THE JOURNAL 0 1991 by The American Society for Biochemistry and Molecular Bioloa,

OF BlOLOClCAL CHEMISTRY Inc.

Vol ,266. No. 1, Issue of January 5, pp. 609-614,1991 Printed in U. S. A .

Proliferative Action of Erythropoietin Is Associated with Rapid Protein Tyrosine Phosphorylation in Responsive B6SUt.EP Cells*

(Received for publication, June 28, 1990)

Frederick W. Quelle and Don M. WojchowskiS From the Department of Molecular and Cell Biology, Pennsylvania State University, University Park, Pennsylvania 16802

Erythropoietin is a prime regulator of the growth and terminal differentiation of erythroid blood cells. However, little is understood concerning its molecular mechanism of action. Presently it is shown in the re- sponsive, factor-dependent murine cell line B6SUt.EP that erythropoietin induces the tyrosine phosphoryla- tion of six plasma membrane-associated proteins in a time- and concentration-dependent fashion (i.e. phos- phoproteins PY 153, PY 140, PY 100, PY93, PY74, and PY54). Among these, PY153 was prominent. For all proteins, maximal levels of phosphorylation were in- duced within 3-7 min at low factor concentrations (100-500 PM). These findings establish tyrosine kinase activation as a novel candidate pathway of erythro- poietin-induced proliferation. In addition, the tyrosine phosphorylation of six proteins with identical M,, as well as a M, 104,000 protein, was induced in B6SUt.EP cells by interleukin 3. In contrast, no in- duced tyrosine phosphorylation was detectable in the erythropoietin-responsive, leukemic erythroid cell line, Rauscher Red 1, yet proteins of M, 153,000 and 54,000 were shown to be phosphorylated constitu- tively at relative levels greater than those observed in B6SUt.EP cells. A possible role for these phosphopro- teins in hematopoietic cell transformation is consid- ered.

Erythropoietin is a glycopeptide hormone that promotes the proliferation of erythroid progenitor cells in marrow (I), spleen (2), and fetal liver (3) and is required for their subse- quent terminal differentiation to circulating red blood cells (4). This factor has been well characterized through its puri- fication (5), cloning (6), expression as an active recombinant product in various heterologous cell lines (7-lo), and use as a therapeutic agent in clinical situations with associated red cell anemia (11,12). In contrast, little is understood regarding the primary biochemical events which are activated following the binding of erythropoietin to cell surface receptors. Mod- ulation of CAMP levels (13-16), arachidonic acid metabolism (17), a serine kinase activity (18), and the mobilization of intracellular calcium stores (19-23) have been reported to be associated with erythropoietin action in responsive cells, or cell lines. However, these effects either are delayed relative to the time of hormone-receptor interaction (17, 18) or have been shown to be associated primarily with erythropoietin- induced differentiation (22). Thus, to date no rapidly modu-

* This work was supported in part by National Institutes of Health Grant R29 DK40242. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ To whom correspondence should be addressed.

lated transduction event has been identified that is associated immediately with erythropoietin-induced proliferation.

For an increasing number of growth factor receptor systems, the immediate activation of plasma membrane-associated ty- rosine kinases has been implicated in signal transduction. This includes not only receptors which themselves encode kinase activity (24-27) but also a subclass of unrelated recep- tors which act to modulate distinct tyrosine kinases (28-33). These include, for example, the lymphocyte CD4/CD8 recep- tor-lck kinase (34) and 1L’-2 (35) receptor systems in which this modulation has been implicated to involve direct, non- covalent association between receptor and kinase. In the instance of the erythropoietin receptor, no recognizable pro- tein kinase domains are encoded by a recently cloned (36), functional receptor cDNA (37). Nonetheless, the present study shows that erythropoietin rapidly induces the tyrosine phosphorylation of several plasma membrane-associated pro- teins in the responsive, factor-dependent murine cell line B6SUt.EP. Notably, this is the first report of a positively modulated biochemical event which is associated immediately with the activation of erythropoietin receptors in non-trans- formed cells. In addition, a possible role for two phosphoty- rosine-containing proteins in leukemic growth is considered based on their observed constitutive phosphorylation in an erythropoietin-responsive yet transformed cell line, Rauscher Red 1 (38-40).

MATERIALS AND METHODS

Cell Lines and Growth Factors-B6SUt.EP cells’ are an erythroid subclone of a previously established murine cell line B6SUtA (41) and were isolated following growth in soft agar in the presence of erythropoietin. B6SUt.EP and DA1 (42) cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum, M 2-mercaptoethanol, and 5% conditioned medium from WEHI-3 cells. Rauscher murine erythroleukemia cells (clone Red 1) (38) were maintained in this medium lacking WEHI-3 CM. Recombinant human erythropoietin was produced and purified as described previously (10) or was provided by R. W. Johnson Pharmaceutical Research Laboratories, La Jolla, CA. Purified syn- thetic murine IL-3 (Biomedical Research Center, Vancouver, British Columbia) or purified recombinant murine IL-3 (DNAX, Palo Alto, CA) was used as indicated.

