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[Cancer Research 60, 1097-1103, February 15, 2000]
© 2000 American Association for Cancer Research

Molecular Biology and Genetics

The c-Fes Protein-Tyrosine Kinase Suppresses Cytokine-independent Outgrowth of Myeloid Leukemia Cells Induced by Bcr-Abl1

Jack M. Lionberger and Thomas E. Smithgall2

Eppley Institute for Research in Cancer and Department of Pathology and Microbiology University of Nebraska Medical Center, Omaha, Nebraska 68198 [J. M. L., T. E. S.], and Department of Molecular Genetics and Biochemistry University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261 [T. E. S.]


   ABSTRACT
Top
 ABSTRACT
Introduction
 Materials and methods
 Results
 DISCUSSION
 REFERENCES
 
The c-Fes protein-tyrosine kinase exhibits strong expression in myeloid hematopoietic cells. Previous studies have shown that Fes induces differentiation in the chronic myelogenous leukemia-derived cell line K-562, suggesting that the Fes signal for differentiation is dominant to the Bcr-Abl signal for transformation in these cells. In addition, Fes has been shown to associate with and phosphorylate Bcr on NH2-terminal sequences retained within Bcr-Abl. To determine whether Fes interacts directly with Bcr-Abl, kinase-inactive Bcr-Abl was coexpressed with Fes in 293T cells, and phosphorylation was assessed by anti-phosphotyrosine immunoblotting. Bcr-Abl was strongly phosphorylated by Fes under these conditions, suggestive of direct interaction. Similarly, tyrosine phosphorylation of kinase-inactive Fes was observed after coexpression with active Bcr-Abl. To test for the interaction of Fes with Bcr-Abl under physiological conditions, wild-type and kinase-defective Fes were stably expressed in the cytokine-dependent myeloid leukemia cell line, DAGM. Expression of either form of Fes alone did not affect the proliferation or interleukin 3 dependence of these cells. The DAGM/Fes cells were then infected with Bcr-Abl retroviruses, and their rates of cytokine-independent outgrowth were compared. Fes dramatically suppressed Bcr-Abl-induced DAGM cell outgrowth relative to a cell line expressing ß-galactosidase as a negative control. This effect required Fes tyrosine kinase activity, because the kinase-inactive form of Fes did not affect Bcr-Abl-induced cell outgrowth. The phosphotyrosine content of both wild-type and kinase-inactive Fes was strongly enhanced after coexpression with Bcr-Abl in DAGM cells, similar to the 293T result. Phosphorylation of wild-type Fes correlated with stimulation of Fes tyrosine kinase activity in the presence of Bcr-Abl. These results show that Fes and Bcr-Abl interact in myeloid cells, leading to Fes activation and suppression of Bcr-Abl-induced conversion to cytokine independence.


   Introduction
Top
 ABSTRACT
 Introduction
Materials and methods
 Results
 DISCUSSION
 REFERENCES
 
The Philadelphia chromosome translocation is associated with several types of human leukemias, most notably CML3 and one form of ALL (reviewed in Refs. 1 and 2 ). This translocation involves the c-abl locus on chromosome 9 and the bcr locus on chromosome 22, resulting in the expression of a family of chimeric Bcr-Abl oncoproteins (3, 4, 5, 6) . CML results from the Mr 210,000 form of Bcr-Abl (p210), whereas ALL is associated with the Mr 185,000 form (p185; Refs. 7 and 8 ). Fusion to NH2-terminal Bcr sequences leads to constitutive activation of the adjacent Abl tyrosine kinase, which is essential for the transforming function of the chimeric oncoprotein (9, 10, 11) . Both forms of Bcr-Abl have been shown to transform cells in culture and to produce CML- and ALL-like syndromes in transgenic mice, providing strong evidence that Bcr-Abl is responsible for the development of these leukemias (12, 13, 14) .

The human c-fes locus encodes a Mr 93,000 cytoplasmic protein-tyrosine kinase (Fes) that exhibits strong expression in hematopoietic cells of the myeloid lineage (reviewed in Ref. 15 ). Fes consists of a unique NH2-terminal region with coiled-coil homology domains, a central SH2 domain, and a COOH-terminal kinase domain. Fes is activated by multiple hematopoietic cytokines, suggesting that it participates in normal myeloid growth regulation (16, 17, 18, 19) . Introduction of Fes into the CML cell line K-562 induces terminal differentiation, indicating that the Fes signal for differentiation is dominant to the Bcr-Abl signal for transformation in this cell line (20, 21, 22) . These results implicate Fes in normal signaling events controlling myeloid differentiation and also suggest that Fes may act to regulate CML progression.

