the hematopoietic growth factor receptor family (4), and as described for all the members of this receptor family, no enzymatic activities could be deduced from ...
THEJOURNALOF BIOLOGICAL CHEMISTRY 8 1992 by The American Society for Bioehemistry and Molecular Biology, Inc.
Vol. 267, No. 15, Issue of May 25, PP. 10670-10675.1992 Printed in U.S.A.
Erythropoietin Inducesthe Tyrosine Phosphorylation of Its Own Receptor in Human Erythropoietin-responsive Cells* (Received for publication, December 30,1991)
Isabelle Dusanter-Four@, Nicole Casadevall, Catherine Lacombe, Odile Muller, Claudine Billat$, Sigmund Fischerll, and Patrick Mayeux From the llznstitut National de la Sante et de la Recherche Medicale, Unites 152 and 332, ZCGM, Hopital Cochin, 27 rue du Faubourg Saint-Jacques, 75014 Paris, France and IUniversite de Reins-Champagne-Ardenne,UFR Sciences, Laboratoire de Biochimie, 51062 Reins, France
Using the human erythropoietin-responsive hemaA large number of growth factor receptors contain tyrosine topoietic cell line UT-7, we showed that erythropoietinkinase activity involved in the mechanism of cell activation (Epo)rapidlyand specifically inducedthetyrosine and proliferation. These include the receptors for the epiderphosphorylation of its own receptor (M. 75,000) and mal growth factor, the platelet derived growth factor, the increased the tyrosine phosphorylation of other pro- macrophage colony-stimulating factor, the insulin growth facteinsofM, 140,000,120,000,95,000,60,000,57,000, tors and the kit ligand (also called stem cell factor or steel and 42,000. Neither granulocyte-macrophage colony- factor) (for reviews, see Refs. 5 and 6). Immediate activation stimulating factor, interleukin 3, interleukin 6, nor of tyrosine kinases has also been implicated in signal transthe kit ligand induced the phosphorylation of the M, duction of hematopoietic growth factors whose receptors do 76,000 receptor protein, although these growth factors induced the phosphorylationof other proteins. Cross- not contain an intrinsic tyrosine kinase activity: granulocytelinking experiments using 12’I-Epo indicated that the macrophage colony-stimulating factor (GM-CSF), interleukin UT-7 cells expressed three Epo receptor subunits, of (IL) 2,3, and4,and more recently, IL7, were shown to induce M, 100,000, 85,000, and 75,000, among which only rapid and transient tyrosine phosphorylations of common the M, 75,000 subunit was tyrosine-phosphorylated and/or distinct sets of proteins (7-12). In addition, IL2 and IL3 were reported to induce the tyrosine phosphorylation of following activation with Epo. their own receptors very rapidly (9,11, 12-14). Also, the IL2 receptor /3 chain was reported to be directly and noncovalently associated with a tyrosine kinase activity in different cell lines Erythropoietin (Epo)’ isthe main hormone controlling (12-14), and the k k tyrosine kinase was selectively coimmuerythropoiesis. It acts on committed erythroid progenitors by noprecipitated with the IL2 receptor in NK and CTLL-2 sustaining theirsurvival, stimulating theirgrowth, and induc- lymphoid cells (15). Epo has also recently been reported to ing their differentiation (for a review, see Ref. 1).The biolog- induce rapid tyrosine phosphorylation of several proteins in ical effects of Epo are initiated by the interaction of the an Epo-responsive erythroid murine cell line (16). Although hormone with specific high affinity Epo receptors expressed this report did not precisely identify any of the Epo-induced on the surface of target cells (reviewed in Ref. 2). However, phosphorylated proteins, it showed that Epo and IL3 induced despite extensive studies, little isknown about the immediate similar patterns of tyrosine phosphorylated proteinsand mechanism of intracellular action of Epo, in part because of pointed out