Analyses of Tyrosine Phosphorylation-In Western analyses of phosphotyrosine-containing proteins, cells were washed in DMEM, incubated in DMEM plus 1% fetal calf serum for 6-10 h, admixed with 0.05 mM Na3V04, and exposed to hematopoietic growth factors (2 X lo6 cells/ml) a t specified concentrations and intervals. Incuba- tions with vanadate were uniform for all samples (30 min) and only the concentration or length of exposure to growth factor was varied. Incubations were halted by rapid freezing a t -70 “C. Cells then were thawed directly into sample buffer (100 “C) for sodium dodecyl sul-

The abbreviations used are: IL, interleukin; DMEM, Dulbecco’s modified Eagle’s medium; SDS, sodium dodecyl sulfate; PAGE, poly- acrylamide gel electrophoresis.

D. M. Wojchowski, R. C. Hardison, D. E. Quelle, R. Burkert- Smith, L. Zon, and J . Schrader, manuscript in preparation.

609

610 Erythropoietin-induced Protein Tyrosine Phosphorylation

fate-polyacrylamide gel electrophoresis (SDS-PAGE) and were boiled for 10 min. SDS-PAGE (7.5% gels) was performed as described by Laemmli (43). Phosphotyrosine-containingproteins were detected by transfer to nitrocellulose and staining with '2"I-protein A and affinity purified antibodies specific for phosphotyrosine (44). Essentially identical procedures were used in analyses of phosphoproteins con- tained in subcellular fractionations. Autoradiographs were analyzed quantitatively using an optical scanning system (LeMont Scientific OASys System).

In immunoprecipitations, washed cells initially were incubated in phosphate-free DMEM, 1% fetal calf serum for 3 h a t 2 X lofi cells/ ml and subsequently for 1 h in this medium containing 0.5 mCi/ml ['"P]orthophosphate (285 Ci/mg). Incubations with erythropoietin were performed as described above. Cells were collected by microcen- trifugation (2 min a t 1000 X g) and were lysed in 10 mM Tris, pH 7.6, 1% Triton x-100,50 mM NaCI, 30 mM Na4P20i, 50 mM NaF, 0.1 mM NanV04, 0.5 pg/ml leupeptin, 0.7 pg/ml pepstatin, 50 pg/ml phenyl- methylsulfonyl fluoride, and 0.1% bovine serum albumin. ["'PIPhos- phoproteins were precipitated using antibodies to phosphotyrosine (2.5 h of incubation a t 4 "C), and protein G-Sepharose (Pharmacia LKB Biotechnology Inc.) (30 min of incubation a t 4 "C). Antigens were eluted by incubation for 10 min a t 4 "C in the above buffer containing 0.1 M phenylphosphate and 0.01% bovine serum albumin and were analyzed by SDS-PAGE. Phosphoamino acid analyses of eluted proteins were performed essentially as described by Cooper et al. (45).

Subcellular Fractionation-Washed cells were disrupted in lysis buffer (200 mM sucrose, 0.05 mM NanVO,, 5 mM NaF, 0.5 pg/ml leupeptin, 0.7 pg/ml pepstatin, 50 pg/ml phenylmethylsulfonyl fluo- ride, 0.5 mM EDTA, and 20 mM Tris, pH 7.5) with 10 strokes of a stainless steel Dounce homogenizer. Nuclei were removed by centrif- ugation a t 600 X g for 10 min. Membranes were subfractionated from post-nuclear supernatants using discontinuous sucrose gradients pre- pared in lysis buffer (7, 15, and 45%; 5-ml gradients, 2 h a t 200,000 X g). Alternatively, cytosol was prepared from post-nuclear superna- tants by centrifugation for 2 h a t 200,000 X g. Membrane subfractions were washed in lysis buffer prior to analyses for phosphotyrosine or marker enzyme content. Fractionations and washes were performed at 0-4 "C.

