-1 mRNA in the epithelial cells surrounding the tubules of the initial epididymal segment (Fig. 2, A–E). More distal segments exhibit low level signals. Thus, SHP-1 expression mirrors the one of Ros in this part of the epididymis (Sonnenberg-Riethmacher et al. 1996). Strong SHP-1 expression is also seen in the corpus with highest levels in the most distal segments. These distal epithelia are devoid of Ros (Sonnenberg-Riethmacher et al. 1996). SHP-1 expression could also be detected by immunoblotting in the epididymis of mice (not shown); immunostaining failed with the available antibodies. However, in human epididymis, strong epithelial SHP-1 expression could be visualized by immunohistochemistry (Fig. 2F and Fig. G). Thus, SHP-1 is expressed in epithelial cells of the epididymis and exhibits an overlapping expression domain with Ros.


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 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.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2199605&req=5

Figure 2: 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.
Mentions: To assess whether SHP-1 might be relevant for epididymal function, we histologically examined the epididymis of male mev/mev mice and compared them to heterozygous controls. Macroscopically, the epididymis of mev/mev mice was smaller, though the overall shape appeared normal. This size reduction was proportional to the reduced body weight of the mev/mevmice. Histological sections revealed that the tall columnar epithelial cells, which form the tubules of the proximal segment in heterozygous animals, are replaced by considerably flatter epithelial cells in the epithelium of mev/mevmice (Fig. 1). In heterozygous animals, these epithelial cells are clearly structured. The nucleus is located above a clearly defined basal cytoplasmic zone. The cytoplasmic zone above the nucleus is broad and the apical surface is rough. In contrast, the nuclei in epithelial cells of mev/mevare often located closer to the basal membrane, and the apical surfaces appear smooth (Fig. 1). The epithelium in the more distal segments of the epididymis exhibited little differences in mutant and control animals (not shown). Histologically, the epithelium of the proximal segment in mev/mevmice has similarities to that of Ros−/− mice (Fig. 1). We also analyzed the epithelium of other organs. Epithelial cells in the intestine of mev/mev mice were smaller than their counterparts in heterozygous animals, but appeared fully differentiated. No differences between mev/mevand heterozygous animals were detectable in epithelia of stomach and pancreas (not shown). In summary, the epididymal epithelium of mev/mevmice exhibits signs of aberrant differentiation. This phenotype may be related to a loss of function of SHP-1 in the epididymal epithelial cells, since SHP-1 is clearly expressed in these cells (Fig. 2). In situ hybridization with an antisense RNA generated from a 1,184-bp fragment of murine SHP-1 cDNA revealed expression of SHP-1 mRNA in the epithelial cells surrounding the tubules of the initial epididymal segment (Fig. 2, A–E). More distal segments exhibit low level signals. Thus, SHP-1 expression mirrors the one of Ros in this part of the epididymis (Sonnenberg-Riethmacher et al. 1996). Strong SHP-1 expression is also seen in the corpus with highest levels in the most distal segments. These distal epithelia are devoid of Ros (Sonnenberg-Riethmacher et al. 1996). SHP-1 expression could also be detected by immunoblotting in the epididymis of mice (not shown); immunostaining failed with the available antibodies. However, in human epididymis, strong epithelial SHP-1 expression could be visualized by immunohistochemistry (Fig. 2F and Fig. G). Thus, SHP-1 is expressed in epithelial cells of the epididymis and exhibits an overlapping expression domain with Ros.

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