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Cell Growth & Differentiation Vol. 11, 581-592, November 2000
© 2000 American Association for Cancer Research


Articles

Src Homology 2 Domain Substitution Modulates the Kinase and Transforming Activities of the Fes Protein-Tyrosine Kinase1

Jim A. Rogers2, 3, Haiyun Y. Cheng2 and Thomas E. Smithgall4

Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261

Abstract

The c-fes proto-oncogene encodes a Mr 93,000 protein-tyrosine kinase (Fes) that is strongly expressed in myeloid cells and has been implicated in myelomonocytic differentiation. Fes autophosphorylation and transforming activity are highly restrained after ectopic expression in fibroblasts, indicating tight negative regulation of Fes kinase activity in vivo. Here we investigated the regulatory role of the Fes Src homology 2 (SH2) domain by producing a series of chimeric constructs in which the Fes SH2 domain was replaced with those of the transforming oncogenes v-Fps and v-Src or by the NH2-terminal SH2 domain of the Ras GTPase-activating protein. Wild-type and chimeric Fes proteins readily underwent tyrosine autophosphorylation in vitro and produced identical cyanogen bromide phosphopeptide cleavage patterns, indicating that the SH2 substitutions did not influence overall kinase activity or autophosphorylation site selection. However, metabolic labeling of Rat-2 fibroblasts expressing each construct showed that only the Fes/Src SH2 chimera was active in vivo. Consistent with this result, the Fes/Src SH2 domain chimera exhibited potent transforming activity in fibroblasts and enhanced differentiation-inducing activity in K-562 myeloid leukemia cells. In addition, the Fes/Src SH2 chimera exhibited constitutive localization to focal adhesions in Rat-2 fibroblasts and induced the attachment and spreading of TF-1 myeloid cells. These data demonstrate a central role for the SH2 domain in the regulation of Fes kinase activity and biological function in vivo.

Introduction

The human c-fes proto-oncogene encodes a Mr 93,000 cytoplasmic protein-tyrosine kinase (Fes) that is expressed strongly in mature myeloid hematopoietic cells [reviewed by Smithgall et al. (1) ]. This pattern of expression implicates Fes in the regulation of myeloid growth and differentiation, a hypothesis supported by several lines of evidence:

(a) The differentiation responsiveness of myeloid leukemia cell lines correlates with Fes expression (2, 3, 4, 5) . One myeloid progenitor cell line, K-562, completely lacks Fes expression and is resistant to most myeloid differentiation inducers (6) . However, K-562 cells transfected with c-fes terminally differentiate to macrophage-like cells in a CSF5 -independent manner, demonstrating that Fes is sufficient for differentiation in this cellular context (7 , 8) .

(b) Fes has been implicated in signal transduction for a number of hematopoietic cytokines. Fes associates with and is activated by the receptors for GM-CSF, erythropoietin, and interleukins 3, 4, and 6 (9, 10, 11, 12, 13) . Macrophages from mice with a targeted inactivating mutation in the c-fes locus are unable to activate Stat3 in response to GM-CSF treatment, providing genetic evidence that Fes is required for cytokine signaling in some cell types (14) .

(c) Suppression of Fes expression in myeloid leukemia cell lines with antisense oligonucleotides blocks differentiation and induces apoptosis in some cases (15 , 16) . These results not only demonstrate a requirement for Fes in the differentiation response but also implicate Fes in antiapoptotic signaling, an important function of cytokines.

More recent work has demonstrated Fes expression in extrahematopoietic sites including the vascular endothelium and neurons of adults (17) . Fes expression is even more widespread in embryonic tissues, occurring in cells derived from all three germ layers (17 , 18) . Early yolk sac blood islands were found to be an area of strong Fes expression in the embryo, which correlates with the observation that transgenic mice expressing a membrane-targeted form of Fes develop vascular tumors (19) . Fes has also been linked to FGF-2-induced chemotaxis and tube formation of capillary endothelial cells (20) . These results point to an important function for Fes in angiogenesis.

Despite its multiple biological functions, the mechanism of Fes kinase regulation is not well understood. In vitro studies have provided evidence that the noncatalytic domains of Fes, which include a large, unique NH2-terminal region and a central SH2 domain, both contribute to the regulation of Fes kinase activity (1) . Recent work shows that the active form of Fes is oligomeric, and that the NH2-terminal region is required for oligomerization (21, 22, 23) . The NH2-terminal region contains two motifs with strong homology to the heptad repeats associated with coiled-coil oligomerization domains. Intermolecular interaction of the coiled-coil domains may mediate Fes oligomerization, leading to autophosphorylation by a trans mechanism. Autophosphorylation of Tyr-713 in the kinase domain is required for full kinase activity in vitro and thus represents an important step in the activation mechanism (8 , 21 , 24) .

Other work suggests that the SH2 domain may also contribute to the regulation of Fes tyrosine kinase activity. Deletion of the SH2 domain from either Fes or its avian transforming homologue v-Fps substantially reduces kinase activity in vitro, suggesting that an intact SH2 domain is required for full kinase activity (24 , 25) . Although these results indicate that the Fes SH2 domain is involved in regulating the kinase domain, it may also contribute to interaction with substrates and influence subcellular localization (26, 27, 28) . Therefore, SH2 mutations may interfere with Fes signaling by a variety of mechanisms.

In this report, we describe novel constructs in which the SH2 domain of Fes was substituted with the SH2 domains of v-Fps, v-Src, or Ras Gap. All of the resulting SH2 chimeras retained kinase activity in vitro, allowing us to investigate the role of SH2 specificity on signaling in vivo independently of effects on intrinsic kinase activity. We found that Src SH2 domain substitution strongly up-regulated Fes kinase activity in fibroblasts, leading to oncogenic transformation. The Fes/Src chimera also demonstrated enhanced differentiation-inducing activity in myeloid leukemia cells, suggesting that Src SH2 substitution does not interfere with physiological function. Using GFP-Fes fusion proteins, we observed that the Fes/Src SH2 chimera relocalized from the cytoplasm to focal adhesions, consistent with earlier reports that Fes activation is associated with this subcellular compartment (27 , 28) . The chimera induced attachment and spreading of TF-1 myeloid leukemia cells and localized to focal sites in these cells as well. These results provide new evidence that the SH2 domain plays a critical role in the regulation of Fes tyrosine kinase activity and biological function in vivo.