Previous work from our laboratory has identified the cellular Bcr protein as a binding partner and substrate for c-Fes (23, 24, 25) . In these studies, the major sites of Fes-mediated tyrosine phosphorylation were localized to NH2-terminal Bcr sequences retained in both forms of Bcr-Abl. These results suggest that Fes may also associate with Bcr-Abl and raise the possibility that the suppressive actions of Fes on K-562 cell growth may result from interactions with Bcr-Abl. In this report, we provide evidence that the NH2-terminal region of Bcr found in Bcr-Abl is sufficient for interaction with Fes, and that this interaction requires the Fes unique NH2-terminal domain. Coexpression of Fes and Bcr-Abl leads to potent reciprocal trans-phosphorylation. Fes was also observed to suppress Bcr-Abl-induced outgrowth of a cytokine-dependent myeloid leukemia cell line. Suppression correlated with strong phosphorylation of Fes by Bcr-Abl in vivo, suggesting that Bcr-Abl may act upstream to activate Fes, leading to the generation of signals for differentiation. These data suggest that Fes-induced differentiation may suppress CML progression as a result of direct interaction with Bcr-Abl.


   Materials and methods
Top
 ABSTRACT
 Introduction
 Materials and methods
Results
 DISCUSSION
 REFERENCES
 
Baculovirus Expression Constructs
Construction of baculovirus expression vectors for Bcr-Abl, Bcr, GST fusion proteins containing Bcr NH2-terminal amino acids 1–413 and 162–413, along with wild-type and mutant forms of Fes, have been described elsewhere (23, 24, 25, 26, 27, 28) .4 To create GST-Bcr 1–71, which contains the Bcr-Abl oligomerization domain (10) , the corresponding Bcr coding sequence was amplified by PCR and subcloned into the baculovirus transfer vector pVL-GST (27) . The resulting construct was used to generate a recombinant baculovirus using Baculogold DNA and the manufacturer’s protocol (PharMingen, San Diego, CA).

GST Fusion Protein Coprecipitation Assays
Sf-9 insect cells were coinfected with recombinant baculoviruses carrying GST-Bcr NH2-terminal fusion proteins and target Fes constructs. Forty-eight h later, infected cells were sonicated in 1.0 ml of lysis buffer (40 mM HEPES, pH 7.0, 150 mM NaCl, 5 mM EDTA, and 1% Triton X-100) supplemented with 25 µg/ml aprotinin, 50 µg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride, 20 mM NaF, 1 mM Na3VO4, and 50 µM Na2MoO4. The lysate was clarified by centrifugation; 20 µl of glutathione-agarose (50% slurry) were added, and the mixture was rotated for 1 h at 4°C. The beads were washed with three 1.0-ml aliquots of lysis buffer, and precipitated proteins were solubilized in SDS sample buffer and resolved by SDS-PAGE. Associated Fes proteins were visualized by immunoblotting with the anti-FLAG monoclonal antibody M2, which recognizes a COOH-terminal FLAG epitope on each of the Fes expression constructs. Duplicate gels were stained with Coomassie blue to verify that equivalent levels of the GST-Bcr fusion proteins were precipitated in each reaction.

Coexpression of Fes and Bcr-Abl in 293T Cells
Mammalian cell expression vectors for wild-type and kinase-defective Fes as well as Bcr have been described previously (23) . To construct expression vectors for p210 Bcr-Abl, cDNAs encoding kinase-active and -inactive forms of p210 Bcr-Abl were subcloned into the mammalian expression vector pCDNA3 (InVitrogen). Human 293T cells were transfected with each construct either alone or in various combinations using a calcium phosphate protocol described elsewhere (28) . Forty-eight h later, transfected cells were lysed directly in 1.0 ml of SDS sample buffer and resolved by SDS-PAGE. Expression of Fes, Bcr, and Bcr-Abl proteins as well as the extent of protein-tyrosine phosphorylation was determined by immunoblotting (24) .

Retroviral Constructs
Subcloning of wild-type and kinase-inactive Fes cDNAs into the retroviral expression vector pSR{alpha}MSVtkneo (9) has been described elsewhere (22 , 29) . The coding sequence for bacterial ß-galactosidase was subcloned from the commercial expression vector pCMVSport (Life Technologies, Inc.) into pSR{alpha}MSVtkneo. Similar pSR{alpha}MSVtkneo constructs containing the coding sequences for the p210 and p185 forms of Bcr-Abl were obtained from Dr. Owen Witte (University of California, Los Angeles, CA). To make retroviral stocks, subconfluent 100-mm dishes of 293T cells were transfected with 30 µg each of the retroviral constructs and an ecotropic packaging vector using the calcium phosphate method described elsewhere (22 , 29 , 30) . Viral supernatants were collected 48, 72, and 96 h after transfection, pooled, filtered with 0.45 µ filters, and stored at -80°C.

