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Stem Cells, Vol. 14, No. 6, 714-724, November 1996
© 1996 AlphaMed Press

ORIGINAL PAPER

Analysis of Fes Kinase Activity in Myeloid Cell Growth and Differentiation

Karen E. Yatesa, Gay M. Crooksb, Judith C. Gassona

a Departments of Biological Chemistry and Medicine, and the Molecular Biology Institute and Jonsson Comprehensive Cancer Center, UCLA School of Medicine, Los Angeles, California, USA;
b Division of Research Immunology and Bone Marrow Transplantation, Children's Hospital of Los Angeles, Los Angeles, California, USA

Key Words. c-Fes. • Tyrosine kinase • Signal transduction • Cytokine • Myeloid cells

Dr. Judith C. Gasson, 11-934 Factor, Division of Hematology-Oncology, Department of Medicine, UCLA School of Medicine, Los Angeles, CA 90095-1678, USA.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
Fes is a nonreceptor protein tyrosine kinase that has been implicated in a variety of cytokine signal transduction pathways, as well as differentiation of myeloid cells. To address the role of Fes in these processes, we overexpressed a kinase-defective Fes protein in the factor-dependent cell-lines, TF-1 and 32D. Proliferative responses to GM-CSF and interleukin 3, and the induction of differentiation by G-CSF were not altered by expression of the kinase mutant Fes protein, indicating that Fes kinase activity is not critical for these biological events in these cell lines.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
Fes is a nonreceptor protein tyrosine kinase distantly related to members of the Src family. Human c-Fes is expressed in hematopoietic cells of the myeloid lineage [1, 2], CD34+ hematopoietic progenitors [3] and also vascular endothelial cells [4]. Fes mRNA or Fes protein has been detected in a variety of primary malignancies, including chronic myeloid leukemia [5,6], acute myeloid leukemia [6, 7], acute lymphocytic leukemia [5, 8] and carcinoma of the lung [9]. Fes has also been observed in cell lines established from primary hematopoietic malignancies such as erythroleukemia [10, 11], chronic myeloid leukemia, acute myeloid leukemia [11], acute lymphocytic leukemia [12] and lymphoma [8, 13].

Fes has recently been implicated in cytokine signal transduction pathways, although its role is somewhat controversial. Using the TF-1 human erythroleukemia cell line, Hanazono et al. found that tyrosine phosphorylation of Fes, as well as its kinase activity, was increased when cells were stimulated with GM-CSF, interleukin 3 (IL-3) [14] or erythropoietin [10]. These investigators used immunoprecipitation to show that Fes was physically associated with the common beta subunit of the GM-CSF and IL-3 receptors [14, 15]. Similar observations were made by Rao and Mufson [16], who demonstrated that the endogenous Fes protein in TF-1 cell lysates was able to bind GST-beta subunit fusion proteins in vitro. Brizzi et al. [17] used immunoprecipitation to show that Fes associated with the beta subunit in primary human polymorphonuclear cells.

However, other investigators have made contrary observations. In studies of Janus family kinases in cytokine-mediated signal transduction, Fes was not affected by GM-CSF stimulation of TF-1 cells [18] or by IL-3 stimulation of the murine cell line, DA3 [19]. In their study of human polymorphonuclear cells, Brizzi et al. [17] found that Fes kinase activity was activated by GM-CSF but not IL-3. Finally, a protein that is similar but not identical to Fes has been reported to be tyrosine phosphorylated upon GM-CSF stimulation in TF-1 cells [20].

Recently, Fes has been shown to associate with gp130, the signal-transducing subunit of the IL-6 receptor, and to be phosphorylated upon IL-6 stimulation [21]. A similar interaction has been demonstrated between Fes and the IL-4 receptor [22]. The region of the receptor shown to interact with Fes has also been shown to be necessary for IL-4-mediated signal transduction [22]. Fes has also been implicated in myeloid cell differentiation. DMSO- and retinoic acid-induced differentiation of the leukemic cell line, HL-60, was inhibited when the cells were treated with Fes antisense oligonucleotides [23-25]. Rather than differentiate, the cells underwent programmed cell death. This effect was also observed in primary leukemia cells [23, 25]. Glazer and colleagues observed upregulation of c-Fes protein during DMSO-induced granulocytic differentiation of HL-60 cells [11]. They also showed that K562 cells are induced to differentiate to granulocytes when they are stably transfected with the c-fes gene [26, 27].

