Proteomic analysis of JAK2V617F-induced changes identifies potential new combinatorial therapeutic approaches.

This corrects the article DOI: 10.1038/leu.2017.143.

Proteomic analysis of JAK2V617F-induced changes identifies potential new combinatorial therapeutic approaches.

In excess of 90% of patients with polycythaemia vera (PV) express a mutated form of Janus kinase 2 (JAK2), JAK2V617F. Such aberrant proteins offer great potential for the treatment of these diseases; however, inhibitors to JAK2 have had limited success in the clinic in terms of curing the disease. To understand the effects of this oncogene in haematopoietic cells with the aim of improving treatment strategies, we undertook a systematic evaluation of the effects of JAK2V617F expression using proteomics. The effects of JAK2V617F on over 5000 proteins and 2000 nuclear phosphopeptide sites were relatively quantified using either SILAC or eight-channel iTRAQ mass spectrometry. Pathway analysis of the proteins identified as changing indicated disruption to the p53 and MYC signalling pathways. These changes were confirmed using orthogonal approaches. The insight gained from this proteomic analysis led to the formation of hypothesis-driven analysis on inhibitor-mediated effects on primary cells from patients with a JAK2V617F mutation. Simultaneous inhibition of MYC and upregulation of p53 led to the preferential extinction of JAK2V617F-positive CD34+ cells, illustrating a potential therapeutic benefit from combined targeting of p53 and MYC.

Transcriptional regulation of immediate-early gene response by THOC5, a member of mRNA export complex, contributes to the M-CSF-induced macrophage differentiation.

Hematopoiesis and commitment to a restricted lineage are guided by a timely expressed set of cytokine receptors and their downstream transcription factors. A member of the mRNA export complex, THOC5 (suppressors of the transcriptional defects of hpr1 delta by overexpression complex 5) is a substrate for several tyrosine kinases such as macrophage colony-stimulating factor (M-CSF) receptor and various leukemogenic tyrosine kinases, such as Bcr-Abl, or NPM-ALK. THOC5 tyrosine phosphorylation is elevated in stem cells from patients with chronic myeloid leukemia, suggesting that THOC5 may be involved in leukemia development. THOC5 is also an essential element in the maintenance of hematopoiesis in adult mice. In this report, we show that THOC5 is located in the nuclear speckles, and that it is translocated from the nucleus to cytoplasm during M-CSF-induced bone marrow-derived macrophage differentiation. Furthermore, we have identified THOC5 target genes by trancriptome analysis, using tamoxifen-inducible THOC5 knockout macrophages. Although only 99 genes were downregulated in THOC5-depleted macrophages, half of the genes are involved in differentiation and/or migration. These include well-known regulators of myeloid differentiation inhibitor of DNA binding (Id)1, Id3, Smad family member 6 (Smad6) and Homeobox (Hox)A1. In addition, a subset of M-CSF-inducible genes, such as Ets family mRNAs are THOC5 target mRNAs. Upon depletion of THOC5, unspliced v-ets erythroblastosis virus E26 oncogene homolog (Ets1) mRNA was accumulated in the nucleus. Furthermore, THOC5 was recruited to chromatin where Ets1 was transcribed and bound to unspliced and spliced Ets1 transcripts, indicating that THOC5 has a role in processing/export of M-CSF-inducible genes. In conclusion, regulation of immediate-early gene response by THOC5, a member of mRNA export complex contributes to the M-CSF-induced macrophage differentiation.

A caspase-3 'death-switch' in colorectal cancer cells for induced and synchronous tumor apoptosis in vitro and in vivo facilitates the development of minimally invasive cell death biomarkers.