RESULTS

In order to initially evaluate possible regulatory effects of erythropoietin on intracellular tyrosine kinase activities, B6SUt.EP cells were exposed to erythropoietin (25 units/ml) for varying intervals (see "Materials and Methods"), and total cellular phosphotyrosine-containing proteins were analyzed by Western blotting. Eight major phosphoproteins were de- tected, Le. PY153, PY140, PY100, PY93, PY74, PY54, PY50, and PY41 (Fig. 1). Among these PY153, PY140, PY100, PY93, PY74, and PY54 showed significant, rapid increases in phos- photyrosine content in response to erythropoietin. Apparent maximal levels of tyrosine phosphorylation of PY153 gener- ally were attained within 3 min of exposure to hormone, while maximal phosphorylation of PY140, PY100, PY93, PY74, and PY54 occurred subsequently (i.e. within 7 min). This early induction of PY153 was observed consistently in repeated time course studies.

The six phosphoproteins modulated by erythropoietin in the above time course studies also were modulated in a con- centration-dependent fashion as shown in Fig. 2. Maximal induction of phosphorylation was attained between 0.5 and 2.5 units/ml erythropoietin. For comparison, the proliferative responsiveness of B6SUt.EP cells to the same preparation of purified erythropoietin, and to IL-3, is shown in Fig. 3. Since B6SUt.EP cells proliferate in response to, and show factor dependence for both erythropoietin and IL-3, tyrosine phos- phorylation as induced by IL-3 also was studied. Exposure to IL-3 induced a similarly rapid increase in the phosphotyrosine content of six proteins with M , equivalent to PY153, PY140, PY100, PY93, PY74, and PY54 (Fig. 1). In addition, IL-3 stimulated the tyrosine phosphorylation of a M , 104,000 pro- tein (PY104) which was not observed in control cells, or in

E l I 0' I 3' 7 12'1 3'

11-3 E';, 12' I

68-

"r1153

-?1140

"n104 "rY100 "rv93

-?Y 74

4 1 5 4

42.7-

27.4- I

rlll h

FIG. 1. Time-dependent erythropoietin (Epo)- and IL-3-in- duced protein tyrosine phosphorylation in B6SUt.EP cells. B6SUt.EP cells were exposed to either erythropoietin (25 units/ml) or synthetic murine IL-3 (100 nM) for varying intervals and then were lysed and immunoblotted using antibodies to phosphotyrosine as described under "Materials and Methods."

"11153 -?VI48

U- -?114

42.1- - w g --1154

FIG. 2. Concentration dependence of erythropoietin-in- duced protein tyrosine phosphorylation in B6SUt.EP cells. Following exposure of B6SUt.EP cells to erythropoietin a t varying concentrations (5.5 min of exposure), phosphotyrosine-containing proteins were analyzed by immunoblotting as described above.

Erylhropoielm (U/ml) Inlerleukln 3 (nu)

FIG. 3. Erythropoietin- versus IL-3-induced growth of B6SUt.EP cells. Following the exposure of B6SUt.EP cells to factor for 20 h, growth rates were estimated quantitatively by the relative rate of incorporation (2 h) of ["Hlthymidine.

Erythropoietin-induced Protein Tyrosine Phosphorylation 611

B6SUt.EP cells exposed to erythropoietin. In order to establish the cellular localization of proteins

that are phosphorylated upon exposure to erythropoietin, phosphotyrosine-containing proteins in subcellular fractions of B6SUt.EP cells were analyzed. All six erythropoietin- modulated phosphoproteins copurified with the low-density, plasma membrane-containing fraction (Fig. 4). The associa- tion of a minor amount of each of these phosphoproteins (15% by optical densitometry) with membranes of densities 21.20 g/ml likely is due to the presence of plasma membranes in this fraction since an essentially equivalent portion of alkaline phosphodiesterase activity (i.e. 15%) copurified with the high density fraction (Table I). No detectable amounts of these specified phosphoproteins were observed in cytosolic frac- tions.

Confirmation that phosphotyrosine epitopes comprise the basis for the binding of antibodies to blotted proteins was provided by phosphoamino acid analyses of proteins labeled metabolically with [:"P]orthophosphate and immunoprecipi- tated from lysed B6SUt.EP cells. Immunoadsorption was shown to result in a substantial, selective increase in phos-

I Hih tknsltv Membrane I Membrane

224-

I C v l o r ~ l I 224 -

109-

71.8-

45.8-

109 -

71.8-

45.8-

FIG. 4. Subcellular localization of erythropoietin-modu- lated phosphotyrosine-containing proteins in B6SUt.EP cells. B6SUt.EP cells were incubated with erythropoietin (30 units/ml) for 9 min. Cytosol, plasma membranes (i.e. the low density membrane fraction, 1.06-1.20 g/ml), and high density membranes ( ~ 1 . 2 0 g/ml) then were isolated and were analyzed by immunoblotting using anti- bodies to phosphotyrosine. The volumes of samples were adjusted to provide equivalent loading (50 pg protein/lane). Panel A, high density membranes uersus plasma membranes (i.e. low density membrane fraction); panel R, cytosol.