We sought to determine the role of Fes kinase activity in myeloid cell proliferation and differentiation. Other tyrosine kinases such as Abl and Lck have been shown to function in cell growth and development by use of a dominate-negative strategy in which a kinase-defective mutant is expressed at a high level in cells which have the endogenous protein [28, 29]. Thus, we made a mutation in the ATP binding site of the Fes kinase domain to abrogate enzymatic activity, and stably expressed the mutant protein in two factor-dependent myeloid cell lines: TF-1 and 32Dc13.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Acknowledgements
 References
 
Plasmids
plEl contains a full-length wild-type c-fes cDNA sequence in the Okayama-Berg vector [30]. SR{alpha}MSVtkNeo is a retroviral expression vector which contains the SV40 origin of replication (generously provided by Owen Witte, UCLA) [31]. The retroviral vector LXSN was provided by D. Miller (Fred Hutchinson Cancer Research Center; Seattle, WA). Polymerase chain reaction (PCR) mutagenesis was used to substitute an arginine for lysine 590 of the human fes cDNA (fesR). The wild human c-fes cDNA was cloned into the Eco RI site of LXSN (L-fes-SN). However, the mutant Fes was unstable in the LXSN vector; therefore, it was cloned into SR{alpha}MSVtkNeo (ConFesR). Both constructs were used to make amphotropic retrovirus, which was used to transduce the human erythroleukemia cell line, TF-1, and the murine leukemia cell line, 32Dc13.

Antibodies
c-Fes antisera #61 and #70 have been previously described [30]. We purchased 4G10 (UBI; Lake Placid, NY) and PY20 (ICN; Irvine, CA), which are mouse monoclonal antibodies that recognize phosphotyrosine.

Cells
32Dc13 cells were maintained in Iscove's modified Dulbecco's medium (IMDM) containing 10% fetal bovine serum (FBS) and 200 pM recombinant murine GM-CSF (Amgen; Thousand Oaks, CA). Cells were washed three times with phosphate buffered saline (PBS), then plated at 5 x 104 cells/ml in 200 pM murine GM-CSF, 200 pM murine IL-3 (Genyzme; Cambridge, MA) or 100 ng/ml G-CSF (Amgen). To determine the percent of 32D cells differentiated to neutrophils, cells were cytospun onto glass slides and stained with Diff-Quik (Baxter; Miami, FL). The slides were examined under a light microscope, using nuclear morphology as a marker of granulocytic differentiation. Three to four hundred cells on each slide were examined. Stock cultures of TF-1, TF-1/Fes, TF-1/FesR and Tf-1/SR{alpha} cells were maintained in 0.5 nM GM-CSF (generously provided by Larry Souza, Amgen). Cells were shifted to 0.5 nM IL-3 or 0.2 nM PIXY321 (generously provided by Linda Park, Immunex; Seattle, WA) three to five days prior to processing, as indicated. For experiments involving erythropoietin (EpoGEN) (Amgen), cells were carried in GM-CSF, factor-starved for 13-18 h, then stimulated with 10 U/ml of erythropoietin. Interferon {alpha}2a (Roferon-A) was purchased from Roche Laboratories, Nutley, NJ.

Gel Electrophoresis and Immunoblotting
Samples were electrophoresed over 7.5% acrylamide Tris-glycine gels. Proteins were electroblotted onto Hybond-ECL nitrocellulose (Amersham; Arlington Heights, IL). Membranes were blocked with Tris-buffered saline containing 0.1% Tween-20 (Sigma; St. Louis, MO) and 4% nonfat dried milk or 4% bovine serum albumin (Sigma). Immune complexes were visualized with biotinylated goat-antirabbit Ig antibody and streptavidin-horseradish peroxidase conjugate or an antimouse Ig horseradish peroxidase conjugate and ECL chemiluminescent detection reagents (Amersham). To strip immunoblots, membranes were rinsed with distilled water and incubated in 0.2 M glycine (pH 2.8) for 30 min at room temperature. Membranes were rinsed again with water and reblocked prior to subsequent antibody incubations.