Novel anticancer drugs targeting key apoptosis regulators have been developed and are undergoing clinical trials. Pharmacodynamic biomarkers to define the optimum dose of drug that provokes tumor apoptosis are in demand; acquisition of longitudinal tumor biopsies is a significant challenge and minimally invasive biomarkers are required. Considering this, we have developed and validated a preclinical 'death-switch' model for the discovery of secreted biomarkers of tumour apoptosis using in vitro proteomics and in vivo evaluation of the novel imaging probe [(18)F]ML-10 for non-invasive detection of apoptosis using positron emission tomography (PET). The 'death-switch' is a constitutively active mutant caspase-3 that is robustly induced by doxycycline to drive synchronous apoptosis in human colorectal cancer cells in vitro or grown as tumor xenografts. Death-switch induction caused caspase-dependent apoptosis between 3 and 24 hours in vitro and regression of 'death-switched' xenografts occurred within 24 h correlating with the percentage of apoptotic cells in tumor and levels of an established cell death biomarker (cleaved cytokeratin-18) in the blood. We sought to define secreted biomarkers of tumor apoptosis from cultured cells using Discovery Isobaric Tag proteomics, which may provide candidates to validate in blood. Early after caspase-3 activation, levels of normally secreted proteins were decreased (e.g. Gelsolin and Midkine) and proteins including CD44 and High Mobility Group protein B1 (HMGB1) that were released into cell culture media in vitro were also identified in the bloodstream of mice bearing death-switched tumors. We also exemplify the utility of the death-switch model for the validation of apoptotic imaging probes using [(18)F]ML-10, a PET tracer currently in clinical trials. Results showed increased tracer uptake of [(18)F]ML-10 in tumours undergoing apoptosis, compared with matched tumour controls imaged in the same animal. Overall, the death-switch model represents a robust and versatile tool for the discovery and validation of apoptosis biomarkers.

A pathway from leukemogenic oncogenes and stem cell chemokines to RNA processing via THOC5.

THOC5 is a member of the THO complex that is involved in processing and transport of mRNA. We have shown previously that hematopoietic stem cells have an absolute requirement for THOC5 for survival and that THOC5 is phosphorylated on tyrosine 225 as a consequence of leukemogenic protein tyrosine kinase (PTK) action. We have investigated pathways for THOC5 phosphorylation to develop an understanding of THO complex modulation by tyrosine kinase (TK) oncogenes in leukemias. We demonstrate that THOC5 phosphorylation is mediated by Src PTK and CD45 protein tyrosine phosphatase action and that this event is sensitive to oxidative status. We show that THOC5 phosphorylation is elevated in stem cells from patients with chronic myeloid leukemia (CML) and that this phosphorylation is sensitive to the frontline drugs used in CML treatment. Further we show that THOC5 Y225 phosphorylation governs mRNA binding. In addition, CXCL12 is shown to induce THOC5 Y225 phosphorylation, and site-directed mutagenesis demonstrates that this modulates motile response. In conclusion, we delineate a signaling pathway stimulated by leukemogenic PTKs, chemokines and oxidative stress that can affect THO complex mediation of gene expression describing mechanisms for post-transcriptional regulation of protein levels.

Proteomic analysis reveals a novel mechanism induced by the leukemic oncogene Tel/PDGFRß in stem cells: activation of the interferon response pathways.

Objective proteomic analysis offers opportunities for hypothesis generation on molecular events associated with pathogenesis in stem cells. Relative quantification mass spectrometry was employed to identify pathways affected by Tel/PDGFRß, an oncogene associated with myeloproliferative neoplasia (MPN). Its effects on over 1800 proteins were quantified with high confidence. Of those up-regulated by Tel/PDGFRß several were involved in the interferon gamma (IFNγ) response. To validate these observations we employed embryonic and myeloid stem cells models which revealed Tel/PDGFRß-induced STAT1 up-regulation and activation was responsible for modulating the interferon response. A STAT1 target highly up-regulated was ICSBP, a transcriptional regulator of myeloid and eosinophilic differentiation. ICSBP interacts with CBP/p300 and Ets transcription factors, to promote transcription of additional genes, including the Egr family, key regulators of myelopoiesis. These interferon responses were recapitulated using IFNγ stimulation of stem cells. Thus Tel/PDGFRß induces aberrant IFN signaling and downstream targets, which may ultimately impact the hematopoietic transcriptional factor network to bias myelomonocytic differentiation in this MPN.

A proof-of-principle gel-free proteomics strategy for the identification of predictive biomarkers for the onset of pre-eclampsia.

OBJECTIVE: Progress in the prevention and treatment of women at risk of pre-eclampsia (PE) still remains hindered by the lack of clinical screening tools that can accurately predict which mothers are at risk. The identification and validation of predictive biomarkers is therefore seen as a critical milestone towards improved healthcare provision and the clinical testing of new therapeutic strategies. Gel-free proteomic technologies offer the capability of analysing hundreds of plasma proteins simultaneously, but as yet these methods have not been applied to pregnancy complications. To assess the feasibility of such an approach to plasma biomarker research in pregnancy we have applied the technique to samples from women with PE to gestation-matched controls. SAMPLE: Pooled plasma samples taken at time of disease from women with PE (n = 23) and gestation-matched controls (n = 23). METHODS: Proteomics strategy for relative quantification of proteins using mass spectrometry. RESULTS: We identified several differences, including elevated levels of endoglin, PAPP-A and PSG1 in PE plasma. Increased levels of endoglin were validated using immunoassay analysis of individual plasma samples. CONCLUSIONS: Although at a relatively early stage, this mass spectrometry-based approach shows promise as a tool to identify global protein changes in plasma. The application of these methods to pre-disease samples is the next step in the identification of clinically useful biomarkers.

