Complex interactions in EML cell stimulation by stemcell factor and IL-3Zhi-jia Yea,b, Erol Gulcicekc, Kathryn Stonec, Tukiet Lamc, Vincent Schulzd, and Sherman M. Weissmana,1

aDepartment of Genetics, Yale University, New Haven, CT 06519; bCollege of Preventive Medicine, Third Military Medical University, Chongqing400038, China; cKeck Center, Yale University, New Haven, CT 06510; and dDepartment of Pediatrics, Yale University School of Medicine, Yale University,New Haven, CT 06510

Contributed by Sherman M. Weissman, December 15, 2010 (sent for review November 2, 2010)

Erythroid myeloid lymphoid (EML) cells are an established multi-potent hematopoietic precursor cell line that can be maintained inmedium including stem cell factor (SCF). EML cultures contain a het-erogeneous mixture of cells, including a lineage-negative, CD34+

subset of cells that propagate rapidly in SCF and can clonally re-generate the mixed population. A secondmajor subset of EML cellsconsists of lineage-negative. CD34− cells that can be propagated inIL-3 but grow slowly, if at all, in SCF, although they express the SCFreceptor (c-kit). The response of these cells to IL-3 is stimulatedsynergistically by SCF, and we present evidence that both the syn-ergy and the inhibition of c-kit responses may be mediated bydirect interaction with IL-3 receptor. Further, the relative level oftyrosine phosphorylation of various substrates by either cytokinealone differs from that produced by the combination of the twocytokines, suggesting that cell signaling by the combination of thetwo cytokines differs from that produced by either alone.

The erythroid myeloid lymphoid (EML) cell line is a multi-potent hematopoietic cell line that can be differentiated in

vitro into cells of various lineages including erythroid, myeloid,or lymphoid cells (1). EML cells were derived originally bytransfection of murine bone marrow with a dominant negativeretinoic acid receptor and then selecting for cells that expandedin medium containing stem cell factor (SCF). EML cells can besubcloned as single cells that expand to produce populations withthe same properties as the original culture and can be passagedrepeatedly without losing their multipotency. Thus, these cellsprovide an interesting model of a self-renewing and spontane-ously differentiating, niche-independent cell system.A suspension culture of EML cells passaged in SCF contains

a complex mixture of cells at various stages of differentiation. Thelineage-negative portion of the culture can be separated roughlyinto a CD34+, stem cell antigen 1 (Sca-1)–high population anda CD34−, Sca-1–low population. The CD34+ subfraction of thecells grows rapidly in medium containing SCF, reconstitutinga mixed population of EML cells. Growth of these cells is stim-ulated synergistically by IL-3, a cytokine capable of stimulatinggrowth of a variety of hematopoietic cell types, but the cells willnot grow in IL-3 medium without SCF. Conversely, the CD34−,lineage-negative cells grow in IL-3 medium, and growth is stim-ulated synergistically by SCF, but this fraction of cells will notgrow, or grows only very slowly, in SCF alone (2).The SCF receptor c-kit is a member of the tyrosine kinase

receptor family (3). SCF plays critical roles in regulating therenewal, growth, and differentiation of hematopoietic stem cells(4–7). SCF activates a tyrosine phosphorylation cascade medi-ated by c-kit resulting in the creation of a complex network af-fecting multiple biological processes (5, 8, 9). The synergy of SCFwith other growth factors or cytokines initiates specific differ-entiation of hematopoietic stem cells into definite lineages (10–12). The IL-3 receptor (IL-3R) also is a tyrosine kinase con-sisting of a heteromer of two types of chains, a common β chainshared with the IL-5 receptor and GM-CSF receptor, and an IL-3–specific α chain (13). Changes in tyrosine phosphorylation ofc-kit or the IL-3R β chain parallel the effects of the cytokines on

cell growth and show clearly the synergistic effect of treatmentof either CD34+ or CD34− cells with a combination of the twocytokines. Remarkably, this differential response to cytokinesoccurs even though the CD34+ and CD34− lines have aboutequal amounts of c-kit mRNA, and c-kit protein is present andexpressed on the cell surface in about equal amounts in the twocell populations (2).In the present study we confirmed the synergistic action of IL-

3 and SCF and show this synergy can occur in nonhematopoieticcells after transfection of the appropriate receptors. We alsofound that an excess of the IL-3R α chain can prevent c-kit re-sponse to SCF. Proteomic analysis of tyrosine phosphorylationproducts shows that many of the tyrosine phosphorylation eventsoccur with treatment by either cytokine. The results confirm thesynergistic action of the two cytokines, but the level of synergisticphosphorylation varies with the substrate, so that treatment withcombined cytokines could create a balance of phosphorylatedsubstrates different from that produced by treatment with eithercytokine alone.

