Interactions of CBL with BCR-ABL and CRKL in BCR-ABL-transformed Myeloid Cells*

The Philadelphia chromosome, detected in virtually all cases of chronic myelogenous leukemia (CML), is formed by a reciprocal translocation between chromosomes 9 and 22 that fusesBCR-encoded sequences upstream of exon 2 of c-ABL. The BCR-ABL fusion creates a gene whose protein product, p210BCR-ABL, has been implicated as the cause of the disease. Although ABL kinase activity has been shown to be required for the transforming abilities of BCR-ABL and numerous substrates of the BCR-ABL tyrosine kinase have been identified, the requirement of most of these substrates for the transforming function of BCR-ABL is unknown. In this study we mapped a direct binding site of the c-CBL proto-oncogene to the SH2 domain of BCR-ABL. This interaction only occurs under conditions where c-CBL is tyrosine-phosphorylated. Despite the direct interaction of c-CBL with the SH2 domain of BCR-ABL, deletion of the SH2 domain of BCR-ABL did not result in an alteration in the complex formation of BCR-ABL and c-CBL, suggesting that another site of direct interaction between c-CBL and BCR-ABL exists or that another protein mediates an indirect interaction of c-CBL and BCR-ABL. Since CRKL, an SH2, SH3 domain-containing adapter protein is known to bind directly to BCR-ABL and also binds to tyrosine-phosphorylated c-CBL, the ability of CRKL to mediate a complex between c-CBL and BCR-ABL was examined.

Chronic myelogenous leukemia is a hematopoietic stem cell malignancy that is associated with a reciprocal translocation between chromosomes 9 and 22, known as the Philadelphia chromosome translocation (1,2). This balanced translocation juxtaposes the breakpoint cluster region (BCR) 1 from chromosome 22 with the c-ABL tyrosine kinase on the long arm of chromosome 9 (3)(4)(5). The BCR-ABL fusion creates a gene whose protein product has been shown to transform hematopoietic progenitor cells in bone marrow culture (6 -8), to transform interleukin-3-dependent myeloid cell lines to growth factor independence (9,10), and to cause a syndrome resembling chronic myelogenous leukemia in syngeneic mice (11,12). The BCR-ABL tyrosine kinase activity is increased severalfold over the normal c-ABL gene product (13)(14)(15). Although ABL kinase activity has been shown to be required for transformation of myeloid cell lines by BCR-ABL (15,16), and numerous substrates of the BCR-ABL tyrosine kinase have been identified, the requirement of most of these substrates for the transforming function of BCR-ABL is unknown.
Two of the substrates of BCR-ABL on which we have focused are CRKL and c-CBL. CRKL is a 39-kDa SH2, SH3 domaincontaining adapter protein that is related to the CRK oncogene of the avian sarcoma virus, CT10 (17). Two human homologs of CRK, in addition to CRKL, have been identified, termed CRK I and CRK II. CRKL is most similar to CRK II in that both contain two SH3 domains, whereas CRK I contains only one SH3 domain as a result of alternative splicing (18). We have previously demonstrated that the N-terminal SH3 domain of CRKL binds directly to a proline-rich region in the C terminus of BCR-ABL, and this direct binding can be disrupted by deletion of this region (19). However, this deletion mutant of BCR-ABL remains transformation competent, and CRKL is tyrosine-phosphorylated and binds to BCR-ABL through indirect interactions in cells expressing this deletion mutant (19).
One of the candidates for mediating indirect binding of CRKL and BCR-ABL is c-CBL. c-CBL is the cellular homolog of v-CBL, the transforming protein of the Cas NS-1 retrovirus that induces pre-B cell lymphomas and myeloid leukemias in mice (20). c-CBL is a common substrate of tyrosine kinases and is tyrosine-phosphorylated in a variety of signaling pathways associated with cellular proliferation or activation (21)(22)(23)(24)(25). c-CBL is known to be tyrosine-phosphorylated in cells expressing activated ABL oncoproteins and has been shown to bind to CRK proteins when tyrosine-phosphorylated (26 -29). In this study we have mapped a direct binding site of tyrosine-phosphorylated c-CBL to the SH2 domain of BCR-ABL. The effects of deletion of this domain on complex formation between c-CBL, BCR-ABL, and CRKL are reported.

