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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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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).
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Figure 4: 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).

Mentions: Constructs and the method for determining the interaction strength of a panel of receptor tyrosine kinase cytoplasmic domains with various molecules containing phosphotyrosine interaction domains were described earlier (Weidner et al. 1996; Bai et al. 1998; Tamura et al. 1999; Vayssiere et al. 2000). Interaction strength was assigned qualitatively by visual inspection of yeast growth in the appropriate selection medium compared to other interaction partners as “strong,” “weak,” or “not detectable” (see Fig. 4 A and Table ). For quantitative β-galactosidase assays, cDNA fragments representing the entire cytoplasmic domain of human PDGFβ receptor (Claesson-Welsh et al. 1988; amino acid [aa] 558–1,106) and murine c-Ros (Riethmacher et al. 1994; aa 1,880–2,339) were cloned into the pLexA vector (CLONTECH Laboratories, Inc.). cDNA sequences corresponding to the tandem SH2 domains and hinge domain of human SHP-1 (aa 1–270) or SHP-2 (aa 1–271) were cloned into pB42AD (CLONTECH Laboratories, Inc.). Expression of all fusion proteins and autophosphorylation activity of kinase constructs were verified by immunoblotting. Expression constructs for the tested interaction partners were cotransformed into Saccharomyces cereviseae strain EY48 (CLONTECH Laboratories, Inc.), and individual colonies were spotted multiply on agar plates with “induction medium” (synthetic dropout medium, Gal, Raf, −His, −Trp, −Ura) containing X-gal (5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside). Growth was allowed for several hours, then the plates were scanned with a flat-bed scanner, and the mean intensity of color development was determined by densitometric evaluation with the program NIH Image 1.57. Signals in the presence of both interaction partners were corrected by subtraction of signals obtained with yeast transfected with expression constructs for the kinase baits and empty pB42AD.


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)

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).
© Copyright Policy
Related In: Results  -  Collection

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

Figure 4: 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).
Mentions: Constructs and the method for determining the interaction strength of a panel of receptor tyrosine kinase cytoplasmic domains with various molecules containing phosphotyrosine interaction domains were described earlier (Weidner et al. 1996; Bai et al. 1998; Tamura et al. 1999; Vayssiere et al. 2000). Interaction strength was assigned qualitatively by visual inspection of yeast growth in the appropriate selection medium compared to other interaction partners as “strong,” “weak,” or “not detectable” (see Fig. 4 A and Table ). For quantitative β-galactosidase assays, cDNA fragments representing the entire cytoplasmic domain of human PDGFβ receptor (Claesson-Welsh et al. 1988; amino acid [aa] 558–1,106) and murine c-Ros (Riethmacher et al. 1994; aa 1,880–2,339) were cloned into the pLexA vector (CLONTECH Laboratories, Inc.). cDNA sequences corresponding to the tandem SH2 domains and hinge domain of human SHP-1 (aa 1–270) or SHP-2 (aa 1–271) were cloned into pB42AD (CLONTECH Laboratories, Inc.). Expression of all fusion proteins and autophosphorylation activity of kinase constructs were verified by immunoblotting. Expression constructs for the tested interaction partners were cotransformed into Saccharomyces cereviseae strain EY48 (CLONTECH Laboratories, Inc.), and individual colonies were spotted multiply on agar plates with “induction medium” (synthetic dropout medium, Gal, Raf, −His, −Trp, −Ura) containing X-gal (5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside). Growth was allowed for several hours, then the plates were scanned with a flat-bed scanner, and the mean intensity of color development was determined by densitometric evaluation with the program NIH Image 1.57. Signals in the presence of both interaction partners were corrected by subtraction of signals obtained with yeast transfected with expression constructs for the kinase baits and empty pB42AD.

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