Proc. Natl. Acad. Sci. USA Vol. 89, pp. 6237-6241, July 1992 Physiology
Association of the erythropoietin receptor with protein tyrosine kinase activity (signal transduction/protein phosphorylation)
DIANA LINNEKIN*, GERALD A. EVANSt, ALAN
D'ANDREAt, AND WILLIAM L. FARRAR*§
*Laboratory of Molecular Immunoregulation, Biological Response Modifiers Program, and tBiological Carcinogenesis and Development Program, Program Resources, Inc./Dyn Corp., National Cancer Institute, Frederick Cancer Research and Development Center, Frederick, MD 21702-1201; and tDivision of Hematology-Oncology, The Children's Hospital, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02115
Communicated by Avram Goldstein, April 14, 1992
with the Epo receptor. The dominant phosphotyrosylprotein observed in the Epo receptor complex after treatment with Epo was 97 kDa. Furthermore, a 97-kDa ATP binding protein was also observed in the Epo receptor complex. These results demonstrate association of the Epo receptor with protein tyrosine kinase activity and suggest that p97 is a protein tyrosine kinase in the Epo receptor complex.
We have examined the signal transduction ABSTRACT mechanism of the hematopoietic growth factor erythropoietin (Epo). Epo stimulation of Ba/F3 cells transfected with the Epo receptor resulted in increases in tyrosine phosphorylation of proteins of 97, 75, and 55 kDa. Epo-induced increases in tyrosine phosphorylation of a 97-kDa protein were also detected within the Epo receptor complex, suggesting that a protein tyrosine kinase is associated with the Epo receptor. Protein tyrosine kinase activity was found within the Epo receptor complex and modulation of this activity was observed after treatment of cells with Epo. Furthermore, constitutively high amounts of protein kinase activity were observed in Epo receptor complexes isolated from autonomously growing cells coexpressing the Epo receptor and the leukemogenic glycoprotein gp55. The dominant phosphotyrosylprotein found associated with the Epo receptor was 97 kDa. An Epo receptorassociated protein of identical molecular mass was also found to bind ATP, a characteristic critical for protein kinases. Collectively, these data demonstrate that the Epo receptor is associated with protein tyrosine kinase activity and further suggest that a 97-kDa phosphotyrosylprotein associated with the Epo receptor is a protein tyrosine kinase involved in Epo-mediated signal transduction.
MATERIALS AND METHODS Cell Lines and Growth Factors. The cell lines Ba/F3, Ba/F3-ER (Ba/F3 transfected with the murine Epo receptor), MEL, and Ba/F3-ER/GP55 (Ba/F3-ER infected with gp55) were grown as described (24). Radiolabeling and Phosphmino Acid Hydrolysis. Radiolabeling, electrophoresis, and phosphoamino acid hydrolysis of phosphotyrosylproteins were performed as described (25). Immunoprecipitations of phosphotyrosylproteins were performed with PY20 (ICN), a monoclonal antibody recognizing phosphotyrosine. Immunoblotting of Phosphotyrosylproteins. Cells were factor stimulated and lysed with extraction buffer (1% Triton X-100/50 mM NaCI/10 mM Tris-HC1, pH 7.6/5 mM EDTA/30 mM sodium pyrophosphate/50 mM sodium fluoride/100 AM sodium orthovanadate/1 mM phenylmethylsulfonyl fluoride). Epo receptor complexes to be probed for phosphotyrosylproteins were prepared by immunoprecipitation with antiserum directed against the N terminus of the Epo receptor (24). Phosphotyrosylproteins from total cell lysates were immunoblotted with PY20 (ICN) and then visualized with iodinated antiserum directed against mouse immunoglobulin (Amersham). Immunoblotting of phosphotyrosylproteins in the Epo receptor complex was performed with the 4G10 monoclonal antibody (UBI, Lake Placid, NY) and visualized by using the ECL Western blotting detection system (Amersham). Immune Complex Assays. Immunoprecipitates were incubated 15 min at 300C in kinase buffer (25 mM Hepes/10 mM MgCl2/10 mM MnCl2, pH 7.5) containing 100 ,Ci of [y-32P]ATP per ml (1 Ci = 37 GBq), washed, and eluted from the protein A-Sepharose with SDS sample buffer. Azido-ATP Binding. Immunoprecipitates immobilized on protein A-Sepharose were washed; resuspended in buffer containing 40 mM Hepes (pH 7.5), 1 mM MgCl2, 0.005% Triton X-100, and 2 ,uCi of 8-azidoadenosine 5'-[a32P]triphosphate (ICN); incubated 5 min with UV light; washed; and removed from the protein A-Sepharose with SDS sample buffer. Proteins were resolved by SDS/PAGE.