MATERIALS AND METHODS
Cells and Cell Culture-The 32Dcl3 cell line (30) was obtained from Joel Greenberger, University of Massachusetts Medical Center, Worcester, MA. Cells were cultured in RPMI 1640 medium (Life Technologies, Inc.) supplemented with 10% fetal bovine serum (UBI) and 15% WEHI-3B conditioned media as a source of interleukin-3. Sublines of 32Dc13 expressing p210BCR-ABL, the SH2 domain deletion of ABL (⌬SH2), a tyrosine kinase inactive mutant of BCR-ABL, and a deletion of a proline-rich region in the C terminus of ABL that mediates direct binding of CRKL to ABL (⌬P1,P2) were generated as described previously (16,19,31). K562 cells are a BCR-ABL-positive leukemic cell line (32) that was cultured in RPMI 1640 supplemented with 10% fetal calf serum at 37°C, 5% CO 2 .
Generation of Baculovirus Constructs-Full-length p210BCR-ABL and the kinase domain of ABL were cloned into a modified version of the pBluBac vector (InVitrogen). This vector, pBluBac3C (gift of R. Maurer, Oregon Health Sciences University, Portland, OR) contains a 12CA5 antibody binding site, a hexa-histidine sequence for Ni-Sepharose purification, a factor X cleavage site, and a BamHI cloning site. Fulllength BCR-ABL was modified to remove untranslated 5Ј sequences and was cloned in-frame into the BamHI site of pBluBac3C. The kinase domain of ABL was obtained by polymerase chain reaction using a BCR-ABL plasmid as the template. The 5Ј primer was 5Ј CAGCG-GATCCAAAGCGCAACAAGCCC 3Ј, and the 3Ј primer was 5Ј TAC-TAGGATCCTTATCAGGATTCCTGGAACATTGT 3Ј. The BamHI restriction site is underlined, and the stop codons are in italics. The polymerase chain reaction product was digested with BamHI, ligated into pBluBac3C, and sequenced to confirm that no errors were generated from the polymerase chain reaction amplification.
The full-length BCR-ABL and the ABL kinase domain were transfected into Sf9 cells as described (InVitrogen). Propagation, identification, and isolation of plaques, generation of high titer stocks, and infection of Sf9 cells for expression were performed as described (33). Both the full-length BCR-ABL and the kinase domain were shown to be of correct predicted size and functional by an immune complex kinase assay. For large scale production of BCR-ABL and the ABL kinase domain, Sf9 cells were grown in Grace's insect media (Life Technologies, Inc.), supplemented with 10% heat-inactivated fetal bovine serum, 0.33% yeastolate, 0.33% lactalbumin hydrolysate, and 50 mg/ml gentamicin, at 27°C in spinner flasks to a density of 1-2 ϫ 10 6 cells/ml. The cells were infected with plaque-purified virus at a multiplicity of infection of 2 and harvested 72 h post-infection in the case of BCR-ABL and 48 h post-infection in the case of kinase domain-infected cells.
Antisera-Rabbit polyclonal antisera against CRKL were generated against a peptide corresponding to amino acids 204 -225 of CRKL. This antiserum was used exclusively for immunoblotting experiments. For other experiments, anti-CRKL antibody was purchased from Santa Cruz Biotechnology. Antibodies recognizing p120 CBL, ABL proteins (24 -11 and K-12), and anti-GST were purchased from Santa Cruz Biotechnology. The anti-hemagglutinin antibody 12CA5 was obtained from Boehringer Mannheim. The anti-phosphotyrosine monoclonal antibody, 4G10, was generated using KLH-phosphotyramine as the immunogen and was used as described (34,35).