the role of tyrosine kinase activation as a candithe very low number of Epo receptors expressed on erythroid date pathway for Epo-induced proliferation. More recently, Miura et al. (17), using an IL3-dependent lymphoblastoid cell progenitors andthe difficulty of obtainingsubstantial amounts of pure Epo-dependent erythroid cell population. line transfected with the murine Epo receptor cDNA, estabThe Epo receptor has been recently cloned from a murine lished a correlation between the ability to stimulate prolifererythroleukemic cell line (3). The deduced nucleotide se- ation and to induce tyrosine phosphorylation by Epo. Howquence encodes a protein of molecular weight 55,000, with a ever, the precise identity of the phosphorylated proteins insingle membrane-spanning domain, which yielded a protein duced by Epo was still a matterof discussion. Using the newly of M , 66,000 (p66) when expressed in COS cells. The Epo described UT-7 human leukemia cell line, which has been receptor primary structure exhibits characteristic motifs of shown to be highly responsive to GM-CSF, IL3, Epo, or IL6 the hematopoietic growth factor receptor family (4),and as for proliferation (18) and for differentiation (19), wenow described for all the members of this receptor family, no report that Epo specifically induces the phosphorylation of enzymatic activities could be deduced from the Epo receptor its own receptor on tyrosine residues in a rapid and transient manner. This Epo-induced phosphorylation of the Epo recepamino acid sequence. tor is one of the earliest events reported to be activated in * This work was supported by Contracts 6327 and 6580 from the Epo-responsive cells. Association pour la Recherche sur le Cancer and a grant from the F. E.G.E.F.L.U.C. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solelyto indicate this fact. $ Supported by the InstitutNational de la Recherche Agronomique. The abbreviations used are: Epo, erythropoietin; 2,4-DNP, 2,4dinitrophenol; GM-CSF, granulocyte-macrophage colony stimulating factor; IL, interleukin; PAGE, polyacrylamide gel electrophoresis; SDS, sodium dodecyl sulfate.
MATERIALS ANDMETHODS
Hormones-Highly purified recombinant human erythropoietin (rhEpo, 200,000 units/mg; 1 unit/ml = 270 p ~ ) GM-CSF , (5 X lo7 units/mg), IL3 (10’units/mg), IL6 (lo7units/mg) were used. Cell Culture-The UT-7 cell line was originally derived from a bone marrow sample of a patient with acute megakaryoblastic leukemia (18). Continuous cell cultures were performed in a-medium (GIBCO-BRL) supplemented with 10% fetal calf serum and 2.5 ng/
10670
Phosphorylation of Erythropoietin Receptor mlrhGM-CSF at 37 "C in a 5% CO, humidifiedatmosphere, as reported (18). The cells were washed and maintained in a-medium without growth factor for 16-18 h before incubation withthe indicated growth factor and processed as described below. The erythropoietinresponsive Rauscher Reds 5cells (20) were maintained in a-modified Eagle's medium containing 5% fetal calf serum, as described (21). Antibodies-Anti-erythropoietin antibodies were obtained from rabbits injected with rhEpo. Antibodies against the murine erythropoietin receptor were obtained by immunizing rabbits againsta fusion protein corresponding to the entire Escherichia coli maltose transporter (MalE) and most of the cytoplasmic part of the murine Epo receptor, as reported previously (21). Antibodies against MalE and against 2.4-dinitrophenol (2,4-DNP) were similarly raisedas controls. All antibodies were purified by protein A-Sepharose (Pharmacia LKB Biotechnology Inc.) chromatography and coupled on a preactivated CNBr-agarose matrix (Pharmacia LKB Biotechnology Inc.) according to themanufacturer's instructions. Analysis of Phosphotyrosyl Proteins-Cells (5-10 X 106/ml) were incubated for various times at 37 "C with Epo or any growth