Results

Construction of the Fes SH2 Domain Chimeras.
Previous studies have shown that Fes tyrosine kinase activity is tightly regulated in fibroblasts. As a result, Fes has been shown to exhibit little or no transforming activity in this cell type (29 , 30) . To evaluate the role of the SH2 domain in kinase regulation, we replaced the SH2 domain of wild-type Fes with the SH2 domains of two different transforming kinases (v-Fps and v-Src) using a PCR-based approach. The SH2 sequence of the avian oncogenic tyrosine kinase v-Fps is very similar to that of human Fes, with only four amino acid substitutions. The v-Src SH2 domain shows a greater divergence from the Fes SH2 at the amino acid level. However, phosphopeptide library screening experiments suggest that the Fes and Src SH2 domains select tyrosine phosphorylated binding partners with very similar sequences, although the Src SH2 may interact with these target sequences more strongly (see "Discussion"). An additional chimera was constructed using the NH2-terminal SH2 domain of p120 Ras Gap; this SH2 domain has been shown previously to release the tyrosine kinase and transforming activities of c-Abl (31) . Like Fes, wild-type c-Abl is nontransforming in fibroblasts and exhibits restrained tyrosine kinase activity. Unlike the other two chimeras, substitution with the Gap SH2 domain was expected to change the profile of interacting partners for Fes. The structures of the Fes/SH2 domain chimeras used in this study are shown in Fig. 1Citation .



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Fig. 1. Structures of Fes constructs used in this study. A, the structure of wild-type human Fes is shown at the top, which includes a unique NH2-terminal region, an SH2 domain, and a COOH-terminal kinase domain. {Delta}SH2, a Fes mutant lacking the SH2 domain (amino acids 450–540), and K590E, a kinase-defective mutant with a Glu substitution for the conserved Lys in the ATP-binding site, are also shown. Myr-Fes is an activated form of Fes in which the v-Src myristylation signal sequence has been added to the NH2-terminal region. A myristylated form of {Delta}SH2 was also generated (Myr-{Delta}SH2). For subcellular localization experiments, GFP was fused to the NH2 terminus of Fes. B, using a PCR-based strategy, Fes SH2 domain chimeras were created in which the wild-type SH2 domain was replaced with the SH2 domain of v-Fps, v-Src, or Ras Gap to produce the chimeras indicated. Membrane-targeted versions of the chimeras were produced by NH2-terminal addition of the coding sequence for the v-Src myristylation signal. The corresponding GFP-Fes SH2 domain chimeras were also created. All of the Fes cDNA clones used in this study encode the FLAG epitope tag at their COOH terminus.

 
SH2 Domain Substitution Does Not Alter Fes Kinase Activity in Vitro.
To investigate whether SH2 domain substitution affected kinase activity in vitro, the wild-type and chimeric Fes proteins were expressed in Sf-9 insect cells, immunoprecipitated, and analyzed by anti-phosphotyrosine immunoblotting. As shown in Fig. 2Citation , anti-phosphotyrosine immunoblots of equivalent amounts of the wild-type and chimeric Fes proteins demonstrate that the SH2 substitutions did not alter the overall extent of tyrosine autophosphorylation. To verify that SH2 domain substitution did not alter the sites of tyrosine autophosphorylation, wild-type and chimeric Fes proteins were immunoprecipitated from Sf-9 cells, incubated in vitro with [{gamma}-32P]ATP, and subjected to CNBr cleavage analysis. Each of the SH2 domain chimeras produced the same CNBr cleavage pattern as wild-type Fes, indicating that the same sites of autophosphorylation are used by the chimeras (Tyr-713 and Tyr-811; see legend to Fig. 2Citation for details). These results show that substitution of the Fes SH2 domain does not alter the activity of the kinase domain in terms of autophosphorylation or change the pattern of tyrosine autophosphorylation in vitro.



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Fig. 2. SH2 domain substitution does not affect Fes autophosphorylation in vitro. A, Wild-type Fes and the three SH2 domain chimeras indicated were expressed in Sf-9 cells as described in "Materials and Methods." Recombinant Fes proteins were immunoprecipitated from clarified cell extracts and analyzed by immunoblotting with antibodies to the Fes protein (top) or to phosphotyrosine (P-Tyr; bottom). B, to determine the pattern of autophosphorylation site usage in vitro, recombinant Fes proteins were immunoprecipitated from infected Sf-9 cell extracts, incubated with [{gamma}-32P]ATP, and subjected to CNBr cleavage analysis. The resulting peptides were separated on 10–20% SDS-tricine gradient gels and visualized by storage phosphor imaging. The primary structure of wild-type Fes is shown with all of the potential sites of CNBr cleavage indicated by the arrows. Previous studies have mapped the sites of Fes autophosphorylation to tyrosines 713 and 811 in the COOH-terminal portion of the Fes kinase domain (21) , which give rise to the Mr 10,000 (Tyr-713; peptide 1) and Mr 4000 (Tyr-811; peptide 2) CNBr cleavage fragments as shown. The lower panel shows the CNBr cleavage analysis of wild-type Fes and the SH2 domain chimeras. All four proteins yielded tyrosine-phosphorylated peptides of Mr 10,000 (peptide 1) and Mr 4000 (peptide 2) as indicated by the arrows.

 
Substitution with the v-Src SH2 Domain Releases Fes Tyrosine Kinase Activity in Vivo.
We next tested the kinase activity of the SH2 domain chimeras in Rat-2 fibroblasts, a cell type in which Fes kinase activity is tightly regulated (29) . Rat-2 fibroblasts were infected with recombinant retroviruses carrying wild-type Fes, the SH2 domain chimeras, an SH2 deletion mutant ({Delta}SH2), as well as transforming (Myr-Fes) and kinase-defective (K590E) mutants as controls (see Fig. 1Citation ). After infection, the cells were plated in soft agar, and individual G418-resistant clones were isolated and used to establish cell lines stably expressing equal levels of each Fes protein. Expression of each of the Fes proteins was verified by immunoblotting (Fig. 3Citation , top panel). The cell lines were then labeled with 32PO4, and the Fes proteins were immunoprecipitated, resolved by SDS-PAGE, and subjected to CNBr cleavage analysis, which permits a clear separation of phosphoserine- and phosphotyrosine-containing fragments (21) . As shown in Fig. 3Citation , the Fes/Src SH2 chimera exhibited low molecular weight phosphotyrosine-containing peptides, indicative of autophosphorylation and kinase activation in vivo. In contrast, none of the other chimeras or Fes proteins exhibited tyrosine phosphorylation in vivo. Similar results were obtained when the same Fes proteins were blotted with anti-phosphotyrosine antibodies (data not shown). Thus, Src SH2 substitution is permissive for Fes kinase activation in vivo in the absence of other mutations.