photyrosine content (Fig. 523). Using this procedure, it also was possible to demonstrate the erythropoietin-induced phos- phorylation of proteins similar in size to PY153, PY140, PY100, and PY74 (Fig. 5A). Although separation is incom- plete in this gel system, PY153 apparently resolved as M, 157,000 and 150,000 peptides. In contrast to Western anal- yses, erythropoietin-induced tyrosine phosphorylation of a Mr 66,000 protein was detected through :32P labeling, while no phosphoproteins similar in size to PY93 or PY54 were ob- served. These differences possibly are due to differential re- activity of antibodies with blotted versus solubilized phospho- proteins. Also, phosphoamino acid analyses (Fig. 5B) suggest that at least certain of these proteins contain substantial amounts of phosphoserine, and erythropoietin-modulated phosphorylation at these residues is possible. Nonetheless, the additional demonstration that immunoadsorption is gen- erally inhibited by phenylphosphate (Fig. 5C) provides further evidence that antibody binding to each of the above phospho- proteins occurs a t phosphotyrosine.

In studies aimed at further investigating the apparent as- sociation between erythropoietin-mediated proliferation and protein tyrosine phosphorylation, analyses of phosphopro- teins also were performed using an alternate erythropoietin- responsive, yet transformed murine erythroid cell line, Rauscher Red 1. No modulation of tyrosine phosphorylation in Red 1 cells was observed in response to either erythropoi- etin or IL-3. However, high relative levels of constitutive tyrosine phosphorylation of M , 153,000 and 54,000 proteins were observed (Fig. 6). As a control, and a point of further comparison, identical analyses were performed using an IL- 3-dependent murine cell line, DA1, which is not responsive to erythropoietin and does not express detectable levels of eryth- ropoietin receptors or receptor transcripts. In these cells, IL- 3 induced the tyrosine phosphorylation of proteins with M , identical to proteins PY153, PY140, PY100, PY74, and PY54 of B6SUt.EP cells. Phosphorylation of a M , 41,000 protein also was stimulated, while no phosphorylation of a protein corresponding to PY93 of B6SUt.EP cells was detected. No modulation of protein phosphorylation in DA1 cells was ob- served in response to erythropoietin.

DISCUSSION

Although erythropoietin (6-9), its cell surface receptor (36, 46-51), and its biological role in erythropoiesis (1-4) have been well characterized, little is understood concerning pri- mary biochemical pathways which are activated by this factor. Several intracellular events recently have been reported to be modulated by erythropoietin, yet none of these appear to comprise an initial step in the mechanism of erythropoietin- induced proliferation. Studies using heterogeneous prepara- tions of marrow cells have indicated that erythropoietin may

TABLE I CoDurification of ervthrowoietin-modulated Dhosahowroteins with olasma membranes

~

High density membrane Plasma membrane z1.20glml 1.06-1.20g/ml

Relative lane intensity",h 1 f 0.9 5.7 f 3.0 Alkaline phosphodiesterase".' 5.1 f 1.4 units/mg protein 30.1 -C 3.8 units/mg protein Cytochrome oxidase",d 36.7 a 6.6 units/mg protein 2.7 f 0.5 units/mg protein

Six independent samples from membrane fractions as analyzed in Fig. 38. Determined by full-lane optical scanning of samples immunoblotted for phosphotyrosine. The volume of each

'. One unit corresponds to the hydrolysis of 1 pmol of thymidine-5'-monophospho-p-nitrophenyl ester/min, as

Measured by the oxidation of reduced cytochrome. One unit corresponds to a change of one log in absorbance

sample was adjusted to provide for equivalent loading (50 pg protein/lane).

determined by absorbance at 400 nM.

at 550 nm/min.

612 Erythropoietin-induced Protein Tyrosine Phosphorylation

106- " 1 1 1 0 0

I DA1 I Red1 I c IL-3 EDO c IL-3 EDO

7l.8- 71- 3 1p"'4

P 1 6 6

44-

B Pi[ * 0

p-s*r[ 9 0

C

c 106-

71-

44-

w J, c D i .

