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Src homology 2 protein tyrosine phosphatase (SHPTP2)/Src homology 2 phosphatase 2 (SHP2) tyrosine phosphatase is a positive regulator of the interleukin 5 receptor signal transduction pathways leading to the prolongation of eosinophil survival.

Pazdrak K, Adachi T, Alam R - J. Exp. Med. (1997)

Bottom Line: We found that IL-5 produced a rapid activation and tyrosine phosphorylation of SHPTP2 within 1 min.The association of SHPTP2 with IL-5betacR was reconstituted using a synthetic phosphotyrosine-containing peptide, betac 605-624, encompassing tyrosine (Y)612.The binding to the phosphotyrosine-containing peptide increased the phosphatase activity of SHPTP2, whereas the same peptide with the phosphorylated Y612--> F mutation did not activate SHPTP2.

View Article: PubMed Central - PubMed

Affiliation: University of Texas Medical Branch, Department of Internal Medicine, Allergy and Immunology Division, Galveston, TX 77555-0762, USA.

ABSTRACT
Interleukin-5 (IL-5) regulates the growth and function of eosinophils. It induces rapid tyrosine phosphorylation of Lyn and Jak2 tyrosine kinases. The role of tyrosine phosphatases in IL-5 signal transduction has not been investigated. In this study, we provide first evidence that SH2 protein tyrosine phosphatase 2 (SHPTP2) phosphotyrosine phosphatase plays a key role in prevention of eosinophil death by IL-5. We found that IL-5 produced a rapid activation and tyrosine phosphorylation of SHPTP2 within 1 min. The tyrosine phosphorylated SHPTP2 was complexed with the adapter protein Grb2 in IL-5-stimulated eosinophils. Furthermore, SHPTP2 appeared to physically associate with beta common (betac) chain of the IL-5 receptor (IL-5betacR). The association of SHPTP2 with IL-5betacR was reconstituted using a synthetic phosphotyrosine-containing peptide, betac 605-624, encompassing tyrosine (Y)612. The binding to the phosphotyrosine-containing peptide increased the phosphatase activity of SHPTP2, whereas the same peptide with the phosphorylated Y612--> F mutation did not activate SHPTP2. Only SHPTP2 antisense oligonucleotides, but not sense SHPTP2, could inhibit tyrosine phosphorylation of microtubule-associated protein kinase, and reverse the eosinophil survival advantage provided by IL-5. Therefore, we conclude that the physical association of SHPTP2 with the phosphorylated betac receptor and Grb2 and its early activation are required for the coupling of the receptor to the Ras signaling pathway and for prevention of eosinophil death by IL-5.

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Coimmunoprecipitation of SHPTP2 and Grb2 in IL-5–stimulated eosinophils. (A) Cell lysates from IL-5–stimulated and unstimulated  eosinophils were immunoprecipitated (IP) with anti-Grb2 antibody. IL-5  (−) and (+) indicate cells incubated with medium or IL-5 for 5 min, respectively. Immunoblotting with antiphosphotyrosine antibody revealed a  70-kD protein, suggesting the presence of SHPTP2 in the immunoprecipitate of Grb2 (left). Western blotting with anti-SHPTP2 antibody confirmed the identity of the phosphatase. (B) Eosinophils lysates were immunoprecipitated with anti-Grb2 antibody and anti-SHPTP2 antibody.  Western blotting with anti-Grb2 antibody revealed the presence of Grb2  in its own immunoprecipitates as well as in immunoprecipitates of  SHPTP2 from IL-5–stimulated eosinophils.
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Figure 3: Coimmunoprecipitation of SHPTP2 and Grb2 in IL-5–stimulated eosinophils. (A) Cell lysates from IL-5–stimulated and unstimulated eosinophils were immunoprecipitated (IP) with anti-Grb2 antibody. IL-5 (−) and (+) indicate cells incubated with medium or IL-5 for 5 min, respectively. Immunoblotting with antiphosphotyrosine antibody revealed a 70-kD protein, suggesting the presence of SHPTP2 in the immunoprecipitate of Grb2 (left). Western blotting with anti-SHPTP2 antibody confirmed the identity of the phosphatase. (B) Eosinophils lysates were immunoprecipitated with anti-Grb2 antibody and anti-SHPTP2 antibody. Western blotting with anti-Grb2 antibody revealed the presence of Grb2 in its own immunoprecipitates as well as in immunoprecipitates of SHPTP2 from IL-5–stimulated eosinophils.

Mentions: As shown in Fig. 3 A, we detected a band of a 70-kD tyrosine phosphorylated protein in the immunoprecipitate of Grb2 from IL-5–stimulated cells. Reprobing the same membrane with the anti-SHPTP2 antibody confirmed the presence of SHPTP2 in immunoprecipitate of Grb2 (Fig. 3 A). In a next set of experiments, we looked for the presence of Grb2 in the immunoprecipitates of SHPTP2. As shown in Fig. 3 B, Grb2 was detectable in the anti-SHPTP2 immunoprecipitates and predictably, in the anti-Grb2 immunoprecipitate from IL-5–stimulated eosinophils. These experiments suggest a physical association of SHPTP2 and Grb2 that occurs in an IL-5–dependent manner.