Erythropoietin (Epo) is a growth factor that has an important role in proliferation and differentiation of erythroid progenitor cells (1). The receptor for Epo has been cloned and is a member of the* hematopoietin receptor superfamily (2-10). Similar to other members of this superfamily, the intracellular portion of the Epo receptor contains no homology with enzymes associated with receptor-mediated signal transduction such as protein tyrosine kinases, protein serine kinases, or guanyl cyclase (2-10). Although little is known of the biochemical mechanism of action of this growth factor, increases in protein tyrosine phosphorylation are a rapid response in cells stimulated with Epo (11, 12). These observations raise the possibility that the receptor for Epo may be associated with one or more protein tyrosine kinases. Indeed, a number of cell-surface recognition molecules such as the T-cell receptor, surface immunoglobulin of B cells, the Fc receptor for IgE, CD4, and CD8 do not contain intrinsic protein tyrosine kinase activity and are associated with protein tyrosine kinases (13-19). Notably, within the hematopoietin receptor superfamily, the P chains of the interleukin (IL) 2 receptor as well as the receptor for human growth hormone both are associated with protein tyrosine kinase activity (20-23). We have found that treatment of cells with Epo rapidly induces increases in protein tyrosine phosphorylation, and we have identified protein tyrosine kinase activity associated
Abbreviations: Epo, erythropoietin; IL, interleukin. §To whom reprint requests should be addressed at: Building 560, Room 21-89A, National Cancer Institute, Frederick Cancer Research and Development Center, Frederick, MD 21702-1201.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
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RESULTS Epo-Induced Increases in Tyrosine Phosphorylation. One of the few intracellular events associated with signal transduction of a number of members of the hematopoietin receptor superfamily is an increase in protein tyrosine phosphorylation (11, 12, 25-29). To examine events associated with Epo signal transduction, we used Ba/F3-ER cells. While parental Ba/F3 cells are dependent on IL-3 for survival, Ba/F3-ER cells proliferate in response to either Epo or IL-3. Phosphotyrosylproteins immunoprecipitated from 32P1-labeled Ba/ F3-ER cells incubated 10 min with either medium or Epo were resolved by two-dimensional gel electrophoresis. Epo stimulation of Ba/F3-ER cells resulted in tyrosine phosphorylation of proteins of 97, 75, 70, and 55 kDa (Fig. 1A). A more minor phosphotyrosylprotein of 58-60 kDa was also observed in some but not all experiments. IL-3 treatment of parental Ba/F3 cells resulted in tyrosine phosphorylation of proteins of 140, 97, 70, and 55 kDa, while no changes were observed in these cells when treated with Epo (Fig. 1B). Immunoblotting with monoclonal antibody directed against phosphotyrosine was a second means to evaluate phosphotyrosylproteins involved in signal transduction by Epo. Ba/F3-ER cells were stimulated with Epo and lysed; proteins were resolved by SDS/PAGE and transferred to Immobilon membranes. As shown in Fig. 1C, stimulation of Ba/F3-ER cells with Epo resulted in increases in phosphotyrosylproteins of 97, 75, and 55 kDa. Epo-induced increases in tyrosine phosphorylation were observed as rapidly as 30 sec after treatment and were maximal by 3 min. To assess the potential for interaction of a protein tyrosine kinase with the Epo receptor, we examined Epo-induced phosphotyrosylproteins in the Epo receptor complex (Fig. 2). The dominant phosphotyrosylprotein consistently observed in the Epo-stimulated receptor complexes was -97 kDa (Fig. 2), while p97 was not present in the control immunoprecipitates (data not shown). Protein Tyrosine Kinase Activity Associated with the Epo Receptor. To test the possibility that one or more protein A
acid
-
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-
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FIG. 2. Detection of phosphotyrosylproteins in the Epo receptor complex. Antiphosphotyrosine immunoblot of proteins in the Epo receptor complex. Ba/F3-ER cells were stimulated with Epo for 5 min and lysed; Epo receptor complexes were prepared by immunoprecipitation. Immunoprecipitates were resolved by SDS/PAGE, transferred to Immobilon, and tested for the presence of phosphotyrosylproteins by immunoblotting with antiphosphotyrosine monoclonal antibody. Phosphotyrosylproteins were visualized with the Amersham ECL kit.
tyrosine kinases were associated with the Epo receptor, we performed immune complex assays on immunoprecipitates of the Epo receptor isolated from Ba/F3-ER cells. The dominant phosphoprotein specific for the Epo receptor complex was -97 kDa and increases in phosphorylation of p97 were noted after a 5-min stimulation with Epo (Fig. 3A). Phosphoproteins of approximately 130 and 55 kDa were also observed in the Epo receptor complex in a number of experiments. A comparison of phosphoproteins in immunoprecipitates performed with preimmune control antiserum demonstrated that p97 was specifically associated with the Epo receptor. Furthermore, p97 was not observed in antiEpo receptor immunoprecipitates performed on lysates from
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(B) One-dimensional gel electrophoresis of phosphotyrosylproteins from Ba/F3 cells stimulated 10 min with either Epo or IL-3. (C) Antiphosphotyrosine immunoblot of lysates from Ba/F3-ER cells stimulated with Epo.