FIG. 5. Immunoprecipi ta t ion and phosphoamino acid analy- sis of phosphotyrosine-containing proteins in erythropoietin- exposed B6SUt.EP cells. Panel A, BGSUt.EP cells were labeled with [:"P]orthophosphate and were exposed to erythropoietin (20 units/ml) for the intervals specified. Cell lysates then were immuno- precipitated using antibodies to phosphotyrosine. Phosphotyrosine- containing proteins were eluted with phenylphosphate and were ana- lyzed by SDS-PAGE. Panel H , in an identical experiment, R6SUt.EP cells were exposed to erythropoietin (20 units/ml, 9 min of exposure) and were immunoprecipitated with anti-phosphotyrosine antibodies. Eluted proteins (lane I ) versus total cellular proteins (lane 2) then were analyzed for phosphoamino acid content. Panel C, inhibition by phenylphosphate of the precipitation of B6SUt.EP phosphoproteins

FIG. 6. Protein tyrosine phosphorylation in DA1 and Rauscher Red 1 cells. Following exposure for 4 min to purified recombinant murine 11,-3 (5 nM), erythropoietin (Epo) (25 units/ml), or no growth factor (Control, C) phosphotyrosine-containing proteins in DA1 and Rauscher Red 1 cell lysates were analyzed by immuno- blotting as described above.

stimulate CAMP production (13,14). However, analyses using responsive clonal cell lines have failed to demonstrate any similar direct modulation (15, 16). Erythropoietin-dependent increases in metabolites of arachidonic acid also have been detected in isolated erythroid tissues (e.g. fetal liver), yet this effect is delayed with a maximum response occurring a t 21 h of exposure (17). Similarly, a reported inhibition by erythro- poietin of the phosphorylation of phosphoserine proteins in Rauscher cells likewise is delayed, requiring 230 min and the use of factor at high concentrations (2800 units/ml) (18). The single previous indication of a rapidly transduced signal is the erythropoietin-induced mobilization of intracellular calcium within 3-10 min of hormone exposure in Friend virus-infected marrow cells (19) and isolated mononuclear erythroid progen- itor cells (20, 21). However, recent studies using partially purified erythroid cells have shown that calcium mobilization apparently occurs in mature erythroblasts (i.e. partially he- moglobinized cells) and not in immature progenitor cells (i.e. burst and colony forming units-erythroid) (22, 23). This sug- gests a primary association with erythroid differentiation, as opposed to erythropoietin-induced proliferation. In contrast, the present study demonstrates erythropoietin-dependent ty- rosine phosphorylation of six plasma membrane proteins within 3-7 min of exposure to factor in murine B6SUt.EP cells, an erythroid subline of B6SUtA cells (41) which displays factor dependence for erythropoietin and IL-3. Thus, this constitutes the first demonstration of a positively modulated biochemical event which is associated immediately with eryth- ropoietin-induced proliferation. Since the phosphotyrosine phosphatase inhibitor orthovanadate (52) was used in all experiments, this transduction event likely involves the acti- vation of tyrosine kinases rather than phosphatase inhibition. Although no recognizable kinase domain is encoded by a recently cloned, functional receptor cDNA (36, 37), a role for tyrosine kinases in mediating erythropoietin-induced growth has been suggested on the basis of efficient transformation of erythroid cells by activated forms of ab1 (53) and src (54) kinases, and the action of an inhibitor of tyrosine kinases, herbimycin A. Herbimycin inhibits erythropoietin-induced

with antibodies to phosphotyrosine. '"P-Labeling and immunoprecip- itations were performed as above in the absence (lane I ) or presence of 0.1 M phenylphosphate (lane 2). Eluted proteins then were analyzed by SDS-PAGE.

Erythropoietin-induced Protein Tyrosine Phosphorylation 613

growth, but not differentiation, in murine TSA8 (55) cells and induces erythroid differentiation in human K562 cells (56). While the interpretation that tyrosine kinase activity neces- sarily is associated selectively with erythropoietin-induced proliferation (55) is considered speculative, the presently dem- onstrated association between erythropoietin action and ty- rosine kinase activation in B6SUt.EP cells is consistent with these earlier reports.

