Association of the Erythropoietin Receptor with Protein Tyrosine Kinase Activity (Signal Transduction/Protein Phosphorylation)

Home , Erythropoietin receptor, Protein kinase, Protein phosphorylation

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

ABSTRACT We have examined the signal transduction with the Epo receptor. The dominant phosphotyrosylprotein mechanism of the hematopoietic growth factor erythropoietin observed in the Epo receptor complex after treatment with (Epo). Epo stimulation ofBa/F3 cells transfected with the Epo Epo was 97 kDa. Furthermore, a 97-kDa ATP binding protein receptor resulted in increases in tyrosine phosphorylation of was also observed in the Epo receptor complex. These proteins of 97, 75, and 55 kDa. Epo-induced increases in results demonstrate association of the Epo receptor with tyrosine phosphorylation of a 97-kDa protein were also de- protein tyrosine kinase activity and suggest that p97 is a tected within the Epo receptor complex, suggesting that a protein tyrosine kinase in the Epo receptor complex. 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 MATERIALS AND METHODS after treatment of cells with Epo. Furthermore, constitutively Cell Lines and Growth Factors. The cell lines Ba/F3, high amounts of protein kinase activity were observed in Epo Ba/F3-ER (Ba/F3 transfected with the murine Epo recep- receptor complexes isolated from autonomously growing cells tor), MEL, and Ba/F3-ER/GP55 (Ba/F3-ER infected with coexpressing the Epo receptor and the leukemogenic glycopro- gp55) were grown as described (24). tein gp55. The dominant phosphotyrosylprotein found associ- Radiolabeling and Phosphmino Acid Hydrolysis. Radio- ated with the Epo receptor was 97 kDa. An Epo receptor- labeling, electrophoresis, and phosphoamino acid hydrolysis associated protein of identical molecular mass was also found ofphosphotyrosylproteins were performed as described (25). to bind ATP, a characteristic critical for protein kinases. were Collectively, these data demonstrate that the Epo receptor is Immunoprecipitations of phosphotyrosylproteins per- associated with protein tyrosine kinase activity and further formed with PY20 (ICN), a monoclonal antibody recognizing suggest that a 97-kDa phosphotyrosylprotein associated with phosphotyrosine. the Epo receptor is a protein tyrosine kinase involved in Immunoblotting of Phosphotyrosylproteins. Cells were fac- Epo-mediated signal transduction. tor stimulated and lysed with extraction buffer (1% Triton X-100/50 mM NaCI/10 mM Tris-HC1, pH 7.6/5 mM Erythropoietin (Epo) is a growth factor that has an important EDTA/30 mM sodium pyrophosphate/50 mM sodium fluo- role in proliferation and differentiation of erythroid progen- ride/100 AM sodium orthovanadate/1 mM phenylmethylsul- itor cells (1). The receptor for Epo has been cloned and is a fonyl fluoride). Epo receptor complexes to be probed for member of the* hematopoietin receptor superfamily (2-10). phosphotyrosylproteins were prepared by immunoprecipita- Similar to other members ofthis superfamily, the intracellular tion with antiserum directed against the N terminus of the portion of the Epo receptor contains no homology with Epo receptor (24). Phosphotyrosylproteins from total cell enzymes associated with receptor-mediated signal transduc- lysates were immunoblotted with PY20 (ICN) and then tion such as protein tyrosine kinases, protein serine kinases, visualized with iodinated antiserum directed against mouse or guanyl cyclase (2-10). Although little is known of the immunoglobulin (Amersham). Immunoblotting of phospho- biochemical mechanism of action of this growth factor, tyrosylproteins in the Epo receptor complex was performed increases in protein tyrosine phosphorylation are a rapid with the 4G10 monoclonal antibody (UBI, Lake Placid, NY) response in cells stimulated with Epo (11, 12). These obser- and visualized by using the ECL Western blotting detection vations raise the possibility that the receptor for Epo may be system (Amersham). associated with one or more protein tyrosine kinases. Indeed, Immune Complex Assays. Immunoprecipitates were incu- a number of cell-surface recognition molecules such as the bated 15 min at 300C in kinase buffer (25 mM Hepes/10 mM T-cell receptor, surface immunoglobulin of B cells, the Fc MgCl2/10 mM MnCl2, pH 7.5) containing 100 ,Ci of receptor for IgE, CD4, and CD8 do not contain intrinsic [y-32P]ATP per ml (1 Ci = 37 GBq), washed, and eluted from protein tyrosine kinase activity and are associated with the protein A-Sepharose with SDS sample buffer. protein tyrosine kinases (13-19). Notably, within the he- Azido-ATP Binding. Immunoprecipitates immobilized on matopoietin receptor superfamily, the P chains of the inter- protein A-Sepharose were washed; resuspended in buffer leukin (IL) 2 receptor as well as the receptor for human containing 40 mM Hepes (pH 7.5), 1 mM MgCl2, 0.005% growth hormone both are associated with protein tyrosine Triton X-100, and 2 ,uCi of 8-azidoadenosine 5'-[a- kinase activity (20-23). 