Proc. Natl. Acad. Sci. USA 89 (1992)
Physiology: Linnekin et al. parental Ba/F3 cells (Fig. 3A). Analysis of immune complex preparations by two-dimensional gel electrophoresis was consistent with results using one-dimensional gel electrophoresis (Fig. 3B). Interestingly, the migration characteristics of p97 phosphorylated in the immune complex assay were very similar to the 97-kDa phosphotyrosylprotein identified in Fig. 1A. The broad band characteristic of the migration of p97 in both Figs. 1 and 3 is likely due to phosphorylation at multiple sites. In addition, glycosylation may also play a role; however, further characterization of the protein will be necessary to be certain. Also evident in Fig. 3B are phosphoproteins of approximately 55 and 60 kDa. These correspond to proteins observed by one-dimensional gel electrophoresis. Longer exposure of the autoradiograph also revealed a 130-kDa protein in the two-dimensional gels but it appears that this
6239
protein does not resolve as readily by two-dimensional as by one-dimensional electrophoresis. The kinetics of the Epo-induced increase in receptorassociated protein kinase activity were next examined. Increases in protein kinase activity were observed within 30 sec of Epo stimulation and returned to baseline levels by 10 min (Fig. 3C). Amino acid hydrolysis of proteins phosphorylated in the immune complex assay revealed phosphorylation on tyrosine residues as well as serine and threonine (Fig. 3D). Stimulation of cells with Epo increased phosphorylation on all amino acid residues. Collectively, these data demonstrate that the Epo receptor is associated with protein kinase activity, that this activity is modulated after treatment with Epo, and that one or more protein tyrosine kinases are in the receptor
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FIG. 3. Protein tyrosine kinase activity associated with the Epo receptor complex. (A) Immune complex assays of Epo receptor (Epo R) complexes. Ba/F3 or Ba/F3-ER cells were incubated in the presence or absence of Epo for 5 min. Cells were then lysed, clarified, and immunoprecipitated with either control antiserum or that recognizing the N terminus of the Epo receptor. Immune complex assays were performed as described. Proteins were resolved by onedimensional SDS/PAGE. I.P., immunoprecipitate. (B) Two-dimensional gel electrophoresis of proteins phosphorylated in immune complex assays of the Epo receptor isolated from control or Epo-stimulated Ba/F3-ER cells. (C) Kinetics of Epo-induced protein phosphorylation detected with immune complex assays performed on Epo receptors isolated from Ba/F3-ER cells. (D) Phosphoamino acid analysis of proteins phosphorylated in immune complex assays of the Epo receptor.
Physiology: Linnekin et al.
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Proc. Nadl. Acad. Sci. USA 89 (1992)
cells that grow in the absence of Epo (30, 31). To address the possibility that interaction of gp55 with the Epo receptor resulted in activation of receptor-associated protein kinase activity, we performed immune complex assays on Epo receptor complexes isolated from Ba/F3-ER cells infected with gp55 (Ba/F3-ER/GP55) as well as the murine erythroleukemia cell line MEL. As shown in Fig. SA, dramatic amounts of protein kinase activity were evident in receptor complexes isolated from both cell lines and the dominant phosphoprotein observed was =97 kDa. Amino acid hydrolysis ofp97 demonstrated phosphorylation on tyrosine as well as serine/threonine residues (Fig. 5B).
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FIG. 4. Identification of a 97-kDa ATP binding protein in the Epo receptor (Epo R) complex. Lysates from Ba/F3-ER cells were immunoprecipitated with either control antiserum or that directed against the N terminus of the Epo receptor. Immunoprecipitates (IP) were incubated 5 min with UV light in buffer containing 2 ,uCi of 8-azidoadenosine-5'-[a-32P]triphosphate in the presence or absence of 5 mM unlabeled ATP. Immunoprecipitates were then washed and removed from the Sepharose with SDS sample buffer; proteins were resolved by one-dimensional SDS/PAGE.
complex. In addition, the presence of serine phosphorylation in the receptor complex also suggests the association of protein serine kinases with the Epo receptor. Identification of an ATP Binding Protein in the Epo Receptor Complex. One feature critical to the phosphotransferase function of protein kinases is the capacity to bind ATP. To identify potential protein kinases in the Epo receptor complex, we evaluated the Epo receptor complex for ATP binding proteins. Epo receptor complexes were isolated from lysates of growing Ba/F3-ER cells, incubated in the presence of a radiolabeled azido derivative of ATP and the azido group crosslinked to the proteins with which it interacted through exposure to UV light. As shown in Fig. 4, an ATP binding protein of ':97 kDa was identified in the Epo receptor complex and not in control immunoprecipitates. Furthermore, the specificity of the interaction was demonstrated by the capacity of excess unlabeled ATP to compete with binding of the radiolabeled azido-ATP to p97. In contrast, a band of -50 kDa was also observed in both control and Epo receptor immunoprecipitates. As is evident from the absence of competition by excess nonradioactive ATP, this is a nonspecific interaction and likely represents binding of the azido-ATP to immunoglobulins used for immu-
noprecipitation. Constitutively Elevated Protein Kinase Activity Associated with the Epo Receptor Isolated from Cells Infected with gpS5. The Friend spleen focus-forming virus contains an env gene, which encodes a 55-kDa glycoprotein previously shown to interact with the Epo receptor (30). Spleen focus-forming virus infection of cells expressing the Epo receptor results in BalF3-ER GP55
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DISCUSSION We have examined the relationship between Epo-induced increases in protein tyrosine phosphorylation and receptorassociated protein tyrosine kinase activity. Epo stimulation of Ba/F3-ER cells resulted in tyrosine phosphorylation of proteins of 97, 75, 70, and 55 kDa (Fig. 1 A and C). We have also demonstrated association of the Epo receptor with protein tyrosine kinase activity and modulation of this activity after treatment of cells with Epo (Fig. 3). Similar results were obtained with the erythroid cell line HCD-57, suggesting similarities in stimulus response coupling mechanisms of endogenously expressed Epo receptors (data not shown). Of particular note was identification of an Epo-modulated 97kDa phosphotyrosylprotein in the Epo receptor complex (Figs. 2 and 3). Our studies have also demonstrated that an ATP binding protein of =97 kDa is present in the Epo receptor complex (Fig. 4). Considered together, these findings support the postulate that the Epo receptor is associated with one or more protein tyrosine kinases and further suggest that the 97-kDa phosphotyrosylprotein in the receptor complex is a protein tyrosine kinase. Demonstration of p97 tyrosine kinase activity still remains to be done. Our data clearly demonstrate the association of p97 with the stimulated Epo receptor (Figs. 2 and 3). The results from immune complex assays show a 97-kDa phosphoprotein associated with the Epo receptor in quiescent cells and increased phosphorylation of p97 in response to Epo (Fig. 3). In contrast, antiphosphotyrosine immunoblotting of the Epo receptor complex only detected p97 after cells were stimulated with Epo (Fig. 2). It is likely that the differences between the immunoblotting results and those of the immune complex assay stem from the use of 32p in the latter procedure. This isotope is detectable in extremely small amounts, thus generating greater sensitivity than the immunoblotting procedure. Furthermore, the immune complex assay detects phosphoserine, phosphothreonine, and phosphotyrosine as compared to detection of only phosphotyrosine in the immunoblotting experiment. These results suggest that p97 is constitutively associated with the Epo receptor.