Although the identity of erythropoietin-modulated kinases and their substrates in B6SUt.EP cells presently is not known, the apparent equivalence between at least certain of the substrates as phosphorylated in response to erythropoietin and IL-3 suggests that overlap exists in the molecular path- ways which are activated by these two growth factors. IL-3- induced tyrosine phosphorylation has been studied previously in several alternate cell lines. In FDC-P1 and DA1 cells, proteins of M , 38,000,56,000,70,000,100,000 and a membrane glycoprotein of M , 140,000 or 150,000 have been identified as substrates for IL-%mediated tyrosine kinases (28, 29). The M , of the erythropoietin- and IL-3-modulated phosphopro- teins presently identified in B6SUt.EP (and DA1) cells ( i e . PY40, PY54, PY74, PY100, PY140, and PY153) are in close apparent correspondence with these previously identified phosphoproteins. Similarly, in a study of IC2 cells, IL-3- induced tyrosine phosphorylation of proteins with M , approx- imating PY100, PY93, PY74, and PY54 of B6SUt.EP has been identified (30). Finally, in a recent study of an IL-3- and granulocyte-macrophage colony-stimulating factor-respon- sive subclone of B6SUt which is not responsive to erythro- poietin ( i e . BGSUtAJ, phosphorylation events similar to those presently demonstrated in B6SUt.EP cells were induced by IL-3 with the exception that proteins corresponding to PY153 and PY104 were not identified (31). It is recognized that certain of these specified differences may be attributable to use of different cell lines, alternate sources of growth factors, and antibodies with potentially different specificities. Likewise, uncertainty exists as to whether specified substrates of apparently equivalent M , in fact represent equivalent pro- teins. For example, in FDC-P1 cells phosphotyrosine-contain- ing proteins of M, 74,000 and 54,000 have been identified as primarily cytosolic (29) whereas in BGSUtA, (31) and B6SUt.EP cells, modulated phosphoproteins of these approx- imate M , are associated with the plasma membrane. None- theless, the direct comparison of erythropoietin- and IL-3- modulated phosphoproteins in the present study suggests that in B6SUt.EP cells, at least certain of these proteins are modulated commonly by both factors. In contrast, the inabil- ity of erythropoietin to stimulate phosphorylation of PY104 in B6SUt.EP cells underlines the likelihood that the pathways activated by these growth factors are, at least in part, distinct. In the IL-3 system, cross-linking and Western analyses have led to the suggestions that phosphotyrosine-containing pro- teins of 140,000 (29,57,58) or 150,000 (28) may be associated with, or in part comprise, the murine IL-3 receptor. However, based on cDNA cloning and expression, Itoh et al. (59) deduce a M , of approximately 95,000 for this receptor. Studies of erythropoietin binding and cross-linking have identified two putative cell surface receptor proteins of M, 65,000-95,000 and 95,000-120,000 (36, 46-51). Although the M , of several of the presently identified membrane phosphoproteins of B6SUt.EP are within this range, all except the IL-3-induced protein PY104 apparently also occur in lymphoid DA1 cells. In addition, it has not been possible to demonstrate reactivity of antibodies to the erythropoietin receptor with any of the presently identified phosphoproteins of B6SUt.EP cells.'

'I F. W. Quelle, unpublished results.

Thus, it is considered unlikely that any of these erythropoie- tin-modulated phosphoproteins correspond to erythropoietin receptors.

With regard to their capacity for factor-independent growth, erythropoietin-responsive Rauscher erythroleukemia cells (e.g. Rauscher Red 1) (38-40) differ from the above factor-dependent cell lines, including B6SUt.EP. Thus, the presently observed lack of regulated tyrosine kinase activity in Rauscher cells, as well as the constitutively high level of tyrosine phosphorylation of M , 153,000 and 54,000 proteins, are consistent with an association between this activity and erythropoietin-induced growth. Interestingly, constitutive ty- rosine phosphorylation of a M, 150,000 membrane protein in factor-independent subclones of IL-3-dependent IC2 and DA1 cells previously has been reported (28). Likewise, in a trk- transformed FDC-P1 cell line which proliferates independ- ently of IL-3, tyrosine phosphorylation of a M , 56,000 protein is constitutive (29). Taken together, these findings suggest that PY153 and PY54 possibly may play a role in maintaining proliferation in various hematopoietic cells. However, the possibility obviously exists that increased phosphorylation of these specified substrates may be only associated with factor- independent growth.

By establishing a relationship between erythropoietin-in- duced proliferation and the tyrosine phosphorylation of plasma membrane-associated proteins in responsive B6SUt.EP cells, the present study serves to identify a novel candidate signal transduction mechanism which is associated immediately with erythropoietin receptor activation and erythropoietin-induced proliferation. The eventual isolation and identification of these phosphoproteins and their associ- ated kinases should advance an understanding of their pro- spective roles in regulated hematopoietic cell growth.

Acknowledgments-We thank Drs. H. Hanafusa (Rockefeller Uni- versity) and J . Y. J . Wang (University of California at San Diego), Drs. A. Miyajima (DNAX, Inc.) and 1 . 4 . Lewis (Biomedical Research Center, British Columbia), and the R. W. Johnson Pharmaceutical Research Laboratories (La Jolla, CA) respectively, for their generous provisions of anti-pbosphotyrosine antibodies, murine interleukin 3, and erythropoietin.

REFERENCES

1. Krantz, S. B., Gallien-Lartigue, O., and Goldwasser, E. (1963) J.

2. Krystal, G. (1983) Exp. Hematol. 11, 649-660 3. Dunn, C. D. R., Jarvis, J. H., and Greenman, J. M. (1975) Exp.

4. Nigon, V., and Godet, J. (1976) Znt. Reu. Cytol. 46, 79-175 5. Miyake, T., Kung, C. K.-H., and Goldwasser, E. (1977) J. Biol.

