The Shc-binding site of the betac subunit of the GM-CSF/IL-3/IL-5 receptors is a negative regulator of hematopoiesis

Abstract

Tyrosine and serine phosphorylation of the common beta chain (beta(c)) of the granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), and IL-5 receptors is widely viewed as a general mechanism that provides positive inputs by coupling the receptor to signaling pathways that stimulate several cellular functions. We show here that despite the known action of Tyr577 in beta(c) to recruit Shc-PI-3 kinase (PI3K) pathway members, Tyr577 plays, surprisingly, a negative regulatory role in cell function, and that this is mediated, at least in part, through the uncoupling of SH2-containing inositol 5'-phosphatase (SHIP) from beta(c). Fetal liver cells from beta(c)/beta(IL-3)(-/-) mice expressing human GM-CSF receptor alpha chain and beta(c) Tyr577Phe mutant showed enhanced colony formation and expansion of progenitor cells in response to GM-CSF. Dissection of these activities revealed that basal survival was increased, as well as cytokine-stimulated proliferation. As expected, the recruitment and activation of Shc was abolished, but interestingly, Gab-2 and Akt phosphorylation increased. Significantly, the activation of PI3K was enhanced and prolonged, accompanied by loss of SHIP activity. These results reveal a previously unrecognized negative signaling role for Tyr577 in beta(c) and demonstrate that uncoupling Shc from cytokine receptors enhances PI3K signaling as well as survival and proliferation.

Figure 1

Mutation of Tyr577 in β_c of the GM-CSF/IL-3/IL-5 receptors enhances hematopoietic colony formation in response to GM-CSF. FL cells from β_c^−/−/β_IL-3^−/− mice were transduced with a retrovirus encoding the GM-CSF receptor α chain and either wild type β_c or mutants of β_c for 72 hours in a cocultivation system with ψ2 retroviral producer cells. Following transduction and quantification of transduction efficiency by immunofluorescence, 100 000 transduced cells were plated in 35-mm dishes containing 0.3% agar with human GM-CSF (100 ng/mL) or a cocktail of IL-6 (50 ng/mL), SCF (100 ng/mL), and Epo (4U/mL). (A) Comparison of wtβ_c with mutants in which all 8 tyrosines had been replaced by phenylalanine (F8), and the single-residue mutants Tyr577Phe or Ser585Gly in mediating colony formation in response to GM-CSF. This experiment is representative of 5 similar experiments. Error bars represent SEM from 3 to 5 replicate plates. ▨ depicts average colony formation of all groups in response to the cocktail stimulation. (B) TheTyr577Phe mutation (□) consistently enhanced colony formation from FL cells in response to 100 ng/mL GM-CSF compared with wtβ_c (■). Error bars represent SEM from 3 to 5 replicate plates. (C) Titration of human GM-CSF for its ability to stimulate colony formation in cells transduced with wtβ_c (■) or Tyr577Phe mutant (squlo]). Pooled data from 3 separate colony assays are shown. Error bars represent SEM from 3 to 11 plates per group. (D) Bone marrow (BM) cells from β_c^−/−/β_IL-3^−/− mice were transduced and plated in agar as above. Colony formation was stimulated with GM-CSF or the IL-6, SCF, and Epo cocktail as described in “Biological assays”. Colony formation was assessed at day 7, and in the experiment shown, error bars represent SEM from 6 replicate plates from 1 experiment that was representative of 3 performed. (E) FL cells transduced with either Tyr577Phe β_c (□ and ) or wt β_c (■/▨) were cultured for the indicated times in liquid media of IMDM plus 15% HI-FCS supplemented with GM-CSF at 100 ng/mL (□ and ■) or a cytokine cocktail (50 ng/mL IL-6, 100 ng/mL SCF, and 10 ng/mL G-CSF; or ▨). At the indicated days, cells were counted, assessed for viability, and plated in agar for a further 10 to 14 days in cocktail containing SCF, Epo, and IL-6. Error bars represent SEM from 3 to 5 replicate plates.