ResultsDynamic Phosphorylation of c-kit and Akt. Stimulation of SCF leadsto dimerization of the c-kit receptor and subsequent activationof its intrinsic tyrosine kinase (14). The phosphorylation of c-kithappens rapidly, and the activated c-kit is internalized, followedby degradation mediated by the ubiquitin pathway (15). To testthe dynamic phosphorylation of c-kit and thymoma viral proto-oncogene 1 (Akt), we checked phosphorylation of c-kit and Aktunder different stimuli at several time points. As shown in Fig. 1,strongly phosphorylated c-kit and Akt were detected as early as 2min after stimulation. Transphosphorylation of c-kit induced byIL-3 was observed at early time points. Compared with SCF, IL-3induced less phosphorylated c-kit or Akt. The PI3K–Akt pathwayplays critical roles in regulating cell proliferation and differenti-ation (16). The differential phosphorylation of c-kit and Akt in-duced by IL-3 and SCF was consistent with the differentproliferation response of EML cells to the two cytokines. Themost intensely phosphorylated c-kit was detected at 5 min. Aktwas maximally phosphorylated at 2 min. Synergy between SCFand IL-3 was prominent and also was seen early after cytokinetreatment, peaking by 5 min.Our previous data showed synergistic phosphorylation and

transphosphorylation between c-kit and the IL-3R β chain inlineage-negative EML cells. Because the phosphorylation of c-kitis dynamic, we also checked phosphorylation of c-kit with dif-ferent doses of SCF at 5 min. As shown in Fig. 2, a low dose ofSCF induced phosphorylation of c-kit at 5 min. The phosphor-

Author contributions: Z.-j.Y. and S.M.W. designed research; Z.-j.Y. performed research;E.G., K.S., and T.L. contributed new reagents/analytic tools; Z.-j.Y., E.G., K.S., and V.S.analyzed data; and Z.-j.Y. and S.M.W. wrote the paper.

The authors declare no conflict of interest.1To whom correspondence should be addressed. E-mail: sherman.weissman@yale.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1018002108/-/DCSupplemental.

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ylation of c-kit was increased as the SCF concentration increasedup to but not beyond 50 ng/mL Phosphorylated c-kit decreasedwith the highest dose of SCF (100 ng). The synergistic phos-phorylation of c-kit, Akt, Mek, and Erk by IL-3 and SCF also wasobserved with lower doses of SCF. Synergistic phosphorylationof the IL-3Rβ also occurred when SCF and IL-3 were addedtogether to cells (Fig. 3).

Antibody Ab-1 Eliminates Synergistic Proliferation and Phosphorylationof c-kit by SCF and IL-3.We confirmed that synergistic proliferationof EML cells is induced by the combination of SCF and IL-3. Thecell proliferation induced by SCF was eliminated when the cellswere pretreated by the inhibitory antibody Ab-1. Antibody Ab-1targets the fourth Ig domain of c-kit and is a dimerization-inhibitory monoclonal antibody. Ab-1 inhibits the phosphoryla-tion of c-kit induced by SCF by preventing c-kit dimerization.Synergistic proliferation also was inhibited by antibody Ab-1.EML proliferation induced by IL-3 was not inhibited significantlyby antibody Ab-1 (P > 0.05) (Fig. 4A). To investigate further themechanism behind the synergistic effects on proliferation, wechecked the activation of the PI3K–Akt and MAPK–ERKpathways in EML cells with different treatments. The PI3K–Aktand MAPK–ERK pathways are critical for inducing cell pro-liferation and inhibiting cell apoptosis (16–18). Significant syn-ergistic phosphorylation of c-kit, Akt, mitogen-activated proteinkinase kinase 1 (Mek), and mitogen-activated protein kinase 1(ERK) induced by SCF plus IL-3 was observed (Fig. 4B). Theantibody Ab-1 not only inhibited phosphorylation of c-kit, Akt,Mek, and ERK by SCF alone but also eliminated synergisticphosphorylation of these proteins by the combination of SCF andIL-3. These results indicate that the synergy between c-kit andIL-3R is dependent on the c-kit dimerization domain, if not onthe dimerization of c-kit itself. Surprisingly, the pretreatmentwith Ab-1 induced detectable phosphorylation of Akt, Mek, andERK compared with an IgG control. Ab-1 is not able to inhibitthe transphosphorylation of c-kit induced by IL-3. Therefore, thelow level of c-kit phosphorylation induced by IL-3 did not occurthrough c-kit kinase activation.

