Alternative modes of GM-CSF receptor activation revealed using activated mutants of the common beta-subunit

Abstract

Granulocyte/macrophage colony-stimulating factor promotes growth, survival, differentiation, and activation of normal myeloid cells and plays an important role in myeloid leukemias. The GM-CSF receptor (GMR) shares a signaling subunit, beta(c), with interleukin-3 and interleukin-5 receptors and has recently been shown to induce activation of Janus kinase 2 (JAK2) and downstream signaling via formation of a unique dodecameric receptor complex. In this study we use 2 activated beta(c) mutants that display distinct signaling capacity and have differential requirements for the GMR alpha-subunit (GMR-alpha) to dissect the signaling pathways associated with the GM-CSF response. The V449E transmembrane mutant selectively activates JAK2/signal transducer and activator of transcription 5 and extracellular signal-regulated kinase (ERK) pathways, resulting in a high level of sensitivity to JAK and ERK inhibitors, whereas the extracellular mutant (FIDelta) selectively activates the phosphoinositide 3-kinase/Akt and IkappaKbeta/nuclear factorkappaB pathways. We also demonstrate a novel and direct interaction between the SH3 domains of Lyn and Src with a conserved proline-rich motif in GMR-alpha and show a selective requirement for Src family kinases by the FIDelta mutant. We relate the nonoverlapping nature of signaling by the activated mutants to the structure of the unique GMR complex and propose alternative modes of receptor activation acting synergistically in the mature liganded receptor complex.

Figure 1

The hβ_c FIΔ mutant generates JAK2- and ERK1/2-independent signals. (A) FDB1 cell populations were cultured without growth factor for 16 hours and then stimulated with mIL-3 or mGM-CSF for 5 minutes. Whole-cell lysates were subjected to Western blot analysis with the indicated antibodies. (B) FDB1 cells were cultured in the absence of growth factor (NF), mIL-3, or mGM-CSF and then fixed, permeabilized, and stained with the indicated primary antibodies (open histograms) or isotype-matched control (gray histograms). (C-D) FDB1 cells cultured in 500 BMU mIL-3 and FDB1 cells expressing FIΔ or V449E cultured in the absence of growth factor were treated for 24 hours with dimethyl sulfoxide (DMSO), (C) JAK2 inhibitor II (J2InhII) or AG490, and (D) U0126 or PD98059 (PD). Viability was measured by 7-amino-actinomycin D staining and flow cytometry. Error bars represent SEM, where n = 3; statistical significance was calculated by the use of a Student t test, *P < .05 **P < .01, ***P < .001.

Figure 2

The hβ_c FIΔ mutant signals through Akt and NFκB pathways. (A) FDB1 cells were cultured in the absence of growth factor for 16 hours and then stimulated with 500 BMU of mIL-3 or mGM-CSF for 5 minutes. Whole-cell lysates were subjected to Western blot analysis with the indicated antibodies. (B) FDB1 cells cultured in 500 BMU mIL-3 and FDB1 cells expressing FIΔ or V449E cultured in the absence of growth factor were treated with DMSO, LY294002 (LY), or wortmannin (Wort) for 24 hours. Viability was measured by 7-amino-actinomycin D staining and flow cytometry. Error bars represent SEM, where n = 2; statistical significance was calculated by the use of a Student t test, *P < .05 **P < .01.

Figure 3

IκKβ and p85 interact with hGMR-α. (A) Flow cytometric analysis to detect hGMR-α and hGMR-α-APVA after transfection of HEK293T cells. hGMR-α was detected with 4H1 antibody (open histograms). An isotype-matched control is shown for comparison (gray histograms). (B) hGMR-α, p85, and IκKβ detected in whole-cell lysates (WCL) from HEK293T cells expressing hGMR-α and in 4H1 immunoprecipitates (4H1 IP). (C) hGMR-α and p85 detected in WCL from HEK293T cells expressing hGMR-α or hGMR-α-APVA and in 4H1 immunoprecipitates (4H1 IP) of the same cells. Control IPs minus antibody (−Ab), or with IgG1 (IgG₁) are also shown. (D) hGMR-α and IκB detected in WCL from HEK293T cells expressing hGMR-α or hGMR-α-APVA, and in 4H1 immunoprecipitates (4H1 IP). Control IPs minus antibody (−Ab), or with IgG₁ are also shown.

Figure 4

Association of Lyn and Src SH3 domains with hGMR-α. (A) Detection of hGMR-α and Lyn in whole-cell lysates (WCL) from HEK293T cells expressing hGMR-α, and 4H1 immunoprecipitates (4H1 IP) of the same cells. (B) Detection of hGMR-α and Lyn in WCL from HEK293T cells expressing hGMR-α or hGMR-α-APVA, and in 4H1 (4H1 IP) and control IgG1 (IgG₁) immunoprecipitates of the same cells. (C) Detection of hGMR-α and Src in WCL from HEK293T cells expressing hGMR-α or hGMR-α-APVA, and in 4H1 (4H1 IP) and control IgG1 (IgG₁) immunoprecipitates of the same cells. (D) Fluorescence polarization analysis of a fluorescein-conjugated mGMR-α peptide alone, with GST, or with p85, Lyn and Src SH3 domain GST fusions proteins. The change in millipolarization (Δmp) is given, where emissions of the mGMR-α peptide alone have been subtracted from the other samples. (E-F) FDB1 cells cultured in 500 BMU of mIL-3 and FDB1 cells expressing FIΔ or V449E cultured in the absence of growth factor were treated with DMSO, (E) 0.5μM SU6656 or 25μM PP1, or (F) 10μM and 25μM Lyn peptide inhibitor (LI) for 24 hours. Viability was measured by trypan blue exclusion. Error bars represent SEM, where n = 2; statistical significance was calculated by the use of a Student t test, *P < .05 **P < .01.

Figure 5

Model for assembly and activation of hβ_c mutants FIΔ and V449E. The high-affinity complex of the GM-CSF receptor (GMR) is a dodecamer structure (center) comprising 2 ligand bound hexamers. The central structure in the dodecamer complex enables JAK2 transphosphorylation and activation of STAT5- and Shc-mediated pathways. Proposed signaling through GMR-α occurs in αβ heterodimers (outer structures in the dodecamer complex), which initiate activation of Akt and NFκB pathways. Also indicated is a possible negative feedback mechanism whereby activation of SHIP downstream of JAK2 may suppress Akt activation. The proposed V449E structure is represented as a β_c tetramer, which does not require GMR-α and initiates a subset of the signals generated by the dodecameric GMR complex. V449E-activated pathways are confined to those downstream of JAK2 and receptor tyrosine phosphorylation and support survival and proliferation in the FDB1 cells. The proposed FIΔ structure comprises an αβ tetramer that precludes JAK2 transphosphorylation. This complex generates signals predominantly through GMR-α and results in activation of Akt and NFκB pathways, supporting FDB1 survival and differentiation.