FIG. 5. Constitutively elevated protein tyrosine kinase activity associated with Epo receptors isolated from cells infected with gp55. (A) Immune complex assays of Epo receptor (Epo R) complexes obtained from Ba/F3-ER/ GP55 or MEL cells. I.P., immunoprecipitate. (B) Phosphoamino acid analysis of p97 phosphorylated in immune complex assays of the Epo receptor.
Physiology: Linnekin et al. Ba/F3 cells expressing both the Epo receptor and gp55 (Ba/F3-ER/GP55) have been shown to grow in the absence of exogenous growth factors. Fig. 5 demonstrates elevated amounts of protein kinase activity in Epo receptor complexes isolated from these cells as well as a murine erythroleukemia cell line, MEL. These data provide strong support for the hypothesis that interaction of gp55 with the Epo receptor leads to activation of receptor-associated protein kinases and subsequent cellular proliferation through a receptor-initiated signal transduction cascade. Thus, constitutive activation of protein kinases associated with the Epo receptor may be relevant in spleen focus-forming virus transformation of cells; however, further studies in cell lines such as HCD-57 are necessary for conclusive evidence of the physiological relevance of these observations in leukemogenesis of erythroid cells. Our results confirm previous work demonstrating the relationship between treatment of Epo responsive cells and increases in protein tyrosine phosphorylation (11, 12). Similar to the work of Miura et al. (12), we have identified phosphotyrosylproteins of 97, 75, 70, and 55 kDa in Epostimulated cells. Studies from both laboratories have suggested that the 75-kDa phosphotyrosylprotein observed in Epo-stimulated cells is either the Epo receptor itself or a protein associated with the Epo receptor (ref. 12; D.L., A.D., and W.L.F., unpublished data). Our data extend previous studies of Epo-mediated signal transduction by demonstrating association of the Epo receptor with protein tyrosine kinase activity, modulation of this activity by Epo and constitutively elevated protein kinase activity in cells infected with gp55. Furthermore, we have identified a 97-kDa phosphotyrosylprotein in the Epo receptor complex that may represent a protein tyrosine kinase. Interestingly, a 97-kDa phosphotyrosylprotein with characteristics consistent with a protein tyrosine kinase is a point of convergence in the signal transduction pathways of cytokines, which include granulocyte-macrophage colony-stimulating factor (CSF), granulocyte-CSF, IL-3, IL-2, as well as Epo (D.L., G.A.E., and W.L.F., unpublished data). These observations raise the provocative possibility that p97 represents a protein tyrosine kinase associated with multiple cytokine receptors. In further support of this possibility is our recent observation, which found that a 97-kDa protein tyrosine kinase is associated with the p75 IL-2 receptor chain (32). The presence of p97 in Ba/F3 cells, a pre-B-lymphoid cell line, makes it unlikely to be the protooncogene fes. It is, however, possible that p97 may represent an unidentified member of the fes/fer/fps family (33). It will be important to purify and identify the 97-kDa phosphotyrosylprotein associated with the Epo and IL-2 receptors. Clearly, our preliminary knowledge of the signal transduction mechanisms of Epo suggests unusual receptor-effector coupling mechanisms and warrants further efforts directed at better understanding the biochemical basis for the powerful biological actions of this important growth factor. The authors would like to thank Dr. Doug Ferris, Dr. Joe Oppenheim, and Dr. Dan Longo for reviewing this manuscript. This project has been funded, at least in part, with federal funds from the Department of Health and Human Services under Contract N01CO-74102 with Program Resources, Inc./Dyn Corp. 1. Krantz, S. (1991) Blood 77, 419-434. 2. D'Andrea, A. D., Lodish, H. F. & Wong, G. G. (1989) Cell 57, 277-285.
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Association of the erythropoietin receptor with protein tyrosine kinase activity (signal transduction/protein phosphorylation)
DIANA LINNEKIN*, GERALD A. EVANSt, ALAN
D'ANDREAt, AND WILLIAM L. FARRAR*§
*Laboratory of Molecular Immunoregulation, Biological Response Modifiers Program, and tBiological Carcinogenesis and Development Program, Program Resources, Inc./Dyn Corp., National Cancer Institute, Frederick Cancer Research and Development Center, Frederick, MD 21702-1201; and tDivision of Hematology-Oncology, The Children's Hospital, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02115
Communicated by Avram Goldstein, April 14, 1992
with the Epo receptor. The dominant phosphotyrosylprotein observed in the Epo receptor complex after treatment with Epo was 97 kDa. Furthermore, a 97-kDa ATP binding protein was also observed in the Epo receptor complex. These results demonstrate association of the Epo receptor with protein tyrosine kinase activity and suggest that p97 is a protein tyrosine kinase in the Epo receptor complex.