factor assayed. In some cases, the cells were preincubated in the presence of 50 pM sodium orthovanadate for 30 min a t 37 "C before growth factor addition. The reaction was stopped by the additionof an excess of cold phosphate-buffered saline solutionand immediate pelleting of the cells. Total cell lysates were obtained by boiling cells for 10 min in SDS lysis buffer (22) containing 1 mM orthovanadate and 5% 0mercaptoethanol (reducing conditions) when indicated. Aliquots (lofi cells) were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)and immediately transferredonto a nitrocellulose filter (BA-85, Schleicher & Schuell) using ahigh intensity field electrotransfer apparatus (Bio-Rad). Nitrocellulose filters were blocked by a 2-4-h incubation with 3% bovine serum albumin (fraction V, Miles Laboratories, Inc.) in 20 mM Tris, pH 7.5, 150 mM NaCI, 0.1% Tween 20 (TBS-Tween). Filters were sequentially incubated with a monoclonal anti-phosphotyrosine antibody (2.5 pg/ml 4G10, Upstate Biotechnology Inc.) and '*"-rabbit anti-mouse F(ab'), antibodies (0.1 pCi/ml) (Amersham Corp.) and intensively washed in TBS-Tween before autoradiography. The specificity of theantiphosphotyrosine antibody was determined by adding 10 mM phenyl phosphate to the filters. In these conditions, no signal of tyrosinephosphorylated proteins was obtained (data notshown). Immunoprecipitations-After incubationwith theappropriate growth factor and washes as indicated above, cells (10'/point) were resuspended in a mild lysis buffer (Buffer A 20 mM Tris, pH 8.0, 137 mM NaCI, 2.7 mM KCI, 1% NonidetP-40, 10% glycerol, 1 mM orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 1mM leupeptin, 1 mM aprotinin, and 1 mM pepstatin) and incubated for 30 min a t 4 "C under constant stirring. Unsolubilized material was removed by centrifugation for 20 min at 20,000 x g at 4 "C. Supernatants were 1 h a t 4 "C with an irrelevant precleared by apreincubationfor antibody (anti-2,4-DNP antibody) coupled to agarose beads or with protein A beads (Pharmacia) and next incubated with the indicated antibody either coupled to agarose beads or in solution (for the antiphosphotyrosineantibodies), as previously reported (21). All the immunoprecipitates were intensively washed before resuspension in SDS lysis buffer and solubilized by boiling for 10 min. When indicated, thesolubilized and denaturedimmunoprecipitates were further diluted 1 to 10 in the Buffer A and incubated with a second antibody. Samples were all subjected to SDS-PAGE and electrotransferred onto nitrocellulose filters as indicated above. Cross-linking Experiments-Cross-linking experimentsandimmunoprecipitation of ""I-Epo cross-linked complexes were done as previously described (21).
10671
18-24 h. After an overnight growth factor deprivation, the viability of the UT-7 cells was 95-loo%, as monitored by trypan blue exclusion, and their cloning efficiency in the presence of GM-CSF, IL3, or Epo was unchanged (not shown). After a 18-h deprivation of growth factors, UT-7 cells were incubated with Epo (1unit/ml) for 10 min and total cellular phosphotyrosyl proteins were analyzed. As shown in Fig. 1, various proteins, of M , 140,000-150,000,115,000-120,000, 95,000-100,000,75,000-78,000,58,000-62,000, and 38,00042,000 (often more weakly detected), were tyrosine-phosphorylated following incubation withEpo. These phosphoproteins will be abbreviated p140, p120, p95, p75, p60, and p42 throughout this paper. Preincubating the cells for 30 min at 37 "C in the presence of orthovanadate (50 p ~ or) genistein (30 pglml), respectively, enhanced or decreased the overall level of Epo-induced phosphorylations on tyrosine (data not shown). As shown in Fig. 2, this Epo-dependent induction of tyrosinephosphorylation was rapid andtransient, nearly maximal a t 2 min, reached a plateau between 5 and 15 min, and decreased thereafter toreach a pattern of tyrosine-phosphorylated proteins similar to unstimulated cells at 30 min. In this particularexperiment, p95 was resolved as a doublet, and p120 and p140 were only weakly detected. When UT-7 cells were incubated for 10 min a t 37 "C in the presence of Ep0