c-Kit and IL-3 Receptor Form a Complex in EML cells. After con-firming the synergistic activation of c-kit by SCF and IL-3, wewere interested in the mechanisms underlying the synergy. Theassociation of c-kit and the erythropoietin receptor (EpoR) leadsto the synergistic activation and trans-activation between thesetwo receptors (10). To determine if c-kit associates with IL-3R, weimmunoprecipitated c-kit or IL-3Rβ from EML cells. Theimmunocomplex was resolved using SDS/PAGE and transferredonto an Immobilon P membrane. As shown by Fig. 5A, antibodyspecific for c-kit immunoprecipitated both c-kit and IL-3Rβ. Theantibody for IL-3Rβ also coimmunoprecipitated IL-3Rβ andc-kit. To investigate further if the complex of c-kit and IL-3Rβ isdependent on the stimulation of the cytokines, we immunopre-cipitated c-kit or IL-3Rβ from EML cells without treatment orwith different treatments. As shown by Fig. 5D, c-kit is constitu-tively associated with IL-3Rβ even without stimulation by cyto-kines. To confirm the specificity of the coimmunoprecipitation,we checked other proteins known to be associated with c-kit in thepellets of the complex immunoprecipitated with c-kit antibody.PI3K is present in the pellets, but PDGF receptor (PDGFR) wasabsent. IL-3Rα also was detected in the immunoprecipitationcomplex obtained with c-kit antibody (Fig. 5D).

Synergy Between IL-3 and SCF also Can Be Demonstrated in a Non-hematopoietic Cell in the Presence of the Appropriate Receptors. Tocheck whether the synergistic phosphorylation of c-kit induced bySCF and IL-3 is specific to a hematopoietic cell line, we cotrans-fected a human embryonic kidney cell line, 293T, with c-kit andIL-3R (α chain and β chain). There was no detectable endogenousc-kit or IL-3R in 293T cells, but both receptors were detected onthe surface of 293T cells transfected with the relevant expressionplasmids. As shown in Fig. 6, the combination of IL-3 and SCFinduced a significant synergistic phosphorylation of c-kit in 293Tcells. The data indicated that the synergistic activation of c-kitinduced by IL-3 and SCF is not limited to EML cells.

Overexpressed IL-3Rα Inhibits Activation of c-kit. The data profileof RNA expression in lineage-negative, CD34+ and lineage-negative, CD34− EML cells showed that the expression of IL-3Rα in lineage-negative, CD34− cells was around threefold thatof lineage-negative, CD34+ cells. We confirmed the microarraydata by Western blotting, shown in Fig. 7A. In our previousstudies we found the growth of lineage-negative, CD34− cells didnot respond to SCF even though the levels of c-kit mRNA,protein, and surfaced antigen expression are rather similar in theCD34− and CD34+ cells (2). We asked whether the overex-pression of IL-3Rα inhibits c-kit signaling. To answer this ques-tion, we transduced EML cells with lentivirus particles expressingthe α chain or β chain of IL-3R fused with internal ribosome entrysite (IRES)-GFP. Surprisingly, no GFP+ cells were detected inEML cells transduced by IL-3Rα–IRES-GFP. Approximately20% GFP+ cells were present in EML cells transduced with equalamounts of virus particles expressing IRES-GFP control or IL-3Rβ–IRES-GFP, as shown in Fig. 7B. These data suggested that

2 min 5 min 10 min 20 min

IL-3(4ng/ml) – + – + – + – + – + – + – + – + SCF(50g/ml) – – + + – – + + – – + + – – + +

pKit(Tyr719)

Kit

pAkt(Ser473)

ABC

Fig. 1. Dynamic phosphorylation of c-kit and Akt induced by IL-3, SCF, or thecombination of SCF and IL-3. Unfractionated EML cells (2 × 106/2 mL) werestarved for 3 h and then stimulated with SCF (50 ng/mL), IL-3 (4 ng/mL), or thecombination of SCF and IL-3 for 2 min, 5 min,10 min, and 20 min. Total celllysates were separated by SDS/PAGE and electrotransferred to Immobilon P,followed by immunoblotting with the indicated antibodies. (A) Phospho-c-kit(Y719) antibody. (B) Membrane from A was stripped and reprobed with c-kitantibody. (C) Immunoblotting with phospho-Akt (ser473) antibody.