32P]triphosphate (ICN); incubated 5 min with UV light; We have found that treatment of cells with Epo rapidly washed; and removed from the protein A-Sepharose with induces increases in protein tyrosine phosphorylation, and SDS sample buffer. Proteins were resolved by SDS/PAGE. we have identified protein tyrosine kinase activity associated Abbreviations: Epo, erythropoietin; IL, interleukin. The publication costs ofthis article were defrayed in part by page charge §To whom reprint requests should be addressed at: Building 560, payment. This article must therefore be hereby marked "advertisement" Room 21-89A, National Cancer Institute, Frederick Cancer Re- in accordance with 18 U.S.C. §1734 solely to indicate this fact. search and Development Center, Frederick, MD 21702-1201. 6237 Downloaded by guest on September 23, 2021 6238 Physiology: Linnekin et al. Proc. Natl. Acad. Sci. USA 89 (1992) RESULTS - + Epo Epo-Induced Increases in Tyrosine Phosphorylation. One of kDa the few intracellular events associated with signal transduction of a number of members of the hematopoietin receptor superfamily is an increase in protein tyrosine phosphoryla- 97- - p97 tion (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 66- cells proliferate in response to either Epo or IL-3. Phospho- tyrosylproteins immunoprecipitated from 32P1-labeled Ba/ F3-ER cells incubated 10 min with either medium or Epo were resolved by two-dimensional gel electrophoresis. Epo 45- stimulation of Ba/F3-ER cells resulted in tyrosine phosphorylation ofproteins of97, 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 FIG. 2. Detection ofphosphotyrosylproteins in the Epo receptor proteins of 140, 97, 70, and 55 kDa, while no changes were complex. Antiphosphotyrosine immunoblot of proteins in the Epo observed in these cells when treated with Epo (Fig. 1B). receptor complex. Ba/F3-ER cells were stimulated with Epo for 5 Immunoblotting with monoclonal antibody directed min and lysed; Epo receptor complexes were prepared by immuno- precipitation. Immunoprecipitates were resolved by SDS/PAGE, against phosphotyrosine was a second means to evaluate transferred to Immobilon, and tested for the presence of phospho- phosphotyrosylproteins involved in signal transduction by tyrosylproteins by immunoblotting with antiphosphotyrosine mono- Epo. Ba/F3-ER cells were stimulated with Epo and lysed; clonal antibody. Phosphotyrosylproteins were visualized with the proteins were resolved by SDS/PAGE and transferred to Amersham ECL kit. Immobilon membranes. As shown in Fig. 1C, stimulation of Ba/F3-ER cells with Epo resulted in increases in phosphotyrosine kinases were associated with the Epo receptor, we tyrosylproteins of97, 75, and 55 kDa. Epo-induced increases performed immune complex assays on immunoprecipitates in tyrosine phosphorylation were observed as rapidly as 30 of the Epo receptor isolated from Ba/F3-ER cells. The sec after treatment and were maximal by 3 min. dominant phosphoprotein specific for the Epo receptor com- To assess the potential for interaction of a protein tyrosine plex was -97 kDa and increases in phosphorylation of p97 kinase with the Epo receptor, we examined Epo-induced were noted after a 5-min stimulation with Epo (Fig. 3A). phosphotyrosylproteins in the Epo receptor complex (Fig. 2). Phosphoproteins of approximately 130 and 55 kDa were also The dominant phosphotyrosylprotein consistently observed observed in the Epo receptor complex in a number of in the Epo-stimulated receptor complexes was -97 kDa (Fig. experiments. A comparison of phosphoproteins in immuno- 2), while p97 was not present in the control immunoprecip- precipitates performed with preimmune control antiserum itates (data not shown). demonstrated that p97 was specifically associated with the Protein Tyrosine Kinase Activity Associated with the Epo Epo receptor. Furthermore, p97 was not observed in anti- Receptor. To test the possibility that one or more protein Epo receptor immunoprecipitates performed on lysates from