The rho-kinase inhibitors Y-27632 and fasudil act synergistically with imatinib to inhibit the expansion of ex vivo CD34(+) CML progenitor cells.

Evidence from cell line-based studies indicates that rho-kinase may play a role in the leukaemic transformation of human cells mediated by the BCR/ABL tyrosine kinase, manifest clinically as chronic myeloid leukaemia (CML). We therefore employed two separate inhibitors, Y-27632 and fasudil, to inhibit the activity of rho-kinase against ex vivo CD34(+) cells collected from patients with CML. We compared the effects of rho-kinase inhibition in those cells with the effects of direct inhibition of BCR/ABL using the specific inhibitor imatinib. We found that inhibition of rho-kinase inhibited the effective proliferation, and reduced survival of CML progenitor cells. When combined with imatinib, rho-kinase inhibition added to the anti-proliferative and pro-apoptotic effects of the BCR/ABL inhibitor. Our studies may indicate therapeutic benefit in some cases for the combination of rho-kinase inhibitors with imatinib.

FMIP controls the adipocyte lineage commitment of C2C12 cells by downmodulation of C/EBP alpha.

Fms interacting protein (FMIP) is a substrate for Fms tyrosine kinase, and a nuclear/cytoplasm shuttling protein with a leucine zipper. As the phosphorylation of FMIP is observed in insulin-stimulated preadipocytes, we examined the role of FMIP in adipocyte differentiation, using the mesenchymal multipotent stem cells, C2C12 cells, that can differentiate into adipocytes, muscle cells and osteoblasts. Ectopic expression of FMIP in C2C12 impairs the adipocyte differentiation induced by treatment with insulin, dexamethasone and 3-isobutyl-1-methylxanthine. These cells exhibit muscle phenotype with multinuclear morphology. Furthermore, knockdown of endogenous FMIP expression by small interfering RNA improves adipocytic lineage commitment of C2C12 cells, while impairing muscle differentiation. Upon stimulation with insulin, CCAAT/enhancer binding protein (C/EBP)beta, but not C/EBPalpha, is upregulated in cells expressing ectopic FMIP, whereas in FMIP knockdown cells, C/EBPalpha is constitutively expressed. Ectopic expression of C/EBPalpha counteracts the effects of FMIP, whereas C/EBPalpha knockdown partially mimics the effects of FMIP in this system. Northern blot analysis and reverse transcriptase-polymerase chain reaction study reveal that ectopic FMIP-expressing cells do not contain the polyadenylated C/EBPalpha mRNA, but contain the C/EBPalpha pre-mRNA, suggesting that FMIP plays a role in RNA processing and/or export. Indeed, a member of the THO complex that plays a role in mRNA export, THOC1, is co-precipitated with FMIP. The data we have acquired on FMIP suggest that it is a target for tyrosine kinase receptors that potentiate mRNA export.

Role of the C-terminal actin binding domain in BCR/ABL-mediated survival and drug resistance.

Philadelphia chromosome-positive, chronic myeloid leukaemia (CML) stem and progenitor cells have a survival and growth advantage compared with their normal counterparts. The mechanisms through which the BCR/ABL protein tyrosine kinase (PTK) induces these effects and the important domains within this protein are not fully defined. The F- and G-actin binding region of the BCR/ABL C-terminus may be important in BCR/ABL-mediated events, and we have investigated this by expressing a C-terminus deletion mutant of the temperature-sensitive BCR/ABL PTK, in a haemopoietic progenitor cell line, which models the chronic phase of CML. The truncated BCR/ABL PTK displayed similar levels of PTK activity when compared with wild type and activation of second messenger formation (in the form of sn-1,2-diacylglycerol) remains intact. On fibronectin substrata, localisation of the protein to the periphery of the cell was, however, dependent on the C-terminus of BCR/ABL PTK. Deletion of the C-terminus reversed both BCR/ABL-mediated apoptotic suppression and drug resistance although the progenitor cells did retain a proliferative advantage at low concentrations of growth factor. These results demonstrated that the C-terminal actin-binding domain of BCR/ABL is important for some of BCR/ABL PTK-mediated leukaemogenic effects.

