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

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.

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.

Figure 2

SHPTP2 activity in eosinophils stimulated with IL-5. Eosinophils were stimulated with IL-5 for 0, 1, 5, and 30 min at 37°C. Cell lysates were immunoprecipitated with anti-SHPTP2 antibody, and immunoprecipitates were assayed for phosphatase activity using para-nitrophenyl phosphate as a substrate. Results are the means ± SD of three experiments. *Significantly different from the control at P <0.05 (Student's t test).

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.

Figure 4

Coimmunoprecipitation of SHPTP2 and IL-5β_cR in IL-5– stimulated eosinophils. (A) Eosinophil lysates were immunoprecipitated with a polyclonal anti-SHPTP2 antibody and subjected to Western blot analysis with anti-SHPTP2 (left) and anti–IL-5β_cR (right) antibodies. The band of IL-5β_cR was present in the immunoprecipitate of SHPTP2 obtained from IL-5–stimulated cells. (B) Anti–IL-5β_cR was used to immunoprecipitate lysates from eosinophils treated with (+) and without (−) IL-5 (5 min). The resulting immunoblot was screened with anti–IL-5β_cR antibody showing the presence of equal amounts of the receptor and with anti-SHPTP2 antibody revealing the presence of phosphatase in immunoprecipitate of IL-5β_cR from stimulated eosinophils. Different intensity of IgH is due to different isotypes of Abs used for immunoprecipitation reacting with Western blotting secondary Ab.

Figure 5

The binding of SHPTP2 to pY⁶¹² peptide. We synthesized a phosphotyrosine-based ITIM motif-containing peptide (peptide 1) derived from IL-5β_cR (residues 605–624). The controls were a nonphosphorylated F⁶¹² peptide (peptide 2) and a β_c 450–465 peptide (peptide 3). The peptides were biotinylated and used in a peptide binding assay using the HL-60 cell lysate. The bound proteins were precipitated with streptavidin-agarose, followed by immunoblotting with anti-SHPTP2. The first lane, (L, cell lysate) shows the position of the 70-kD SHPTP2. The identity of the upper band is unknown. The buffer control is shown in the last lane (0).

Figure 6

SHPTP2 activity is stimulated by pY⁶¹² peptide. HL-60 cell lysates were incubated with the control F⁶¹² (peptide 1) and phosphorylated pY⁶¹² peptide (peptide 2), and then immunoprecipitated with anti-SHPTP2 and subjected for phosphatase assay. Shown are the means ± SD of six independent determinations from two separate experiments. *Significantly different from the control (peptide 1) at P <0.05 (Student's t test).

Figure 7

Effect of SHPTP2 oligodeoxynucleotides on SHPTP2 and Erk2 expression. Whole cell lysates were prepared from eosinophils treated with 7.5 μM SHPTP2 antisense (AS), sense (SS), and nonsense (NS) ODNs or medium (0 ) for 6 h. The lysates with 10 μg of protein/lane were resolved by SDS-PAGE. Anti-SHPTP2 and anti–Erk-2 Abs were used in Western blot analysis to assess expression of proteins. Pretreatment with SHPTP2 antisense ODN, but not with SS or NS, significantly decreased expression of SHPTP2 in eosinophils. Lower p42 band shows equal amounts of Erk2 kinases not affected by treatment with ODNs.

Figure 8

SHPTP2 antisense oligonucleotides block the survival promoting effect of IL-5. Eosinophils were cultured in the presence of 7.5 μM SHPTP2 antisense (AS), sense (SS), and nonsense (NS) ODNs or medium for 6 h. Cells were then further incubated with or without IL-5 (10⁻¹⁰ M) for 2 h, and then washed and incubated without ODNs and IL-5 for an additional 24 h. No inhibition of eosinophil death by IL-5 was observed in SHPTP2 AS-treated cells. In contrast, SHPTP2 SS- and NS-treated cells demonstrated a significant prolongation of eosinophil survival by IL-5. None of the ODNs at 7.5 μM concentration was toxic to eosinophils. Shown are the means ± SD of five independent experiments. *Significantly different from the cells not treated with IL-5 at P <0.05 (Student's t test).

Figure 9

SHPTP2 antisense oligodeoxynucleotides inhibit IL-5– induced tyrosine phosphorylation of MAP/Erk2 kinase. Eosinophils were cultured in the presence of 7.5 μM SHPTP2 antisense (AS) or sense (SS) ODNs for 6 h, and then stimulated with IL-5 for 10 min. The cell lysates were immunoprecipitated with anti-Erk2 Ab, resolved by SDS-PAGE, and immunoblotted by an antiphosphotyrosine mAb (left) followed by reprobing with anti-Erk-2 Ab (right). An increase in tyrosine phosphorylation of Erk2 was observed in eosinophils pretreated with sense ODN, whereas pretreatment with antisense SHPTP2 significantly inhibited IL-5–induced phosphorylation of MAP/Erk2 kinase. The blot is representative of three independent experiments.