+ -
- +
, FIG. 1. Epo-induced tyrosine phosphorylations. UT-7 cells (106/point) were incubated for 10 min at 37 "C in the presence of 1 unit/ml of Epo, solubilized by boiling in SDS lysis buffer, and subjected to SDS-PAGE underreducing conditions. Proteins were transferred onto nitrocellulose and incubated with anti-phosphotyrosine antibodies.Phosphotyrosine proteins were revealed by '2sI-labeled anti-mouse antibody. The immunoblot was exposed for either 1 (left of the pointed panel)or 3 (rightpanel) days a t -70"C. M,( X bands is indicated. 0
2' 5' 10' 15' 30' 6 0
RESULTS
Epo Induces Rapid Phosphorylation of a Limited Number of Proteins on Tyrosine Residues-The human leukemic cell line UT-7 was reported to be highly responsive to Epo (18).An important number of Epo receptors, about 7000 receptors/ cell, was indeed expressed at the surface of these cells. All these receptors exhibited a high affinity for Epo (one classof Epo receptors, Kd = 180 PM) (data not shown). Such characteristics allowed the use of UT-7 cells to investigate intracellular mechanisms of Epo. Although these cells were shown to be strictly growth-factor dependent for their proliferation (18),they could be deprived of any of these growth factors for
FIG.2. Time dependence of the Epo-induced tyrosine phosphorylations. UT-7 cells (106/point) were incubated at 37 "C in the presence of Epo (1unit/ml) for the indicated times and then solubilized and subjected to SDS-PAGEunder reducing conditions. Cellular phosphotyrosine containing proteins was analyzed by immunoblotof the pointed tingas described inthe legend to Fig. 1. M, (X band is indicated.
Phosphorylation of Erythropoietin Receptor
10672
various amounts of Epo (Fig. 3), the presence of Epo-dependent tyrosine-phosphorylated proteins was detected with a concentration of Epo as low as 0.1 unit/ml. The proteins p140, p120, p95, p75, p60, and p42 mentioned above were all phosphorylated following Epo action, with similar concentration dependence (Fig. 3). In addition to Epo, the human UT-7cells were reported to be responsive to various growth factors, such as GM-CSF, IL3, IL6(18), andthe kitligand(19). We compared the pattern of cellular proteins induced to tyrosine phosphorylation by these growth factors with that observed with Epo. As shown in Fig. 4, all five growth factors induced protein tyrosine phosphorylations in UT-7-deprived cells: phosphoproteins within M , of 95,000-100,000 and 140,000 were induced by all the factors. In addition, the kit ligand induced large phosphorylation of a M , 150,000 protein, which might represent thekit receptor itself (33). However, only Epo specifically induced the phosphorylation of the p75, which represented one of the major phosphoproteins induced. The Major Tyrosine Phosphorylated Protein Induced by Epo Is the Epo Receptor Itself-since only Epo induced the tyrosine phosphorylation of p75, we tested whether this protein was associated to the Epo receptor or was the receptor itself. UT-7 cells were incubated with or without Epo (1 unit/ml) for 10 min,gently solubilized usingBuffer A to maintain ERO : U/ml
0 0.01 0.1 0.3 1
10 100