We have examined the signal transduction ABSTRACT mechanism of the hematopoietic growth factor erythropoietin (Epo). Epo stimulation of Ba/F3 cells transfected with the Epo receptor resulted in increases in tyrosine phosphorylation of proteins of 97, 75, and 55 kDa. Epo-induced increases in tyrosine phosphorylation of a 97-kDa protein were also detected within the Epo receptor complex, suggesting that a protein tyrosine kinase is associated with the Epo receptor. Protein tyrosine kinase activity was found within the Epo receptor complex and modulation of this activity was observed after treatment of cells with Epo. Furthermore, constitutively high amounts of protein kinase activity were observed in Epo receptor complexes isolated from autonomously growing cells coexpressing the Epo receptor and the leukemogenic glycoprotein gp55. The dominant phosphotyrosylprotein found associated with the Epo receptor was 97 kDa. An Epo receptorassociated protein of identical molecular mass was also found to bind ATP, a characteristic critical for protein kinases. Collectively, these data demonstrate that the Epo receptor is associated with protein tyrosine kinase activity and further suggest that a 97-kDa phosphotyrosylprotein associated with the Epo receptor is a protein tyrosine kinase involved in Epo-mediated signal transduction.
MATERIALS AND METHODS Cell Lines and Growth Factors. The cell lines Ba/F3, Ba/F3-ER (Ba/F3 transfected with the murine Epo receptor), MEL, and Ba/F3-ER/GP55 (Ba/F3-ER infected with gp55) were grown as described (24). Radiolabeling and Phosphmino Acid Hydrolysis. Radiolabeling, electrophoresis, and phosphoamino acid hydrolysis of phosphotyrosylproteins were performed as described (25). Immunoprecipitations of phosphotyrosylproteins were performed with PY20 (ICN), a monoclonal antibody recognizing phosphotyrosine. Immunoblotting of Phosphotyrosylproteins. Cells were factor stimulated and lysed with extraction buffer (1% Triton X-100/50 mM NaCI/10 mM Tris-HC1, pH 7.6/5 mM EDTA/30 mM sodium pyrophosphate/50 mM sodium fluoride/100 AM sodium orthovanadate/1 mM phenylmethylsulfonyl fluoride). Epo receptor complexes to be probed for phosphotyrosylproteins were prepared by immunoprecipitation with antiserum directed against the N terminus of the Epo receptor (24). Phosphotyrosylproteins from total cell lysates were immunoblotted with PY20 (ICN) and then visualized with iodinated antiserum directed against mouse immunoglobulin (Amersham). Immunoblotting of phosphotyrosylproteins in the Epo receptor complex was performed with the 4G10 monoclonal antibody (UBI, Lake Placid, NY) and visualized by using the ECL Western blotting detection system (Amersham). Immune Complex Assays. Immunoprecipitates were incubated 15 min at 300C in kinase buffer (25 mM Hepes/10 mM MgCl2/10 mM MnCl2, pH 7.5) containing 100 ,Ci of [y-32P]ATP per ml (1 Ci = 37 GBq), washed, and eluted from the protein A-Sepharose with SDS sample buffer. Azido-ATP Binding. Immunoprecipitates immobilized on protein A-Sepharose were washed; resuspended in buffer containing 40 mM Hepes (pH 7.5), 1 mM MgCl2, 0.005% Triton X-100, and 2 ,uCi of 8-azidoadenosine 5'-[a32P]triphosphate (ICN); incubated 5 min with UV light; washed; and removed from the protein A-Sepharose with SDS sample buffer. Proteins were resolved by SDS/PAGE.
Erythropoietin (Epo) is a growth factor that has an important role in proliferation and differentiation of erythroid progenitor cells (1). The receptor for Epo has been cloned and is a member of the* hematopoietin receptor superfamily (2-10). Similar to other members of this superfamily, the intracellular portion of the Epo receptor contains no homology with enzymes associated with receptor-mediated signal transduction such as protein tyrosine kinases, protein serine kinases, or guanyl cyclase (2-10). Although little is known of the biochemical mechanism of action of this growth factor, increases in protein tyrosine phosphorylation are a rapid response in cells stimulated with Epo (11, 12). These observations raise the possibility that the receptor for Epo may be associated with one or more protein tyrosine kinases. Indeed, a number of cell-surface recognition molecules such as the T-cell receptor, surface immunoglobulin of B cells, the Fc receptor for IgE, CD4, and CD8 do not contain intrinsic protein tyrosine kinase activity and are associated with protein tyrosine kinases (13-19). Notably, within the hematopoietin receptor superfamily, the P chains of the interleukin (IL) 2 receptor as well as the receptor for human growth hormone both are associated with protein tyrosine kinase activity (20-23). We have found that treatment of cells with Epo rapidly induces increases in protein tyrosine phosphorylation, and we have identified protein tyrosine kinase activity associated
Abbreviations: Epo, erythropoietin; IL, interleukin. §To whom reprint requests should be addressed at: Building 560, Room 21-89A, National Cancer Institute, Frederick Cancer Research and Development Center, Frederick, MD 21702-1201.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
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Proc. Natl. Acad. Sci. USA 89 (1992)
