Phosphorylation of a Fes-related Protein in Response to Granulocyte-Macrophage Colony Stimulating Factor

Previous work has suggested that a 97-kDa protein (p97) is involved in the signal transduction pathway of granulocyte-macrophage colony stimulating factor (GM-CSF) as well as interleukin 3, erythropoietin, and interleukin 2. We have examined the relationship of p97 to the protein tyrosine kinase Fes in the GM-CSF signal transduction pathway in erythroid and myeloid cell lines. GM-CSF stimulation of three different cell lines induced tyrosine phosphorylation of p97 as well as a number of other phosphotyrosylproteins. Although each cell line expressed the proto-oncogene product Fes, antisera specific for Fes did not recognize p97 in immunoblotting experiments. Furthermore, immunodepletion of Fes did not reduce the amount of p97 in GM-CSF-treated cells. Two-dimensional gel electrophoresis demonstrated that p97 and Fes have similar charge to mass ratios, and limited proteolytic mapping of p97 and Fes suggested that these proteins may be related but are not identical. Our studies demonstrate that p97 is not Fes but is probably a Fes-related protein. Previous work has suggested that a 97-kDa protein (p97) is involved in the signal transduction pathway of granulocyte-macrophage colony stimulating factor (GM-CSF) as well as interleukin 3, erythropoietin, and interleukin 2. We have examined the relationship of p97 to the protein tyrosine kinase Fes in the GM-CSF signal transduction pathway in erythroid and myeloid cell lines. GM-CSF stimulation of three different cell lines induced tyrosine phosphorylation of p97 as well as a number of other phosphotyrosylproteins. Although each cell line expressed the proto-oncogene product Fes, antisera specific for Fes did not recognize p97 in immunoblotting experiments. Furthermore, immunodepletion of Fes did not reduce the amount of p97 in GM-CSF-treated cells. Two-dimensional gel electrophoresis demonstrated that p97 and Fes have similar charge to mass ratios, and limited proteolytic mapping of p97 and Fes suggested that these proteins may be related but are not identical. Our studies demonstrate that p97 is not Fes but is probably a Fes-related protein. INTRODUCTIONGM-CSF 1The abbreviations used are: GM-CSFgranulocyte-macrophage colony stimulating factorILinterleukinPAGEpolyacrylamide gel electrophoresis. stimulates proliferation and differentiation of myeloid progenitor cells as well as activation of neutrophils(1Metcalf D. Science. 1991; 254: 529-535Crossref PubMed Scopus (330) Google Scholar, 2Gasson J.C. Blood. 1991; 77: 1131-1145Crossref PubMed Google Scholar). The receptor for GM-CSF consists of an α subunit of 85 kDa which binds ligand with low affinity and a β subunit of approximately 120 kDa which does not bind ligand but converts the receptor complex to high affinity binding(3Gearing D. King J. Gough N. Nicola N. EMBO J. 1988; 8: 3667-3676Crossref Scopus (521) Google Scholar, 4Hayashida K. Kitamura T. Gorman D. Arai K. Yokota T. Miyajima A. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 9655-9659Crossref PubMed Scopus (518) Google Scholar). Similar to other members of the hemapoietin receptor superfamily, the components of the GM-CSF receptor do not contain intrinsic tyrosine or serine/threonine kinase activity. Recent work has suggested that the rapid increases in protein tyrosine phosphorylation stimulated by GM-CSF may be mediated through activation of multiple tyrosine kinases. A number of Src-like tyrosine kinases (Hck, Lyn, and Yes), as well as JAK2, are stimulated in response to GM-CSF(5Witthuhn B. Quelle F. Silvernnoinen O. Yi T. Tang B. Miura O. Ihle J. Cell. 1993; 74: 227-236Abstract Full Text PDF PubMed Scopus (1001) Google Scholar, 6Silvennoinen O. Witthuhn B. Quelle F. Cleveland J. Yi T. Ihle J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8429-8433Crossref PubMed Scopus (437) Google Scholar, 7Torigoe T. O'Connor R. Santoli D. Reed J. Blood. 