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Fig. 3. Src SH2 domain substitution releases Fes tyrosine kinase activity in vivo. Clones of Rat-2 fibroblasts expressing the indicated Fes constructs were labeled with 32PO4 as described in "Materials and Methods." Cells expressing only the G418 resistance marker were included as a negative control (Neo). Labeled Fes proteins were isolated by immunoprecipitation, and aliquots were analyzed for Fes protein levels by immunoblotting (top panel). The remainder of the immunoprecipitates were resolved by SDS-PAGE, and the incorporation of 32P was determined by storage phosphor imaging (second panel). The 32P-labeled proteins were extracted from the gel and subjected to CNBr cleavage analysis, and the resulting peptides were resolved on 10–20% SDS-tricine gradient gels; phosphopeptides were visualized by storage phosphor imaging. A long exposure (third panel) reveals labeled fragments corresponding to the major sites of Fes tyrosine autophosphorylation in the Fes/Src SH2 chimera (P-Tyr, far right lane; Ref. 21 ). The incorporation of radioactive phosphate into the other constructs was limited to a Mr 34,000 peptide that was readily visible after a short exposure (P-Ser, bottom panel). This phosphopeptide has been shown previously to contain only phosphoserine (21) . Two important exceptions include {Delta}SH2, in which this peptide is smaller, suggesting that the Mr 34,000 fragment contains the SH2 domain, and the Fes/Gap SH2 chimera, which was refractory to 32P-labeling in vivo. Also note that Myr-Fes does not produce detectable tyrosine phosphopeptides in this assay. This finding is consistent with previous work from our laboratory showing that addition of a myristylation signal is sufficient to induce transformation without a large increase in tyrosine kinase activity when assessed by anti-phosphotyrosine immunoblotting (23) .

 
Previous work from our laboratory and others has shown that the major phosphoamino acid present in Fes after isolation from 32P-labeled cells is phosphoserine (4 , 21 , 29) . In our previous studies, CNBr digestion produced a major phosphoserine-containing fragment of Mr 34,000 (21) . This fragment was also prominent in the CNBr digests of the chimeras and other Fes proteins after isolation from the Rat-2 fibroblasts (Fig. 3)Citation . One interesting exception is the Fes/Gap SH2 domain chimera, in which very little 32P incorporation was observed. This result suggests that the SH2 domain may control the interaction of Fes with the cellular serine kinase responsible for this phosphorylation event.

Substitution with the v-Src SH2 Domain Releases Fes Transforming Activity.
To determine whether the tyrosine kinase activity of the Fes/Src SH2 domain chimera observed in vivo was sufficient to induce a biological response, transformation assays were conducted with this chimeric Fes construct. Cell lines expressing equivalent levels of the Fes/Src and other SH2 chimeras were plated in focus-forming assays. The Fes/Src SH2 chimera induced irregularly growing cell clusters similar to those observed with Myr-Fes, a membrane-targeted, transforming variant of Fes used as a positive control (19 , 23 , 32) . Fig. 4Citation shows the morphology of Rat-2 cells expressing the Fes/Src SH2 chimera, which grew in large clumps and did not adhere strongly to the culture dish. In contrast, cells expressing wild-type Fes, the SH2 deletion mutant, the kinase-inactive mutant, or the other SH2 domain chimeras grew in smooth monolayers indistinguishable from control cells expressing only the drug selection marker. Similar results were obtained with a soft agar colony assay for transformation, in which only the Fes/Src SH2 domain chimera and the Myr-Fes-positive control produced loose macroscopic colonies of transformed cells (data not shown).



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Fig. 4. Src SH2 domain substitution releases Fes transforming activity. Wild-type and mutant forms of Fes were expressed in Rat-2 fibroblasts using recombinant retroviruses. Infected cells were selected with G418 for 2 weeks and stained with Wright-Giemsa, and a representative field of each culture was photographed under phase-contrast microscopy. Panels shown include cells expressing wild-type Fes (Fes), an SH2 domain deletion mutant ({Delta}SH2), Fes with an NH2-terminal myristylation signal (Myr-Fes), a kinase-inactive mutant (K590E), and the three SH2 domain chimeras (Fes/Fps SH2, Fes/Gap SH2, and Fes/Src SH2). Cells infected with an empty retrovirus served as a negative control (Neo). x40.

 
To verify that Fes tyrosine kinase activity is required for transformation by the Fes/Src SH2 domain chimera, two additional chimeras were made with mutations in the Fes kinase domain (Y713F and K590E). The Y713F mutation of the major autophosphorylation site greatly reduces Fes kinase activity, whereas the K590E mutation maps to the conserved Lys in the ATP-binding site and abolishes kinase activity entirely (21 , 24) . Neither of these constructs transformed Rat-2 cells, despite strong protein expression, indicating that both the Src SH2 domain and a fully active Fes kinase domain are required for transformation (data not shown). These results demonstrate that substitution of the Fes SH2 domain with that of Src is sufficient to activate the Fes kinase domain, generating a signal for transformation.

Gap SH2 Substitution Suppresses Transformation by an Activated Form of Fes.
Data presented above indicate that substitution with the Src SH2 domain releases Fes kinase activity and induces Rat-2 cell transformation, whereas Fps and Gap SH2 domain substitution appear to be without effect. However, another possibility is that altered SH2 specificity may interfere with kinase activation and transformation, which would not be observed in the context of wild-type Fes because of its lack of transforming activity in Rat-2 cells. To test for possible suppressive actions of SH2 domain substitution, each of the SH2 chimeras was fused to the NH2-terminal myristylation signal from v-Src. This modification has been shown previously to target wild-type Fes to the membrane and release its transforming potential (19 , 32) . Myristylated forms of wild-type Fes (Myr-Fes), the SH2 domain chimeras (Myr-Fes/Fps SH2, Myr-Fes/Gap SH2, and Myr-Fes/Src SH2), and a myristylated form of the SH2 deletion mutant (Myr-{Delta}SH2) were expressed in Rat-2 cells, and their transforming activities were compared in the focus-forming assay. As shown in Fig. 5Citation A, Myr-Fes produced a strongly transformed phenotype, as did the myristylated forms of the Src and Fps SH2 domain chimeras. However, substitution with the Gap SH2 domain completely blocked the transforming activity of Myr-Fes, as did deletion of the Fes SH2 domain. Soft agar colony assays for transformation produced similar results, and cloned cell lines expressing Myr-Fes/Gap SH2 did not transform, even when passaged for more than three weeks (data not shown). This finding is in marked contrast to previous studies with a c-Abl SH2 chimera, in which substitution of the same Gap SH2 sequence released transforming activity (31) . This result suggests that very different mechanisms are responsible for the regulation of Fes and c-Abl tyrosine kinase activity (see "Discussion"). Expression of the nontransforming Myr-Fes/Gap SH2 chimera as well as the other constructs was verified by immunoblotting (Fig. 5B)Citation .