Chem. 252,5558-5564 6. Jacobs, K., Shoemaker, C., Rudersdorf, R., Neill, S. D., Kaufman,

R. J., Mufson, A., Seehra, J., Jones, S. S., Hewick, R., Fritsch, E. F., Kawakita, M., Shimizu, T., and Miyake, T. (1985) Nature

7. Davis, J. M., Arakawa, T., Strickland, T. W., and Yphantis, D.

8. Sasaki, H., Bothner, B., Dell, A,, and Fukuda, M. (1987) J. Biol.

9. Recny, M. A., Scoble, H. A., and Kim, Y. (1987) J . Biol. Chem.

10. Quelle, F. W., Caslake, L. F., Burkert, R. E., and Wojchowski, D.

11. Winearls, C. G., Oliver, D. O., Pippard, M. J., Reid, C., Downing,

12. Eschbach, J. W., Egrie, J . C., Downing, M. R., Browne, J. K., and

13. Chiuini, F., Torre, G. D., Fano, G., and Viti, A. (1979) Acta

14. Bonanou-Tzedaki, S. A,, Setchenska, M. S., and Arnstein, H. R.

15. Tsuda, H., Sawada, T., Sakaguchi, M., Kawakita, M., and Tak-

Biol. Chem. 238,4085-4090

Hematol. 3, 65-78

313,806-810

A. (1987) Biochemistry 26, 2633-2638

Chem. 262,12059-12076

262, 17156-17163

M. (1989) Blood 74, 652-657

M. R., and Cotes, P. M. (1986) Lancet 2, 1175-1178

Adamson, J . W. (1987) N. Engl. J . Med. 316, 73-78

Huematol. 61, 251-257

V. (1986) Eur. J . Biochem. 155, 363-370

614 Erythropoietin-induced Prott

atsuki, K. (1989) Exp. Hematol. 17 , 211-217 16. Fukumoto, H., Matsui, Y., and Obinata, M. (1989) Deuelopment

17. Beckman, B. S., Mason-Garcia, M., Nystuen, L., King, L., and Fisher, J. W. (1987) Biochem. Biophys. Res. Commun. 147 ,

18. Choi, H.-S., Wojchowski, D. M., and Sytkowski, A. J. (1987) J.

19. Sawyer, S. T., and Krantz, S. B. (1984) J. Biol. Chem. 259,2769-

20. Miller, B. A., Scaduto, R. C., Tillotson, D. L., Botti, J. J., and

21. Mladenovic, J., and Kay, N. E. (1988) J. Lab. Clin. Med. 112,

22. Miller, B. A., Cheung, J. Y., Tillotson, D. L., Hope, S. M., and

23. Imagawa, S., Smith, B. R., Palmer-Crocker, R., and Bunn, H. F.

24. Ullrich, A., Bell, J. R., Chen, E. Y., Herrera, R., Petruzzelli, L. M., Dull, T. J., Gray, A., Coussens, L., Liao, Y.-C., Tsubokawa, M., Mason, A., Seeburg, P. H., Grunfeld, C., Rosen, 0. M., and Ramachandran, J. (1985) Nature 313 , 756-761

25. Scherr, C. J., Rettenmier, C. W., Sacca, R., Roussel, M. F., Look, A. T., and Stanley, E. R. (1985) Cell 4 1 , 665-676

26. Yarden, Y., Escobedo, J. A., Kuang, W-J., Yang-Feng, T. L., Daniel, T. O., Tremble, P. M., Chen, E. Y., Ando, M. E., Harkins, R. N., Francke, U., Fried, V. A., Ullrich, A., and Williams, L. T. (1986) Nature 323, 226-232

27. Ullrich, A., Coussens, L., Hayflick, J. S., Dull, T. J., Gray, A., Tam, A. W., Lee, J., Yarden, Y., Libermann, T. A., Schlessin- ger, J., Downward, J., Mayes, E. L. V., Whittle, N., Waterfield, M. D., and Seeburg, P. H. (1984) Nature 309 , 418-425

28. Koyasu, S., Tojo, A,, Miyajima, A., Akiyama, T., Kasuga, M., Urabe, A., Schreurs, J., Arai, K.-I., Takaku, F., and Yahara, I.