p c-kit(Y719)

p Akt(S473)

p Mek(S217/221)

p Erk(T202/Y204)

-actin

IL-3(4ng/ml)SCF(ng/ml)

− − − − − − − + + + + + +

0 10 25 50 75 100 0 0 10 25 50 75 100

Fig. 2. Phosphorylation of c-kit, Akt, Mek, and ERK induced by different doses of SCF. EML cells were starved for 3 h and then were stimulated with differentamounts of SCF or IL-3 (4 ng/mL) plus different amounts of SCF for 5 min. The cells were lysed, and equal amounts of protein from each lysate were separatedby SDS/PAGE and electrotransferred to Immobilon P, followed by immunoblotting with the indicated antibodies. The results show that IL-3 phosphorylatesc-kit weakly but synergizes with SCF to cause phosphorylation of c-kit and its downstream kinases.

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overexpression of IL-3Rα is toxic to EML cells, perhaps by de-priving the cells of SCF stimulation. To confirm this hypothesis,we transformed c-kit and IL-3Rα into 293T cells and checked ifoverexpressed IL-3Rα inhibits phosphorylation of c-kit in a non-EML cell. As shown in Fig. 7C, the phosphorylation of c-kit in-duced by SCF was reduced when the IL-3Rα and c-kit were addedto the cells. These results indicated that the programmed responseof hematopoietic precursors to SCF might be determined byquantitative changes in gene expression of the relevant receptors.

Mass Spectrometry-Based Proteomics Analysis of EML Cells Inducedby SCF, IL-3, and the Combination of SCF and IL-3. To compare thevery early responses of activation by SCF, IL-3, or the combi-nation of the two cytokines in EML cells, we analyzed thechanges in the levels of tyrosine phosphorylated proteins afterbrief stimulation of EML cells. We used a stable isotope labelingby amino acids in cell culture (SILAC) procedure in which thecytokine-treated cells were grown in medium containing lysineand arginine labeled with heavy isotopes of carbon and nitrogen,and control cells were grown in medium with unsubstituted(light) amino acids.The results showed, as expected, that there was a considerable

overlap between proteins phosphorylated in response to IL-3 or toSCF (Table S1). Because of the limits in sensitivity of mass spec-troscopy, we cannot conclude that proteins detected with only oneof the two treatments were differentially phosphorylated in re-sponse to one or the other cytokine. However, there was prom-

inent difference in the amount of synergistic phosphorylation ofdifferent substrates compared with the effect of either cytokinealone. For example, both c-kit and inositol polyphosphate-5-phosphatase D (INPP5D) showed markedly increased levels ofphosphotyrosine when cells were treated with the combination ofthe two cytokines, but a number of other substrates showed es-sentially no difference in phosphorylation when the cells weretreated with the two combined cytokines or with either one alone.SILAC profile patterns of phosphorylation of the key proteinsmediating phosphatidylinositol 3′-kinase and Ras/MAPK path-ways are consistent with the Western blotting data.

DiscussionEML cells consist of a heterogeneous population that will prop-agate in medium containing SCF. The CD34+, lineage-negativesubset of EML cells is the only substantial subset of cells thatgrows rapidly in SCF medium in the absence of other cytokines.This observation strongly suggests that the CD34+ populationrapidly regenerates the mixed culture in response to SCF. Al-though this subset of cells expresses both subunits of IL-3R, thecells are refractory to treatment with IL-3 alone, as seen both incell growth and in phosphorylation of IL-3R.The CD34− cells continue to express levels of c-kit mRNA,

total cell protein, and cell-surface protein similar to those of theCD34+ cells. However, the CD34− cells no longer respond to SCFby receptor phosphorylation or cell growth. In both subsets ofEML cells SCF and IL-3R have a marked synergistic effect oncell growth and phosphorylation when the two cytokines arepresent simultaneously. This effect indicates that in both celltypes the receptors remain able to recognize their cognate ligands,although the consequences of this recognition differ drastically,so that SCF stimulates the less differentiated cells and IL-3 themore differentiated cells.Renewal and expansion in the hematopoietic system depend

on cytokines that promote survival and division of cells of variouslineages or stages of differentiation (19–21). In particular, SCFpromotes the expansion of early hematopoietic precursor cells,whereas IL-3 is a multilineage cytokine that stimulates growth ofearly, partly differentiated cells (7, 22, 23). The effects of thesecytokines on EML cells is consistent with this difference, becausethe population of lineage-negative, CD34+ cells shows lowerlevels of expression of a number of lineage-specific genes, and