A acid base Control 'p. kDa 's .... 116 - . 97 -- BafF3-ER .b B 66 - Arw...,.. .Aqp- OBO A4s Iq P." ...'.NC. 45

B C Minutes Control Epo IL-3 Post 0 .5 1 2 3 1 40 k~a Epo 10 5 20

200 kDa

200 - p140

97-- 116 --

- p70 97 - 66- pi7 FIG. 1. Stimulation of tyrosine phosphorylation by -- p55 Epo. (A) Two-dimensional gel electrophoresis of phos- 66 -- photyrosylproteins from Ba/F3-ER cells stimulated with 45- -.. 4W__ Epo for 10 min. Isoelectric focusing and SDS/PAGE p55 were used to resolve purified phosphotyrosylproteins. (B) One-dimensional gel electrophoresis of phosphoty- 45- rosylproteins from Ba/F3 cells stimulated 10 min with either Epo or IL-3. (C) Antiphosphotyrosine immunoblot oflysates from Ba/F3-ER cells stimulated with Epo. Downloaded by guest on September 23, 2021 Physiology: Linnekin et al. Proc. Natl. Acad. Sci. USA 89 (1992) 6239 parental Ba/F3 cells (Fig. 3A). Analysis of immune complex protein does not resolve as readily by two-dimensional as by preparations by two-dimensional gel electrophoresis was one-dimensional electrophoresis. consistent with results using one-dimensional gel electropho- The kinetics of the Epo-induced increase in receptor- resis (Fig. 3B). Interestingly, the migration characteristics of associated protein kinase activity were next examined. In- p97 phosphorylated in the immune complex assay were very creases in protein kinase activity were observed within 30 sec similar to the 97-kDa phosphotyrosylprotein identified in Fig. of Epo stimulation and returned to baseline levels by 10 min 1A. The broad band characteristic of the migration of p97 in (Fig. 3C). both Figs. 1 and 3 is likely due to phosphorylation at multiple Amino acid hydrolysis of proteins phosphorylated in the sites. In addition, glycosylation may also play a role; how- immune complex assay revealed phosphorylation on tyrosine ever, further characterization ofthe protein will be necessary residues as well as serine and threonine (Fig. 3D). Stimula- to be certain. Also evident in Fig. 3B are phosphoproteins of tion ofcells with Epo increased phosphorylation on all amino approximately 55 and 60 kDa. These correspond to proteins acid residues. Collectively, these data demonstrate that the observed by one-dimensional gel electrophoresis. Longer Epo receptor is associated with protein kinase activity, that exposure of the autoradiograph also revealed a 130-kDa this activity is modulated after treatment with Epo, and that protein in the two-dimensional gels but it appears that this one or more protein tyrosine kinases are in the receptor A BalF3-ER Ba/F3- Parental