Retrovirus Production and Transduction
Amphotropic retroviruses were produced in two ways: helper plasmid and SR{alpha}MSVtkNeo or ConFesR were transiently transfected into 107 COS-1 cells in 0.5 ml 1.2 x RPMI by electroporation using a Bio-Rad Gene Pulsar at 250V and 960 µF. Cell culture supernatants were collected from days 3 to 6 post-transfection, then pooled and filtered through 0.45 µm filters. Alternatively, virus-containing medium was harvested from cultures of PA317 packaging cell lines that stably produced LXSN, L-fes-SN, Sr{alpha}MSVtkNeo and ConFesR retroviruses. For transduction, viral supernatants were added to cells in culture and incubated 4 h in the presence of 20 µg/ml protamine (Elkins-Sinn, Inc; Cherry Hill, NJ). The cells were washed twice with PBS, then cultured for 72 h before addition of 0.8-1.2 active mg/ml geneticin (GIBCO; Grand Island, NY) (determined empirically for each cell line). Transduced cells were maintained in GM-CSF throughout selection.

Cell Lysis, Immunoprecipitation and Immune Complex Kinase Assays
Whole-cell lysates were prepared by pelleting cells and lysing them directly in 1 x SDS sample loading buffer. For antiphosphotyrosine immunoblotting, cells were factor-starved for 13-18 h, stimulated with cytokines for 5 min, then lysed in 20 mM Tris pH 7.4, 1 mM EGTA, 75 µM sodium vanadate, 50 mM NaF, plus proteinase inhibitors, as described [30]. Lysates were sonicated twice for 5 sec, then cleared by centrifugation for 5 min at top speed in a microcentrifuge. For immunoprecipitation, cells were lysed for 60 min on ice in 20 mM Tris pH 7.4, 150 mM NaCl2, 1 mM EDTA, 1% NP40, 0.5% sodium deoxycholate, 75 µM sodium orthovanadate, 50 mM NaF, plus proteinase inhibitors. Cleared lysates were incubated with the indicated antiserum and immune complexes precipitated with Staph A cells (Calbiochem; LaJolla, CA), then washed with lysis buffer. For immune complex kinase assays, immuno-precipitates were washed once with kinase buffer (10 mM MnCl2, 20 mM Tris pH 7.4, 75 µM vanadate, 50 mM NaF, plus protease inhibitors), resuspended in kinase buffer, then incubated with 10 µCi {gamma}32P-ATP for 30 min at room temperature. Reactions were stopped by adding lysis buffer, then washing twice with kinase buffer containing 10 mM EDTA.

Colony Assays
Cells were plated in 0.5 ml IMDM containing 0.3% agar, 20% FBS and cytokines, as indicated. Colonies (>40 cells) were counted after 14 days. Aliquots of cells prepared for colony assays were also plated in liquid culture and counted after five days to ensure that equal numbers of cells were in the starting dilution.

Cell Cycle Analysis
DNA content was assessed by flow cytometric analysis of propidium iodide. Cells were washed and resuspended in 0.01% propidium iodide staining buffer [32] immediately prior to flow cytometry. Samples were run on a FACScan flow cytometer (Becton-Dickinson Immunocytometry Systems; San Jose, CA) equipped with a 15 mW 488 nM air-cooled argonion laser. Ten thousand events were acquired using Lysys II software, and they were analyzed using the SFIT model in the CellFIT program (Becton-Dickinson).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Acknowledgements
 References
 
PCR mutagenesis was used to substitute an arginine for lysine 590 of the human fes cDNA (fesR); this substitution has been shown to inhibit kinase activity in v-Fes [33] and other kinases [28, 29, 34]. This mutation also introduced a novel Bgl II restriction enzyme site into the fes cDNA sequence.