DAGM Cell Proliferation Assays
DAGM myeloid leukemia cells (31) were cultured in RPMI 1640 medium containing 10% fetal bovine serum, 50 µg/ml gentamicin, and 2.5 ng/ml IL-3. Cells were infected with ß-gal, Fes-WT, or Fes-KE retroviruses and selected with G-418 at 800 µg/ml. Expression of the Fes proteins was confirmed in the drug-resistant cell population by immunoblotting with the anti-FLAG monoclonal antibody. Fes and ß-gal control cells were then superinfected with p210 Bcr-Abl or ß-gal control retroviruses as follows. Cells (106) were resuspended in 5 ml of the viral supernatant in the presence of 4 µg/ml Polybrene. To enhance retroviral gene transfer, plates were centrifuged at 2400 rpm for 4 h at 20°C (32) . The virus was replaced with fresh medium containing G-418 and IL-3, and the cultures were incubated for 72 h. Cells were washed twice with 10 ml of RPMI 1640 and resuspended in complete medium plus G-418 at 4 x 105 cells/ml. For analysis of IL-3-independent outgrowth, cells were washed free of cytokine, and 4 x 104 cells were plated in 100 µl of complete medium in each well of a 96-well plate (six wells/time point). Plates were sealed with tape until analysis to prevent evaporation. To quantitate cell outgrowth, cells in each well were combined with 25 µl of MTT reagent (5 mg/ml in H2O) and incubated for 4 h at 37°C. The reaction was stopped by adding 100 µl of MTT lysis buffer (50% dimethylformamide in H2O containing 20% SDS, 2.5% glacial acetic acid, and 2.5% 1 N HCl, pH 4.7). Plates were resealed with tape and incubated overnight at 37°C, and absorbance of each well was read at 570 nm. Positive controls were analyzed in a similar manner in the presence of IL-3; however, only three wells were used per time point. For all plates, a well containing only culture medium and no cells served as a background control for the absorbance readings.

Analysis of Fes and Bcr-Abl Expression and Phosphorylation in DAGM Cells
For analysis of Fes tyrosine phosphorylation, aliquots of cells were washed twice with RPMI 1640 and replated in RPMI 1640 containing 1% FBS 12–18 h prior to lysis. Cells were pelleted, washed with PBS, and lysed in 1 ml of RIPA buffer [50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 1 mM EDTA, and 1% sodium deoxycholate]. Fes or Bcr-Abl proteins were immunoprecipitated from clarified cell lysates using 15 µl of anti-FLAG monoclonal antibody resin (Sigma) or 1 µg of anti-Abl antibody (K-12; Santa Cruz Biotechnology) and 15 µl of protein G-Sepharose (50% slurry; Pharmacia), respectively. Immunoprecipitates were washed with RIPA buffer, resolved by SDS-PAGE, and immunoblotted for Fes with the anti-FLAG monoclonal antibody M2, for Bcr-Abl with the anti-Abl monoclonal antibody 8E9, or for phosphotyrosine with the anti-phosphotyrosine monoclonal antibody, PY99 (Santa Cruz).

For in vitro kinase assays, the cells were lysed in NP40 buffer [40 mM Tris-HCl (pH 7.4), 120 mM NaCl, 5 mM EDTA, 2 mM EGTA, and 1% NP40] supplemented with 25 µg/ml aprotinin, 50 µg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride, 20 mM NaF, 1 mM Na3VO4, and 50 µM Na2MoO4. Fes was immunoprecipitated from clarified cell lysates with 15 µl of M2 antibody resin, washed twice with RIPA buffer, twice with kinase buffer [50 mM HEPES (pH 7.4), 10 mM MgCl2], and incubated with 10 µCi [{gamma}-32P]ATP for 10 min at 30°C. Reactions were stopped by heating in SDS sample buffer, resolved by SDS-PAGE, transferred to polyvinylidene difluoride membranes, and incorporation of 32P was quantitated by storage phosphor technology (Molecular Dynamics Phosphorimager). Fes proteins were visualized on the same membrane with the M2 anti-FLAG antibody, and relative protein levels were quantitated by laser densitometry to allow normalization of 32P incorporation.


   Results
Top
 ABSTRACT
 Introduction
 Materials and methods
 Results
DISCUSSION
 REFERENCES
 
The Fes NH2-terminal region binds to the Bcr-derived sequence of Bcr-Abl
Previous studies from our laboratory have demonstrated that Fes associates with the normal full-length Bcr protein (24 , 25) . To determine whether the NH2-terminal region of Bcr retained in the various forms of Bcr-Abl is sufficient for interaction with Fes, we coexpressed wild-type and mutant forms of Fes with a GST-Bcr fusion protein containing Bcr NH2-terminal amino acids 1–413 in Sf-9 cells. The structures of the Fes and GST-Bcr proteins are shown in Fig. 1Citation . The GST-Bcr fusion protein was precipitated from the cells with glutathione-agarose beads, and associated Fes proteins were detected by immunoblotting. As shown in Fig. 2Citation , GST-Bcr 1–413 readily bound to wild-type Fes, indicating that the Bcr 1–413 region is sufficient to recognize the Fes protein. A kinase-inactive form of Fes (Fes-KE) was also strongly bound, indicating that Fes-Bcr interaction does not require Fes tyrosine autophosphorylation. Three deletion mutants of Fes were used to identify the domain of Fes responsible for interaction with Bcr: {Delta}SH2, which lacks the central SH2 domain; {Delta}N, which lacks the unique NH2-terminal region; and {Delta}K, which lacks the COOH-terminal kinase domain (Fig. 1)Citation . Fig. 2Citation shows that although both {Delta}SH2 and {Delta}K were strongly coprecipitated, {Delta}N was unable to bind to GST-Bcr 1–413. These data indicate that the Bcr 1–413 region interacts with Fes through its unique NH2-terminal domain.