°- cc cc: cc cc o o kDa 0) C I.P. I.P. kDa j cLi 200- - -- 200 - 116- ^ p130

97- On --- p97 97- 86- '.4.s* -55 66-_ 45- 45- Epo - - +

Epo - +

B acid base ON"

kDa

97- 1,",MR -Is W.. N.Z- - 66-

FIG. 3. Protein tyrosine kinase activity associated with the Epo receptor com- 45- plex. (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. C D Cells were then lysed, clarified, and im- Minutes munoprecipitated with either control Post Epo 0 .5 1 2 3 10 antiserum or that recognizing the N ter- kDa minus of the Epo receptor. Immune I ^ * ~~~Serine complex assays were performed as described. Proteins were resolved by one- Threonine dimensional SDS/PAGE. I.P., immunoprecipitate. (B) Two-dimensional gel electrophoresis of proteins phosphory- - p130 lated in immune complex assays of the Tyrosine Epo receptor isolated from control or _ p97 *w Epo-stimulated Ba/F3-ER cells. (C) Ki- p130 p97 p55 netics of Epo-induced protein phosphorylation detected with immune complex assays performed on Epo receptors isolated from Ba/F3-ER cells. (D) Phos- p55 phoamino acid analysis of proteins phosphorylated in immune complex assays of the Epo receptor. Downloaded by guest on September 23, 2021 6240 Physiology: Linnekin et al. Proc. Nadl. Acad. Sci. USA 89 (1992)

0 I-1- cc cc cells that grow in the absence of Epo (30, 31). To address the C C o 0 IP possibility that interaction of gp55 with the Epo receptor O 0 a CL kDa OO LJ Lu resulted in activation of receptor-associated protein kinase 200 activity, we performed immune complex assays on Epo receptor complexes isolated from Ba/F3-ER cells infected 116 -- with gp55 (Ba/F3-ER/GP55) as well as the murine erythro- 97- leukemia cell line MEL. As shown in Fig. SA, dramatic amounts of protein kinase activity were evident in receptor 66 complexes isolated from both cell lines and the dominant phosphoprotein observed was =97 kDa. Amino acid hydro- - Ig lysis ofp97 demonstrated phosphorylation on tyrosine as well 45 -- as serine/threonine residues (Fig. 5B). - + - + Unlabeled ATP DISCUSSION FIG. 4. Identification ofa 97-kDa ATP binding protein in the Epo We have examined the relationship between Epo-induced receptor (Epo R) complex. Lysates from Ba/F3-ER cells were increases in protein tyrosine phosphorylation and receptor- immunoprecipitated with either control antiserum or that directed associated protein tyrosine kinase activity. Epo stimulation against the N terminus ofthe Epo receptor. Immunoprecipitates (IP) were incubated 5 min with UV light in buffer containing 2 ,uCi of of Ba/F3-ER cells resulted in tyrosine phosphorylation of 8-azidoadenosine-5'-[a-32P]triphosphate in the presence or absence proteins of97, 75, 70, and 55 kDa (Fig. 1 A and C). We have of 5 mM unlabeled ATP. Immunoprecipitates were then washed and also demonstrated association of the Epo receptor with removed from the Sepharose with SDS sample buffer; proteins were protein tyrosine kinase activity and modulation of this activ- resolved by one-dimensional SDS/PAGE. ity after treatment of cells with Epo (Fig. 3). Similar results were obtained with the erythroid cell line HCD-57, suggesting complex. In addition, the presence of serine phosphorylation similarities in stimulus response coupling mechanisms of in the receptor complex also suggests