Immunoprecipitation and Immunoblotting-Cells were lysed in Nonidet P-40 lysis buffer (20 mM Tris, pH 8.0, 1 mM EDTA, 150 mM NaCl, 1% Nonidet P-40, 10% glycerol) containing 10 mg/ml aprotinin, 1 mM Na 3 VO 4 , and 1 mM phenylmethylsulfonyl fluoride. The 32D lysates were normalized in preliminary experiments by immunoblotting for c-ABL, and these amounts were used for subsequent experiments. Normalized lysates were either immunoprecipitated with 25 l of CRKL or ABL antisera followed by SDS-PAGE or were analyzed as whole cell lysates by SDS-PAGE. Proteins were transferred onto polyvinylidene difluoride (PVDF, Immobilon-P, Millipore) membranes in a buffer containing 25 mM Tris, 192.5 mM glycine, 20% MeOH for 4 h at 0.45 A. Residual binding sites were blocked by incubation in TBS (10 mM Tris, pH 8.0, 150 mM NaCl) containing 3% bovine serum albumin for 60 min at 25°C. The blots were incubated for 4 -16 h at room temperature with primary antibody. Antibody reactions were detected using enhanced chemiluminescence (Pierce). Sf9 lysates expressing ABL kinase or full-length BCR-ABL were bound to 12CA5 antibody at 4°C for 6 -8 h with Protein A-Sepharose (Pharmacia Biotech Inc.), washed three times with ice-cold PBS, and then incubated with 32D, 32Dp210, or K562 lysates for 4 h. The beads were then washed three more times with PBS before being boiled in SDS-sample buffer and analyzed by SDS-PAGE.
Glutathione S-Transferase (GST) Binding Assays-The GST-CRKL constructs were previously described (19). GST-ABL SH2, GST-ABL SH3, and GST-ABL SH2, SH3 were gifts from W. Haser (Dana-Farber Cancer Institute, Boston). GST-fusion constructs were expressed in isopropylthio-␤-D-galactosidase-induced Escherichia coli (DH-5␣). The GST-fusion proteins were isolated from sonicated bacterial lysates using glutathione-Sepharose beads (Pharmacia). Coomassie-stained gels of the GST-fusion proteins were used to normalize for the expression of the various GST-fusion proteins. Between 2.5 and 5 g of GST-fusion proteins were incubated with 50 l of glutathione-Sepharose beads in bacterial lysis buffer (150 mM NaCl, 16 mM Na 2 HPO 4 , 4 mM NaH 2 PO 4 , pH 7.3) containing 10 g/ml aprotinin, 1 mM Na 3 VO 4 , 1 mM phenylmethylsulfonyl fluoride, and 0.1% ␤-mercaptoethanol. The beads were washed four times in ice-cold PBS and incubated for 4 h with normalized cell lysates. The beads were washed three times with PBS and boiled in SDS-sample buffer. Proteins were separated by SDS-PAGE and transferred onto PVDF membranes for immunoblot analysis.
Gel Overlay Assay-Purified GST, GST-ABL SH2, and GST-ABL SH3 fusion proteins were used for this assay. PVDF membranes were blocked overnight at 4°C in PBS with 0.05% Tween 20 and 5% non-fat dry milk. The blots were washed twice in PBS with 0.05% Tween 20 (PBS-T) and incubated for 2 h at room temperature with either GST Immunoblots were stripped and reprobed with CBL antisera and demonstrated that equal amounts of CBL were present in the immunoprecipitates from each lysate (data not shown). C, lysates were immunoprecipitated with a monoclonal ABL antisera and immunoblotted with CBL antisera.
FIG. 2. In vitro association of CBL and ABL. A, ABL kinase and BCR-ABL were expressed as fusion proteins containing a 12CA5 antibody binding site. Lysates from K562 or 32D cells were run over columns containing 12CA5 antibody, or 12CA5 antibody bound to the kinase domain of ABL or full-length BCR-ABL. CBL binding was detected by immunoblotting with CBL antisera. B and C, lysates of K562 or 32D cells were analyzed as in A for CBL binding to the indicated fusion proteins. Glutathione beads and bacterially expressed GST were used as controls. WCL indicates whole cell lysate to determine the presence and migration of c-CBL.
only or GST-ABL SH2 or SH3 fusion proteins (at 2 mg/ml) in binding buffer (25 mM sodium phosphate, pH 7.20, 150 mM NaCl, 0.1% Tween 20, 2.5 mM EDTA, 20 mM NaF, 1% non-fat milk, 1 mM dithiothreitol, 10 g/ml leupeptin, and 10 mg/ml aprotinin). Bound GST protein was detected by incubation with anti-GST antibody diluted 1:500 in binding buffer excluding milk, dithiothreitol, and protease inhibitors. Antibody reactions were developed using enhanced chemiluminescence. The blots were washed for 1 h in PBS-T between each of the steps.