RESULTS Epo-Induced Increases in Tyrosine Phosphorylation. One of the few intracellular events associated with signal transduction of a number of members of the hematopoietin receptor superfamily is an increase in protein tyrosine phosphorylation (11, 12, 25-29). To examine events associated with Epo signal transduction, we used Ba/F3-ER cells. While parental Ba/F3 cells are dependent on IL-3 for survival, Ba/F3-ER cells proliferate in response to either Epo or IL-3. Phosphotyrosylproteins immunoprecipitated from 32P1-labeled Ba/ F3-ER cells incubated 10 min with either medium or Epo were resolved by two-dimensional gel electrophoresis. Epo stimulation of Ba/F3-ER cells resulted in tyrosine phosphorylation of proteins of 97, 75, 70, and 55 kDa (Fig. 1A). A more minor phosphotyrosylprotein of 58-60 kDa was also observed in some but not all experiments. IL-3 treatment of parental Ba/F3 cells resulted in tyrosine phosphorylation of proteins of 140, 97, 70, and 55 kDa, while no changes were observed in these cells when treated with Epo (Fig. 1B). Immunoblotting with monoclonal antibody directed against phosphotyrosine was a second means to evaluate phosphotyrosylproteins involved in signal transduction by Epo. Ba/F3-ER cells were stimulated with Epo and lysed; proteins were resolved by SDS/PAGE and transferred to Immobilon membranes. As shown in Fig. 1C, stimulation of Ba/F3-ER cells with Epo resulted in increases in phosphotyrosylproteins of 97, 75, and 55 kDa. Epo-induced increases in tyrosine phosphorylation were observed as rapidly as 30 sec after treatment and were maximal by 3 min. To assess the potential for interaction of a protein tyrosine kinase with the Epo receptor, we examined Epo-induced phosphotyrosylproteins in the Epo receptor complex (Fig. 2). The dominant phosphotyrosylprotein consistently observed in the Epo-stimulated receptor complexes was -97 kDa (Fig. 2), while p97 was not present in the control immunoprecipitates (data not shown). Protein Tyrosine Kinase Activity Associated with the Epo Receptor. To test the possibility that one or more protein A
acid
-
+
Epo
kDa 97-
-
p97
66-
45-
FIG. 2. Detection of phosphotyrosylproteins in the Epo receptor complex. Antiphosphotyrosine immunoblot of proteins in the Epo receptor complex. Ba/F3-ER cells were stimulated with Epo for 5 min and lysed; Epo receptor complexes were prepared by immunoprecipitation. Immunoprecipitates were resolved by SDS/PAGE, transferred to Immobilon, and tested for the presence of phosphotyrosylproteins by immunoblotting with antiphosphotyrosine monoclonal antibody. Phosphotyrosylproteins were visualized with the Amersham ECL kit.
tyrosine kinases were associated with the Epo receptor, we performed immune complex assays on immunoprecipitates of the Epo receptor isolated from Ba/F3-ER cells. The dominant phosphoprotein specific for the Epo receptor complex was -97 kDa and increases in phosphorylation of p97 were noted after a 5-min stimulation with Epo (Fig. 3A). Phosphoproteins of approximately 130 and 55 kDa were also observed in the Epo receptor complex in a number of experiments. A comparison of phosphoproteins in immunoprecipitates performed with preimmune control antiserum demonstrated that p97 was specifically associated with the Epo receptor. Furthermore, p97 was not observed in antiEpo receptor immunoprecipitates performed on lysates from
base
Control
'p.
kDa
's ....
116
-
97
--
66
-
. BafF3-ER .b
..
OBO
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A4s
Iq
...'
B
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.NC.
45
B
C
Minutes
Control Epo IL-3
Post
k~a 200
Epo
0
.5
1
2
3
10
15
20 40
kDa 200
-
116
--
97
-
p140
97--
p70
--
p55
66-
pi7
66
photyrosylproteins from Ba/F3-ER cells stimulated with Epo for 10 min. Isoelectric focusing and SDS/PAGE
--
45-..
FIG. 1. Stimulation of tyrosine phosphorylation by Epo. (A) Two-dimensional gel electrophoresis of phos-
4W__
p55 45-
were
used to resolve
purified phosphotyrosylproteins.
(B) One-dimensional gel electrophoresis of phosphotyrosylproteins from Ba/F3 cells stimulated 10 min with either Epo or IL-3. (C) Antiphosphotyrosine immunoblot of lysates from Ba/F3-ER cells stimulated with Epo.
Proc. Natl. Acad. Sci. USA 89 (1992)
Physiology: Linnekin et al. parental Ba/F3 cells (Fig. 3A). Analysis of immune complex preparations by two-dimensional gel electrophoresis was consistent with results using one-dimensional gel electrophoresis (Fig. 3B). Interestingly, the migration characteristics of p97 phosphorylated in the immune complex assay were very similar to the 97-kDa phosphotyrosylprotein identified in Fig. 1A. The broad band characteristic of the migration of p97 in both Figs. 1 and 3 is likely due to phosphorylation at multiple sites. In addition, glycosylation may also play a role; however, further characterization of the protein will be necessary to be certain. Also evident in Fig. 3B are phosphoproteins of approximately 55 and 60 kDa. These correspond to proteins observed by one-dimensional gel electrophoresis. Longer exposure of the autoradiograph also revealed a 130-kDa protein in the two-dimensional gels but it appears that this
6239
protein does not resolve as readily by two-dimensional as by one-dimensional electrophoresis. The kinetics of the Epo-induced increase in receptorassociated protein kinase activity were next examined. Increases in protein kinase activity were observed within 30 sec of Epo stimulation and returned to baseline levels by 10 min (Fig. 3C). Amino acid hydrolysis of proteins phosphorylated in the immune complex assay revealed phosphorylation on tyrosine residues as well as serine and threonine (Fig. 3D). Stimulation of cells with Epo increased phosphorylation on all amino acid residues. Collectively, these data demonstrate that the Epo receptor is associated with protein kinase activity, that this activity is modulated after treatment with Epo, and that one or more protein tyrosine kinases are in the receptor
A BalF3-ER
kDa 200-
11697-
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C
-
Ba/F3- Parental
I.P. kDa
--
^ On
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o
o
j
cLi
I.P.