Figure 2

Mutation of Tyr 577 in β_c enhances GM-CSF–responsive survival. FL cells transduced with α chain and wtβ_c or Tyr577Pheβ_c were washed and plated at 1.5 × 10⁵/mL in IMDM containing either 0.5% HI-FCS or 10% HI-FCS. Groups contained either no added factor (□), 50 ng/mL hGM-CSF (■), or a prosurvival cytokine cocktail containing IL-6 (50 ng/mL), SCF (100 ng/mL), and G-CSF (10 ng/mL) (▧). After 48 hours, cells were stained with the 4H1 anti-GMRα monoclonal antibody followed by an anti-mouse IgG-PE antibody, and with annexin V–FLUOS. Viability analysis was performed by flow cytometry as described in “Materials and methods.” The average of duplicate samples (± SD) is indicated. *P < .001; **P = .001. This experiment is representative of 3 performed.

Figure 3

FL cells transduced with Tyr577Pheβ_c show enhanced Gab2 tyrosine phosphorylation in the absence of Shc phosphorylation. (A) FL cells transduced with either wtβ_c or Tyr577Phe were cultured for 12 hours in RPMI containing 0.5% HI-FCS and stimulated with GM-CSF for 5 minutes prior to lysis. Cell lysates were cleared and immunoprecipitated with antibodies to β_c, Shc, SHP2, or Gab2. Immunoprecipitates were subjected to SDS-PAGE and immunoblotting with the antibodies as shown in Figure 1A. (B) Mutation of Tyr577 does not abolish SHP2 coimmunoprecipitation with β_c. CTL-EN cells expressing either wtβ_c or Tyr577Phe were factor deprived for 12 hours before stimulation with 50 ng/mL GM-CSF for 1, 2, 5, 15, 30, or 45 minutes. Following stimulation, β_c was immunoprecipitated with the anti-β_c antibody 1C1, and the immunoprecipitates were subjected to SDS-PAGE and immunoblotted with anti-phosphoTyr577, antiphosphotyrosine 4G10, anti-β_c, or anti-SHP2 antibodies. A vertical line has been inserted to indicate where a gel lane was cut. These were from separate gels run from a single experiment. (C) FL cells transduced with either wtβ_c or Tyr577Phe were cultured for 12 hours before stimulation with 50 ng/mL GM-CSF for 5, 15, or 30 minutes. Following stimulation, the cells were lysed, and the lysates were subjected to SDS-PAGE and immunoblotted with phosphospecific antibodies to JAK2, STAT5A, Akt, and Erk. An anti-Erk antibody was used as a loading control. The data shown is representative of 3 separate experiments performed using transduced FL cells.

Figure 4

Enhanced and prolonged PI3K activation in CTL-EN cells expressing β_c Tyr577Phe in response to GM-CSF. CTL-EN cells expressing either wtβ_c or Tyr577Phe were factor deprived for 12 hours in RPMI containing 0.5% HI-FCS before stimulation with 50 ng/mL GM-CSF for 0, 15, 30, or 60 minutes. The cells were then lysed, and the lysates cleared and subjected to immunoprecipitation with anti-β_c antibodies. (A) Immunoprecipitates were then tested in an in vitro kinase assay. (B) Quantification of counts from panel A as measured in a liquid scintillation counter. Experiment shown is representative of 3 separate assays that were performed. (C) FL cells transduced with either wtβ_c or Tyr577Phe were stimulated with GM-CSF for 0, 5, 15, 30, 60, 120, 240, or 360 minutes before lysis. Cell lysates were cleared and immunoprecipitated with antibodies to β_c. Immunoprecipitates were subjected to SDS-PAGE and immunoblotting with the antibodies as shown. Anti-β_c antibody 1C1 and anti-p85 were used as a loading controls for immunoprecipitations and lysates, respectively.

Figure 5

Mutation of Tyr577Phe in β_c abolishes the hydrolysis of PI-(3,4,5)P3 in response to GM-CSF. CTL-EN cells expressing either wtβ_c or Tyr577Phe were factor deprived for 12 hours in RPMI containing 0.5% HI-FCS before stimulation with either 0 or 50 ng/mL GM-CSF. Following stimulation, the cells were lysed, and the lysates were cleared and subjected to immunoprecipitation with anti-Shc antibody or anti-β_c antibody. (A) Shc immunoprecipitates were subjected to SDS-PAGE and immunoblotting with antibody to phospho-SHIP. (B) β_c immunoprecipitates subjected to a PtdIns(3,4,5)P₃ 5-phosphatase assay. (C) Quantification of PtdIns(3,4,5)P₃ 5-phosphatase activity in each immunoprecipitate. The bars represent the mean (± SD) from 2 independent experiments.