protein-receptor interactions, and immunoprecipitated using either anti-Epo, anti-Epo receptor, or anti-phosphotyrosine antibodies. As shown in Fig. 5A, in cell extracts incubated withEpo, both anti-Epo and anti-Epo receptorantibodies specifically immunoprecipitated onemajor protein of M, 75,000 (lanes 4 and 6), which was absent in cell extracts incubated in the absence of Epo (lanes 3 and 5). The antiEpo receptor antibodies we used were raisedagainst the murine Epo receptor. However, they still recognized the human Epo receptor but with a low efficiency. Two other proteins of M , 95,000 and 120,000 were also specifically immunoprecipitated by anti-Epo and anti-Epo receptor antibodies in these Epo-treatedcells. In some experiments, an additional protein of M, 140,000 was also specifically immunoprecipitated (see Fig. 5B, lane 2 ) . When using anti-phosphotyrosine antibodies, we foundthatthe p75 was one of the major tyrosine-phosphorylated proteins after Epo stimulation (Fig. 5A, lane 8). Similarly, p95 and p120 were also detected. None of these phosphoproteinswere immunoprecipitated by irrelevant antibodies (lunes 1-2). Interestingly, a shorter exposure of the autoradiogram shown in Fig. 5A indicated that the p75 band highly visible in lanes 4 and 8 was in fact a doublet (not shown). We tested then whether the human tyrosine-phosphorylated protein p75 corresponded to therecently cloned chain of the murine Epo receptor (p66). UT-7 cells incubated with or without Epowere either gently lysed and immunoprecipitated with anti-Epo antibodies as indicated above or resuspended in SDS lysis buffer and boiled for 10 min to disrupt any noncovalent association between proteins prior to immunoprecipitation with anti-Epo receptor antibodies. As shown in Fig. 5B, lane 4 , even when protein-protein interactions were disrupted, anti-Epo receptor antibodies still recognized the Epo-induced p75, indicating that this phosphoprotein was in B
A 1
2
1 3
2 4
3 5
4 6
7
8
"
FIG. 3. Concentration dependence of Epo-induced protein tyrosine phosphorylation in UT-7 cells.Growth factor-deprived UT-7 cells were incubated for 10 min a t 37 "C with various concentrations of Epo. Cell samples(106/point) were subjected to SDSPAGE under reducing conditions, andcellular phosphotyrosine-containing proteinswere analyzed by immunoblotting as described in the legend to Fig. 1. 1
2
3
4
5
-. 4
120.
4
95.
a
75.
a
V
-
140 120 95 75
6
150
*140 4 120 a
95
I
75
FIG. 4. Protein tyrosine phosphorylation induced by various growth factors in UT-7 cells. Growth factor-deprived UT-7 cells were incubated for 10 min a t 37 "C with various growth factors, and total cellular phophotyrosine-containing proteins were analyzed as indicated in the legend to Fig. 1. Lane 1, no growth factor added; lane 2, IL6 (10 ng/ml); lane 3,kit ligand (50 ng/ml); lane 4, IL3 (10 units/ml); lane 5 , GM-CSF (1 ng/ml); lane 6, Epo (1 unit/ml). The lower level of radioactivity seen in lane 5 was due toa loading artifact.
FIG. 5. The major Epo-induced tyrosine-phosphorylated protein p75 is the Epo receptor. A, UT-7 cells (lO'/point) were preincubated with 50 P M orthovanadate and then incubated with (lanes 2,4,6, and 8) or without (lanes I, 3 , 5 , and 7) Epo (1 unit/ml) for 10 min. Cells were solubilized under mild conditions and immu(lanes I-Z), noprecipitated with controlanti-2,4-DNPantibodies anti-Epo antibodies (lanes 3-4), anti-Epo receptor antibodies (lanes 5-6), or anti-phosphotyrosine antibodies(lanes 7-8). Immunoprecipitates were analyzed by SDS-PAGE, andphosphotyrosine-containing proteins were detected by immunoblotting, as described above. Lanes 1-6 corresponded to a 3-day exposure, whereas lanes 7-8 corresponded to a 1-day exposure. B, UT-7 cells (lO'/point) preincubated with 50 PM orthovanadate were incubated with (lanes 2 and 4 ) or without (lanes I and 3)Epo. Samples were solubilized under mild conditions (Buffer A) and immunoprecipitated with anti-Epo antibodies (lanes 1-2). Alternatively, samples were solubilized by boiling in SDS lysis buffer and immunoprecipitated with anti-Epo receptorantibodies (lanes 3-4). Phosphotyrosine-containing proteins present in the immunoprecipitates were analyzed by immunoblotting.