200 -
p130
---
cc
p97
97-
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'.4.s* -55 45-
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Epo
acid
B
-
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base
ON" kDa 97-
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-Is
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-
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C
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* ~~~Serine
Threonine -
p130
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*w p130
Tyrosine
p97
p55
FIG. 3. Protein tyrosine kinase activity associated with the Epo receptor complex. (A) Immune complex assays of Epo receptor (Epo R) complexes. Ba/F3 or Ba/F3-ER cells were incubated in the presence or absence of Epo for 5 min. Cells were then lysed, clarified, and immunoprecipitated with either control antiserum or that recognizing the N terminus of the Epo receptor. Immune complex assays were performed as described. Proteins were resolved by onedimensional SDS/PAGE. I.P., immunoprecipitate. (B) Two-dimensional gel electrophoresis of proteins phosphorylated in immune complex assays of the Epo receptor isolated from control or Epo-stimulated Ba/F3-ER cells. (C) Kinetics of Epo-induced protein phosphorylation detected with immune complex assays performed on Epo receptors isolated from Ba/F3-ER cells. (D) Phosphoamino acid analysis of proteins phosphorylated in immune complex assays of the Epo receptor.
Physiology: Linnekin et al.
6240
Proc. Nadl. Acad. Sci. USA 89 (1992)
cells that grow in the absence of Epo (30, 31). To address the possibility that interaction of gp55 with the Epo receptor resulted in activation of receptor-associated protein kinase activity, we performed immune complex assays on Epo receptor complexes isolated from Ba/F3-ER cells infected with gp55 (Ba/F3-ER/GP55) as well as the murine erythroleukemia cell line MEL. As shown in Fig. SA, dramatic amounts of protein kinase activity were evident in receptor complexes isolated from both cell lines and the dominant phosphoprotein observed was =97 kDa. Amino acid hydrolysis ofp97 demonstrated phosphorylation on tyrosine as well as serine/threonine residues (Fig. 5B).
0 I1-
kDa 200 116
C
C
O
0
cc
cc
o
0
Lu
LJ
OO
IP
CL
a
--
97-
66
Ig
45 --
+
+
-
Unlabeled ATP
FIG. 4. Identification of a 97-kDa ATP binding protein in the Epo receptor (Epo R) complex. Lysates from Ba/F3-ER cells were immunoprecipitated with either control antiserum or that directed against the N terminus of the Epo receptor. Immunoprecipitates (IP) were incubated 5 min with UV light in buffer containing 2 ,uCi of 8-azidoadenosine-5'-[a-32P]triphosphate in the presence or absence of 5 mM unlabeled ATP. Immunoprecipitates were then washed and removed from the Sepharose with SDS sample buffer; proteins were resolved by one-dimensional SDS/PAGE.
complex. In addition, the presence of serine phosphorylation in the receptor complex also suggests the association of protein serine kinases with the Epo receptor. Identification of an ATP Binding Protein in the Epo Receptor Complex. One feature critical to the phosphotransferase function of protein kinases is the capacity to bind ATP. To identify potential protein kinases in the Epo receptor complex, we evaluated the Epo receptor complex for ATP binding proteins. Epo receptor complexes were isolated from lysates of growing Ba/F3-ER cells, incubated in the presence of a radiolabeled azido derivative of ATP and the azido group crosslinked to the proteins with which it interacted through exposure to UV light. As shown in Fig. 4, an ATP binding protein of ':97 kDa was identified in the Epo receptor complex and not in control immunoprecipitates. Furthermore, the specificity of the interaction was demonstrated by the capacity of excess unlabeled ATP to compete with binding of the radiolabeled azido-ATP to p97. In contrast, a band of -50 kDa was also observed in both control and Epo receptor immunoprecipitates. As is evident from the absence of competition by excess nonradioactive ATP, this is a nonspecific interaction and likely represents binding of the azido-ATP to immunoglobulins used for immu-
noprecipitation. Constitutively Elevated Protein Kinase Activity Associated with the Epo Receptor Isolated from Cells Infected with gpS5. The Friend spleen focus-forming virus contains an env gene, which encodes a 55-kDa glycoprotein previously shown to interact with the Epo receptor (30). Spleen focus-forming virus infection of cells expressing the Epo receptor results in BalF3-ER GP55
MEL
°2cr
2 c
A
o
kDa 200 116 97--;. 66-
0 °0 o. 0 o ujCl 0 w
B P. i:;i
0..