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  		Fig. 1. Fes and Bcr expression constructs used in this study. Wild-type Fes is shown at the top and consists of 822 amino acids comprising a unique NH2-terminal domain (amino acids 1–450), an SH2 domain (amino acids 451–540), and a COOH-terminal kinase domain. The Fes-KE mutant contains a Glu substitution for the conserved Lys in the ATP binding pocket. The {Delta}N, {Delta}SH2, and {Delta}K mutants lack the NH2-terminal, SH2, or kinase domains, respectively. All Fes expression constructs are fused to the eight-amino acid FLAG epitope tag at their COOH termini. The cellular Bcr gene encodes a Mr 160,000 protein with an NH2-terminal Ser/Thr kinase domain (Kin), a central region with homology to the Dbl family of guanine nucleotide exchange factors (Dbl), an adjacent pleckstrin homology domain (PH), and a COOH-terminal GAP domain for Rac (GAP). In this study, the NH2-terminal Ser kinase domain (amino acids 1–413) was expressed as a fusion protein with GST (GST-Bcr 1–413). Smaller fusion proteins containing portions of the Bcr NH2-terminal region were also expressed as GST fusions. These include GST-Bcr 1–71, which contains the NH2-terminal coiled-coil oligomerization domain, and GST-Bcr 162–413, which includes several tyrosine residues that are phosphorylated by Fes.

 


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  		Fig. 2. GST-Bcr 1–413 binds Fes through its unique NH2-terminal region. A, GST-Bcr 1–413 was coexpressed with wild-type, full-length Fes (WT), kinase-inactive Fes (KE), or deletion mutants lacking the NH2-terminal unique domain ({Delta}N), SH2 domain ({Delta}SH2), or COOH-terminal kinase domain ({Delta}K) in Sf-9 cells. Protein complexes were precipitated from clarified cell lysates with GSH-agarose and washed, and associated Fes proteins were visualized by immunoblotting. B, expression levels of the Fes proteins were determined by immunoblotting aliquots of the whole-cell lysates. Arrows (right), positions of the Fes proteins.

 
The Bcr-Abl coiled-coil oligomerization domain is not sufficient for interaction with Fes
Previous studies have shown that the active form of Bcr-Abl is oligomeric (10 , 33) . Oligomerization of Bcr-Abl requires a coiled-coil domain encoded by the first 63 amino acids of the Bcr-derived portion of the protein (10) . Recent studies from our laboratory have identified at least two coiled-coil forming motifs in the NH2-terminal region of c-Fes, which appear to mediate Fes oligomerization and regulate its kinase activity (22 , 27) . These findings suggest that interaction of Fes with Bcr and Bcr-Abl may involve interaction of their coiled-coil domains. To test this possibility, two additional GST-Bcr fusion proteins were constructed: one encodes the first 71 amino acids of Bcr and encompasses the coiled-coil oligomerization domain, whereas the second encodes Bcr amino acids 162–413, a region that includes the major sites of Fes-induced Bcr tyrosine phosphorylation (23 , 25) . These Bcr GST fusion proteins, as well as GST-Bcr 1–413, were coexpressed with full-length Fes in Sf-9 cells. The resulting protein complexes were precipitated with glutathione-agarose, and associated Fes was visualized by immunoblotting. As shown in Fig. 3Citation , the GST-Bcr 1–71 fusion protein did not coprecipitate Fes, suggesting that the NH2-terminal oligomerization domain of Bcr-Abl is not sufficient for Fes binding in this system. However, GST-Bcr 1–413 and 162–413 both formed stable complexes with Fes, localizing at least one binding domain to Bcr residues 162–413.



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  		Fig. 3. Identification of Bcr NH2-terminal sequences mediating interaction with Fes. Top, GST-Bcr fusion proteins containing the entire Bcr kinase domain (amino acids 1–413), the coiled-coil domain (amino acids 1–71), or the region of Fes tyrosine phosphorylation (amino acids 162–413) were coexpressed with full-length wild-type Fes in Sf-9 cells. Fes was coexpressed with GST as a negative control. Protein complexes were precipitated with GSH-agarose and washed, and associated Fes proteins were visualized by immunoblotting. Middle, a duplicate gel of the precipitated proteins was stained with Coomassie blue to visualize GST and the GST-Bcr fusion proteins. Arrows, positions of GST and the GST-Bcr fusion proteins. Bottom, presence of Fes in each lysate was verified by immunoblotting.