10673
Phosphorylation of Erythropoietin Receptor fact thehuman Epo receptor itself. To confirm this observation, a different experimental approach was used. We incubated the growth factor-deprived UT-7 cells with '251-Ep0 and tested whether the '251-Eporeceptor complexes were phosphorylated on tyrosine residues. As indicated in Fig. 6, anti-phosphotyrosine antibodies immunoprecipitated '251-Epobound to its receptor in a dosedependent manner. Nearly 30%of the totall2'1-Epo-receptor complexes present in the solubilized extracts were immunoprecipitated by the anti-phosphotyrosine antibodies. These "'I-Epo-labeled proteins were eluted from the immunoprecipitates by phenyl phosphate (0.1 M), confirming that the immunoprecipitation was due to specific recognition of phosphotyrosine residues in Epo-receptor complexes. The p75 Was the Only Subunit Phosphorylated in the EpoReceptor Complex-Previous studies have shown that the murine Epo receptor is a multimeric complex composed of at least three subunits of M, 100,000,85,000, and 66,000 (21). In order to identify the Epo receptor subunits expressed in the human UT-7 cells and to characterize the receptor subunits that specifically reacted with anti-phosphotyrosine antibodies, we incubated the UT-7 cells with Iz5I-Epo and crosslinked the Iz5I-Epobound complexes with disuccinimidyl suberate before immunoprecipitation with anti-Epo antibodies. As shown in Fig. 7A, the three receptor subunits, p100, p85, and p66, previously described in the murine Epo-responsive Reds cells (lanes 1-2) were expressed in UT-7 cells (lane 4 ) . However, the relative molecular weight of the lower receptor subunit expressed in UT-7 cells seemed to be slightly higher than the p66 detected in Reds cells; from four experiments, we determined a M , of 71,000 & 4,000 in UT-7 cells. When '"I-Epo cross-linked complexes were immunoprecipitated with anti-phosphotyrosine antibodies insteadof anti-Epo antibodies and analyzed by SDS-PAGE, the same three labeled bands were identified (Fig. 7B, lane 1). However, if crosslinked samples were first denatured by boiling in SDS lysis buffer before being immunoprecipitated by anti-Epo or antiphosphotyrosine antibodies, the anti-phosphotyrosine antibodies only recognized the receptor subunit p75 (Fig. 7B, lane
B 1 2 3
A 1 2
3 4
4
5
6
205-
*PlOO 4 p85 4 ~ 7 1
c c
45-i
FIG. 7. A single "'I-Epo cross-linked protein is tyrosinephosphorylated. A , Reds cells (lanes 1-2) or UT-7 cells (lanes 3-4) were incubated with "'I-Epo (1 nM) in the absence (lanes 1 , 2, and 4 ) or the presence (lane 3 ) of an excess of unlabeled Epo and cross(lane 1 ) linked with l-ethyl-3-(3-dimethylaminopropyl)carbodiimide or disuccinimidyl suberate (lanes 2-4). Cellsweresolubilized and immunoprecipitated using anti-Epo antibodies, and immunoprecipitates were analyzed by SDS-PAGE. p100, p85, and p66 correspond to of the murine receptor subunits present the molecular weight (X in the pointed '2'II-Epobound complex, after subtractingthe apparent molecular weight of the Epo molecule (34,000).B, 1251-Epocrosslinked UT-7 cells were immunoprecipitated with either anti-Epo(lane I ) , anti-MalE (lane 2), or anti-phosphotyrosine antibodies (lane 3) and further subjected to SDS-PAGE. Alternatively, Iz5I-Epocrosslinked cells were immunoprecipitated with anti-Epo antibodies and further denatured by boiling in SDS lysis buffer, before being reimmunoprecipitated using anti-phosphotyrosine (lane 4 ) , anti-MalE (lane 5),or anti-Epo (lane 6 ) antibodies and subjected to SDS-PAGE and autoradiography. Molecular weight markers are indicated on the left of part B ) . 4 ) , although under the same conditions, the anti-Epo anti-
bodies still immunoprecipitated the three receptor subunits (Fig.7B, lane 6). Therefore, in the human Epo-dependent UT-7 cells, among the threeEpo receptor subunits identified, only the receptor subunit p75 was tyrosine-phosphorylated following activation by Epo.
~
DISCUSSION
/
/ 0
0'. 4
I 4
I
40
ANTI PTyr : Fg/mL FIG. 6. Immunoprecipitation of 1261-Epo-receptorcrosslinked complexes by anti-phosphotyrosine antibodies. UT-7 cells (lo7 cells/point) were incubated with "'1-Epo, solubilized in
1.5% Triton X-100, and immunoprecipitated using various concentrations of anti-phosphotyrosine antibodies, followed by protein ASepharose precipitation. Immunoprecipitates were washed and treated with 0.1 M phenyl phosphate for 10 min. The eluted radioactivity was determined by y counting.