Tyrosine Phosphorylation of CBL and Association of CBL with BCR-ABL in BCR-ABL-expressing Cells-Previous stud-
ies have demonstrated that p120 c-CBL is tyrosine-phosphorylated in cells expressing activated ABL oncoproteins, including BCR-ABL. Furthermore, BCR-ABL and c-CBL form a complex as demonstrated by co-immunoprecipitation (26 -29). These results were confirmed in 32D cells expressing p210BCR-ABL and in K562 cells, a BCR-ABL-positive cell line derived from a chronic myelogenous leukemia patient (32). Fig. 1 shows that c-CBL is tyrosine-phosphorylated in BCR-ABL expressing cells but not in control, interleukin-3-dependent 32D cells. A number of tyrosine-phosphorylated proteins co-immunoprecipitate with c-CBL in BCR-ABL-expressing cells, including proteins of relative molecular mass of 210, 170, 135-140, 68 -70, and 56 kDa (data not shown). As shown in Fig. 1, B and C, BCR-ABL is present in c-CBL immunoprecipitates, and c-CBL is present in ABL immunoprecipitates. The tyrosine phosphorylation of c-CBL and association with BCR-ABL is dependent on ABL kinase activity as it is not seen in cells expressing a tyrosine kinase inactive mutant of BCR-ABL (Fig. 3).
Localization of Domains of BCR-ABL That Interact with c-CBL-The data presented above demonstrated that c-CBL and BCR-ABL co-immunoprecipitate, and this association requires tyrosine kinase activity of BCR-ABL. To assist in localization of a possible binding sequence of BCR-ABL to CBL, full-length BCR-ABL and the kinase domain of ABL were expressed in the baculovirus system, tagged with an N-terminal hemagglutinin (12CA5) epitope. 12CA5 immunoprecipitates from lysates of SF9 cells infected with these constructs were incubated with lysates from K562 cells and analyzed for c-CBL binding. As shown in Fig. 2A, full-length BCR-ABL but not the kinase domain was capable of binding to c-CBL. No binding of c-CBL to the full-length BCR-ABL was seen using lysates from 32D cells, again suggesting that tyrosine phosphorylation of c-CBL is required for binding to BCR-ABL and that the binding of c-CBL to BCR-ABL requires a domain outside of the kinase domain of ABL.
A possibility raised by the data presented above is that c-CBL may be binding to the SH2 domain of ABL in a phosphotyrosine-dependent fashion. To test this, bacterially expressed GST-fusion proteins containing the ABL SH2 or SH3 domains were evaluated for their ability to bind c-CBL from cellular lysates. As seen in Fig. 2, B and C, the ABL SH2 but not ABL SH3 domain was capable of binding CBL from BCR-ABL lysates but not from 32D cells.
Although these experiments suggested that the SH2 domain of ABL was binding tyrosine-phosphorylated CBL, these experiments could not determine whether the interaction was direct or indirect. To assess whether the SH2 domain of ABL could bind directly to CBL, a gel overlay assay was performed. c-CBL immunoprecipitates from 32D and BCR-ABL-expressing cells were separated by SDS-PAGE and transferred onto PVDF membranes. The membranes were incubated with GST-ABL SH2 or SH3 domains, and binding was detected with a GST antiserum. The data presented in Fig. 3 show that equal amounts of c-CBL were immunoprecipitated from all cells examined and that the ABL SH2 domain binds directly to c-CBL. This binding of c-CBL to the ABL SH2 domain is only seen under conditions where c-CBL is tyrosine-phosphorylated (Fig. 3).  (16, 36 -38) have previously constructed BCR-ABL mutants with a deletion of the SH2 domain. This mutant remains capable of rendering myeloid cells factor-independent for growth but is defective in fibroblast transformation assays. Myeloid cells expressing this mutant were examined for defects in c-CBL tyrosine phosphorylation or BCR-ABL association. As shown in Fig. 1, c-CBL is tyrosine-phosphorylated in cells expressing the SH2 domain mutant of BCR-ABL and also co-immunoprecipitates with BCR-ABL.

