Recruitment of SH2-containing protein tyrosine phosphatase SHP-1 to the interleukin 2 receptor; loss of SHP-1 expression in human T-lymphotropic virus type I-transformed T cells - PubMed (original) (raw)

Recruitment of SH2-containing protein tyrosine phosphatase SHP-1 to the interleukin 2 receptor; loss of SHP-1 expression in human T-lymphotropic virus type I-transformed T cells

T S Migone et al. Proc Natl Acad Sci U S A. 1998.

Abstract

Interleukin 2 (IL-2) rapidly induces tyrosine phosphorylation of intracellular substrates, including the IL-2 receptor beta chain (IL-2Rbeta), Janus kinase 1 (Jak1), Jak3, signal transducer/activator of transcription proteins, and Shc, but the mechanism underlying dephosphorylation of these proteins is not known. The src homology 2 (SH2) containing tyrosine phosphatase 1 (SHP-1) is recruited by several hematopoietic surface receptors indicating that this phosphatase plays an important role as a regulator of signaling. We have found that IL-2 induces association of SHP-1 with the IL-2 receptor complex, and that once SHP-1 is recruited to the activated receptor it is able to decrease tyrosine phosphorylation of IL-2Rbeta and the associated tyrosine kinases Jak1 and Jak3. This dephosphorylation is specific as expression of a catalytically inactive form of SHP-1, or expression of the related phosphatase SHP-2 did not result in dephosphorylation of the IL-2 receptor components. Furthermore, we have found that SHP-1 expression is greatly decreased or undetectable in a number of IL-2 independent HTLV-I transformed T cell lines that exhibit constitutive Jak/signal transducer/activator of transcription activation. In HTLV-I infected T cells, down-regulation of SHP-1 expression was also found to correlate with the acquisition of IL-2 independence. These observations suggest that SHP-1 normally functions to antagonize the IL-2 signal transduction pathway and that HTLV-I infection and oncogenic transformation can lead to loss of SHP-1 expression resulting in constitutive activation of IL-2 regulated T cell responses.

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Figures

Figure 1

Figure 1

Time course of tyrosine phosphorylation of IL-2 receptor components after IL-2 stimulation. Molt4β cells were stimulated for 10′ with 2nM IL-2 at 37°C. Cells were washed in PBS and resuspended in 10% FBS/RPMI 1640 medium and left in culture for the indicated time. In control lanes (marked c), cells were not exposed to IL-2. Cells were then washed, lysed and immunoprecipitations were performed with the indicated antibodies: (A) anti-Jak 1; (B) anti-Jak3; (C) anti-IL-2Rβ and were followed by Western blot analysis with mAb 4G10. Immunoblots were stripped and reprobed to control for equal protein loading.

Figure 2

Figure 2

IL-2 induces association of SHP-1 with IL-2Rβ. (A) Molt4β cells were either stimulated with 2nM IL-2 for the indicated time or (B) stimulated for 10′ with 2nM IL-2, washed and resuspended in 10% FBS/RPMI 1640 medium in the absence of IL-2 for the indicated time. Cells were then washed, lysed and immunoprecipitations were performed with mAb 561 to hIL-2Rβ, followed by immunoblotting with anti-SHP-1 (Upstate Biotechnology). Immunoblots were then stripped and reprobed with an antiserum to human IL-2Rβ (Santa Cruz Biotechnology). (C) Freshly isolated peripheral blood mononuclear cells and Kit-225 cells were rested for 4 hr and then stimulated for 10′ with 2nM IL-2 at 37°C. Immunoprecipitations and Western blot analyses were performed as above.

Figure 3

Figure 3

SHP- 1 expression down-regulates tyrosine phosphorylation of IL-2 receptor components. (A) 293T+ cells were transfected with the indicated combination of plasmids; 48 hr posttransfection cells were washed, lysed and proteins were immunoprecipitated with mAb 561 to IL-2Rβ or (B) with antibodies to Jak1 and Jak3, followed by Western blot analysis with antiphosphotyrosine mAb. Also shown are controls for equal expression of the transfected plasmids. (C) A catalytically inactive form of SHP-1 does not affect tyrosine phosphorylation of the IL-2 receptor complex. 293 T+ cells were transfected with cDNAs for IL-2Rβ, γc, Jak1, Jak3, and either empty vector or the indicated forms of SHP-1 or SHP-2. Immunoprecipitations and immunoblotting were performed as described in A. Also shown are controls for equal expression of the transfected plasmids.

Figure 4

Figure 4

SHP-1 expression down regulates IL-2 induced phosphorylation of Jak1 and Jak3 in αβγ-3T3 cells, but does not affect their ability to associate with IL-2Rβ. (A) αβγ-3T3 cells were infected with virus encoding wild-type Jak3 (lanes 1 and 2), kinase inactive Jak3 (lanes 3 and 4), and wild-type Jak3 and SHP-1 (lanes 5 and 6). Cells were stimulated for 30 min with 2nM IL-2 at 37°C. Immunoprecipitations and Western blot analysis were performed as above. (B) The membrane was stripped and reprobed with anti Jak1 and anti-Jak3 antibody to analyze the effect of SHP-1 on IL-2Rβ-Jak1 interactions. Additionally, the same membrane was reprobed with anti-IL-2Rβ antibody as a control for equal protein loading.

Figure 5

Figure 5

IL-2 receptor phosphorylation in HTLV-I-transformed T cells. The HTLV-I-transformed T cell line MT-2 was stimulated as described in Fig. 1. Immunoprecipitations and Western blot analysis were performed as described in Fig. 1.

Figure 6

Figure 6

SHP-1 expression is not detected in some HTLV-I-transformed T cell lines. (A) Lysates (40 μg) from MOLT4β or MT-2 cells probed with antisera to either SHP-1 or SHP-2. (B) Lysates (40 μg) from the HTLV-I-transformed T cell lines MT1, MT2, MT4, MJ, Hut102B, or from MOLT4β, freshly isolated peripheral blood mononuclear cells, phytohemagglutinin-activated peripheral blood lymphocytes, and Jurkat, were immunoblotted with anti-SHP-1. (C) 40 μg of lysates from the HTLV-I-infected T cells MB3–12A, MB3–12B (grown in medium containing 3 units IL-2/ml), MB3–6 (grown in medium containing 20 units IL-2/ml) were compared with lysates from MT-2, MOLT4β, and YT cells by immunoblotting with anti-SHP-1.

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