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Fig. 5. Gap SH2 domain substitution blocks the transforming activity of myristylated Fes. Rat-2 fibroblasts were infected with recombinant retroviruses carrying membrane-targeted variants of wild-type Fes (Myr-Fes), the SH2 domain deletion mutant (Myr-{Delta}SH2), and the three SH2 domain chimeras (Myr-Fes/Fps SH2, Myr-Fes/Gap SH2, and Myr-Fes/Src SH2). Cells infected with an empty retrovirus served as a negative control (Neo). A, infected cells were selected with G418 for 2 weeks and stained with Wright-Giemsa, and a representative field of each culture was photographed under phase-contrast microscopy. x40. B, expression of Fes proteins in cells from A was confirmed by immunoblotting.

 
Src SH2 Domain Substitution Enhances the Ability of Fes to Induce Hematopoietic Differentiation.
Fes is expressed strongly in myeloid hematopoietic cells and has been directly implicated in myeloid differentiation by virtue of its ability to induce functional maturation of the undifferentiated erythroleukemic cell line, K-562 (see "Introduction"). Because Src SH2 domain substitution released both the kinase activity and transforming potential of Fes in fibroblasts, we expressed the chimera in K-562 cells to determine whether the chimera retained the capacity to induce hematopoietic differentiation as well. One hallmark of the Fes-induced differentiation response is the suppression of proliferation, a characteristic that makes it difficult to select and expand a small population of positive cells (8) . We therefore used a strategy of cocultivation of K-562 cells with Fes retrovirus-producing 293T cells to insure initial infection of a large cell population. The infected K-562 cells were harvested from the coculture, plated in proliferation assays, and monitored for 8 days. Expression of the Fes proteins was verified by flow cytometry. As shown in Fig. 6Citation , expression of wild-type Fes in K-562 cells by this method reduced proliferation by ~50% compared with control cells infected with a retrovirus carrying the green fluorescent protein cDNA. This result is in good agreement with previous findings for Fes in this cell line (7 , 8) . Using this approach, we found that the growth-suppressive effect of the Src SH2 domain chimera was even more striking, with growth rates reduced to ~10% of the control value.



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Fig. 6. The Fes/Src SH2 chimera exhibits enhanced growth-suppressive and differentiation-inducing activity in K-562 myeloid leukemia cells. A, growth suppression. K-562 myeloid leukemia cells were incubated with 293T cells producing recombinant retroviruses carrying either the GFP ({square}), wild-type Fes (), or the Fes/Src SH2 domain chimera ({blacksquare}). After 48 h of coculture, K-562 cells were separated from the 293T cells and replated at 105 cells/ml in the presence of G418. Viable cells were counted 4, 6, and 8 days later. Expression of GFP and the Fes proteins was verified by flow cytometry, and the cell numbers were corrected for the percentage of cells positive for expression of each protein. B and C, analysis of myeloid differentiation by flow cytometry. Aliquots of cells infected with recombinant retroviruses carrying wild-type Fes and the Fes/Src SH2 chimera were analyzed for Fes protein expression and for the cell surface myeloid differentiation antigens CD13 and CD33 using flow cytometry on the days indicated. The percentage of CD13- or CD33-positive cells was normalized to the percentage of cells positive for Fes or Fes/Src SH2 chimera expression in each population. Prior to normalization, all values were corrected for background fluorescence observed with a control population of cells cocultured with 293T cells producing a retrovirus carrying only the neo selection marker. Results shown are the average of two independent experiments. , wild-type Fes; {blacksquare}, Fes/Src SH2 domain chimera.

 
To verify that the effect of the chimeric Fes protein on K-562 cells was attributable to differentiation and not toxicity, fixed cells were stained for the myeloid cell surface differentiation markers CD13 and CD33 (8) . The percentage of cells expressing these differentiation markers was then normalized to the percentage of cells expressing the corresponding Fes protein, and the resulting values are presented in Fig. 6, B and CCitation . Cells expressing wild-type Fes showed an increase in CD13 and CD33 expression that paralleled the decrease in proliferative capacity, indicative of terminal differentiation. K-562 cells expressing the Fes/Src SH2 chimera were also strongly positive for the expression of both differentiation antigens, indicating that the Fes/Src SH2 chimera retains the capacity to activate signaling pathways linked to terminal differentiation. Differentiation occurred more rapidly and to a greater extent with the chimera than with wild-type Fes. These findings are consistent with the hypothesis that Src SH2 substitution leads to constitutive activation of Fes without affecting its normal biological activity.

Subcellular Localization of the Fes SH2 Domain Chimeras.
Previous studies have established that overexpression of Fes in macrophages, a physiological site of Fes expression, induces its activation and association with proteins related to cell adhesion and cell-cell contact (27 , 28) . This finding suggested that the transforming Fes/Src SH2 chimera may exhibit altered subcellular localization in fibroblasts. To investigate the effect of SH2 domain substitution on subcellular localization, each chimera was recloned in-frame with GFP and introduced into Rat-2 fibroblasts. As observed with the unmodified chimeras, the GFP-Fes/Src SH2 domain chimera exhibited very strong focus-forming activity in this cell type, whereas little or no transforming activity was observed with wild-type GFP-Fes or any of the other GFP-SH2 domain chimeras (Fig. 7A)Citation . The tyrosine kinase activity of the GFP-Fes fusions was also assessed by immunoprecipitation and anti-phosphotyrosine immunoblotting. As expected, the GFP-Fes/Src SH2 chimera exhibited strong tyrosine phosphorylation in Rat-2 cells, whereas the other chimeras and wild-type GFP-Fes exhibited much lower levels of tyrosine phosphorylation (Fig. 7B)Citation .