1992; 80: 617-624Crossref PubMed Google Scholar, 8Corey S. Eguinoa A. Puyana-Theall K. Bolen J. Cantley L. Mollinedo F. Jackson T. Hawkins P. Stephens L. EMBO J. 1993; 12: 2681-2690Crossref PubMed Scopus (170) Google Scholar, 9Linnekin D. Howard O.M.Z. Park L. Farrar W.L. Ferris D.F. Longo D.L. Blood. 1994; 84: 94-103Crossref PubMed Google Scholar). In addition, the work of Hanazono and co-workers (10Hanazono Y. Chiba S. Sasaki K. Mano H. Miyajima A. Arai K. Yazaki Y. Hirai H. EMBO J. 1993; 12: 1641-1646Crossref PubMed Scopus (144) Google Scholar, 11Hanazono Y. Chiba S. Sasaki K. Mano H. Yazaki Y. Hirai H. Blood. 1993; 81: 3193-3196Crossref PubMed Google Scholar) has suggested that Fes is activated in response to GM-CSF as well as IL-3 and erythropoietin. Interestingly, a number of laboratories have reported a phosphotyrosylprotein similar in size to Fes (90-100 kDa) in GM-CSF-, IL-3-, or erythropoietin-stimulated cells (12Linnekin D. Evans G. Michiel D. Farrar W. J. Biol. Chem. 1992; 267: 23993-23998Abstract Full Text PDF PubMed Google Scholar, 13Linnekin D. Evans G. Michiel D. Farrar W. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6237-6241Crossref PubMed Scopus (43) Google Scholar, 14Quelle F. Quelle D. Wojchowski D. J. Biol. Chem. 1992; 267: 17055-17060Abstract Full Text PDF PubMed Google Scholar, 15Kanakura Y. Druker B. Cannistra S. Furukawa Y. Torimoto Y. Griffin J. Blood. 1990; 76: 706-715Crossref PubMed Google Scholar). Furthermore, we previously have described a putative tyrosine kinase of 97 kDa (p97) phosphorylated on tyrosine residues in response to GM-CSF(12Linnekin D. Evans G. Michiel D. Farrar W. J. Biol. Chem. 1992; 267: 23993-23998Abstract Full Text PDF PubMed Google Scholar). The present study addresses whether p97 is the proto-oncogene product Fes or potentially a Fes-related protein.MATERIALS AND METHODSCell Lines, Growth Factors, and AntibodiesMo7e and TF-1 cells were cultured in RPMI 1640 (Life Technologies, Inc.), 10% fetal calf serum (Inovar), 10-20% conditioned media from A5637 cells (producers of GM-CSF and stem cell factor), 1% L-glutamine, and penicillin-streptomycin. HL-60 cells were cultured in RPMI 1640 (Life Technologies, Inc.), 10% fetal calf serum (Life Technologies, Inc.). For experiments examining the effects of Me2SO on HL-60 response to GM-CSF, cells were cultured 3 days in 1.25% Me2SO (Sigma). Recombinant GM-CSF was obtained from the Biological Response Modifiers Program (Frederick Cancer Research and Development Center, Frederick, MD) and was produced by Peprotech. The antisera used to immunoprecipitate Fes was a rabbit polyclonal antibody directed against a glutathione S-transferase fusion protein, which included amino acids 381-563 of murine Fes. The antisera used to immunoblot Fes was a rat monoclonal antibody purchased from Oncogene Science. Anti-phosphotyrosine monoclonal antibody PY-20 (ICN) was used for immunoprecipitation of phosphotyrosylproteins.In Vivo Radiolabeling, Immunoprecipitation, Electrophoresis, and ImmunoblottingMetabolic labeling with [32P]orthophosphate, immunoprecipitations, one- and two-dimensional electrophoresis, and immunoblotting were performed as described in detail previously(9Linnekin D. Howard O.M.Z. Park L. Farrar W.L. Ferris D.F. Longo D.L. Blood. 1994; 84: 94-103Crossref PubMed Google Scholar).Peptide MappingPeptide mapping was performed as described by Cleveland et al.