29. Isfort, R., Huhn, R. D., Frackelton, A. R., Jr., and Ihle, J. N.

30. Morla, A. O., Schreurs, J., Miyajima, A., and Wang, J . Y. J . (1988)

31. Sorensen, P. H. B., Mui, A. L.-F., Murthy, S. C., and Krystal, G.

32. Farrar, W. L., and Ferris, D. K. (1989) J. Biol. Chem. 264,

33. Saltzman, E. M., Luhowskyj, S. M., and Casnellie, J. E. (1989) J. Biol. Chem. 264,19979-19983

34. Turner, J . M., Brodsky, M. H., Irving, B. A., Levin, S. D., Perlmutter, R. M., and Littman, D. R. (1990) Cell 60,755-765

35. Merida, I., and Gaulton, G. N. (1990) J. Biol. Chem. 2 6 5 , 5690- 5694

105,109-114

392-398

Biol. Chem. 262,2933-2936

2774

Cheung, J. Y. (1988) J. Clin. Znuest. 8 2 , 309-315

23-27

Scaduto, R. C. (1989) Blood 7 3 , 1188-1194

(1989) Blood 73 , 1452-1457

(1987) EMBO J. 6, 3979-3984

(1988) J. Biol. C&m. 2 6 3 , 19203-19209

Mol. Cell. Biol. 8, 2214-2218

(1989) Blood 7 3 , 406-418

12562-12567

?in Tyrosine Phosphorylation

36. D’Andrea, A. D., Lodish, H. F., and Wong, G. G. (1989) Cell 57 ,

37. Li, J.-P., D’Andrea, A. D., Lodish, H. F., and Baltimore, D. (1990) Nature 343 , 762-764

38. de Both, N. J., Vermey, M., van’t Hull, E., Klootwijk-van-Dijke, E., van Griensven, L. J. L. D., Mol, J. N. M., and Stoof, T. J. (1979) Nature 2 7 2 , 626-628

39. Sytkowski, A. J., Salvado, A. J., Smith, G. M., and McIntyre, C. J. (1980) Science 210 , 74-76

40. Weiss, T. L., Barker, M. E., Selleck, S. E., and Wintroub, B. U. (1989) J. Biol. Chem. 264, 1804-1810

41. Greenberger, J . S., Sakakeeny, M. A., Humphries, R. K., Eaves, C. J., and Eckner, R. J . (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 2931-2935

42. Ihle, J. N., Rein, A., and Mural, R. (1984) in Advances in Viral

York Oncology (Klein, G., ed) Vol. 4, pp. 95-137, Raven Press, New

277-285

43. Laemmli, U. K. (1970) Nature 227,680-685 44. Wang, J. Y. J. (1985) Mol. Cell. Biol. 5, 3640-3643 45. Cooper, J. A., Sefton, B. M., and Hunter, T. (1983) Methods

46. Sawyer, S. T., Krantz, S. B., and Luna, J. (1987) Proc. Natl. Acad.

47. Mayeux, P., Billat, C., and Jacquot, R. (1987) J. Biol. Chem. 262 ,

48. Sasaki, R., Yanagawa, S., Hitomi, K., and Chiba, H. (1987) Eur. J . Biochem. 168, 43-48

49. Tojo, A., Fukamachi, H., Kasuga, M., Urabe, A., and Takaku, F. (1987) Biochem. Biophys. Res. Commun. 148, 443-448

50. Todokoro, K., Kanazawa, S., Amanuma, H., and Ikawa, Y. (1987) Proc. Natl. Acad. Sci. U. S. A. 84,4126-4130

51. McCaffery, P. J., Fraser, J. K., Lin, F-K., and Berridge, M. V. (1989) J. Biol. Chem. 264 , 10507-10512

52. Swamp, G., Cohen, S., and Garbers, D. L. (1982) Biochem. Biophys. Res. Commun. 107,1104-1109

53. Waneck, G., and Rosenberg, N. (1981) Cell 26, 79-89 54. Anderson, S. M., Klinken, S. P., and Hankins, W. D. (1985) Mol.

55. Noguchi, T., Fukumoto, H., Mishina, Y., and Obinata, M. (1988)

56. Honma, Y., Okabe-Kado, J., Hozumi, M., Uehara, Y., and Mi-

57. Isfort, R. J., Stevens, D., May, W. S., and Ihle, J. N. (1988) Proc.

58. Sorensen, P., Mui, A. L.-F., and Krystal, G. (1989) J. Biol. Chem.

59. Itoh, N., Yonehara, S., Schreurs, J., Gorman, D. M., Maruyama, K., Ishii, A,, Yahara, I., Arai, K., and Miyajima, A. (1990) Science 247,324-327

Enzymol. 9 9 , 387-402

Sci. U. S. A. 84, 3690-3694

13985-13990

Cell. Biol. 5, 3369-3375

Mol. Cell. Biol. 8, 2604-2609

zuno, S. (1989) Cancer Res. 49,331-334

Natl. Acad. Sci. U. S. A. 8 5 , 7982-7986

264,19253-19258