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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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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.
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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.

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)

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.
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Related In: Results  -  Collection

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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.
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