(16Cleveland D.W. Fischer S. Kirschner M. Laemmli U. J. Biol. Chem. 1977; 252: 1102-1106Abstract Full Text PDF PubMed Google Scholar). In brief, anti-Fes or anti-phosphotyrosine immunoprecipitates from GM-CSF-stimulated myeloid cells radiolabeled with [32P]orthophosphate were resolved using one-dimensional gel electrophoresis. The gels were dried and subjected to autoradiography. The band corresponding to either Fes or p97 was excised from the gel, rehydrated in water, and incubated for 30 min in Cleveland equilibration buffer containing 125 mM Tris-HCl (pH 6.8) and 0.1% SDS. The gel slice was then placed in a preformed well of a stacking gel, underlayered with equilibration buffer containing 20% glycerol, then overlaid with equilibration buffer containing 10% glycerol, 0.001% bromphenol blue, and 0.05 μg of V8 protease (Boehringer Mannheim). Electrophoresis was performed using a 1.5-mm, 15% polyacrylamide gel as described previously(16Cleveland D.W. Fischer S. Kirschner M. Laemmli U. J. Biol. Chem. 1977; 252: 1102-1106Abstract Full Text PDF PubMed Google Scholar).RESULTSTwo factor-dependent human cell lines widely used for study of the GM-CSF signal transduction pathway are Mo7e and TF-1 cells. Stimulation of either of these cell lines results in increases in proliferative response ranging from 20-200 fold over control (data not shown). Another factor-responsive cell line used for study of human GM-CSF are HL-60 cells cultured in Me2SO for 3 days. These cells, although approaching a differentiated phenotype, have a transient capacity to proliferate in response to GM-CSF and have been used for study of GM-CSF-induced tyrosine phosphorylation(9Linnekin D. Howard O.M.Z. Park L. Farrar W.L. Ferris D.F. Longo D.L. Blood. 1994; 84: 94-103Crossref PubMed Google Scholar). Shown in Fig. 1A are the results of GM-CSF stimulation of the Mo7e and HL-60/Me2SO cells. Cells were radiolabeled for 3 h with [32P]orthophosphate, stimulated with GM-CSF for 10 min, and lysed, and phosphotyrosylproteins were immunoprecipitated using PY-20, a monoclonal antibody specific for phosphotyrosine. Phosphotyrosylproteins were resolved using SDS-PAGE and visualized using autoradiography. As shown in Fig. 1A, GM-CSF-induced phosphorylation of a 97-kDa protein as well as phosphotyrosylproteins of 140, 120, 70, 55, and 42 kDa in the Mo7e and HL-60/Me2SO cells. Identical results were also obtained with the TF-1 cell line (data not shown).Expression of Fes was next examined in the Mo7e, TF-1, and HL-60/Me2SO cell lines. Shown in Fig. 1B are results of a Fes immunoblot of Fes immunoprecipitates performed on equivalent amounts of protein isolated from the cells indicated. As expected, Fes was present in the erythroid and myeloid cells (Mo7e, HL-60/Me2SO, and TF-1) but not in the lymphoid line, Jurkat.Previous work has suggested that GM-CSF induces tyrosine phosphorylation of Fes as well as increases its autophosphorylation (10Hanazono Y. Chiba S. Sasaki K. Mano H. Miyajima A. Arai K. Yazaki Y. Hirai H. EMBO J. 1993; 12: 1641-1646Crossref PubMed Scopus (144) Google Scholar, 11Hanazono Y. Chiba S. Sasaki K. Mano H. Yazaki Y. Hirai H. Blood. 