Poly A+RNA was prepared from TF-1 cells transduced with SR{alpha}MSVtkNeo (TF-1/SR{alpha}) and ConFesR (TF-1/FesR) retroviruses. Reverse-transcribed PCR products of fes mRNA were digested with the restriction enzyme, Bgl II. Only the PCR product from TF-1/FesR cells was digested by the enzyme, indicating that fes RNA was being transcribed from the retroviral construct (data not shown).

Anti-Fes immunoblots of whole-cell lysates demonstrated that TF-1/Fes and TF-1Fes R cells overexpress their respective Fes proteins, relative to parental TF-1 or control TF-1/SR{alpha} cells (Fig. 1AGo). Immune complex kinase assays confirmed that the overexpressed protein in TF-1/FesR cells was kinase-inactive. The level of Fes kinase activity was approximately equal in immunoprecipitates from TF-1/SR{alpha} and TF-1/FesR cells (Fig. 2AGo), while the amount of protein was greater (~10-fold) in the TF-1/FesR cells (Fig. 2BGo). To ensure that all of the Fes protein had been immunoprecipitated, the remaining cell lysates were assayed by antiFes immunoblot and found to be negative. Interestingly, when the blots containing immunoprecipitated Fes were probed with antiphosphotyrosine antibody 4G10, the kinase-negative Fes appeared to be phosphorylated on tyrosine to the same degree as the endogenous protein (Fig. 2CGo). Similar results were observed in two additional independent experiments.



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  		Figure 1. Expression of Fes proteins in transduced TF-1 and 32Dc13 cell lines. A) Anti-Fes (#61) immunoblot of whole cell lysates prepared from 105 parental TF-1, TF-/SR{alpha}, TF-1/Fes or TF-1/FesR cells. B) Anti-Fes (#61) immunoblot of whole-cell lysates prepared from 32D/SR{alpha} (lanes 1 and 3-5) or 32D/FesR (lanes 2 and 6-8) cells. Cells in lanes 3-8 were grown in the presence of 100 ng/ml G-CSF (lanes 3 and 6), 0.5 nM GM-CSF (lanes 4 and 7), or 0.5 nM IL-3 (lanes 5 and 8) for four days prior to preparing the lysates.

 


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  		Figure 2. In vitro kinase activity of Fes in TF-1/FesR and TF-1/SR{alpha}cells. A) Autoradiograph of immune complex kinase assay. "IP" is immunoprecipitating antiserum #70, and "NRS" is normal rabbit serum. B) Anti-Fes immunoblot (antiserum #61) of the same immune complexes shown in panel A. C) Antiphosphotyrosine immunoblot (antibody 4G10) of the same immune complexes as in panel A.

 
We examined cytokine-induced tyrosine phosphorylation in TF-1, TF-1/SR{alpha} and TF-1/FesR cells. As we had observed previously, induction of tyrosine phosphorylation was maximal at 5 min post-stimulation (data not shown). Cells were factor-starved for 13-18 h, then stimulated with cytokines for 5 min. Immunoblots probed with antiphosphotyrosine antibodies, 4G10 (Fig. 3Go) or PY20 (data not shown), did not reveal any differences between the major species of tyrosine-phosphorylated proteins in these cells, although the phosphorylation of a number of proteins was clearly induced. There are some minor differences between the bands in the parental TF-1 cells (labeled "A") and in both of the transduced cell lines (labeled "B" and "C"). The transduced cells were age-matched in culture and were processed at the same time. TF-1 cells were processed on different days and had been in culture longer at the time of the experiment, which may account for the differences. TF-1 cells require serum to proliferate [35]; therefore, we examined cytokine-induced tyrosine phosphorylation in factor-and serum-starved cells, but again, did not observe any differences between TF-1/FesR and TF-1/SR{alpha} cells (data not shown).



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  		Figure 3. Cytokine-stimulated tyrosine phosphorylation in TF-1, TF-1/SR{alpha} and TF-1/FesR cells. Antiphosphotyrosine immunoblot (4G10) of cleared lysates from cells stimulated with indicated cytokines for 5 min. A is TF-1, B is TF-1/SR{alpha}, C is TF-1/FesR cells, IFN{alpha} is interferon-{alpha}, and EPO is erythropoietin.