Glucose transport regulation by p210 Bcr-Abl in a chronic myeloid leukaemia model.

The regulation of nutrient transport by both cytokines and oncogenes has been linked to haemopoietic cell survival. In this study, we found that activation of Bcr--Abl protein tyrosine kinase was associated with the stimulation of glucose transport in the multipotent haemopoietic cell line FDCP-mix, a cell model for chronic-phase chronic myeloid leukaemia (CML). Bcr--Abl upregulation of glucose transport was mediated by phosphatidylinositol-3-kinase. The observation that Bcr--Abl can regulate glucose transport in a CML cell model raises the possibility that glucose transport regulation may have a role to play in the aberrant survival of stem cells in the chronic phase of CML.

v-Abl protein-tyrosine kinase up-regulates p21WAF-1 in cell cycle arrested and proliferating myeloid cells.

v-Abl protein-tyrosine kinase (PTK) promotes cell survival without cell proliferation in interleukin (IL)-3-deprived IC.DP premast cells (1). We now show that in these conditions v-Abl PTK transcriptionally up-regulated the cyclin-dependent kinase inhibitor (CDKI) p21(WAF-1) and inhibited CDK2 and CDK4. When readdition of IL-3 stimulated cell proliferation, p21(WAF-1) was inactivated as a CDKI despite maintenance of elevated protein level. p21(WAF-1) was also up-regulated yet was nonfunctional as a CDKI when v-Abl PTK was activated in cells maintained in IL-3, but this occurred without increased p21(WAF-1) transcription. Using a C-terminal epitope-specific p21(WAF-1) antibody, v-Abl PTK-mediated increase in p21(WAF-1) could be detected in intact cells only in the presence of IL-3. This indicated different binding partners of p21(WAF-1) and/or protein conformation in nondividing or proliferating cells, respectively. The binding of CDK2, CDK4, or proliferating cell nuclear antigen to p21(WAF-1) and its subcellular localization were unchanged in the presence or absence of IL-3. However, two-dimensional analysis revealed different forms of up-regulated p21(WAF-1) in IL-3-deprived, nondividing cells compared with IL-3-stimulated proliferating cells. These data demonstrate that elevation of the CDKI p21(WAF-1) is not always sufficient for cell cycle arrest and indicate an IL-3-sensitive pathway for the inactivation of p21(WAF-1) function as a CDKI.

The specific enhancement of interferon alpha induced growth inhibition by BCR/ABL only occurs in multipotent cells.

INTRODUCTION: The causative oncogene in CML is the BCR/ABL protein tyrosine kinase. This stem cell disease is often treated with interferon alpha (IFN-alpha) which can initiate haematological and cytological remission which is associated with increased survival. There is however no clear indication of why CML cells are more responsive to IFN-alpha. MATERIALS AND METHODS: To establish if BCR/ABL increases the sensitivity of multipotent cells to IFN-alpha a temperature sensitive mutant of BCR/ABL was expressed in the multipotent haemopoietic stem cell line FDCP-Mix. The effect of IFN-alpha in terms of proliferation, induction of apoptosis, changes in cell cycle inhibitor proteins, and differentiation was assessed by [3H]thymidine incorporation, Annexin V and Western blot analysis. RESULTS: When the BCR/ABL tyrosine kinase was activated, the IFN-alpha-induced inhibition on the growth rate of the FDCP-Mix cell population was more marked than in control populations. The BCR/ABL-mediated effect was due to decreased rates of DNA synthesis. There was no IFN-alpha-mediated induction of apoptosis. This enhanced BCR/ABL mediated growth inhibition occurred over a range of growth factor concentrations and was independent of changes in p21(Cip1) and p27(Kip) levels. When FDCP-Mix cells were induced to differentiate into mature macrophages and neutrophils in the presence of IFN-alpha, there was increased sensitivity to IFN-alpha that was independent of BCR/ABL activity. CONCLUSION: BCR/ABL PTK expression in this primitive multipotent haematopoietic cell line results in an enhanced response to IFN-alpha. In contrast, the more mature myeloid progenitor cells are equally responsive to this growth inhibitor. This data may explain some of the clinical effects of IFN-alpha.

Bcr-Abl protein tyrosine kinase activity induces a loss of p53 protein that mediates a delay in myeloid differentiation.