1993; 81: 3193-3196Crossref PubMed Google Scholar). In addition, our prevous work demonstrated that p97 has a number of features consistent with those of a tyrosine kinase(12Linnekin D. Evans G. Michiel D. Farrar W. J. Biol. Chem. 1992; 267: 23993-23998Abstract Full Text PDF PubMed Google Scholar). To address whether p97 was the proto-oncogene product Fes, we performed immunoblotting of radiolabeled phosphotyrosylproteins from untreated or GM-CSF-treated Mo7e cells. As shown in the autoradiograph in Fig. 2A, GM-CSF stimulated tyrosine phosphorylation of p97; however, the Fes antisera did not recognize p97 in the immunoblot shown in Fig. 2B. The identical results were obtained in each of the three cell lines tested. These experiments were done with phosphotyrosylproteins immunoprecipitated from 10-fold more cells (108 cells/point) then the studies shown in Fig. 1 to reduce the probability that the amount of p97 in the immunoprecipitate was below the threshold of detection of the antibody used to detect Fes. As another approach to address any potential relationship between p97 and Fes, we performed immunodepletion studies. Using conditions in which Fes could clearly be immunodepleted (Fig. 2C), we found no reduction in the amount of radiolabeled p97 in anti-phosphotyrosine immunoprecipitates from GM-CSF-stimulated cells (Fig. 2D).Figure 2:Fes-specific antibody does not immunoblot p97. Panel A, GM-CSF-induced tyrosine phosphorylation in Mo7e cells. Radiolabeled phosphotyrosylproteins were immunoprecipitated using the PY-20 antibody, resolved using SDS-PAGE, and transferred to Immobilon. Shown in panel A is the autoradiograph from this experiment. PanelB, Western blot analysis of Fes in GM-CSF-induced phosphotyrosylproteins from panelAversus a Fes immunoprecipitate. Enhanced chemiluminescence (ECL) was used for visualization. The abbreviation IP designates immunoprecipitation, and the abbreviation IB designates immunoblotting. PanelC, immunodepletion of Fes from Mo7e cells. Lysates from Mo7e cells were immunoprecipitated with antibody to Fes. The Fes-depleted supernatants were saved and reimmunoprecipitated with more Fes antibody. Shown are the Fes immunoblots before and after immunodepletion. PanelD, immunodepletion of Fes does not reduce the levels of p97. Mo7e cells were radiolabeled with [32P]orthophosphate as described in Fig. 1. Cells were incubated in the presence or absence of GM-CSF and lysed, and lysates clarified. The indicated lysate was depleted of Fes through immunoprecipitation and phosphotyrosylproteins from all the lysates were immunoprecipitated as described in Fig. 1. Phosphotyrosylproteins were visualized using autoradiography.View Large Image Figure ViewerDownload Hi-res image Download (PPT)We next examined whether we could detect GM-CSFinduced phosphorylation of Fes in vivo (Fig. 3). Tyrosine phosphorylation of Fes was examined in both Mo7e (Fig. 3A) and Me2SO-treated HL-60 cells (Fig. 3B) after stimulation with GM-CSF. Although no GM-CSF-induced tyrosine phosphorylation of Fes was observed in either cell line, the Fes immunoblots in Fig. 3(C and D) demonstrate that Fes was present in each of the immunoprecipitates. We also examined the effects of GM-CSF on phosphorylation of Fes using [32P]orthophosphate and found no changes at any time point examined (Fig. 3E). Thus, using anti-phosphotyrosine immunoblotting as well as radiolabeling procedures, we could not detect GM-CSFinduced phosphorylation of Fes.Figure 3:GM-CSF does not induce phosphorylation of Fes. Mo7e (panelsA and C) or Me2SO-treated HL-60 cells (panels B and D) were resuspended at 2 × 107/ml in RPMI 1640. Cells were stimulated for the indicated times, lysed, and immunoprecipitated with antibody to Fes or a control. Immunoprecipitates were resolved using SDS-PAGE, transferred to Immobilon, and Western blots performed using either antibodies specific for phosphotyrosine (panelsA and B) or Fes (panelsC and D). PanelE, Mo7e radiolabeled with [32P]orthophosphate were stimulated with GM-CSF, lysed, and immunoprecipitated with antibody specific for Fes. Immunoprecipitates were resolved using SDS-PAGE and proteins visualized using autoradiography.