 
The transduced TF-1 cell lines were plated in soft agar to assess the proliferative response to IL-3 and GM-CSF (Fig. 4Go). Two experiments are shown. In experiment #2, the level of transduction by the LXSN virus was low, and the cell population did not survive G418 selection. In both experiments, TF-1/Fes cells had a slight increase in the number of colonies formed in the presence of IL-3 compared to TF-1/LXSN cells; however, TF-1/FesR cells also showed an increase in the number of colonies compared to the control retrovirus. When the cells were plated in the presence of GM-CSF no significant difference in the number of colonies was observed. No colonies grew in the absence of growth factor (data not shown).



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  		Figure 4. Soft agar colony formation of transduced TF-1 cells. Cells were plated in triplicate in 0.5 ml Iscove's medium containing 20% FBS and 0.3% agar in the presence of cytokines, as indicated. The data are expressed as the mean number of colonies formed, with the standard deviation indicated by error bars. Two separate experiments, using independently transduced populations of cells, are shown. Two hundred cells per well were plated in Experiment #1, and 250 cells/well were plated in Experiment #2.

 
We also used the murine factor-dependent cell line, 32D, to examine the role of Fes kinase activity in cytokine-stimulated proliferation and differentiation. 32D cells were transduced with the ConFesR and SR{alpha}MSVtkNeo retroviruses, and the overexpression of the mutant Fes protein confirmed in the population of stably transduced cells (Fig. 1BGo, lanes 1 and 2). At the start of the experiment (day 0), 32D/SR{alpha} and 32D/FesR cells were washed twice in PBS and plated in GM-CSF, IL-3 or G-CSF. Cells were counted at 24 h intervals (Fig. 5Go), and cell cycle analysis was performed on days 0, 2 and 4 of the experiment (Fig. 6Go). In addition, the cells were cytospun onto glass slides, stained and evaluated for differentiation by nuclear morphology (Table 1Go). The level of expression of Fes proteins at the end of the experiment (day 4) was determined by immunoblot (Fig. 1BGo, lanes 3-8). The experiment was repeated several times; the data presented here are from a representative experiment.



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  		Figure 5. Growth of transduced 32D cell lines on GM-CSF, IL-3 and G-CSF. 32D/SR{alpha} and 32D/FesR cells were grown on GM-CSF, then washed twice in PBS and plated at 5 x 104 cells/ml in IMDM containing 10% FBS and 200 pM GM-CSF, 200 pM IL-3 or 100 ng/ml G-CSF, as indicated.

 


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  		Figure 6. Cell cycle analysis of transduced 32D cell lines. 32D/SR{alpha} and 32D/FesR cells were grown on GM-CSF, then washed in PBS and plated in cytokines as in Figure 5.Go. DNA content was analyzed on days 0,2 and 4 by propidium iodide. The percentages of each cell population in various phases of the cell cycle are shown.

 

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  		Table 1. Percentage of cells differentiated to neutrophils
 
We did not observe differences in the growth characteristics of 32D/SR{alpha} and 32D/FesR cells that were grown on GM-CSF, IL-3 or G-CSF (Fig. 5Go). The slight increase in cell number of 32D/FesR cells on days 3 and 4 of G-CSF treatment was not seen in subsequent experiments (data not shown). In an independent experiment, we looked at the growth of 32Dc13 cells overexpressing wild-type Fes protein, as well as 32D/SR{alpha} and 32D/FesR cells, and found that the 32D/Fes cells had similar growth characteristics (data not shown).

We examined the transduced 32Dc13 cells microscopically, using nuclear morphology as a marker of granulocytic differentiation. 32D/FesR and control cell populations exhibited similar amounts of differentiation (Table 1Go). Thirty-five percent of the cells maintained in G-CSF had differentiated to neutrophils by day 4. The level of spontaneous differentiation in GM-CSF and IL-3 cultures remained less than 5% (Table 1Go).

