©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Phosphorylation of a Fes-related Protein in Response to Granulocyte-Macrophage Colony Stimulating Factor (*)

(Received for publication, September 15, 1994; and in revised form, December 19, 1994)

Diana Linnekin (1)(§) Sherry M. Mou (2) Peter Greer (3) Dan L. Longo (1) Douglas K. Ferris (2)

From the  (1)Laboratory of Leukocyte Biology, Biological Response Modifiers Program, Division of Cancer Treatment, and the (2)Biological Carcinogenesis and Development Program, Program Recourses, Inc./DynCorp, Frederick Cancer Research and Development Center, NCI, Frederick, Maryland 21702, and (3)Cancer Research Laboratories, Departments of Pathology and Biochemistry, Queens University, Ontario K7L 3N6, Canada

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

GM-CSF (^1)stimulates proliferation and differentiation of myeloid progenitor cells as well as activation of neutrophils(1, 2) . The receptor for GM-CSF consists of an alpha subunit of 85 kDa which binds ligand with low affinity and a beta subunit of approximately 120 kDa which does not bind ligand but converts the receptor complex to high affinity binding(3, 4) . 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(5, 6, 7, 8, 9) . In addition, the work of Hanazono and co-workers (10, 11) 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 (12, 13, 14, 15) . Furthermore, we previously have described a putative tyrosine kinase of 97 kDa (p97) phosphorylated on tyrosine residues in response to GM-CSF(12) . The present study addresses whether p97 is the proto-oncogene product Fes or potentially a Fes-related protein.


MATERIALS AND METHODS

Cell Lines, Growth Factors, and Antibodies

Mo7e 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 Me(2)SO on HL-60 response to GM-CSF, cells were cultured 3 days in 1.25% Me(2)SO (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 Immunoblotting

Metabolic labeling with [P]orthophosphate, immunoprecipitations, one- and two-dimensional electrophoresis, and immunoblotting were performed as described in detail previously(9) .

Peptide Mapping

Peptide mapping was performed as described by Cleveland et al.(16) . In brief, anti-Fes or anti-phosphotyrosine immunoprecipitates from GM-CSF-stimulated myeloid cells radiolabeled with [P]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(16) .


RESULTS

Two 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 Me(2)SO 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(9) . Shown in Fig. 1A are the results of GM-CSF stimulation of the Mo7e and HL-60/Me(2)SO cells. Cells were radiolabeled for 3 h with [P]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/Me(2)SO cells. Identical results were also obtained with the TF-1 cell line (data not shown).


Figure 1: GM-CSF induces tyrosine phosphorylation in cell lines expressing Fes. Panel A, tyrosine phosphorylation in GM-CSF-treated cells. Cells were washed in phosphate-free RPMI 1640 and radiolabeled with [P]orthophosphate (1 mCi/ml) for 3 h. One milliliter of cells (10^7/ml) were stimulated with GM-CSF for 10 min, pelleted, lysed, and immunoprecipitated with the anti-phosphotyrosine monoclonal antibody PY-20. Phosphotyrosylproteins were eluted, diluted with SDS sample buffer, resolved using one-dimensional gel electrophoresis, dried, and visualized with autoradiography. Panel B, expression of Fes. Cells were lysed and equivalent amounts of protein immunoprecipitated with anti-Fes. Washed immunoprecipitates were eluted with SDS sample buffer, resolved using one-dimensional gel electrophoresis, and transferred to Immobilon. Shown are the results of immunoblotting with monoclonal antibody against Fes using ECL for visualization. The abbreviation IP designates immunoprecipitation, and the abbreviation IB designates immunoblotting.



Expression of Fes was next examined in the Mo7e, TF-1, and HL-60/Me(2)SO 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/Me(2)SO, 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 (10, 11) . In addition, our prevous work demonstrated that p97 has a number of features consistent with those of a tyrosine kinase(12) . 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 (10^8 cells/point) then the studies shown in Fig. 1to 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 [P]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.