the association of endogenously expressed Epo receptors (data not shown). Of protein serine kinases with the Epo receptor. particular note was identification of an Epo-modulated 97- Identification ofan ATP Binding Protein in the Epo Receptor kDa phosphotyrosylprotein in the Epo receptor complex Complex. One feature critical to the phosphotransferase func- (Figs. 2 and 3). Our studies have also demonstrated that an tion ofprotein kinases is the capacity to bind ATP. To identify ATP binding protein of =97 kDa is present in the Epo potential protein kinases in the Epo receptor complex, we receptor complex (Fig. 4). Considered together, these find- evaluated the Epo receptor complex forATP binding proteins. ings support the postulate that the Epo receptor is associated Epo receptor complexes were isolated from lysates ofgrowing with one or more protein tyrosine kinases and further suggest Ba/F3-ER cells, incubated in the presence of a radiolabeled that the 97-kDa phosphotyrosylprotein in the receptor com- azido derivative ofATP and the azido group crosslinked to the plex is a protein tyrosine kinase. Demonstration of p97 proteins with which itinteracted through exposure to UV light. tyrosine kinase activity still remains to be done. As shown in Fig. 4, an ATP binding protein of ':97 kDa was Our data clearly demonstrate the association of p97 with identified in the Epo receptor complex and not in control the stimulated Epo receptor (Figs. 2 and 3). The results from immunoprecipitates. Furthermore, the specificity ofthe inter- immune complex assays show a 97-kDa phosphoprotein action was demonstrated by the capacity of excess unlabeled associated with the Epo receptor in quiescent cells and ATP to compete with binding ofthe radiolabeled azido-ATP to increased phosphorylation ofp97 in response to Epo (Fig. 3). p97. In contrast, a band of -50 kDa was also observed in both In contrast, antiphosphotyrosine immunoblotting of the Epo control and Epo receptor immunoprecipitates. As is evident receptor complex only detected p97 after cells were stimu- from the absence of competition by excess nonradioactive lated with Epo (Fig. 2). It is likely that the differences ATP, this is a nonspecific interaction and likely represents between the immunoblotting results and those ofthe immune binding of the azido-ATP to immunoglobulins used for immu- complex assay stem from the use of 32p in the latter proce- noprecipitation. dure. This isotope is detectable in extremely small amounts, Constitutively Elevated Protein Kinase Activity Associated thus generating greater sensitivity than the immunoblotting with the Epo Receptor Isolated from Cells Infected with gpS5. procedure. Furthermore, the immune complex assay detects The Friend spleen focus-forming virus contains an env gene, phosphoserine, phosphothreonine, and phosphotyrosine as which encodes a 55-kDa glycoprotein previously shown to compared to detection of only phosphotyrosine in the immu- interact with the Epo receptor (30). Spleen focus-forming noblotting experiment. These results suggest that p97 is virus infection of cells expressing the Epo receptor results in constitutively associated with the Epo receptor. BalF3-ER GP55 MEL A °2cr 2 c B o °00 0 o. P. o ujCl 0 w kDa i:;i Free Phosphate 200 - 0..