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Fig. 7. Transforming and kinase activities of GFP-Fes fusion proteins. Using recombinant retroviruses, wild-type Fes and the SH2 domain chimeras were expressed as NH2-terminal fusion proteins with GFP in Rat-2 cells. A, focus-forming assay. After infection with recombinant retroviruses, Rat-2 cells were selected with G418 for 2 weeks, and transformed foci were visualized by Wright-Giemsa staining. Foci were counted from scanned images of the stained dishes using a Bio-Rad GS710 Scanning Densitometer and Quantity One software. Cells infected with a retrovirus carrying GFP alone served as a negative control. Experiments were performed in triplicate, and the result shown is the mean; bars, SD. B, GFP-Fes tyrosine phosphorylation. GFP-Fes fusion proteins were immunoprecipitated from the Rat-2 cell populations described in A and immunoblotted with anti-FLAG antibodies to detect GFP-Fes protein expression (upper panel) or with anti-phosphotyrosine antibodies to detect autophosphorylation (lower panel). Arrows, positions of the GFP-Fes fusion proteins.

 
The subcellular distribution of each GFP-Fes fusion protein was evaluated by direct fluorescence microscopy of live cells. As shown in Fig. 8Citation , wild-type GFP-Fes and the Fps and Gap SH2 chimeras all exhibited a similar diffuse cytoplasmic localization. On the other hand, the Fes/Src SH2 chimera exhibited concentrated localization, suggestive of focal adhesion targeting. To test this idea further, fibroblasts expressing the GFP-Src SH2 chimera were treated with Colcemid to enhance the formation of focal contacts (33) , which were then visualized with antibodies to the focal adhesion protein talin (34) . As shown in Fig. 9Citation , the talin antibodies produced a staining pattern almost identical to that of GFP-Fes, and merging the images produced nearly complete overlap in localization. We also investigated the phosphorylation state of the focal adhesion molecule paxillin in fibroblasts expressing each of the SH2 domain chimeras. As shown in Fig. 10Citation , paxillin is strongly phosphorylated on tyrosine exclusively in cells expressing the Src SH2 domain chimera, providing further evidence for interaction of this chimera with focal contacts. These results strongly suggest that substitution with the Src SH2 domain stabilizes the association of Fes with focal adhesions and may lead to the generation of signals for transformation from this subcellular compartment.



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Fig. 8. Subcellular localization of GFP-Fes SH2 domain chimeras in Rat-2 fibroblasts. Rat-2 fibroblasts were infected with recombinant retroviruses carrying GFP or GFP-Fes fusion proteins with the wild-type SH2 domain (Fes SH2), the v-Fps SH2 domain (Fps SH2), the Ras Gap SH2 domain (GAP SH2), or the v-Src SH2 domain (Src SH2). Digital images of fluorescent cells were acquired with a Nikon TE300 inverted microscope with epifluorescence capability and Spot digital camera. The GFP-Fes/Src SH2 domain chimera showed strong localization to focal contacts (arrows). x400.

 


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Fig. 9. Colocalization of the GFP-Fes/Src SH2 chimera with talin in Rat-2 fibroblasts. Rat-2 fibroblasts expressing the GFP-Fes/Src SH2 domain chimera were treated with 50 nM Colcemid for 12 h to stabilize and extend focal adhesions (33) . Cells were fixed and stained with an anti-talin monoclonal antibody, followed by a goat antimouse IgG-Texas red conjugate (top panel). The pattern of GFP-Fes/Src SH2 fluorescence is shown in the center, and a merged image is shown at the bottom. x400.

 


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Fig. 10. The Fes/Src SH2 chimera induces tyrosine phosphorylation of paxillin. Paxillin was immunoprecipitated from populations of Rat-2 fibroblasts expressing GFP, GFP-Fes, or the GFP-Fes SH2 domain chimeras. Aliquots of the paxillin immunoprecipitates were immunoblotted with antibodies to phosphotyrosine (P-Tyr, top) or paxillin (bottom). Arrows, position of paxillin in each lane.

 
The GFP-Fes/Src SH2 Chimera Induces TF-1 Myeloid Cell Attachment and Localizes to Focal Sites.
In a final series of experiments, we investigated whether activation of Fes by SH2 domain substitution affected adhesion-related responses in a cell type where Fes is normally expressed. For these experiments, we used the myeloid progenitor cell line TF-1, which requires cytokines for growth and has the potential to differentiate along the macrophage pathway (35) . TF-1 cells were infected with recombinant retroviruses carrying wild-type GFP-Fes, each of the GFP-Fes/SH2 domain chimeras, or GFP alone as a negative control. The cells were selected in the presence of G418 and GM-CSF, and expression of GFP and the GFP-Fes proteins was monitored by fluorescence microscopy. As shown in Fig. 11Citation , a subpopulation of TF-1 cells expressing the Fes/Src SH2 domain chimera attached to the culture plate and spread to form macrophage-like cells. This effect was only observed with the Fes/Src SH2 chimera and did not occur with any of the other infected cell populations. In addition, the GFP-Fes/Src SH2 chimera exhibited a striking redistribution to focal sites in the attached cells, reminiscent of the localization observed in fibroblasts. These data show that the effects of the SH2 substitution are not unique to fibroblasts but occur in a physiologically relevant cell type as well.



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Fig. 11. The Fes/Src SH2 domain chimera induces attachment and spreading of the myeloid cell line TF-1 and localizes to focal sites. Equal numbers of TF-1 cells stably expressing GFP or GFP-Fes fusion proteins with the wild-type SH2 domain (Fes SH2), the v-Fps SH2 domain (Fps SH2), the Ras Gap SH2 domain (GAP SH2), or the v-Src SH2 domain (Src SH2) were plated in 24-well plates and incubated for 7 days. The right-hand column of images shows the live cell fluorescence of the total cell population in each well (attached and unattached cells). The attached cell population was then stained with Giemsa stain and visualized by light microscopy (left-hand images).