_
-7
Free Phosphate
Serine Threonine
Tyrosine 45
DISCUSSION We have examined the relationship between Epo-induced increases in protein tyrosine phosphorylation and receptorassociated protein tyrosine kinase activity. Epo stimulation of Ba/F3-ER cells resulted in tyrosine phosphorylation of proteins of 97, 75, 70, and 55 kDa (Fig. 1 A and C). We have also demonstrated association of the Epo receptor with protein tyrosine kinase activity and modulation of this activity after treatment of cells with Epo (Fig. 3). Similar results were obtained with the erythroid cell line HCD-57, suggesting similarities in stimulus response coupling mechanisms of endogenously expressed Epo receptors (data not shown). Of particular note was identification of an Epo-modulated 97kDa phosphotyrosylprotein in the Epo receptor complex (Figs. 2 and 3). Our studies have also demonstrated that an ATP binding protein of =97 kDa is present in the Epo receptor complex (Fig. 4). Considered together, these findings support the postulate that the Epo receptor is associated with one or more protein tyrosine kinases and further suggest that the 97-kDa phosphotyrosylprotein in the receptor complex is a protein tyrosine kinase. Demonstration of p97 tyrosine kinase activity still remains to be done. Our data clearly demonstrate the association of p97 with the stimulated Epo receptor (Figs. 2 and 3). The results from immune complex assays show a 97-kDa phosphoprotein associated with the Epo receptor in quiescent cells and increased phosphorylation of p97 in response to Epo (Fig. 3). In contrast, antiphosphotyrosine immunoblotting of the Epo receptor complex only detected p97 after cells were stimulated with Epo (Fig. 2). It is likely that the differences between the immunoblotting results and those of the immune complex assay stem from the use of 32p in the latter procedure. This isotope is detectable in extremely small amounts, thus generating greater sensitivity than the immunoblotting procedure. Furthermore, the immune complex assay detects phosphoserine, phosphothreonine, and phosphotyrosine as compared to detection of only phosphotyrosine in the immunoblotting experiment. These results suggest that p97 is constitutively associated with the Epo receptor.
FIG. 5. Constitutively elevated protein tyrosine kinase activity associated with Epo receptors isolated from cells infected with gp55. (A) Immune complex assays of Epo receptor (Epo R) complexes obtained from Ba/F3-ER/ GP55 or MEL cells. I.P., immunoprecipitate. (B) Phosphoamino acid analysis of p97 phosphorylated in immune complex assays of the Epo receptor.
Physiology: Linnekin et al. Ba/F3 cells expressing both the Epo receptor and gp55 (Ba/F3-ER/GP55) have been shown to grow in the absence of exogenous growth factors. Fig. 5 demonstrates elevated amounts of protein kinase activity in Epo receptor complexes isolated from these cells as well as a murine erythroleukemia cell line, MEL. These data provide strong support for the hypothesis that interaction of gp55 with the Epo receptor leads to activation of receptor-associated protein kinases and subsequent cellular proliferation through a receptor-initiated signal transduction cascade. Thus, constitutive activation of protein kinases associated with the Epo receptor may be relevant in spleen focus-forming virus transformation of cells; however, further studies in cell lines such as HCD-57 are necessary for conclusive evidence of the physiological relevance of these observations in leukemogenesis of erythroid cells. Our results confirm previous work demonstrating the relationship between treatment of Epo responsive cells and increases in protein tyrosine phosphorylation (11, 12). Similar to the work of Miura et al. (12), we have identified phosphotyrosylproteins of 97, 75, 70, and 55 kDa in Epostimulated cells. Studies from both laboratories have suggested that the 75-kDa phosphotyrosylprotein observed in Epo-stimulated cells is either the Epo receptor itself or a protein associated with the Epo receptor (ref. 12; D.L., A.D., and W.L.F., unpublished data). Our data extend previous studies of Epo-mediated signal transduction by demonstrating association of the Epo receptor with protein tyrosine kinase activity, modulation of this activity by Epo and constitutively elevated protein kinase activity in cells infected with gp55. Furthermore, we have identified a 97-kDa phosphotyrosylprotein in the Epo receptor complex that may represent a protein tyrosine kinase. Interestingly, a 97-kDa phosphotyrosylprotein with characteristics consistent with a protein tyrosine kinase is a point of convergence in the signal transduction pathways of cytokines, which include granulocyte-macrophage colony-stimulating factor (CSF), granulocyte-CSF, IL-3, IL-2, as well as Epo (D.L., G.A.E., and W.L.F., unpublished data). These observations raise the provocative possibility that p97 represents a protein tyrosine kinase associated with multiple cytokine receptors. In further support of this possibility is our recent observation, which found that a 97-kDa protein tyrosine kinase is associated with the p75 IL-2 receptor chain (32). The presence of p97 in Ba/F3 cells, a pre-B-lymphoid cell line, makes it unlikely to be the protooncogene fes. It is, however, possible that p97 may represent an unidentified member of the fes/fer/fps family (33). It will be important to purify and identify the 97-kDa phosphotyrosylprotein associated with the Epo and IL-2 receptors. Clearly, our preliminary knowledge of the signal transduction mechanisms of Epo suggests unusual receptor-effector coupling mechanisms and warrants further efforts directed at better understanding the biochemical basis for the powerful biological actions of this important growth factor. The authors would like to thank Dr. Doug Ferris, Dr. Joe Oppenheim, and Dr. Dan Longo for reviewing this manuscript. This project has been funded, at least in part, with federal funds from the Department of Health and Human Services under Contract N01CO-74102 with Program Resources, Inc./Dyn Corp. 1. Krantz, S. (1991) Blood 77, 419-434. 2. D'Andrea, A. D., Lodish, H. F. & Wong, G. G. (1989) Cell 57, 277-285.
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