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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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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).
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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).

Mentions: Since eosinophils are terminally differentiated cells with life spans of ∼4 d, the use of antisense oligodeoxynucleotides is the most practical method to specifically alter expression of SHPTP2. Eosinophils were incubated with ODNs without FCS to protect stability of ODNs. As demonstrated in Fig. 7, eosinophils exposed to 7.5 μM antisense ODN for 6 h expressed little or no detectable SHPTP2, whereas sense or nonsense ODNs did not alter SHPTP2 level. Antisense ODNs used in our assay did not alter expression of a closely related phosphatase SHPTP1 (data not shown). The viability of eosinophils assessed at this time (immediately before stimulation with IL-5) always exceeded 90% and was not different from control samples, indicating that at concentration of 7.5 μM the ODNs were not toxic to the cells. After 2 h of stimulation, eosinophils were resuspended in medium without IL-5, ODNs, and FCS. We excluded FCS from this stage of experiment, since in a previous study by Inhorn et al., serum appeared to alleviate the requirement for the Ras pathway for GM-CSF–dependent viability and proliferation in mutant cells with β receptor lacking the 626–763 amino acid residues (23). Viability of the cells cultured without serum was always higher than 55% as assessed 24 h after stimulation with IL-5. In contrast to IL-5–stimulated cells, there was usually <40% viable control (not treated with IL-5) eosinophils at the same time. However, as shown in Fig. 8, SHPTP2 antisense oligonucleotides blocked the ability of IL-5 to prevent eosinophil death (35.0 ± 6.8 versus 66.2 ± 8.0 versus 60.2 ± 5.4% for antisense, sense, and nonsense ODN– treated cells, respectively, P <0.05) indicating critical role of SHPTP2 phosphatase in IL-5–induced survival of eosinophils.


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)

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).
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Related In: Results  -  Collection

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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).
Mentions: Since eosinophils are terminally differentiated cells with life spans of ∼4 d, the use of antisense oligodeoxynucleotides is the most practical method to specifically alter expression of SHPTP2. Eosinophils were incubated with ODNs without FCS to protect stability of ODNs. As demonstrated in Fig. 7, eosinophils exposed to 7.5 μM antisense ODN for 6 h expressed little or no detectable SHPTP2, whereas sense or nonsense ODNs did not alter SHPTP2 level. Antisense ODNs used in our assay did not alter expression of a closely related phosphatase SHPTP1 (data not shown). The viability of eosinophils assessed at this time (immediately before stimulation with IL-5) always exceeded 90% and was not different from control samples, indicating that at concentration of 7.5 μM the ODNs were not toxic to the cells. After 2 h of stimulation, eosinophils were resuspended in medium without IL-5, ODNs, and FCS. We excluded FCS from this stage of experiment, since in a previous study by Inhorn et al., serum appeared to alleviate the requirement for the Ras pathway for GM-CSF–dependent viability and proliferation in mutant cells with β receptor lacking the 626–763 amino acid residues (23). Viability of the cells cultured without serum was always higher than 55% as assessed 24 h after stimulation with IL-5. In contrast to IL-5–stimulated cells, there was usually <40% viable control (not treated with IL-5) eosinophils at the same time. However, as shown in Fig. 8, SHPTP2 antisense oligonucleotides blocked the ability of IL-5 to prevent eosinophil death (35.0 ± 6.8 versus 66.2 ± 8.0 versus 60.2 ± 5.4% for antisense, sense, and nonsense ODN– treated cells, respectively, P <0.05) indicating critical role of SHPTP2 phosphatase in IL-5–induced survival of eosinophils.

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
Related in: MedlinePlus