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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.

Show MeSH
IL-5 induces tyrosine phosphorylation of SHPTP2 in eosinophils. Eosinophils were incubated with IL-5 as indicated for 1, 5, and 30  min. Cells were then lysed, and the cell lysates were immunoprecipitated  (IP) with anti-SHPTP2 antibody. The membranes were Western blotted  (WB) with antiphosphotyrosine (left) and anti-SHPTP2 (right) antibodies.  The left panel shows tyrosine-phosphorylated SHPTP2 bands in stimulated eosinophils. The right panel confirms the position and amount of  SHPTP2. The bottom thick bands are due to the rabbit IgH in the immunoprecipitation.
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Figure 1: IL-5 induces tyrosine phosphorylation of SHPTP2 in eosinophils. Eosinophils were incubated with IL-5 as indicated for 1, 5, and 30 min. Cells were then lysed, and the cell lysates were immunoprecipitated (IP) with anti-SHPTP2 antibody. The membranes were Western blotted (WB) with antiphosphotyrosine (left) and anti-SHPTP2 (right) antibodies. The left panel shows tyrosine-phosphorylated SHPTP2 bands in stimulated eosinophils. The right panel confirms the position and amount of SHPTP2. The bottom thick bands are due to the rabbit IgH in the immunoprecipitation.

Mentions: It has been reported that the tyrosine phosphorylation of SHPTP2 is associated with its increased phosphatase activity (19). Therefore, we first tested whether IL-5 induced tyrosine phosphorylation of SHPTP2. In this analysis, eosinophils were stimulated with IL-5 for various time periods and then lysed and immunoprecipitated with a polyclonal anti-SHPTP2 antibody. Western blotting with an antiphosphotyrosine antibody revealed phosphorylation of SHPTP2 as early as 1 min, reaching the highest level 5 min after the stimulation (Fig. 1). This suggests a relatively early involvement of SHPTP2 in the IL-5 signaling pathway. Reprobing the membrane with the anti-SHPTP2 antibody confirmed the presence of equal amounts of the immunoprecipitated protein.


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)

IL-5 induces tyrosine phosphorylation of SHPTP2 in eosinophils. Eosinophils were incubated with IL-5 as indicated for 1, 5, and 30  min. Cells were then lysed, and the cell lysates were immunoprecipitated  (IP) with anti-SHPTP2 antibody. The membranes were Western blotted  (WB) with antiphosphotyrosine (left) and anti-SHPTP2 (right) antibodies.  The left panel shows tyrosine-phosphorylated SHPTP2 bands in stimulated eosinophils. The right panel confirms the position and amount of  SHPTP2. The bottom thick bands are due to the rabbit IgH in the immunoprecipitation.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199030&req=5

Figure 1: IL-5 induces tyrosine phosphorylation of SHPTP2 in eosinophils. Eosinophils were incubated with IL-5 as indicated for 1, 5, and 30 min. Cells were then lysed, and the cell lysates were immunoprecipitated (IP) with anti-SHPTP2 antibody. The membranes were Western blotted (WB) with antiphosphotyrosine (left) and anti-SHPTP2 (right) antibodies. The left panel shows tyrosine-phosphorylated SHPTP2 bands in stimulated eosinophils. The right panel confirms the position and amount of SHPTP2. The bottom thick bands are due to the rabbit IgH in the immunoprecipitation.
Mentions: It has been reported that the tyrosine phosphorylation of SHPTP2 is associated with its increased phosphatase activity (19). Therefore, we first tested whether IL-5 induced tyrosine phosphorylation of SHPTP2. In this analysis, eosinophils were stimulated with IL-5 for various time periods and then lysed and immunoprecipitated with a polyclonal anti-SHPTP2 antibody. Western blotting with an antiphosphotyrosine antibody revealed phosphorylation of SHPTP2 as early as 1 min, reaching the highest level 5 min after the stimulation (Fig. 1). This suggests a relatively early involvement of SHPTP2 in the IL-5 signaling pathway. Reprobing the membrane with the anti-SHPTP2 antibody confirmed the presence of equal amounts of the immunoprecipitated protein.

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