Chronic myeloid leukaemia is a haemopoietic stem cell disorder, the hallmark of which is the expression of the Bcr-Abl Protein Tyrosine Kinase (PTK). We have previously reported that activation of a temperature sensitive Bcr-Abl PTK in the multipotent haemopoietic cell line FDCP-Mix for short periods resulted in subtle changes including, a transient suppression of apoptosis and no inhibition of differentiation. In contrast, activation of the Bcr-Abl PTK for 12 weeks results in cells that display a delay in differentiation at the early granulocyte stage. Flow cytometric analysis also indicates that the expression of cell surface differentiation markers and nuclear morphology are uncoupled. Furthermore, a significant number of the mature neutrophils display abnormal morphological features. Prolonged exposure to Bcr-Abl PTK results in interleukin-3 independent growth and decreased p53 protein levels. FDCP-Mix cells expressing a dominant negative p53 and p53null FDCP-Mix cells demonstrate that the reduction in p53 is causally related to the delay in development. Returning the cells to the restrictive temperature restores the p53 protein levels, the growth factor dependence and largely relieves the effects on development. We conclude that prolonged Bcr-Abl PTK activity within multipotent cells results in a reduction of p53 that drives a delayed and abnormal differentiation.

The effect of Bcr-Abl protein tyrosine kinase on maturation and proliferation of primitive haematopoietic cells.

BACKGROUND: Chronic Myeloid Leukaemia (CML) is characterised by the chromosomal translocation resulting in expression of the Bcr-Abl protein tyrosine kinase (PTK) in early stem cells and their progeny. However the precise nature of Bcr-Abl effects in primitive CML stem cells remains a matter of active debate. MATERIALS AND METHODS: Extremely primitive Bcr-Abl fusion positive cells were purified from patients with CML using multiparameter flow cytometric analysis of CD34, Thy, and lineage marker (Lin) expression, plus rhodamine-123 (Rh-123) brightness. Progenitor cells of increasing maturity were examined for cycling status by flow cytometry and their proliferative status directly correlated with cell phenotype. The activation status of a key transcription factor, signal transducers and activators of transcription (STAT-5), was also analyzed by immunocytochemistry. RESULTS: The most primitive stem cells currently defined (CD34+Lin-Thy+ Rh-1231o) were present as a lower proportion of the stem cell compartment (CD34+Lin-) of CML patients at presentation than of normal individuals (2.3% +/- 0.4 compared with 5.1% +/- 0.6 respectively). Conversely there was a significantly higher proportion of the more mature cells (CD34+Lin-Thy-Rh-123 hi) in CML patients than in normal individuals (79.3 +/- 1.8 compared with 70.9 +/- 3.3). No primitive subpopulation of CML CD34+Lin- cells was cycling to a significantly greater degree than cells from normal donors, in fact, late progenitor cells (CD34+Lin+) were cycling significantly less in CML samples than normal samples. STAT5, however, was observed to be activated in CML cells. CONCLUSIONS: We conclude that no subpopulation of CML stem cells displays significantly increased cell cycling. Thus, increased cycling cannot be a direct consequence of Bcr-Abl PTK acquisition in highly enriched stem cells from patients with CML. In vivo CML need not be considered a disease of unbridled stem cell proliferation, but a subtle defect in the balance between self renewal and maturation.

Transforming growth factor-beta 1 induces apoptosis independently of p53 and selectively reduces expression of Bcl-2 in multipotent hematopoietic cells.

Transforming growth factor-beta1 (TGF-beta1) can inhibit cell proliferation or induce apoptosis in multipotent hematopoietic cells. To study the mechanisms of TGF-beta1 action on primitive hematopoietic cells, we used the interleukin-3 (IL-3)-dependent, multipotent FDCP-Mix cell line. TGF-beta1-mediated growth inhibition was observed in high concentrations of IL-3, while at lower IL-3 concentrations TGF-beta1 induced apoptosis. The proapoptotic effects of TGF-beta1 occur via a p53-independent pathway, since p53(null) FDCP-Mix demonstrated the same responses to TGF-beta1. IL-3 has been suggested to enhance survival via an increase in (antiapoptotic) Bcl-x(L) expression. In FDCP-Mix cells, neither IL-3 nor TGF-beta1 induced any change in Bcl-x(L) protein levels or the proapoptotic proteins Bad or Bax. However, TGF-beta1 had a major effect on Bcl-2 levels, reducing them in the presence of high and low concentrations of IL-3. Overexpression of Bcl-2 in FDCP-Mix cells rescued them from TGF-beta1-induced apoptosis but was incapable of inhibiting TGF-beta1-mediated growth arrest. We conclude that TGF-beta1-induced cell death is independent of p53 and inhibited by Bcl-2, with no effect on Bcl-x(L). The significance of these results for stem cell survival in bone marrow are discussed.