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 Me(2)SO-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 [P]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 Me(2)SO-treated HL-60 cells (panels B and D) were resuspended at 2 times 10^7/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 [P]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.



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 patterns of p97 and of Fes were quite similar. Both Fes and p97 migrated to a relatively basic position in the isoelectric focusing dimension; however, Fes consistently had a slightly broader and more basic position. Somewhat surprisingly, the molecular sizes of p97 and Fes, originally described as a 92-kDa protein, were close to identical when resolved in the second dimension. The basis for this apparent discrepancy relates to the debate over the actual molecular weight of the phosphorylase b marker used for the molecular mass standards. While phosphorylase b was once thought to be a 92-kDa protein, later work demonstrated it to be 97 kDa. Thus it is likely that the apparent molecular mass of Fes may actually be closer to 97 kDa. We also examined migration of Fer, another member of the Fps/Fes family of tyrosine kinases using two-dimensional gel electrophoresis. Interestingly, similar to p97 and Fes, Fer also migrated toward the basic end of the isoelectric focusing gel (data not shown). In contrast, however, in SDS-PAGE gels, Fer migrated at a molecular weight distinctly greater than p97 or Fes. These data suggest that p97 is not Fes or Fer but may represent a related protein. To further test this postulate, we performed limited peptide mapping of p97 and Fes isolated from GM-CSF-stimulated cells. Comparison of the peptide maps of Fes and p97 shown in Fig. 5demonstrates that several, but not all, of the proteolytic fragments from Fes were the same molecular weight as those generated from the digestion of p97. These data, considered in conjunction with the similarity in migration of p97 and Fes using two-dimensional electrophoresis, strongly suggest that these proteins are related but distinct.


Figure 4: Two-dimensional gel electrophoresis of GM-CSF-induced phosphotyrosylproteins and Fes. Mo7e cells were radiolabeled with [P]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 immunoprecipitation. Proteins were resolved using isoelectric focusing in the first dimension and SDS-PAGE in the second dimension. The abbreviation IP designates immunoprecipitation.




Figure 5: Peptide mapping of p97 and Fes. Proteins were excised from SDS-polyacrylamide gels, rehydrated, equilibrated, digested with 0.05 µg of V8 protease, and subjected to electrophoresis as described under ``Materials and Methods.''




DISCUSSION

The Fes/Fps family of protein tyrosine kinases currently includes Fes/Fps, Fer, and a truncated form of Fer designated FerT(17, 18, 19, 20, 21, 22) . Of these members, Fer is expressed relatively ubiquitously, FerT is testes-specific and Fes is expressed predominantly in myeloid cells and certain erythroid progenitors. Recent work has suggested that Fes may be activated in response to GM-CSF, IL-3, and erythropoietin(10, 11) . Fes has also been suggested to play a role in maintaining viability of granulocytes during differentiation(23) . Previous work from our laboratory identified a putative protein tyrosine kinase of approximately 97 kDa (p97), which was phosphorylated in response to a number of different hematopoietic growth factors(12) . The objective of this study was to address the relationship between Fes and the 97-kDa protein phosphorylated in response to GM-CSF. For this purpose we have used three cell lines of erythroid and myeloid origin which express Fes and proliferate in response to GM-CSF. Consistent with previous work, p97 was one of the dominant phosphotyrosylproteins induced by GM-CSF in each of the cell lines examined (Fig. 1). Several lines of evidence strongly suggest that p97 is not Fes. First, anti-Fes did not recognize p97 in the GM-CSF-induced phosphotyrosylproteins (Fig. 2, A and B). Second, immunodepletion of Fes did not reduce the levels of the radiolabeled p97 in the anti-phosphotyrosine immunoprecipitates (Fig. 2D). Third, we did not observe GM-CSF-induced phosphorylation of Fes using either anti-phosphotyrosine immunoblotting or [P]orthophosphate radiolabeling (Fig. 3). Fourth, peptide mapping experiments of Fes and p97 suggested that these two proteins are distinct but probably related (Fig. 5). In this regard, the similarity in the migration of p97, Fes, and Fer in two-dimensional gel electrophoresis further suggests that these proteins are related (Fig. 4). We have also examined whether p97 is the Fps/Fes family member Fer and found it unlikely based on the difference in molecular weight of p97 and Fer, as well as the absence of cytokine-induced increases in Fer autophosphorylation. (^2)In summary, these studies demonstrate that p97 is not the Fes proto-oncogene product and suggest that it is a member of the Fps/Fes family of tyrosine kinases.