116 - Serine 97--;. _ -7 FIG. 5. Constitutively elevated protein tyrosine kinase activity associated with Epo receptors isolated from cells 66- Threonine infected with gp55. (A) Immune complex assays of Epo receptor (Epo R) complexes obtained from Ba/F3-ER/ Tyrosine GP55 or MEL cells. I.P., immunoprecipitate. (B) Phos- phoamino acid analysis of p97 phosphorylated in immune 4 5 complex assays of the Epo receptor. Downloaded by guest on September 23, 2021 Physiology: Linnekin et al. Proc. Natl. Acad. Sci. USA 89 (1992) 6241 Ba/F3 cells expressing both the Epo receptor and gp55 3. Cosman, D., Lyman, S. D., Idzerda, R. L., Beckman, M. P., (Ba/F3-ER/GP55) have been shown to grow in the absence Park, L. S., Goodwin, R. G. & March, C. J. (1990) Trends of exogenous growth factors. Fig. 5 demonstrates elevated Biochem. Sci. 15, 265-270. 4. Goodwin, R. G., Friend, D., Ziegler, S. F., Jerzy, R., Falk, amounts ofprotein kinase activity in Epo receptor complexes B. A., Gimpel, S., Cosman, D., Dower, S. K., March, C. J. & isolated from these cells as well as a murine erythroleukemia Namen, A. E. (1990) Cell 60, 941-951. cell line, MEL. These data provide strong support for the 5. Itoh, N., Yonehara, S., Scherurs, J., Gorman, D. M., Maru- hypothesis that interaction of gp55 with the Epo receptor yama, K., Ishil, A., Yahara, I., Arai, K. & Miyajima, A. (1989) leads to activation ofreceptor-associated protein kinases and Science 247, 324-326. subsequent cellular proliferation through a receptor-initiated 6. Gorman, D. M., Itoh, N., Kitamura, T., Schreurs, J., Yone- signal transduction cascade. Thus, constitutive activation of hara, S., Yahara, I., Arai, K. E. & Miyajima, A. (1990) Proc. Natl. Acad. Sci. USA 87, 5459-5463. protein kinases associated with the Epo receptor may be 7. Gearing, D. P., King, J. A., Gough, N. M. & Nicola, N. A. relevant in spleen focus-forming virus transformation of (1989) EMBO J. 8, 3667-3676. cells; however, further studies in cell lines such as HCD-57 8. Hayashida, K., Kitamura, T., Gorman, D. M., Arai, K. I., are necessary for conclusive evidence of the physiological Yokota, T. & Miyajima, A. (1990) Proc. Natl. Acad. Sci. USA relevance of these observations in leukemogenesis of eryth- 87, 9655-9659. roid cells. 9. Mosley, B., Beckmann, M., March, C., Idzerda, R., Gimpel, Our results confirm previous work demonstrating the re- S., VandenBos, T., Friend, D., Alpert, A., Anderson, D., Jackson, J., Wignall, J., Smith, C., Gallis, B., Sims, J., Urdal, lationship between treatment of Epo responsive cells and D., Widmer, M., Cosman, D. & Park, L. (1989) Cell 59, increases in protein tyrosine phosphorylation (11, 12). Sim- 335-348. ilar to the work of Miura et al. (12), we have identified 10. Kitamura, T., Sato, N., Arai, K. & Miyajima, A. (1991) Cell 66, phosphotyrosylproteins of 97, 75, 70, and 55 kDa in Epo- 1165-1174. stimulated cells. Studies from both laboratories have sug- 11. Quelle, F. W. & Wojchowski, D. M. (1991) J. Biol. Chem. 266, gested that the 75-kDa phosphotyrosylprotein observed in 609-614. Epo-stimulated cells is either the Epo receptor itself or a 12. Miura, O., D'Andrea, A., Kalbat, D. & Ihle, J. N. (1991) Mol. associated with the Epo receptor (ref. 12; D.L., A.D., Cell. Biol. 11, 4895-4902. protein 13. Samelson, L., Phillips, A., Luong, E. & Klausner, R. (1990) and W.L.F., unpublished data). Our data extend previous Proc. Natl. Acad. Sci. USA 87, 4358-4362. studies of Epo-mediated signal transduction by demonstrat- 14. Yamanashi, Y., Kakiuchi, T., Mizuguchi, J., Yammamoto, T. ing association of the Epo receptor with protein tyrosine & Toyoshima, K. (1990) Science 251, 192-195. kinase activity, modulation of this activity by Epo and 15. Burkhard, A., Brunswick, M., Bolen, J. & Mond, J. (1991) constitutively elevated protein kinase activity in cells in- Proc. Natl. Acad. Sci. USA 