 
Discussion

Data presented in this report demonstrate that SH2 domain substitution has a major impact on the tyrosine kinase and transforming activities of the fes proto-oncogene in fibroblasts. Substitution of the Fes SH2 domain with that of v-Src released kinase activity in this cell type, leading to oncogenic transformation. This effect appears to be specific for the Src SH2 domain, because substitution of the Fes SH2 domain with that of the closely related transforming oncogene v-Fps or with the NH2-terminal SH2 domain of Ras Gap did not release tyrosine kinase or transforming activity. Results with the Ras Gap SH2 domain are particularly interesting in light of a previous report showing that substitution with this SH2 domain released the transforming and tyrosine kinase activities of c-Abl (31) . Not only did Gap SH2 substitution fail to release Fes transforming activity, it also interfered with transformation by a membrane-targeted form of Fes (Myr-Fes; Fig. 5Citation ). In addition, the Fes/Gap SH2 chimera was not subject to serine phosphorylation in vivo (Fig. 3)Citation , suggesting that interaction of Fes with an endogenous serine kinase may be necessary for full kinase activity or generation of signals related to transformation. Sites of serine phosphorylation have been identified within c-Abl, and phosphorylation of these sites can contribute to kinase activation (36) . In the case of Fes, the major site of serine phosphorylation maps to a serine-rich motif NH2-terminal to the SH2 domain.6 However, the identity of the kinase responsible for this phosphorylation event and its biological significance remain unknown.

What is the mechanism by which Src SH2 domain substitution releases the transforming and tyrosine kinase activities of Fes? One possibility is that the Src SH2 domain may stabilize localization of Fes at subcellular sites that promote activation. For example, Fes is activated by a variety of cytokines (see "Introduction"), suggesting that association with cytokine receptors at the plasma membrane is sufficient for activation. Consistent with this idea is the observation that constitutive localization of Fes to cellular membranes with the v-Src myristylation signal results in fibroblast transformation (Figs. 4Citation and 5Citation ; Refs. 19 and 32 ). In the case of the Src SH2 domain chimera, constitutive localization to focal adhesions may account for the transformation result. Analysis of GFP-Fes fusion proteins reveals that the Fes/Src SH2 domain chimera colocalizes with talin, a marker for focal adhesions (Fig. 9)Citation , and induces the tyrosine phosphorylation of paxillin, another focal adhesion-related protein (Fig. 10)Citation . In contrast, wild-type Fes and the other SH2 domain chimeras demonstrated a diffuse cytoplasmic localization and did not affect paxillin tyrosine phosphorylation. Src is also a well-known component of focal adhesions, and localization of Src to this subcellular compartment may be dependent, at least in part, on its SH2 domain (37 , 38) . The Src SH2 domain contributes to the interaction of Src with several components of the signaling machinery of focal complexes, including the focal adhesion kinase, Cas and paxillin, all of which become tyrosine phosphorylated in response to adhesion (34) . Interestingly, previous studies have shown that wild-type Fes interacts with Cas in macrophages, and that this interaction involves the Fes SH2 domain (28) . Replacing the Fes SH2 with that of Src may enhance Fes interaction with Cas in fibroblasts, resulting in focal adhesion targeting, kinase activation, and transformation. Several studies have shown that Src is able to activate the Ras-Erk pathway by creating binding sites for Grb-2/Sos on the focal adhesion kinase (39 , 40) . A similar signaling pathway may be responsible for transformation in fibroblasts expressing the Fes/Src chimera. Whether cell adhesion or cell-cell contact regulates Fes kinase activity under physiological conditions will require further investigation.

Src SH2 substitution not only released Fes transformation signaling in fibroblasts but also enhanced the ability of Fes to induce differentiation marker expression in K-562 cells (Fig. 6)Citation . We also observed that the Fes/Src SH2 domain chimera promotes the attachment and spreading of TF-1 macrophage precursor cells (Fig. 11)Citation . Similar results have been obtained with Fes variants activated by mutations in the first coiled-coil homology domain (but retaining the wild-type SH2), indicating that the differentiation-inducing activity of the chimera is more likely attributable to loss of negative regulation rather than gain of Src SH2 domain function (23) .7 The morphological effects of Src SH2 domain substitution correlate with redistribution of Fes to highly localized sites in TF-1 cells (Fig. 11)Citation , consistent with previous reports of Fes association with cell adhesion-related proteins in macrophages (27 , 28) .

Our results suggest that the specificity of the Fes SH2 domain may not be absolutely critical for interaction with differentiation signaling partners in myeloid cells and instead may have a more prominent role in the regulation of kinase activity. Alternatively, overlap between the Fes and Src SH2 specificity profiles may be sufficient to permit differentiation signaling. Using tyrosine phosphopeptide library screening techniques, the Src SH2 domain has been shown to strongly select the peptide sequence YPEEI (41 , 42) . The Fes SH2 domain showed a preference for the sequence YPEXV/I in parallel experiments, which is very similar to the optimal sequence for Src. Thus, substitution with the Src SH2 domain may be sufficient to disrupt kinase regulation without preventing interactions with target proteins essential for differentiation. A final possibility is that substrate selection by Fes may be more dependent upon regions outside of the SH2 domain, such as the unique NH2-terminal region or the kinase domain. Previous studies have shown that the unique NH2-terminal region of Fes interacts with the breakpoint cluster region (Bcr) protein, leading to tyrosine phosphorylation of Bcr and recruitment of Grb-2/Sos and other SH2-containing proteins (26 , 43 , 44) . Similarly, the Fes-related kinase Fer has been shown to interact with catenin-related proteins through its unique NH2-terminal domain (45 , 46) .

Materials and Methods

Construction of the Fes SH2 Domain Chimeras and Other Fes Mutants.
Replacement of the Fes SH2 domain with the SH2 domain of v-Src used a PCR-based approach. The coding sequence of the v-Src SH2 domain (Gly-151-Leu-241) was amplified using a forward primer that modified codon 150 in the 5' end of the v-Src SH2 domain to create a KpnI restriction site; a unique KpnI site is formed by homologous codons in the Fes SH2 sequence. The 5' end of the v-Src SH2 reverse primer contained sequences complementary to the 5' end of the Fes kinase domain. In a second PCR reaction, the coding sequence of the Fes kinase domain (amino acids 541–822) was amplified using a forward primer with 5' sequences complementary to the 3' end of the v-Src SH2 domain. The products of the first and second PCR reactions were purified and joined in a subsequent overlap-extension PCR reaction (47) . The resulting PCR product, containing the v-Src SH2 domain fused to the Fes kinase domain, was digested with KpnI and BamHI and swapped with the equivalent restriction fragment from wild-type Fes to generate the full-length chimeric protein. A similar strategy was used to substitute the coding sequence of the wild-type SH2 domain with that of v-Fps (Gly-823-Lys-900) and the more NH2-terminal SH2 domain of Ras Gap (Gly-185-Pro-271). The nucleotide sequences of the PCR-derived portions of the Fes SH2 chimeras were confirmed by automated DNA sequence analysis. Construction of human Fes mutants lacking the SH2 domain ({Delta}SH2) or with a Glu substitution for the conserved Lys in the ATP binding site (K590E) has been described elsewhere (24) .