 
To ensure that GST-Bcr 1–71 was functional, we coexpressed this as well as the other GST-Bcr fusion proteins with either full-length Bcr or p185 Bcr-Abl. As shown in Fig. 4Citation , both Bcr and Bcr-Abl readily coprecipitated with Bcr 1–71, providing a positive control for the Fes binding experiments above. In addition, the Bcr 1–413 and to a lesser extent the 162–413 fusion proteins bound to both Bcr and Bcr-Abl. Association of Bcr and Bcr-Abl with the GST-Bcr 162–413 fusion protein was unexpected, because this fragment lacks the coiled-coil domain. This result suggests that the 162–413 region may contribute to Bcr and Bcr-Abl oligomerization in addition to association with Fes.



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  		Fig. 4. The GST-Bcr 1–71 fusion protein binds to Bcr and Bcr-Abl. Top, GST alone or GST-Bcr fusion proteins containing the entire Bcr kinase domain (amino acids 1–413), the coiled-coil domain (amino acids 1–71), or the region of Fes tyrosine phosphorylation (amino acids 162–413) were coexpressed with Bcr (left four lanes) or p185 Bcr-Abl (right four lanes) in Sf-9 cells. Protein complexes were precipitated with GSH-agarose and washed, and associated Bcr or Bcr-Abl proteins were visualized by immunoblotting. Middle, a duplicate gel of the precipitated proteins was stained with Coomassie blue to visualize GST and the GST-Bcr fusion proteins. Arrows, positions of GST and the GST-Bcr fusion proteins. Bottom, presence of Bcr and Bcr-Abl in each lysate was verified by immunoblotting.

 
Reciprocal Trans-phosphorylation of Fes and p210 Bcr-Abl
Previous studies from our laboratory have shown that Bcr is a substrate for the Fes protein both in vitro and in vivo (23, 24, 25) . Phosphorylation of Bcr occurs on NH2-terminal tyrosine residues that are retained in Bcr-Abl, suggesting that Bcr-Abl may be a target for phosphorylation by Fes as well. To investigate the possibility that Bcr-Abl is a substrate for Fes, both kinases were expressed in the human cell line, 293T. To distinguish trans-phosphorylation of Bcr-Abl by Fes from Bcr-Abl autophosphorylation, a kinase-inactive form of Bcr-Abl was used (p210-KR). As shown in Fig. 5Citation , coexpression with wild-type Fes induced potent tyrosine phosphorylation of kinase-inactive Bcr-Abl, as evaluated by anti-phosphotyrosine immunoblotting. The extent of Fes-induced trans-phosphorylation of Bcr-Abl is equivalent to that observed with active Bcr-Abl expressed alone (Fig. 5Citation , compare Lanes 4 and 8). In a reciprocal experiment, the active form of p210 Bcr-Abl was coexpressed with kinase-inactive Fes (Fes-KE). Bcr-Abl phosphorylated Fes-KE to the same extent observed after expression of active Fes alone (Fig. 5Citation , compare Lanes 1 and 7). As a positive control, Fes was also coexpressed with normal Bcr. As reported previously, Bcr was strongly phosphorylated by Fes (23) . Control blots show that Bcr and the kinase-inactive mutants of Fes and Bcr-Abl are not detected by the anti-phosphotyrosine antibody, and that all proteins are expressed at comparable levels. Taken together, these data demonstrate potent, reciprocal trans-phosphorylation between p210 Bcr-Abl and Fes. The effects of phosphorylation of Bcr-Abl by Fes are currently unknown but could modulate Bcr-Abl signal transduction (see "Discussion"). Conversely, direct phosphorylation of Fes by Bcr-Abl may serve to activate Fes; this idea is supported by experiments described in the next section.



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  		Fig. 5. Reciprocal trans-phosphorylation of p210 Bcr-Abl and Fes. Wild-type Fes (Fes-WT), kinase-inactive Fes (Fes-KE), Bcr, kinase-active p210 Bcr-Abl (p210-WT), and kinase-inactive p210 Bcr-Abl (p210-KR) were expressed individually or in the combinations shown in 293T cells. Cells lysates were prepared directly in SDS sample buffer and resolved by SDS-PAGE. Tyrosine phosphorylation was assessed with anti-phosphotyrosine antibodies (P-Tyr; top), anti-FLAG antibodies that recognize Fes (Fes; middle), and anti-Bcr antibodies that recognize both Bcr and Bcr-Abl (Bcr, bottom). Arrows (right), positions of the Fes, Bcr, and p210 Bcr-Abl proteins.