In Vivo Interactions of c-CBL with a BCR-ABL Mutant Lacking the SH2 Domain-We and others
Association of CRKL and CBL-The data for the SH2 domain deletion of BCR-ABL and its interaction with c-CBL are reminiscent of data that we had obtained for CRKL and ABL interactions (19). We previously mapped a direct binding site for the N-terminal SH3 domain of CRKL to a proline-rich region in the C terminus of ABL. Deletion of this region in BCR-ABL did not result in an obvious transformation defect. In cells expressing this mutant (p210⌬P1,P2) CRKL remained tyrosine-phosphorylated and was present in a complex with BCR-ABL as demonstrated by co-immunoprecipitation. A variety of evidence suggested that another protein was mediating an indirect interaction of CRKL and BCR-ABL in this mutant cell line (19).
A possible explanation of these data is that CBL and CRKL form a complex in BCR-ABL-expressing cells. To determine whether CBL and CRKL interact, CRKL immunoprecipitates followed by CBL immunoblots or vice versa were performed. As shown in Fig. 4, these two proteins form a complex in cells expressing kinase active BCR-ABL but not in control 32D cells or cells expressing a kinase inactive BCR-ABL. Co-immunoprecipitation is also seen in cells expressing the proline deletion mutant of BCR-ABL-⌬P1, P2, and the SH2 domain deletion (Fig. 4). Using various GST-CRKL constructs, we have shown that c-CBL binds to the SH2 domain of CRKL, and this inter-action is dependent on c-CBL tyrosine phosphorylation (19). Using a gel overlay assay with GST-CRKL constructs, we find that the CRKL SH2 domain is capable of binding directly to c-CBL under conditions where c-CBL is tyrosine-phosphorylated (Fig. 5).
In Vitro Analysis of ABL, CBL, and CRKL Complex Formation Using ABL Mutants-The data presented thus far are consistent with a model of BCR-ABL interactions shown in Fig.  6A. In this model, BCR-ABL interacts directly and indirectly with both CRKL and CBL. That is, BCR-ABL interacts directly through its SH2 domain with tyrosine-phosphorylated CBL, and tyrosine-phosphorylated CBL also interacts with the SH2 domain of CRKL. Similarly a proline-rich region of BCR-ABL interacts directly with CRKL which in turn interacts with CBL. From this model, several predictions are possible. Using a GST-CRKL SH3 N-terminal domain, we would expect to see binding of BCR-ABL from lysates of cells expressing BCR-ABL. However, no binding should be seen using lysates of cells expressing the proline-rich deletion mutant of BCR-ABL (⌬P1,P2). As seen in Fig. 7A, this is the observed result confirming this site as a binding site between BCR-ABL and the SH3 domain of CRKL. Consistent with a lack of binding of BCR-ABL from the ⌬P1, P2 mutant to the SH3 domain of CRKL, no binding of CBL is seen when lysates from 32Dp210⌬P1, P2-expressing cells are analyzed for binding to the GST-CRKL SH3 construct (Figs. 6B and 7C). However, using the SH2 domain of CRKL, some binding of BCR-ABL was seen from cells expressing BCR-ABL or the ⌬P1, P2 mutant (Fig. 7B). This could be explained by our model with CBL binding to the SH2 domain of CRKL and presumably through another tyrosine residue also binding to BCR-ABL (Fig. 6A). To confirm these findings, lysates of BCR-ABL cells were run over various GST-CRKL fusion proteins and analyzed for CBL binding (Fig. 7C). In these experiments, the SH3 domain of CRKL bound to CBL from lysates of cells expressing full-length BCR- ABL. This would be expected as the SH3 domain of CRKL would interact with BCR-ABL which through its SH2 domain interacts with CBL. A prediction of this model would be that this interaction would be abolished if lysates from cells expressing the SH2 domain deletion of BCR-ABL were used in this experiment. As seen in Fig. 7C, this prediction was confirmed as is modeled in Fig. 6C. DISCUSSION In this article we have demonstrated a direct interaction between c-CBL and BCR-ABL. Our data show that the kinase domain of ABL is not capable of binding to c-CBL, whereas a full-length BCR-ABL does, suggesting that a domain outside of the kinase is required for binding of c-CBL to BCR-ABL. The interaction of BCR-ABL and c-CBL only occurs under conditions where c-CBL is tyrosine-phosphorylated, implying that tyrosine-phosphorylated c-CBL may bind to the SH2 domain of ABL. Direct binding of the SH2 domain of ABL to c-CBL was confirmed using a gel overlay assay and again this binding only occurred in lysates where tyrosine phosphorylation of c-CBL has been demonstrated.