Recent work has suggested a broader role for Fes in mammalian cell physiology than previously thought. Fes has been observed in embryonic tissues, and Fes or a Fes-related protein has been reported in murine lymphoid cell lines(24, 25) . Further more, we have previously noted a protein of approximately 97-kDa which is phosphorylated on tyrosine in response to IL-2(12, 26, 27) . Peptide mapping of the IL-2-induced phosphotyrosylprotein of 97-kDa and a protein of similar size and pI phosphorylated in response to GM-CSF from [P]orthophosphate-labeled lymphoid and myeloid cells has indeed suggested that these proteins are either closely related or identical(12) . Unfortunately, our efforts to determine if these proteins are identical through peptide mapping of p97 from [S]methionine-labeled phosphotyrosylproteins from GM-CSF-stimulated myeloid cells and IL-2-stimulated lymphoid cells have failed. Thus, to date, the experimental designs necessary to further address the relationship of the 97-kDa protein found in cytokine-stimulated myeloid and lymphoid cells have been unsuccessful.

The role of the Fes proto-oncogene product in cytokine signal transduction has been somewhat controversial. Hanazano and colleagues (10, 11) have seen increases in Fes autophosphorylation, as well as increases in its tyrosine phosphorylation after stimulation of TF-1 cells with GM-CSF, IL-3, or erythropoietin. In addition, these investigators have reported that GM-CSF induces Fes to associate with the beta chain of the GM-CSF receptor(10) . In contrast, other groups have not found Fes activated in response to either erythropoietin or GM-CSF(5) . Thus, more work will be necessary to delineate more conclusively the role of Fes in signal transduction of hematopoietic cytokines. A relatively large body of work has also suggested that Fes plays a role in the differentiated phenotype in myeloid cells. Although overexpression of Fes partially abrogated CSF-1 dependence in a macrophage-derived cell line, it appeared to be through a signal transduction pathway distinct from that utilized by CSF-1(28) . Glazer and co-workers (29, 30, 31) reported increases in Fes activity in differentiating myeloid cells and also found that transfection of the erythroid cell line K562 with fes conferred the capacity for these cells to differentiate toward a myeloid phenotype More recently, another group has shown that antisense oligonucleotides specific for fes induced apoptosis in HL-60 cells treated with inducers of granulocytic differentiation(23) . These investigators concluded that Fes played a role in preventing apoptosis during granulopoiesis.

Considered in total, these observations strongly suggest that, as in the Src and JAK family of tyrosine kinases, Fes/Fer have members that perform parallel or similar functions in the signal transduction pathways of multiple stimuli. Our studies suggest that p97 is related to Fes and may represent a new member of the Fps/Fes/Fer family of protein tyrosine kinases. Future work will be directed toward cloning new members of this family of tyrosine kinases and better characterizing their role in receptor-effector coupling of hematopoietic cytokines.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Bldg. 567-1, Laboratory of Leukocyte Biology, Biological Response Modifiers Program, Division of Cancer Treatment, NCI, Frederick Cancer Research and Development Center, Frederick, MD 21702. Tel.: 301-846-1427; Fax: 301-846-5651.

(^1)
The abbreviations used are: GM-CSF, granulocyte-macrophage colony stimulating factor; IL, interleukin; PAGE, polyacrylamide gel electrophoresis.

(^2)
D. K. Ferris, unpublished data.


ACKNOWLEDGEMENTS

We thank Dr. Frank Ruscetti for helpful discussions and review of the manuscript.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.