 
Fes suppresses Bcr-Abl-induced transformation of DAGM cells to cytokine independence
Previous studies have shown that expression of Fes in the CML-derived cell line K-562 leads to growth suppression and terminal differentiation (20, 21, 22) . Previous data from our laboratory (23, 24, 25) as well as data presented above demonstrate that Fes has the potential to interact directly with Bcr-Abl, which may contribute to this suppressive action of Fes in K-562 cells. To determine whether Fes can affect Bcr-Abl signaling in a defined system, we used the cytokine-dependent myeloid leukemia cell line DAGM. Previous studies have shown that expression of the p210 form of Bcr-Abl induces cytokine independence in this cell line, providing a useful model of transformation by Bcr-Abl (31) .

DAGM cells were infected with recombinant retroviruses carrying wild-type Fes, the kinase-inactive Fes mutant (Fes-KE), or ß-gal as a negative control. The cells were then selected with G-418 in the presence of IL-3 for 2 weeks. Immunoblots were performed to verify expression of the Fes proteins (Fig. 6)Citation . Expression of either form of Fes did not alter the growth response of the cells to IL-3 (data not shown). Each of the cell lines was then superinfected with a retrovirus carrying the p210 form of Bcr-Abl, and viable cell outgrowth followed in the absence of cytokine. As shown in Fig. 6Citation , the presence of Fes strongly suppressed Bcr-Abl-induced cell outgrowth relative to the ß-gal control. This difference was most striking on day 8 of the experiment. At this time point, Bcr-Abl-induced control cell outgrowth had saturated the assay, whereas Bcr-Abl-induced growth of the DAGM/Fes cells was barely detectable. The suppressive effect required the kinase activity of Fes, because cells expressing the kinase-inactive Fes mutant grew out at the same rate as the ß-gal control population following introduction of Bcr-Abl. All of the cells grew out at the same rapid rate when incubated in the presence IL-3, indicating that the effect of Fes is specific to transformation by Bcr-Abl and not a general effect on cell proliferation (data not shown). These data indicate that Fes can suppress Bcr-Abl signals for cytokine independence in DAGM cells.



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  		Fig. 6. Suppression of p210 Bcr-Abl-mediated cytokine-independent outgrowth of DAGM cells by Fes. DAGM cells stably expressing wild-type Fes (Fes-WT), kinase-defective Fes (Fes-KE), or ß-gal as a negative control were infected with a recombinant p210 Bcr-Abl retrovirus. After incubation for 3 days in the presence of IL-3 and G-418, cytokine was removed, and 4 x 104 cells were plated in each well of a 96-well plate in 100 µl of growth medium plus G-418. Six replicate wells were plated for each group at each time point. Relative cell viability was determined on the days indicated using the MTT reduction assay described in "Materials and Methods." Absorbance values at 570 nm for each time point were averaged and are plotted; bars, SD. Comparable results were obtained from two independent experiments. {blacktriangleup}, Fes-WT; {blacksquare}, Fes-KE; •, ß-gal. Inset, expression of the Fes proteins was verified by immunoblotting in each of the DAGM cell populations prior to infection with the p210 Bcr-Abl retrovirus.

 
Bcr-Abl enhances Fes tyrosine phosphorylation and kinase activity in DAGM cells
To determine whether the suppressive action of c-Fes on the rate of Bcr-Abl-mediated DAGM cell outgrowth correlated with changes in Fes tyrosine kinase activity, we investigated Fes tyrosine phosphorylation in vivo and kinase activity in vitro. DAGM cells coexpressing Fes and either the p210 or the p185 forms of Bcr-Abl or Fes alone were serum and cytokine starved overnight. Fes was then immunoprecipitated and probed for phosphotyrosine content on anti-phosphotyrosine immunoblots. As shown in Fig. 7Citation , the phosphotyrosine content of Fes was increased in the presence of Bcr-Abl. To determine whether Fes is directly phosphorylated by Bcr-Abl in DAGM cells, similar experiments were performed on the cell populations coexpressing Bcr-Abl and the kinase-defective form of Fes. Fig. 7Citation shows that kinase-inactive Fes was also strongly tyrosine phosphorylated in cells coexpressing Bcr-Abl, whereas no phosphorylation was observed in the control cell line. This result strongly suggests that Fes is a substrate for Bcr-Abl in DAGM cells and is consistent with the coexpression data from 293T cells presented above (Fig. 5)Citation .



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  		Fig. 7. Enhanced Fes phosphotyrosine content in DAGM myeloid leukemia cells expressing Bcr-Abl. DAGM cells stably expressing wild-type Fes (Fes-WT; top two blots) or kinase-inactive Fes (Fes-KE; bottom two blots) were infected with recombinant retroviruses carrying the p185 or p210 forms of Bcr-Abl. Uninfected cells served as a negative control (Con). Fes proteins were immunoprecipitated from clarified cell lysates and blotted with anti-phosphotyrosine antibodies (P-Tyr) or with the M2 anti-FLAG antibody, which recognizes the Fes protein (Fes). Arrows (right), positions of the Fes proteins. Comparable results were obtained in two independent experiments; a representative example is shown.