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The previous experiments suggested that p97 represented a protein distinct from the Fes tyrosine kinase. To address any potential relationship between Fes and p97, we examined the charge to mass ratio of GM-CSF-induced phosphotyrosylproteins and Fes using two-dimensional gel electrophoresis. Samples were subjected to isoelectric focusing in the first dimension and SDS-PAGE in the second dimension. As shown in Fig. 4, the migration of p97 and of Fes were Fes and p97 to a in the isoelectric focusing however, Fes a and more the of p97 and Fes, described as a protein, were to identical resolved in the second dimension. The for this to the over the of the used for the mass was to be a protein, work demonstrated to be 97 is that the mass of Fes may be to 97 We also examined migration of another of the of tyrosine kinases using two-dimensional gel electrophoresis. Interestingly, similar to p97 and Fes, also the of the isoelectric focusing gel (data not shown). In however, in SDS-PAGE at a p97 or Fes. These that p97 is not Fes or but may a related protein. To this we performed limited mapping of p97 and Fes isolated from GM-CSF-stimulated cells. of the of Fes and p97 shown in Fig. that but not of the proteolytic from Fes were the as those from the of p97. These in with the in migration of p97 and Fes using two-dimensional electrophoresis, that these proteins are related but gel electrophoresis of GM-CSF-induced phosphotyrosylproteins and Fes. Mo7e cells were radiolabeled with [32P]orthophosphate as described in Fig. 1. Cells were stimulated with GM-CSF for 10 min and lysed, and either Fes or phosphotyrosylproteins were isolated using were resolved using isoelectric focusing in the first dimension and SDS-PAGE in the second dimension. The abbreviation IP designates Large Image Figure ViewerDownload Hi-res image Download mapping of p97 and Fes. were excised from with 0.05 μg of V8 and subjected to electrophoresis as described and Large Image Figure ViewerDownload Hi-res image Download of protein tyrosine kinases and a of R. J. J. M. H. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, A. J. J. A. H. W. EMBO J. PubMed Scopus Google Scholar, J. T. Cell. Biol. PubMed Scopus Google Scholar, N. J. Cell. Biol. PubMed Scopus Google Scholar, T. K. T. W. N. J. Cell. Biol. PubMed Scopus Google Scholar, A. K. U. Cell. Biol. 1990; PubMed Scopus Google Scholar). these is expressed is and Fes is expressed in myeloid cells and erythroid Recent work has suggested that Fes may be activated in response to GM-CSF, and Y. Chiba S. Sasaki K. Mano H. Miyajima A. Arai K. Yazaki Y. Hirai H. EMBO J. 1993; 12: 1641-1646Crossref PubMed Scopus (144) Google Scholar, 11Hanazono Y. Chiba S. Sasaki K. Mano H. Yazaki Y. Hirai H. Blood. 1993; 81: 3193-3196Crossref PubMed Google Scholar). Fes has also been suggested to a in of R. A. D. P. G. G. U. S. J. 1993; PubMed Scopus Google Scholar). Previous work from our a putative protein tyrosine kinase of approximately 97 kDa which was phosphorylated in response to a number of different D. Evans G. Michiel D. Farrar W. J. Biol. Chem. 1992; 267: 23993-23998Abstract Full Text PDF PubMed Google Scholar). The of this study was to address the relationship between Fes and the 97-kDa protein phosphorylated in response to GM-CSF. For this we have used three cell lines of erythroid and myeloid which Fes and proliferate in response to GM-CSF. with previous p97 was of the phosphotyrosylproteins induced by GM-CSF in each of the cell lines examined (Fig. lines of that p97 is not Fes. anti-Fes did not recognize p97 in the GM-CSF-induced phosphotyrosylproteins (Fig. A and immunodepletion of Fes did not reduce the levels of the