IL-3(4ng/ml)SCF(50ng/ml)

− + − +− − + +

IB:pY

IB: pS

IB:IL-3R

IP: IL-3R

Fig. 3. Phosphorylation of the IL-3R β chain induced by IL-3, SCF, or IL-3 plusSCF. EML cells were starved for 3 h and treated by IL-3 (4 ng/mL), SCF (50ng/mL), or IL-3 plus SCF for 5 min. The IL-3R β chain was immunoprecipitatedwith antibody for IL-3Rβ, and the immunoprecipitated pellets were resolvedby SDS/PAGE and electrotransferred to Immobilon P, followed by immuno-blotting with the indicated antibodies. The results show that SCF acts syn-ergistically with IL-3 to phosphorylate the IL-3R β chain. This effect wasparticularly prominent for serine phosphorylation. IL3-Rβ, common β-chainIL-3R; pS, phosphorylated serine; pY, phosphorylated tyrosine.

02468

101214

Ctl IgG Ab-1 Ctl IgG Ab-1 Ctl IgG Ab-1

IL-3 SCF IL-3+SCF

Cell num

ber(x105)

A

B + + + + − − − −− − − − + + + +− + − + − + − +− − + + − − + +

pc-kit(Y719)c-kitpAkt(Ser308)AktpMEK1/2(Ser217/221)pErk1/2(Thr202/Tyr204)Erk

IgG(5ug/ml)Ab-1(5ug/ml)IL-3(2ng/ml)SCF(50ng/ml)

Fig. 4. Antibody Ab-1 eliminated synergistic prolifera-tion of EML cells and synergistic phosphorylation ofmediators of c-kit signaling induced in EML cells by SCFcombined with IL-3. (A) EML cells were cultured in thepresence of SCF (50 ng/mL), IL-3 (4 ng/mL), or both withan isotype control of antibody Ab-1 (5 μg/mL), or withantibody Ab-1 (5 μg/mL).The initial number of cells forthe different stimuli was 1 × 105 in 1 mL medium. Thecells were cultured for 2 d and counted with Trypan blueto exclude dead cells. (B) Ab-1 blocks synergistic phos-phorylation of c-kit, Akt, and ERK induced by the com-bination of SCF and IL-3. EML cells were starved for 4 hand incubated with Ab-1 or Ig isotype control (IgG1/κ) for30 min, followed by stimulation with IL-3 (4 ng/mL), SCF(50 ng/mL), or the combination of IL-3 and SCF for 5 min.Then cells were lysed. Similar amounts of protein fromdifferent lysates were loaded into SDS/PAGE and elec-trotransferred to an Immobilon P membrane. The mem-brane was probed with the indicated antibodies.

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especially those of the erythroid lineage, than does the CD34−,lineage-negative population (2). The switch from responsivenessto SCF in CD34+ cells to responsiveness to IL-3 and non-responsiveness to SCF in the CD34− cells represents an inter-esting developmental switch.The receptor c-kit has been known for some time to interact

with certain other tyrosine kinase receptors including EpoR,GM-CSF receptor (GM-CSFR), and IL-7 receptor, and thisphysical interaction may be associated with cross-phosphoryla-tion of c-kit by erythropoietin (EPO) or other cytokines (24–27).The physiologic significance of this cross-phosphorylation hasbeen questioned previously.In our studies we did observe ligand-independent coimmuno-

precipitation of c-kit and IL-3R and some cross-phosphorylationbetween the two receptors. However, the level of phosphorylationof the second receptor was quite low and may not be of physio-logic significance. In contrast, there was a marked synergistic ef-fect in both cell growth and tyrosine phosphorylation both incells responsive to SCF alone and in other cells (the CD34−

subpopulation) that showed neither c-kit phosphorylation norgrowth response to SCF. The synergy in tyrosine phosphorylationcould be reproduced in a nonhematopoietic human embryonic

kidney cell line, so that other hematopoiesis-specific proteins arenot necessary for the effect. Synergy did require the presence ofboth the α and β chains of IL-3R as well as c-kit.The shift of c-kit–expressing cells to a state unresponsive to