Addition of the NH2-terminal myristylation signal sequence of v-Src to the NH2-terminal region of Fes to create the transforming oncogene Myr-Fes was accomplished using a PCR-based approach. The 5' end of the Fes cDNA was amplified using a forward primer encoding a unique HindIII site and the v-Src myristylation signal sequence, Met-Gly-Ser-Ser-Lys-Ser-Lys, fused to Fes homologous sequences beginning with codon 3 and a reverse primer that maps to the 3' end of the Fes unique NH2-terminal domain. The resulting PCR product was digested with HindIII and AccI and swapped with the equivalent restriction fragment in wild-type Fes to generate the full-length Myr-Fes cDNA. The transforming activity of the resulting Myr-Fes protein kinase has been described elsewhere (19 , 32) . The same subcloning strategy was used to generate the Myr forms of the Fes/SH2 chimeras.

To create the GFP-Fes expression constructs, the coding sequence of enhanced GFP was amplified from the vector pEGFP-1 (Clontech) by PCR and subcloned into the retroviral expression vector pSR{alpha}MSVtkneo (see below). The cDNA clones encoding wild-type and chimeric forms of Fes were subcloned downstream of and in-frame with GFP in this vector.

Expression of Fes Proteins in Sf-9 Cells and CNBr Cleavage Analysis of Autophosphorylation Sites.
The cDNAs encoding the Fes SH2 domain chimeras were subcloned into the baculovirus expression vector pVL1392, and the resulting constructs were used to generate recombinant baculoviruses as described elsewhere (21 , 48) . For Fes protein expression, subconfluent monolayers of Sf-9 cells were infected with recombinant Fes baculoviruses and incubated for 48 h. Infected cells were sonicated in 1.0 ml of ice-cold Fes lysis buffer [50 mM Tris-HCl (pH 7.4), 50 mM NaCl, 1 mM EDTA, 1 mM MgCl2, and 0.1% 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. Fes proteins were immunoprecipitated from clarified cell lysates with the M2 anti-FLAG monoclonal antibody resin (Sigma). M2 recognizes the COOH-terminal FLAG epitope fused to each of the Fes constructs used in this study. The immunoprecipitates were washed with radioimmune precipitation assay (RIPA) buffer (50 mM Tris-HCl, 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 1 mM EDTA, and 1% sodium deoxycholate), resuspended in SDS-PAGE sample buffer, resolved on 8% SDS-polyacrylamide gels, and visualized by immunoblotting with M2 or anti-phosphotyrosine antibodies (PY20; Transduction Laboratories).

CNBr cleavage analysis was performed essentially as described previously (21) . Briefly, the Fes immunoprecipitates were washed in kinase buffer (50 mM HEPES, pH 7.4, 10 mM MgCl2) prior to addition of [{gamma}-32P]ATP (10 µCi; DuPont-New England Nuclear). After incubation for 15 min at 30°C, the proteins were resolved by SDS-PAGE, eluted from the gel, and resuspended in a solution of CNBr (20 mg/ml in 70% formic acid). After overnight incubation at room temperature, the CNBr-generated fragments were resolved on 10–20% Tricine gradient gels (Novex) and visualized by storage phosphor technology (Molecular Dynamics PhosphorImager).

Production of Recombinant Retroviruses and Infection of Rat-2 Fibroblasts.
Wild-type, mutant, and GFP-tagged forms of Fes were subcloned into the retroviral vector pSR{alpha}MSVtkneo (49) . Recombinant retroviruses were produced by cotransfecting 293T cells with the pSR{alpha}-Fes constructs and an ecotropic packaging vector as described elsewhere (23 , 32 , 50) . The retroviral supernatant was collected every 12 h for 3 days, and the pooled supernatants were aliquoted and stored at -80oC.

Rat-2 cells were obtained from the ATCC and maintained in DMEM supplemented with 5% FBS. For viral infection, Rat-2 cells (2.5 x 105 ) were plated in 60-mm tissue culture dishes and incubated overnight at 37oC. Retroviral supernatants (5 ml) were thawed on ice, supplemented with Polybrene to 4 µg/ml, and added to the cells. After incubation for 4 h at room temperature, the viral supernatants were aspirated and replaced with 4 ml of fresh medium. The infected cells were then incubated for 48 h at 37°C prior to plating in transformation assays.

Transformation Assays.
Rat-2 fibroblast transformation was assessed by both focus-forming and soft agar colony assays. For focus-forming assays, 2 x 104 retrovirally infected cells were plated in 60-mm tissue culture dishes in the presence of 800 µg/ml G418. The cells were incubated at 37oC for 2 weeks, at which time they were stained with Wright-Giemsa and observed by light microscopy. For soft agar colony assays, cells were suspended at 2 x 104 cells/ml in DMEM containing 5% FBS, 0.3% SeaPlaque agarose (FMC BioProducts), and 800 µg/ml G418. The mixture was then plated in 35-mm bacterial culture dishes (Falcon 1008) containing 1 ml of bottom layers of presolidified agarose solution. The dishes were incubated in humidified chambers at 37oC for 1–3 weeks. Cloned cell lines were created by picking individual colonies from each agarose culture and expanding under G418 selection. Expression was verified by immunoblotting with the M2 anti-FLAG antibody as described elsewhere (21) .

Metabolic Labeling of Stably Transfected Rat-2 Cell Lines.
Rat-2 cell lines stably expressing wild-type and mutant Fes proteins were radiolabeled as described previously (21) . Briefly, confluent cells in 100-mm dishes were washed twice and incubated in phosphate-free DMEM for 1 h at 37oC. Proteins were then radiolabeled in vivo by incubating the cells for an additional 4 h at 37oC in phosphate-free DMEM containing 2.0 mCi/ml 32PO4. After incubation, the cells were frozen in situ on liquid nitrogen and lysed in 1.0 ml of Fes lysis buffer. The Fes proteins were immunoprecipitated with the M2 antibody resin, resolved by SDS-PAGE, and subjected to CNBr cleavage analysis as described above.