Role of phosphatidylinositol 3-kinase and specific protein kinase B isoforms in the suppression of apoptosis mediated by the Abelson protein-tyrosine kinase.

Leukemogenic oncogenes, such as the Abelson protein-tyrosine kinases (PTK), disrupt the normal regulation of survival, proliferation, and differentiation in hemopoietic progenitor cells. In the absence of cytokines, hemopoietic progenitor cells die by apoptosis. Abl PTKs mediate suppression of this apoptotic response leading to aberrant survival. To investigate the mechanism of Abl PTK action, we have used an interleukin-3-dependent murine mast cell line that expresses a temperature-sensitive form of the v-ABL PTK, which is active at the permissive temperature of 32 degrees C and inactive at 39 degrees C. At the permissive temperature, these cells are resistant to apoptosis induced both by the withdrawal of the hemopoietic growth factor (interleukin-3) and the addition of cytotoxic drugs. We demonstrate that v-Abl associates with and stimulates activation of phosphatidylinositol 3-kinase (PI3K) and, crucially, that this activation results in enhanced cellular levels of the mass of the second messenger phosphatidylinositol-3,4,5-trisphosphate. Activation of PI3K leads to enhanced activity of PKB and increased levels of the anti-apoptotic protein Bcl-X(L). Transfection of cells with a dominant negative PKB reduces both the Abl-stimulated PKB activity and the survival effect conferred by activation of this oncogene. Thus, PI3K and PKB are required for the anti-apoptotic effects of Abl PTK.

Neuropeptide control of bone marrow neutrophil production is mediated by both direct and indirect effects on CFU-GM.

Noradrenaline- and peptide-containing nerve fibres project into the bone marrow and terminate in association with stromal cells and within the parenchyma. Peptidergic nerve terminals are also associated with antigen-processing and -presenting cells throughout the body and have been shown to be important in leucocyte trafficking and wound healing, as well as haemopoiesis. Here, we tested the in vivo effects of deleting the peripheral neuropeptide network on haemopoiesis and also investigated whether the target cell population for these substances was myeloid progenitor cells (colony-forming unit-granulocyte/macrophage, CFU-GM). Deletion of the neuropeptides, substance P (SP) and calcitonin gene-related peptide (CGRP) by capsaicin abrogates normal blood cell production. These neuropeptides produced significant stimulation of colony formation from unfractionated bone marrow and elicited production of soluble factors capable of stimulating highly enriched CFU-GM. CGRP also had a direct stimulatory effect on highly enriched CFU-GM. Noradrenaline elicited factors that inhibited colony formation and had no direct effect on CFU-GM. We conclude that the neuropeptides form the positive arm of a neural control system and that noradrenaline acts as a negative regulator.

Activation of granulocyte-macrophage colony-stimulating factor and interleukin-3 receptor subunits in a multipotential hematopoietic progenitor cell line leads to differential effects on development.

Activation of specific cytokine receptors promotes survival and proliferation of hematopoietic progenitor cells but their role in the control of differentiation is unclear. To address this issue, the effects of human interleukin-3 (hIL-3) and human granulocyte-macrophage colony-stimulating factor (hGM-CSF) on hematopoietic development were investigated in hematopoietic progenitor cells. Murine multipotent factor-dependent cell-Paterson (FDCP)-mix cells, which can self-renew or differentiate, were transfected with the genes encoding the unique alpha and/or shared beta(c) human hIL-3 receptor (hIL-3 R) or hGM-CSF receptor (hGM R) subunits by retroviral gene transfer. Selective activation of hIL-3 Ralpha,beta(c) or hGM Ralpha,beta(c) transfects by hIL-3 and hGM-CSF promoted self-renewal and myeloid differentiation, respectively, over a range of cytokine (0.1 to 100 ng/mL) concentrations. These qualitatively distinct developmental outcomes were associated with different patterns of protein tyrosine phosphorylation and, thus, differential signaling pathway activation. The cell lines generated provide a model to investigate molecular events underlying self-renewal and differentiation and indicate that the alpha subunits act in combination with the hbeta(c) to govern developmental decisions. The role of the alpha subunit in conferring specificity was studied by using a chimeric receptor composed of the extracellular hIL-3 Ralpha and intracellular hGM Ralpha subunit domains. This receptor promoted differentiation in response to hIL-3. Thus, the alpha subunit cytosolic domain is an essential component in determining cell fate via specific signaling events.