 
To investigate the effect of Bcr-Abl on Fes kinase activity, Fes immunoprecipitates from DAGM cells were incubated in vitro with [{gamma}-32P]ATP, and the extent of autophosphorylation was assessed by storage phosphor imaging. As shown in Fig. 8Citation , Fes autophosphorylation increased from 5- to >15-fold after isolation from DAGM cells expressing p185 Bcr-Abl and from 3- to 7-fold in immunoprecipitates from cells expressing the p210 form of Bcr-Abl, relative to control cells expressing Fes alone. These results support a mechanism in which tyrosine phosphorylation of Fes by Bcr-Abl leads to Fes activation and the generation of downstream signals for differentiation (see "Discussion").



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  		Fig. 8. Coexpression of Fes with Bcr-Abl in DAGM cells enhances Fes kinase activity in vitro. DAGM cells stably expressing wild-type Fes were infected with recombinant retroviruses carrying the p185 or p210 forms of Bcr-Abl. Uninfected cells served as a negative control (Con). Fes proteins were immunoprecipitated from clarified cell lysates, and kinase activity was assayed in vitro by addition of [{gamma}-32P]ATP. Phosphorylated Fes proteins were resolved by SDS-PAGE, transferred to polyvinylidene difluoride membranes, and visualized by storage phosphor imaging (top). The membrane was then probed with the M2 anti-FLAG antibody to determine the level of Fes in each immunoprecipitate (middle). Levels of 32P-incorporation were corrected to Fes protein levels (estimated by two-dimensional densitometry) and are plotted as the fold-increase relative to control cells (bottom). Results for two independent experiments are shown.

 

   DISCUSSION
Top
 ABSTRACT
 Introduction
 Materials and methods
 Results
 DISCUSSION
REFERENCES
 
Experiments reported here provide new evidence for the interaction of Bcr-Abl with the Fes tyrosine kinase at the molecular and cellular levels. At the molecular level, Fes was found to interact with NH2-terminal Bcr sequences found in both forms of Bcr-Abl. This interaction was shown to require the NH2-terminal region of Fes, a region previously shown to be sufficient for interaction with full-length Bcr (24) . The NH2-terminal region of Fes is known to contain regions with homology to coiled-coil forming domains, which mediate Fes oligomerization in vitro and may contribute to the regulation of Fes kinase activity in vivo (22 , 27) . Interestingly, both Bcr and Bcr-Abl are known to form tetramers via Bcr-encoded NH2-terminal coiled-coil domains (10) . However, data presented here suggest that the Bcr-encoded tetramerization domain alone is not sufficient to mediate interaction with Fes. Instead, interaction appears to involve sequences encompassing Bcr NH2-terminal residues 162–413, a region that also contains several tyrosine phosphorylation sites for Fes both in vitro and in vivo (23 , 25) . However, the strongest interaction is observed when the full-length NH2-terminal region is used in the binding assay (amino acids 1–413). Thus, the Bcr coiled-coil domain may contribute indirectly to Fes interaction.

In addition to evidence for physical interaction between Bcr-Abl and Fes, we also observed that Fes and Bcr-Abl can phosphorylate one another in a heterologous expression system (293T cells). By coexpressing kinase-defective mutants of each protein with the corresponding wild-type partner, we observed very strong reciprocal tyrosine phosphorylation. The most likely mechanism for the increased tyrosine phosphorylation of Fes in this defined system is direct phosphorylation by the Bcr-Abl tyrosine kinase domain. Direct tyrosine phosphorylation of Fes by Bcr-Abl at the major site of Fes autophosphorylation (Tyr-713) is predicted to stimulate Fes kinase activity. Mutagenesis studies have demonstrated that this consensus activation loop tyrosine residue is required for full Fes kinase activity both in vitro and in vivo (28 , 34) . In addition, we have observed that coexpression with normal Bcr stimulates Fes autophosphorylation in vivo, possibly by binding to the Fes NH2-terminal region and disrupting negative regulatory interactions between the coiled-coil domains (23) . Bcr-Abl may activate Fes kinase activity by this mechanism as well. Our results are consistent with a previous study demonstrating an increase in endogenous Fes tyrosine phosphorylation in v-Abl- and Bcr-Abl-transformed cells (35) and provide the first evidence that this increase may be the result of a direct interaction between the two kinases.