Src homology 2 protein tyrosine phosphatase (SHPTP2)/Src homology 2 phosphatase 2 (SHP2) tyrosine phosphatase is a positive regulator of the interleukin 5 receptor signal transduction pathways leading to the prolongation of eosinophil survival.

Pazdrak K, Adachi T, Alam R - J. Exp. Med. (1997)

Coimmunoprecipitation of SHPTP2 and Grb2 in IL-5–stimulated eosinophils. (A) Cell lysates from IL-5–stimulated and unstimulated  eosinophils were immunoprecipitated (IP) with anti-Grb2 antibody. IL-5  (−) and (+) indicate cells incubated with medium or IL-5 for 5 min, respectively. Immunoblotting with antiphosphotyrosine antibody revealed a  70-kD protein, suggesting the presence of SHPTP2 in the immunoprecipitate of Grb2 (left). Western blotting with anti-SHPTP2 antibody confirmed the identity of the phosphatase. (B) Eosinophils lysates were immunoprecipitated with anti-Grb2 antibody and anti-SHPTP2 antibody.  Western blotting with anti-Grb2 antibody revealed the presence of Grb2  in its own immunoprecipitates as well as in immunoprecipitates of  SHPTP2 from IL-5–stimulated eosinophils.
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Related In: Results  -  Collection

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Figure 3: Coimmunoprecipitation of SHPTP2 and Grb2 in IL-5–stimulated eosinophils. (A) Cell lysates from IL-5–stimulated and unstimulated eosinophils were immunoprecipitated (IP) with anti-Grb2 antibody. IL-5 (−) and (+) indicate cells incubated with medium or IL-5 for 5 min, respectively. Immunoblotting with antiphosphotyrosine antibody revealed a 70-kD protein, suggesting the presence of SHPTP2 in the immunoprecipitate of Grb2 (left). Western blotting with anti-SHPTP2 antibody confirmed the identity of the phosphatase. (B) Eosinophils lysates were immunoprecipitated with anti-Grb2 antibody and anti-SHPTP2 antibody. Western blotting with anti-Grb2 antibody revealed the presence of Grb2 in its own immunoprecipitates as well as in immunoprecipitates of SHPTP2 from IL-5–stimulated eosinophils.
Mentions: As shown in Fig. 3 A, we detected a band of a 70-kD tyrosine phosphorylated protein in the immunoprecipitate of Grb2 from IL-5–stimulated cells. Reprobing the same membrane with the anti-SHPTP2 antibody confirmed the presence of SHPTP2 in immunoprecipitate of Grb2 (Fig. 3 A). In a next set of experiments, we looked for the presence of Grb2 in the immunoprecipitates of SHPTP2. As shown in Fig. 3 B, Grb2 was detectable in the anti-SHPTP2 immunoprecipitates and predictably, in the anti-Grb2 immunoprecipitate from IL-5–stimulated eosinophils. These experiments suggest a physical association of SHPTP2 and Grb2 that occurs in an IL-5–dependent manner.

Bottom Line: We found that IL-5 produced a rapid activation and tyrosine phosphorylation of SHPTP2 within 1 min.The association of SHPTP2 with IL-5betacR was reconstituted using a synthetic phosphotyrosine-containing peptide, betac 605-624, encompassing tyrosine (Y)612.The binding to the phosphotyrosine-containing peptide increased the phosphatase activity of SHPTP2, whereas the same peptide with the phosphorylated Y612--> F mutation did not activate SHPTP2.

View Article: PubMed Central - PubMed

Affiliation: University of Texas Medical Branch, Department of Internal Medicine, Allergy and Immunology Division, Galveston, TX 77555-0762, USA.

ABSTRACT
Interleukin-5 (IL-5) regulates the growth and function of eosinophils. It induces rapid tyrosine phosphorylation of Lyn and Jak2 tyrosine kinases. The role of tyrosine phosphatases in IL-5 signal transduction has not been investigated. In this study, we provide first evidence that SH2 protein tyrosine phosphatase 2 (SHPTP2) phosphotyrosine phosphatase plays a key role in prevention of eosinophil death by IL-5. We found that IL-5 produced a rapid activation and tyrosine phosphorylation of SHPTP2 within 1 min. The tyrosine phosphorylated SHPTP2 was complexed with the adapter protein Grb2 in IL-5-stimulated eosinophils. Furthermore, SHPTP2 appeared to physically associate with beta common (betac) chain of the IL-5 receptor (IL-5betacR). The association of SHPTP2 with IL-5betacR was reconstituted using a synthetic phosphotyrosine-containing peptide, betac 605-624, encompassing tyrosine (Y)612. The binding to the phosphotyrosine-containing peptide increased the phosphatase activity of SHPTP2, whereas the same peptide with the phosphorylated Y612--> F mutation did not activate SHPTP2. Only SHPTP2 antisense oligonucleotides, but not sense SHPTP2, could inhibit tyrosine phosphorylation of microtubule-associated protein kinase, and reverse the eosinophil survival advantage provided by IL-5. Therefore, we conclude that the physical association of SHPTP2 with the phosphorylated betac receptor and Grb2 and its early activation are required for the coupling of the receptor to the Ras signaling pathway and for prevention of eosinophil death by IL-5.

Show MeSH