Our studies indicate that Epo rapidly and specifically induces the tyrosine phosphorylation of its own receptor in human Epo-responsive cells. Two different experimental approaches led ustothis conclusion. 1) We detected Epoinduced tyrosine phosphorylations in UT-7cells and showed that the major Epo-induced phosphoprotein p75 was specifically immunoprecipitated by anti-Epo receptor antibodies, even after dissociation of any protein-protein interactions (Fig. 5). (2) We also characterized the human Epo receptor subunits expressed in theUT-7 cell line by cross-linking lZ5IEpo and showed among the threereceptor subunits, p75, p85, and p100, cross-linked to '251-Epo, only p75 was tyrosinephosphorylated (Fig. 7). Furthermore, the Epo receptor was one of the major substrates tyrosine-phosphorylated following Epoactionon UT-7 cells. Our report of the human Epo receptor as being a major substrate for Epo-induced tyrosinephosphorylations appears to be in conflict with reports by Quelle et al. (16) who indicates that none of the Epo-induced murine tyrosine-phosphorylated proteins were immunoprecipitated by anti-Epo receptor antibodies and with reports by Miura et al. (17) who observed that only a minor fraction of the Epo receptor was tyrosine-phosphorylated. Such discrepancies are likely to be due, a t least in part, to the different systems and cell lines used, which include either human (the
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Phosphorylation of Erythropoietin Receptor
present report) or murine (16) cell lines expressing endogenous Epo receptors, or a murine cell line expressing transfected Epo receptor gene (17). Using a similar murine cell line transfected with the murine Epo receptor, these same investigators had previously reported that themajority of the Epo receptors were retained in the endoplasmic reticulum and Golgi subcellular compartments of the transfected cells (23). Therefore, only a small portion of the Epo receptors synthesized in these cells reached the plasma membrane and were actually susceptible to Epo activation at the cell surface. Different levels of Epo receptor expression at thecell surface between these different systems, as well as different reactivities of anti-Epo receptor reagents (16), might thus explain these apparent discrepancies. The UT-7 cells express a large number of endogenous high affinity Epo receptors at their surface (7000 sites/cell, Kd = 180 PM, see above), approximately 10-20-fold higher than in other Epo-responsive cells (2), which rendered the detection of Epo receptors more easy. We showedthat this human cell line expressed three Epo receptor subunits of M , 100,000, 85,000, and 71,000 (& 4,000), as detected by cross-linking experiments. However, the lower M , receptor subunit was identified as a protein of M , 75,000 3,000 ( n = 6) in our immunoblotting experiment. This slight difference might be explained in part by the fact that themolecular weight of Epo receptor subunits identified in cross-linking experiments are in fact deduced from the apparent molecular weight of the Epo-receptor complex in SDS gels subtracted of the apparent molecular weight of the Epo molecule. This receptor subunit was recognized by the antibodies raised against the recently cloned murine Epo receptor chain p66 (see Fig. 5) and probably represents the human homologue ofthis chain.Molecular cloning of this human receptor chain had indicated that this chain contained508 amino acids (24-25) and yielded a protein of M , 66,000 when expressed in COS cells (25). Our present results indicate that the endogenous human Epo receptor chain expressed in the UT-7cells is then likely to be further processed to yield a proteinof M, 75,000. Similar results were obtained in another human erythroid cell line TF1 in which the Epo receptor was reported to exhibit a M , of 71,000 (26). The detection of three Epo-binding subunits expressed on the UT-7 cells was not surprising; similar results were recently reported in murine Epo-responsive cells, for which two (p100 and p85) or three (p100, p85,and p66) receptor subunits were identified, depending on the cross-linking agent used (21). We also observed that among the three receptor subunits expressed at the surface of the UT-7 cells, only one, the p75, was tyrosine-phosphorylated following Epo activation. This suggests that the three Epo receptor subunits may not play equivalent roles in the transduction of Epo signaling and/or in thereceptor fate. Similar results were previously found for