radiolabeled p97 in the anti-phosphotyrosine immunoprecipitates (Fig. we did not GM-CSF-induced phosphorylation of Fes using either anti-phosphotyrosine immunoblotting or [32P]orthophosphate radiolabeling (Fig. 3). mapping experiments of Fes and p97 suggested that these proteins are distinct but probably related (Fig. In this the in the migration of p97, Fes, and in two-dimensional gel electrophoresis that these proteins are related (Fig. We have also examined whether p97 is the and found on the in of p97 and as well as the absence of increases in K. In these studies demonstrate that p97 is not the Fes proto-oncogene product and that is a of the of tyrosine work has suggested a for Fes in cell previously Fes has been observed in and Fes or a Fes-related protein has been reported in murine lymphoid cell A. G. G. M. 1994; Google Scholar, K. R. P. N. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar). we have previously a protein of approximately 97-kDa which is phosphorylated on tyrosine in response to D. Evans G. Michiel D. Farrar W. J. Biol. Chem. 1992; 267: 23993-23998Abstract Full Text PDF PubMed Google Scholar, W. Ferris D. J. Biol. Chem. Full Text PDF PubMed Google Scholar, D. J. D. Farrar W. 8: Google Scholar). mapping of the phosphotyrosylprotein of 97-kDa and a protein of similar size and phosphorylated in response to GM-CSF from lymphoid and myeloid cells has suggested that these proteins are either related or D. Evans G. Michiel D. Farrar W. J. Biol. Chem. 1992; 267: 23993-23998Abstract Full Text PDF PubMed Google Scholar). our to these proteins are identical through mapping of p97 from phosphotyrosylproteins from GM-CSF-stimulated myeloid cells and lymphoid cells have Thus, to the to address the relationship of the 97-kDa protein found in myeloid and lymphoid cells have been of the Fes proto-oncogene product in signal transduction has been and (10Hanazono Y. Chiba S. Sasaki K. Mano H. Miyajima A. Arai K. Yazaki Y. Hirai H. EMBO J. 1993; 12: 1641-1646Crossref PubMed Scopus (144) Google Scholar, 11Hanazono Y. Chiba S. Sasaki K. Mano H. Yazaki Y. Hirai H. Blood. 1993; 81: 3193-3196Crossref PubMed Google Scholar) have increases in Fes as well as increases in its tyrosine phosphorylation after stimulation of TF-1 cells with GM-CSF, or erythropoietin. In addition, these have reported that GM-CSF induces Fes to with the β of the GM-CSF Y. Chiba S. Sasaki K. Mano H. Miyajima A. Arai K. Yazaki Y. Hirai H. EMBO J. 1993; 12: 1641-1646Crossref PubMed Scopus (144) Google Scholar). In other have not found Fes activated in response to either or GM-CSF(5Witthuhn B. Quelle F. Silvernnoinen O. Yi T. Tang B. Miura O. Ihle J. Cell. 1993; 74: 227-236Abstract Full Text PDF PubMed Scopus (1001) Google Scholar). Thus, more work be to more the of Fes in signal transduction of A of work has also suggested that Fes a in the differentiated in myeloid cells. Although of Fes in a cell line, to be through a signal transduction pathway distinct from that by L. R. M. R. R. Cell. Biol. 1994; PubMed Scopus Google Scholar). and co-workers G. T. R. J. Biol. Chem. Full Text PDF PubMed Google Scholar, G. R. J. Biol. Chem. Full Text PDF PubMed Google Scholar, T. G. R. J. Biol. Chem. 1988; Full Text PDF PubMed Google Scholar) reported increases in Fes in myeloid cells and also found that of the erythroid cell line with the capacity for these cells to a myeloid another has shown that specific for induced in HL-60 cells with of R. A. D. P. G. G. U. S. J. 1993; PubMed Scopus Google Scholar). These that Fes a in in these as in the and of tyrosine have members that or similar in the signal transduction of multiple Our studies that p97 is related to Fes and may a of the of protein tyrosine kinases. work be directed members of this of tyrosine kinases and in of INTRODUCTIONGM-CSF 1The abbreviations used are: GM-CSFgranulocyte-macrophage colony stimulating factorILinterleukinPAGEpolyacrylamide gel electrophoresis. stimulates proliferation and differentiation of myeloid progenitor cells as well as activation of neutrophils(1Metcalf D. Science. 