To determine the effect of Fes kinase activity on cell cycle regulation in 32D cells, we performed DNA analysis to quantitate cells in the G1, S and G2 + M phases (Fig. 6Go). Cells treated with G-CSF growth-arrest and differentiate (Fig. 5Go and Table 1Go). Thus, a greater percentage of the cells treated with G-CSF arrested in G1/G0 by day 2 of the experiment, compared to cells grown on GM-CSF and IL-3 (Fig. 6Go). As the cells on GM-CSF and IL-3 became more dense, they too began to die, and fewer cells were in mitosis by day 4. The distribution of cells in each cycle phase was similar for 32D/SR{alpha} and 32D/FesR cells.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Acknowledgements
 References
 
We used proliferation in liquid culture and soft agar, tyrosine phosphorylation, differentiation and cell cycle analysis to examine the effect of overexpressing a kinase-negative Fes protein in two factor-dependent myeloid cell lines, TF-1 and 32Dc13. Our results indicate that Fes kinase activity is not essential for the cytokin-mediated proliferation and/or differentiation of the TF-1 and 32Dc13 cell lines, although it is possible that the FesR protein is not acting as a dominant-negative mutant in this system.

Overexpression of the kinase-defective Fes mutant did not change the pattern of cytokine-stimulated tyrosine phosphorylation in TF-1 cells. Our work [36] and that of others [37] have demonstrated that tyrosine phosphorylation of the major protein species is not always correlated to proliferation as one might expect. Indeed, certain mutants of the alpha subunit of the GM-CSF receptor expressed in stable cell lines transduce a proliferative signal indistinguishable from wild-type, yet phosphorylation of protein on tyrosine residues is barely detectable [36]. It is also possible that substrates phosphorylated by Fes are not sufficiently abundant to be detected by antiphosphotyrosine antibodies in immunoblots of whole-cell lysates.

The biological role of a tyrosine kinase can be difficult to reveal. Several groups have used knockout mice to genetically search for function. This approach can yield a great deal of information, as seen in Fyn- [38] or Src-deficient [39] mice. However, the biological effects may manifest only in a subset of the cell types which express the protein [40, 41]. For example, when Greer et al. [4] generated transgenic mice expressing a Fes protein that was tethered to membranes, the mice developed hemangiomas, indicating a role for Fes in vascular endothelium. However, the mice had normal hematopoietic cells.

In some cases, homozygous deletion of a kinase has very subtle defects [42], and multiple knockouts are necessary for a more severe phenotype to be revealed [41, 43]. Thus, there may be redundancy in the cell, where one kinase can substitute for another when necessary. Alternatively, there may be accessory signaling pathways that the cell can utilize. This may be true for our studies, in that we are not able to see the biological effect, if any, of overexpressing the kinase-mutant Fes in myeloid cells because the cells are able to compensate for the blocked pathway.

The conflicting observations regarding the role of Fes in cytokine signal transduction may be a result of cellular variation, even when two laboratories are using the same cell line. For example, Glazer and colleagues saw a dramatic effect of expressing Fes in K562 cells—the cells began to differentiate into granulocytes [27]. This was shown by enzyme assays, cell adherence and induction of lineage-specific cell surface markers such as CD33 [26]. However, when we expressed our Fes constructs in K562 cells, we did not observe these effects. Upon analysis of our parental K562 cells, we found that they were already expressing CD33 (Yates and Gasson, unpublished observations). Therefore, there are differences in the starting population of cells that may be a result of years of growth in tissue culture. Thus, the most compelling evidence for Fes in cytokine signal transduction is that which comes from normal cells that have not been subjected to the stresses of tissue culture [17].


   Acknowledgements
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
References
 
Flow cytometry was performed by Negotia Neagos in the Jonsson Comprehensive Cancer Center Flow Cytometry Core Laboratory. We thank Anne Carlson and Kathleen Sakamoto for critical reading of the manuscript, and Wendy Aft for preparation of the manuscript.

This work was supported by a Shirley McKernan Cancer Research Endowment and NIH grants P01 CA32737-14 (a program project grant) and CA 16042 (the Jonsson Comprehensive Cancer Center core grant).


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 

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Received July 18, 1996; accepted for publication August 12, 1996.




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