Previous studies from our laboratory have shown that normal Bcr is a substrate for the Fes tyrosine kinase (23, 24, 25) . Tryptic phosphopeptide mapping and in vitro SH2 domain binding experiments showed that Fes phosphorylates Bcr on some tyrosines that are also phosphorylated within Bcr-Abl (36) and others that are unique to Fes (23) . These data suggest that Fes may phosphorylate a similar pattern of tyrosines within Bcr-Abl. Fes-mediated phosphorylation of Bcr-Abl on unique sites may alter the recruitment of effector proteins to Bcr-Abl and influence downstream signaling. Bcr-Abl is known to interact with a variety of molecules with phosphotyrosine-binding SH2 and PTB domains, including Grb2 (37 , 38) , Shc (31) , CrkL (39) , Stat factors (40 , 41) , and phosphatidylinositol 3-kinase (42) . Phosphorylation of alternative Tyr residues could influence recruitment of negative regulatory molecules such as SH2-containing protein-tyrosine phosphatases or the Ras GTPase activating protein. Along these lines, a recent study has shown that PTP-1B can suppress transformation signaling by Bcr-Abl and induce differentiation in K-562 cells (43) . Such a mechanism may explain the negative effect of Fes on Bcr-Abl signaling in DAGM cells (see below).

In K-562 cells (44) , which are derived from the blast-crisis phase of CML and express no detectable Fes RNA or protein (20 , 45 , 46) , the introduction of wild-type Fes is associated with reduced proliferative capacity and terminal differentiation (20) . These earlier studies provided the first evidence that Fes may suppress Bcr-Abl transformation signaling in addition to generating a signal for differentiation. Experiments with DAGM cells presented here are consistent with these earlier observations and extend them in several important ways: (a) they provide evidence that Fes can directly interfere with Bcr-Abl signals for transformation in a defined system. Fes was observed to suppress the ability of Bcr-Abl to induce cytokine independence in DAGM cells but had no effect on IL-3 induced outgrowth. This result argues that the effects of Fes are specific for Bcr-Abl; (b) the kinase activity of Fes was shown to be required for the growth-suppressive effect. This result suggests that either phosphorylation of Bcr-Abl by Fes or phosphorylation of other Fes substrates (or both) is required for the growth-suppressive actions observed; and (c) both wild-type and kinase-inactive Fes proteins showed enhanced phosphotyrosine content after coexpression with Bcr-Abl in DAGM cells, consistent with observations in 293T cells that Fes is a direct target for Bcr-Abl in vivo. Enhanced tyrosine phosphorylation of wild-type Fes correlated with increased kinase activity in vitro, suggesting that Bcr-Abl may provide an upstream activating signal for Fes in this system. After activation, Fes may generate signals for differentiation that are dominant to Bcr-Abl signals for cytokine-independent survival. This model is consistent with the changes seen after expression of Fes in K-562 cells and provides a possible mechanism to explain the differentiation observed in granulocytes that are overproduced during the chronic phase of CML. Several other studies have documented that Bcr-Abl can activate differentiation pathways in various model systems including PC-12 cells (47) and M1 myeloid leukemia cells (48) . In addition, we observed that Bcr-Abl-dependent DAGM cells coexpressing wild-type Fes changed their morphology and became adherent to the culture dish, whereas the cytokine-dependent c-Fes cells did not (data not shown). Thus, Fes and Bcr-Abl may cooperate to generate a signal for differentiation in myeloid progenitors.

In summary, data presented here demonstrate a direct physical interaction between Fes and Bcr-Abl, leading to reciprocal trans-phosphorylation on tyrosine residues. Fes activation was observed in a physiologically relevant cell line after coexpression with Bcr-Abl, and activation correlated with delayed Bcr-Abl-dependent outgrowth in the absence of cytokine. These data support a model in which activation of Fes by Bcr-Abl in myeloid progenitors may activate a differentiation pathway that serves to attenuate the tumorigenic capacity of Bcr-Abl in CML. Furthermore, phosphorylation of Bcr-Abl by Fes may affect its signaling properties directly. Future studies will address these possibilities.


   ACKNOWLEDGMENTS
 
We thank Dr. Owen Witte, Howard Hughes Medical Institute, University of California, Los Angeles, for Bcr-Abl cDNA clones, retroviral vectors, and the DAGM cell line, and Dr. Jean Wang, University of California, San Diego, for the anti-Abl antibody, 8E9.


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

1 Supported by NIH Grant CA58667 and ACS Grant RPG-96-052-04-TBE (to T. E. S.). Back

2 To whom requests for reprints should be addressed, at Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, E1240 Biomedical Science Tower, Pittsburgh, PA 15261. Phone: (412) 648-9495; Fax: (412) 624-1401; E-mail: tsmithga{at}pitt.edu Back

3 The abbreviations used are: CML, chronic myelogenous leukemia; ALL, acute lymphocytic leukemia; GST, glutathione S-transferase; ß-gal, ß-galactosidase; IL, interleukin; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; SH2, Src homology 2. Back

4 J. A. Rogers, N. A. Dunham, and T. E. Smithgall. SH2 domain substitution modulates the kinase and transforming activities of the c-Fes protein-tyrosine kinase, submitted for publication. Back

Received 10/ 8/99. Accepted 12/ 9/99.


   REFERENCES
Top
 ABSTRACT
 Introduction
 Materials and methods
 Results
 DISCUSSION
 REFERENCES
 

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