SCF without down-modulation of the SCF receptor correlateswith the extent of differentiation of the EML cells, providinga mechanism that permits selective expansion of stem cells thatmay differentiate spontaneously without expanding the moredifferentiated precursors. At the same time, the synergistic effectswould mean that the later precursor cells become sensitive tosmall amounts of IL-3. The transfection studies with 293T cellssuggest that one mechanism for suppressing the response of c-kit–expressing cells to SCF would be mediated by an excess of the IL-3 α chain, although it is possible that other mechanisms maycontribute in the EML cells. A similar phenomenon was observedfor the interaction of c-kit with the α chain of GM-CSF andEpoR. GM-CSF and SCF synergized in activating the MEK–ERK pathway, but overexpression of the α chain of GM-CSFRinhibited c-kit phosphorylation by SCF (28). The combination ofSCF and EPO synergistically phosphorylates c-kit and EpoR (10,25). However, in a leukemic proerythroblast, EPO induces si-lencing of c-kit making the cells EPO dependent (29), so different

CD117 A

PC

CD123 PE CD131 PE

IL-3(4ng/ml)

SCF(50ng/ml)

- + - + - - + +

p c-Kit(Y719)

c-Kit

CD117 A

PC

CD123 PE CD131 PENon Staining

A

B C

Fig. 6. Phosphorylation of c-kit induced by IL-3 (4 ng/mL),SCF (50 ng/mL), or IL-3 plus SCF in the nonhematopoietic cellline 293T. (A) No c-kit (CD117) or IL-3R α chain (CD123) or IL-3R β chain (CD131) was detected in parental 293T cells. (B)Expressed c-kit, IL-3R α chain, and IL-3Rβ chain were detectedby FACS on the surface of 293T cells that had been trans-fected 3 d previously with vectors expressing these recep-tors. (C) 293T cells expressing c-kit, IL-3R α chain, and IL-3Rβ chain were harvested after stimulation with SCF (50ng/mL), IL-3 (4 ng/mL), or both for 5 min. Whole-cell lysateswere analyzed by SDS/PAGE, followed by immunoblottingwith tyrosine phosphorylated c-kit (Y719) antibody andanti–c-kit antibody. The results show that c-kit responded toSCF in these cells and that the response was increased by theaddition of IL-3 to SCF.

A B C

None IL-3 SCF IL-3+SCF

IP: IL -3Rβ IB: c -Kit

IP: c -Kit IB:IL -3Rβ

150kD

130kD

D

IL-3R β

c-Kit150 kD

IL-3Rβ

130 kD

IgG

IP

IB

150 kD

IgG c-Kit

c-Kit

IL-3Ra

PI3K85

65 kD

85 kD

IP

IB

IL-3Rβ

c-kit150 kD

130 kD

IgG c-Kit

IP

IB

Fig. 5. c-Kit and IL-3 receptor form a complex in EML cells. EML cells were lysed and subjected to immunoprecipitation with c-kit antibody or IL-3R β-chainantibody. The immunoprecipitated pellets were separated by SDS/PAGE and electrotransferred to Immobilon P, followed by immunoblotting with the in-dicated antibodies. (A) The cells were cultured in SCF medium, harvested, washed with PBS, and lysed with lysis buffer B. The lysates were immunoprecipitatedwith c-kit antibody (from goat), and probed with IL-3R β-chain antibody (from rabbit). The membrane then was stripped and reprobed with c-kit antibody(from rabbit). (B) The cells cultured in SCF medium were harvested, washed with PBS, and lysed with lysis buffer B. The lysates were immunoprecipitated withIL-3R β-chain antibody (from rabbit) and probed with c-kit antibody (from goat) and IL-3R β-chain antibody. (C) Cells cultured in SCF medium were harvestedand lysed in lysis buffer B. The lysates were immunoprecipitated with c-kit antibody (from goat) and probed with IL-3R α-chain antibody (from rabbit), 85-kDsubunit of PI3K antibody (frommouse), and c-kit antibody (from rabbit). (D) EML cells were starved for 3 h and induced with IL-3 (4 ng/mL), SCF (100 ng/mL), orboth cytokines for 5 min. The cells were washed with PBS and then lysed. The cell lysates immunoprecipitated with IL-3R β-chain antibody and immunoblottedwith c-kit antibody or were immunoprecipitated with c-kit antibody followed by immunoblotting with IL-3R β-chain antibody. The results show that c-kitcoprecipitates with the IL-3R receptor and that this association is not affected by the presence or absence of the cytokines used in these experiments.