1991; 254: 529-535Crossref PubMed Scopus (330) Google Scholar, 2Gasson J.C. Blood. 1991; 77: 1131-1145Crossref PubMed Google Scholar). The receptor for GM-CSF consists of an α subunit of 85 kDa which binds ligand with low affinity and a β subunit of approximately 120 kDa which does not bind ligand but converts the receptor complex to high affinity binding(3Gearing D. King J. Gough N. Nicola N. EMBO J. 1988; 8: 3667-3676Crossref Scopus (521) Google Scholar, 4Hayashida K. Kitamura T. Gorman D. Arai K. Yokota T. Miyajima A. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 9655-9659Crossref PubMed Scopus (518) Google Scholar). Similar to other members of the hemapoietin receptor superfamily, the components of the GM-CSF receptor do not contain intrinsic tyrosine or serine/threonine kinase activity. Recent work has suggested that the rapid increases in protein tyrosine phosphorylation stimulated by GM-CSF may be mediated through activation of multiple tyrosine kinases. A number of Src-like tyrosine kinases (Hck, Lyn, and Yes), as well as JAK2, are stimulated in response to GM-CSF(5Witthuhn B. Quelle F. Silvernnoinen O. Yi T. Tang B. Miura O. Ihle J. Cell. 1993; 74: 227-236Abstract Full Text PDF PubMed Scopus (1001) Google Scholar, 6Silvennoinen O. Witthuhn B. Quelle F. Cleveland J. Yi T. Ihle J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8429-8433Crossref PubMed Scopus (437) Google Scholar, 7Torigoe T. O'Connor R. Santoli D. Reed J. Blood. 1992; 80: 617-624Crossref PubMed Google Scholar, 8Corey S. Eguinoa A. Puyana-Theall K. Bolen J. Cantley L. Mollinedo F. Jackson T. Hawkins P. Stephens L. EMBO J. 1993; 12: 2681-2690Crossref PubMed Scopus (170) Google Scholar, 9Linnekin D. Howard O.M.Z. Park L. Farrar W.L. Ferris D.F. Longo D.L. Blood. 1994; 84: 94-103Crossref PubMed Google Scholar). In addition, the work of Hanazono and co-workers (10Hanazono Y. Chiba S. Sasaki K. Mano H. Miyajima A. Arai K. Yazaki Y. Hirai H. EMBO J. 1993; 12: 1641-1646Crossref PubMed Scopus (144) Google Scholar, 11Hanazono Y. Chiba S. Sasaki K. Mano H. Yazaki Y. Hirai H. Blood. 1993; 81: 3193-3196Crossref PubMed Google Scholar) has suggested that Fes is activated in response to GM-CSF as well as IL-3 and erythropoietin. Interestingly, a number of laboratories have reported a phosphotyrosylprotein similar in size to Fes (90-100 kDa) in GM-CSF-, IL-3-, or erythropoietin-stimulated cells (12Linnekin D. Evans G. Michiel D. Farrar W. J. Biol. Chem. 1992; 267: 23993-23998Abstract Full Text PDF PubMed Google Scholar, 13Linnekin D. Evans G. Michiel D. Farrar W. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6237-6241Crossref PubMed Scopus (43) Google Scholar, 14Quelle F. Quelle D. Wojchowski D. J. Biol. Chem. 1992; 267: 17055-17060Abstract Full Text PDF PubMed Google Scholar, 15Kanakura Y. Druker B. Cannistra S. Furukawa Y. Torimoto Y. Griffin J. Blood. 1990; 76: 706-715Crossref PubMed Google Scholar). Furthermore, we previously have described a putative tyrosine kinase of 97 kDa (p97) phosphorylated on tyrosine residues in response to GM-CSF(12Linnekin D. Evans G. Michiel D. Farrar W. J. Biol. Chem. 1992; 267: 23993-23998Abstract Full Text PDF PubMed Google Scholar). The present study addresses whether p97 is the proto-oncogene product Fes or potentially a Fes-related protein.