the IL3 receptor for which only the p140, and not the p70 receptor subunit, was tyrosine-phosphorylated following IL3 activation (11).In addition, our results strengthen the hypothesis according to which the p75, on the one hand, and the pl00 andp85, on the other hand, are unrelated proteins, as we previously reported (21). Apart from the Epo receptor, a number of other substrates were also tyrosine-phosphorylated following incubation with Epo. These phosphorylations were rapid, 2 min after Epo addition, and transient, decreasing after 15 min. Similar kinetics of induction of tyrosine phosphorylation were found in cells treated with IL2, IL3, IL4, and IL7 (7-12). Tyrosine phosphorylations were induced with concentrations of Epo as low as 0.1 unit/ml and reached a half-maximal level in the presence of 1 unit/ml of Epo, which corresponded to 270 pM
*
(Fig. 2). Comparison of Epo receptor affinity in these cells (Kd = 180 pM) with activation by Epo of the tyrosine kinase function appeared to correlate perfectly with the occupancy of a substantial fraction of the high affinity Epo receptors on UT-7 cells. The other tyrosine-phosphorylated proteins we identified in Epo-incubated UT-7 cells, namely p140, p120, p95, p60, p57, and p42, may correspond to those reported to be induced by Epo in murine cells (14-15), although uncertainty still exists as to whether specified substrates of apparent equivalent M, in fact represent equivalent proteins. Apart from the p75 that we identified as the Epo receptor, the relationship between any of these Epo-dependent phosphoproteins detected in our studies with known substrates for tyrosine phosphorylation is not known. Studies in platelet-derived growth factor-, epidermal growth factor-, or insulin-stimulated living cells have shown that several tyrosine phosphoproteins with molecular weights similar to those reported in the present study were activated and/or coimmunoprecipitated with their respective hormone receptors. These proteins have been identified as phospholipase C (Mr 140,000), GTPase-activatingprotein( M , 120,000),phosphatidylinositol 3-kinase ( M , 85,000), ruf (Mr74,000), and mitogen-associated protein kinase 2 (Mr 42,000) (27). Similar tyrosine-phosphorylated substrates were reported to be activated by membrane-associated non-receptor tyrosine kinases (30). The association of some of these proteins with the tyrosine kinase receptors was postulated to be vital for the function of the hormonal signal transduction pathway. IL2 receptor was also reported to be associated to phosphatidylinositol 3-kinase (28, 29) and/or to the tyrosine kinase p56 kk (15). The presence of an equivalent Epo-receptor signaling complex in erythroid cells requires further studies. However, it has been recently reported that Epo activates specifically the ruf kinase (31). A tyrosine-phosphorylated substrate of similar molecular weight (74,000), which was not immunoprecipitated by anti-Epo receptor antibodies, was also induced by Epo in murine cells (16, 17) and might represent the ruf kinase. It is noteworthy that, in our experiments, the p75 band appeared as a doublet (Fig. 5A). The lower molecular weight species of this doublet might represent either a less phosphorylated Epo receptor species or an unrelated associated phosphotyrosine protein such as ruf. The proteins p95, p120, and even p140, that were immunoprecipitated with anti-Epo and anti-Eporeceptor antibodies might also be components of the native multimeric Epo receptor previously evidenced by hydrodynamics studies (32). All our results strengthen the hypothesis that Epo acts on cells by inducing the tyrosine phosphorylation of a number of proteins, including its own receptor. Since the human UT7 cells proliferate and differentiate in the presence of Epo (1920), the Epo-induced tyrosine phosphorylations we detected could be related to a proliferative, as well as a differentiating signal. Whetheractivated Epo receptors that seem to be devoid of intrinsic tyrosine kinase activity interact with intracellular tyrosine kinase(s)either directly or througha coupling protein is presently unknown. Alternatively, the binding of the receptor may alter the conformation or location of substrate proteins,allowing their phosphorylation. Further studies are required to answer these questions. Acknowledgments-We are grateful to Sylvie Gisselbrecht, Marc Sitbon, and Ali Turhan for helpful discussions and critical reading of the manuscript.
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