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mechanisms may operate in controlling cytokine responsivenessat the receptor level in different types of cells. Those data suggestthe balance of different cytokine receptors plays an importantrole in lineage proliferation, differentiation, and maturation ofhematopoietic progenitors. Wemay speculate that, in the absenceof IL-3, the IL-3R α chain directly locks the SCF receptor ina nonresponsive configuration, and this block is relieved when IL-3 binds to its receptor, although the two receptors remain asso-ciated with one another.The shift from SCF to IL-3 responsiveness could simply be

a mechanism for limiting growth and cytokine responsiveness toless differentiated cells in the absence of IL-3 or also couldchange the types of signals the cell receives from the cytokineenvironment (30). Study of phosphotyrosine protein formation inresponse to short periods of cytokine treatment, using theSILAC procedure, provides clues in this regard (31, 32). Re-assuringly, the SILAC data show preferential IL-3R β-chainphosphorylation by IL-3, c-kit phosphorylation by SCF, and in-creased phosphorylation of these receptors when both cytokinesare added.An unexpected phenomenon emerged from analysis of tyro-

sine phosphorylation. The synergistic effect of c-kit and IL-3 wasevident in the increased phosphorylation of c-kit in the presenceof IL-3 together with SCF. Some other proteins, such as INPP5D(SH2-containing inositol phosphatase 1, SHIP), showed a simi-lar synergistic phosphorylation. However, there was a group ofproteins in which the phosphotyrosine level did not differ sig-

nificantly whether SCF alone or SCF plus IL-3 was used tostimulate the cells. Thus, the synergistic effect, which is sub-stantially stronger than the effect of either cytokine separately,is not merely an augmentation of responsiveness but showsa different spectrum of phosphorylation stimulation, limited toa subset of genes responding to either cytokine alone. Overallthe synergistic effect is not merely an augmentation of preex-isting signaling activity but provides a new pattern of relativeabundance of phosphotyrosines in the cell and potentially anadditional level of complexity in the response patterns of cells tocytokine stimulation.

MethodsCell Lines, Cytokines, Antibodies, and Other Agents. EML C1 cells were the kindgift of Dr.Schickwann Tsai (University of Utah Health Care) and were culturedas described in the literature (1). The 293T cells were purchased from ATTCand cultured according to the ATCC instructions. Purified recombinantmouse stem cell factor (rmSCF) and purified recombinant mouse interleukin-3 (rmIL-3) were purchased from Peprotech. Antibodies specific for phos-phorylated c-kit at Tyrosine 719 [p c-kit (Tyr719)], phospho-p44/42 MAPK(Thr202/Tyr204), phospho-Akt (Ser473), phospho-Akt (Thr308), and anti-bodies against total c-kit, p44/42 MAPK, Akt, and the PI3K-specific inhibitorLY294002 were purchased from Cell Signaling Technology. Antibodiesagainst the IL-3R α chain (IL-3Rα), IL-3R β chain (IL-3Rβ), phospho-tyrosine,and the p85 subunit of PI3K were purchased from Santa Cruz Biotechnology.Monoclonal antibody Ab-1 specific for the Ig-like domain 4 in the extracel-lular region of c-kit and its isotype control IgG1/κ were purchased fromLab Vision. Phycoerythrin (PE)-conjugated CD123 (IL-3Rα) antibody, CD131

GFP

FL3

MIG-GFP MIG-IL-3Rβ-GFPMIG-IL-3Ra-GFP

A

B

C + + + + + + + +− − + + − − + +− + − + − + − +− − − − + + + +

c-kit

IL-3Ra

GAPDH

c-kitSCF(50ng/ml)IL-3 (4 ng/ml)IL-3Ra

Fig. 7. Overexpression of the IL-3R α chain was toxic to EML cells and inhibited phosphorylation of c-kit in 293T cells. (A) Expression of c-kit, IL-3Rα, andGAPDH in lineage-negative, CD34+ (Lin-CD34+) and lineage-negative, CD34− (Lin-CD34−) EML cells. (B) Flow cytometry analysis of EML cells transduced bythree different kinds of lentivirus particles: expression GFP alone, IL-3R β chain merged with GFP, and IL-3R α chain merged with GFP. The viral transductionwas carried out at the same ratio of virus to EML cells. (C) 293T cells transfected with c-kit expressing plasmid alone or with indicated amount of c-kit andplasmid expressing the IL-3R α chain. The cells were collected 2 d after transfection, lysed, subjected to SDS/PAGE, and then immunoblotted with anti–phospho-c-kit at Y719. Similar amounts of proteins from the indicated cell lysates were loaded.