Hematopoietic Differentiation Assay.
The human erythroleukemia cell line K-562 (6) was obtained from the ATCC and grown in RPMI 1640 containing 10% FBS. K-562 cells were infected with recombinant Fes retroviruses using a coculture approach. Cultures of virus-producing 293T cells were initiated by cotransfection with retroviral and packaging plasmids as described above, except an amphotropic packaging plasmid was used. Two days after transfection, 4 ml of DMEM containing 5% FBS and 3 x 105 K-562 cells were added to the 293T cultures along with 4 µg/ml Polybrene. After incubation for 2 days at 37oC, the K-562 cells were removed from the 293T cell culture by aspiration. Infected K-562 cells were replated on new culture dishes and incubated for an additional 2 days at 37oC, allowing residual 293T cells to re-adhere. The infected K-562 cells were reharvested, counted, and plated at 105 cells/60-mm tissue culture dish in 4 ml of RPMI containing 10% FBS and 800 µg/ml G418. Four, 6, and 8 days later, viable cell counts were performed by trypan blue exclusion, and aliquots were fixed for 20 min in 1% paraformaldehyde and stored at 4oC in PBS prior to staining and flow cytometry. For single-cell analysis of Fes expression, 105 fixed cells were permeabilized with 0.05% saponin in RPMI 1640 containing 5% FBS (RPMI-FS) for 20 min. Cells were resuspended in 200 µl of RPMI-FS containing the M2 anti-FLAG monoclonal antibody (20 µg/ml) for 1 h. The cells were washed twice with RPMI-FS and then incubated with a goat antimouse IgG-FITC conjugate (20 µg/ml in RPMI-FS; Molecular Probes) for 1 h. The cells were then washed three more times prior to FACS analysis. Analysis of CD13 and CD33 expression was performed with direct FITC-conjugated antibodies for these myeloid differentiation markers (Southern Biotechnology Associates; Ref. 8 ). Staining was performed as described above for the M2 antibody, except the saponin was omitted.

Subcellular Localization of GFP-Fes Fusion Proteins.
Rat-2 fibroblasts were infected with recombinant GFP-Fes retroviruses as described above. To enhance the efficiency of infection, cells were centrifuged at 1000 x g during the infection period (51) . Populations of infected cells were selected with G418, and expression was verified by immunoblotting and by immunofluorescence microscopy.

To visualize focal adhesions, cells were plated in 24-well plates (3 x 104 cells/well) and incubated overnight at 37oC. Cells were fixed in situ with 2% paraformaldehyde in PBS for 30 min at room temperature, followed by permeabilization with 0.05% saponin in DMEM containing 5% FBS (DMEM-FS) for 30 min. The cells were incubated with an anti-talin monoclonal antibody (diluted 1:50 in DMEM-FS; Sigma) for 1 h at room temperature. The cells were then washed with DMEM-FS and incubated with a goat antimouse IgG-Texas red conjugate for 1 h. Cells were washed again with PBS, and immunofluorescent images were recorded using a Nikon TE300 inverted microscope with epifluorescence capability and a SPOT cooled CCD high-resolution digital camera (Diagnostic Instruments). For some experiments, cells were treated with 50 nM Colcemid for 12 h prior to staining to induce elongation of the focal contacts and enhance their visualization (33) .

Analysis of GFP-Fes Fusion Protein Expression and Paxillin Phosphorylation.
Rat-2 fibroblasts stably expressing GFP fusions of wild-type Fes and SH2 chimeras were lysed in Fes lysis buffer supplemented with 25 µg/ml aprotinin, 50 µg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride, 20 mM NaF, and 1 mM Na3VO4. Cell lysates were clarified by centrifugation and divided into two aliquots. Fes proteins were immunoprecipitated from one aliquot with the M2 anti-FLAG monoclonal antibody resin. Paxillin was immunoprecipitated from the second aliquot with an anti-paxillin monoclonal antibody (Transduction Laboratories) and protein-G Sepharose beads (Pharmacia). Immunoprecipitates were washed three times with 1.0 ml RIPA buffer, and precipitated proteins were resolved by SDS-PAGE. Proteins were transferred to polyvinylidene difluoride membranes and probed with the M2 anti-Flag antibody to detect Fes protein expression, with the paxillin antibody, or with the anti-phosphotyrosine antibody PY99 (Santa Cruz) to detect Fes and paxillin tyrosine phosphorylation.

Expression of Fes in TF-1 Cells.
The human myeloid leukemia cell line TF-1 (35) was obtained from the ATCC and cultured in RPMI 1640 supplemented with 10% FBS, 50 µg/ml gentamicin, and 1 ng/ml GM-CSF (BioSource International). Recombinant retroviruses for TF-1 cell infection were prepared using the 293T cell protocol described above, except that an amphotropic packaging plasmid was used. For TF-1 cell infection, 2 x 105 cells were plated in each well of a six-well plate and centrifuged at 500 x g for 15 min. The medium was aspirated and replaced with the retroviral supernatant plus Polybrene, and the infection protocol was continued as described above for the Rat-2 cells. After infection, the cells were placed under G418 selection for 10–14 days. Expression of GFP or the GFP-Fes fusion proteins was confirmed by fluorescence microscopy, under which the drug-resistant cell population exhibited uniform green fluorescence (see Fig. 11Citation ). Expression of the GFP-Fes fusion proteins was also confirmed by immunoblotting (data not shown). For attachment experiments, 5 x 104 newly infected cells were plated in 24-well plates in a total volume of 1 ml and returned to the incubator for 7 days. Digital images of live-cell fluorescence were recorded of the entire cell population in each well (attached and unattached cells). The attached cell population was then stained in situ with Giemsa stain and visualized by light microscopy.

Acknowledgments

We thank Nancy Dunham for constructing the Fes SH2 domain chimeras.

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 This work was supported by NIH Grant CA 58667 (to T. E. S.). Back

2 These authors contributed equally to this work. Back

3 Present address: Department of Cell Biology and Anatomy, University of Nebraska Medical Center, Omaha, NE 68198. Back

4 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

5 The abbreviations used are: CSF, colony stimulating factor; GM-CSF, granulocyte/macrophage-CSF; SH2, Src homology 2; Gap, GTPaseactivating protein; Cas, Crk-associated substrate; GFP, green fluorescent protein; CNBr, cyanogen bromide; ATCC, American Type Culture Collection. Back

6 K. Peters and T. Smithgall, unpublished observation. Back

7 H. Cheng and T. Smithgall, unpublished data. Back

Received for publication 4/25/00. Revision received 8/14/00. Accepted for publication 9/25/00.

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