88, 7410-7414. fected with gp55. Furthermore, we have identified a 97-kDa 16. Gold, M., Matsuuchi, L., Kelly, R. & DeFranco, A. (1991) phosphotyrosylprotein in the Epo receptor complex that may Proc. Nat!. Acad. Sci. USA 88, 3436-3440. represent a protein tyrosine kinase. Interestingly, a 97-kDa 17. Sugie, K., Kawakami, T., Maeda, Y., Kawabe, T., Uchida, A. with characteristics consistent with a & Yodoi, J. (1991) Proc. Nat!. Acad. Sci. USA 88, 9132-9135. phosphotyrosylprotein 18. Barber, E., Dasgupta, J., Schlossman, S., Trevillyan, J. & protein tyrosine kinase is a point ofconvergence in the signal Rudd, C. (1989) Proc. Natl. Acad. Sci. USA 86, 3277-3281. transduction pathways of cytokines, which include granulo- 19. Veillette, A., Bookman, M., Horak, E. & Bolen, J. (1988) Cell cyte-macrophage colony-stimulating factor (CSF), granulo- 55, 301-308. cyte-CSF, IL-3, IL-2, as well as Epo (D.L., G.A.E., and 20. Carter-su, C., Stubbart, R., Wang, X., Stred, S., Argetsinger, W.L.F., unpublished data). These observations raise the L. & Shafer, J. (1989) J. Biol. Chem. 264, 18654-18661. provocative possibility that p97 represents a protein tyrosine 21. Hatakeyama, M., Kono, T., Kobayashi, N., Kawahara, A., kinase associated with multiple cytokine receptors. In further Levin, S., Perlmutter, R. & Taniguchi, T. (1991) Science 252, support of this possibility is our recent observation, which 1523-1528. found that a 97-kDa kinase is associated with 22. Michiel, D., Garcia, G., Evans, G. & Farrar, W. (1991) Cyto- protein tyrosine kine 3, 428-438. the p75 IL-2 receptor chain (32). The presence of p97 in 23. Fung, M., Scearce, T., Hoffman, J., Peffer, N., Hammes, S., Ba/F3 cells, a pre-B-lymphoid cell line, makes it unlikely to Hosking, J., Schmandt, R., Kuziel, W., Haynes, B., Mills, G. be the protooncogene fes. It is, however, possible that p97 & Greene, W. (1991) J. Immunol. 147, 1253-1260. may represent an unidentified member of the fes/fer/fps 24. Yoshimura, A., D'Andrea, A. D. & Lodish, H. F. (1990) Proc. family (33). It will be important to purify and identify the Natl. Acad. Sci. USA 87, 4139-4143. 97-kDa phosphotyrosylprotein associated with the Epo and 25. Linnekin, D. & Farrar, W. L. (1990) Biochem. J. 271,317-324. IL-2 receptors. Clearly, our preliminary knowledge of the 26. Farrar, W. L. & Ferris, D. K. (1989) J. Biol. Chem. 264, signal transduction mechanisms of Epo suggests unusual 12562-12567. mechanisms and warrants further 27. Koyasu, S., Tojo, A., Miyaijima, A., Akiyama, T., Kasuga, M., receptor-effector coupling Urabe, A., Schreurs, J., Arai, K., Takaku, F. & Yahara, I. efforts directed at better understanding the biochemical basis (1987) EMBO J. 6, 3979-3984. for the powerful biological actions of this important growth 28. Kanakura, Y., Druker, B., Cannistra, S., Furukawa, Y., To- factor. rimoto, Y. & Griffin, J. (1990) Blood 76, 706-715. 29. Morla, A., Schireurs, J., Miyajima, A. & Wang, J. (1988) Mol. The authors would like to thank Dr. Doug Ferris, Dr. Joe Oppen- Cell. Biol. 8, 2214-2218. heim, and Dr. Dan Longo for reviewing this manuscript. This project 30. Li, J. P., D'Andrea, A. D., Lodish, H. F. & Baltimore, D. has been funded, at least in part, with federal funds from the (1990) Nature (London) 343, 762-764. Department of Health and Human Services under Contract N01- 31. Ruscetti, S., Janesch, N., Chakraborti, A., Sawyer, S. & CO-74102 with Program Resources, Inc./Dyn Corp. Hankins, W. (1990) J. Virol. 63, 1057-1062. 32. Garcia, G., Evans, G., Michiel, D. & Farrar, W. (1992) Bio- 1. Krantz, S. (1991) Blood 77, 419-434. chem. J., in press. 2. D'Andrea, A. D., Lodish, H. F. & Wong, G. G. (1989) Cell 57, 33. Pawson, T., Letwin, K., Lee, T., Hao, Q., Heisterkamp, N. & 277-285. Groffen, J. (1989) Mol. Cell. Biol. 9, 5722-5725. Downloaded by guest on September 23, 2021