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(IL-3Rβ) antibody, and APC-conjugated CD117 (c-kit) antibody were pur-chased from eBioscience.

Immunoprecipitation, Total Cell Lysate, and Immunoblotting Assay. EML cellswere deprived of SCF and horse serum for 4 h, and 293T cells were deprivedof serum for 6 h. This deprivation period was followed by the addition of SCF(50 ng/mL) or IL-3 (4 ng/mL) or the combination of SCF and IL-3. The cells wereinduced for the indicated times and then were collected and washed with PBSbuffer and lysed at 1 × 107cells/mL with ice-cold lysis buffer A (lysis buffer fortotal cell protein analysis) or lysed at 1 × 107cells/mL with ice-cold lysis buffer B(lysis buffer for immunoprecipitation) as described previously (24). For im-munoprecipitation, the lysates were cleared by centrifugation before anti-body application. c-Kit, IL-3Rα, or IL-3Rβ was immunoprecipitated from thecleared lysates with their respective antibodies at 1:50 dilution. The immu-noprecipitated complex or total cell lysates were subjected to SDS/PAGE, andWestern blotting was performed on a PVDF membrane (Millipore) accordingto the indicated manufacturers’ instructions for primary antibodies.

Cell Transfection and Transduction. For stable transfection, cDNA encoding IL-3Rα, the common β chain (IL-3Rβc), or the β chain for IL-3 receptor (IL-3Rβil-3)was cloned into the self-inactivating lentiviral vector plasmid pGK-IRES-GFP.Lentiviral particles were generated by the methods described in ref. 33. GFP+

cells were isolated and analyzed by FACS.For temporary transfection, cDNA encoding c-kit, IL-3Rα, IL-3Rβc, or IL-

3Rβil-3 was inserted into the vector pcDNA3.1 and then was transfected into293T cells by Lipofectamine 2000 (Roche). The expression of ectopic proteinswas confirmed by FACS and Western blotting.

Cell Proliferation Assays. EML cells were washed twice with PBS and resus-pended in Iscove’s Modified Dulbecco’s Media plus 15% horse serum at

105 cells/mL. Then 104 cells per well were seeded in 12-well plates. The cellswere preincubated with antibody Ab-1 or isotype control IgG for 30 minbefore induction of SCF, IL-3, or SCF plus IL-3. Viable cells were counted byTrypan blue staining on day 2.

SILAC Analyses. EML cells were washed twice before being transferred toSILAC medium (Invitrogen), which was prepared according to the manu-facturer’s instructions. Briefly, [14N]arginine and [12C]lysine were added intothe light medium, and [15N]arginine and [13C]lysine were added into theheavy medium. Complete replacement with heavy arginine and lysine wasconfirmed by mass spectrometry after six passages. After six passages, 20million EML cells growing in light and heavy media were harvested, washedtwice, and then starved for 4 h in light or heavy medium without dialyzedFBS or cytokines. After stimulation with SCF (50 ng/mL), IL-3(4 ng/mL), ora combination of SCF (50 ng/mL) and IL-3(4 ng/mL) for 5 min, the cells wereharvested and lysed in 100 μL modified RIPA buffer. Equal amounts of cel-lular lysates from the light and heavy media were mixed together. Totalproteins with phosphotyrosine were precipitated following the protocoldescribed in ref. 34. The phosphotyrosine proteins were released from theimmunoprecipitated pellet by heating (95 °C for 5 min) and were separatedby SDS/PAGE. After SDS/PAGE, the gel was stained with SimplyBlue Safe-Stain (Invitrogen). Each lane containing proteins was sliced into 15 pieces.The proteins in the sliced gel were extracted and digested. The peptideswere enriched by TiO2 chromatography and processed for LC-MS.

ACKNOWLEDGMENTS. We thank Dr. Wen-sheng Chen, Dr. Efim Golub,Jin Lian, and Dr. Zhengping Yu for their technical assistance and sugges-tions. This work was supported by National Institutes of Health GrantR01AG23111.

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