Despite the direct interaction of c-CBL with the SH2 domain of BCR-ABL, deletion of the SH2 domain of BCR-ABL did not result in an alteration in the complex formation of BCR-ABL and c-CBL. This suggests that another site of direct interaction between c-CBL and BCR-ABL exists or that another protein mediates an indirect interaction of c-CBL and BCR-ABL. Numerous proteins have been shown to interact with BCR-ABL, and many have been shown to be tyrosine-phosphorylated in BCR-ABL expressing cells. Besides c-CBL, these proteins include rasGAP (31), GRB-2 (39,40), SHC (41,42), FES (43), SYP (44), CRKL (45,46), the 85-kDa subunit of phosphatidylinositol 3-kinase (47), VAV (48), c-BCR (49), paxillin (50), and various other cytoskeletal proteins (51).
In addition to binding to the SH2 domain of ABL, c-CBL was shown to bind to the SH2 domain of CRKL. c-CBL is also known to bind to the SH2 domain of the 85-kDa subunit of phosphatidylinositol 3-kinase, FYN, LCK, and phospholipase C-␥ (23,52,53). Binding of c-CBL to the SH3 domain of GRB-2 has also been demonstrated; however, this association decreases when c-CBL is tyrosine-phosphorylated (23,52,54). c-CBL has also been shown to co-immunoprecipitate with paxillin (29). Since many of these proteins also bind to BCR-ABL, they are candidates for mediating the indirect interaction of CBL with BCR-ABL. A model of the potential interactions of these proteins with each other and BCR-ABL is presented in Fig. 6. Consistent with this model, c-CBL immunoprecipitates from BCR-ABL cells not only contain BCR-ABL but also contain CRKL (29). As supported by our data, tyrosine-phosphorylated c-CBL binds to the SH2 domain of ABL in BCR-ABL and to the SH2 domain of CRKL. Thus, c-CBL interacts both directly and indirectly with BCR-ABL. That is, tyrosine-phosphorylated CBL interacts directly with BCR-ABL by binding to the SH2 domain of ABL. CBL also interacts indirectly with BCR-ABL through CRKL. We have shown previously that a proline-rich region of BCR-ABL interacts directly with CRKL (19), and as shown here, the SH2 domain of CRKL interacts with c-CBL, thus allowing an indirect interaction of BCR-ABL and CBL.
A prediction of this model would be that BCR-ABL should be capable of binding to the GST-SH2 domain of CRKL by virtue of tyrosine-phosphorylated CBL binding to BCR-ABL and the SH2 domain of CRKL. This prediction was confirmed in our GST-fusion protein experiments. Similarly, the SH3 domain of CRKL bound CBL from lysates of cells expressing full-length BCR-ABL as the SH3 domain of CRKL binds to BCR-ABL which binds through its SH2 domain to c-CBL. This interaction was abolished in cells expressing the SH2 domain deletion of BCR-ABL as modeled in Fig. 6C.
It is possible that more complicated complexes occur or that proteins other than CRKL mediate an indirect interaction between CBL and BCR-ABL. For example, the SH2 domain of GRB-2 is known to bind to Tyr-177 of BCR-ABL, and the SH3 domain of GRB-2 has been shown to bind to CBL (23). We have not seen any defect in tyrosine phosphorylation of c-CBL in cells expressing the tyrosine to phenylalanine mutant of BCR-ABL that abolishes binding of BCR-ABL to GRB-2 (data not shown). However, this mutant would be expected to bind c-CBL through an intact SH2 domain. As tyrosine-phosphorylated CBL also binds to the SH2 domain of CRKL, it is possible that GRB-2 binding to BCR-ABL allows CRKL to associate with BCR-ABL through this interaction with CBL. Given the multitude of complexes induced by BCR-ABL, combinations of mutants (e.g. Tyr-177 to Phe with a deletion of the SH2 domain and a deletion of proline-rich sequences in the C terminus of ABL) will be required to determine the roles of various signaling proteins in BCR-ABL transformation. Results of these experiments may not allow an assessment of the requirement of an individual protein for transformation by BCR-ABL as these mutants might abolish complex formation of several proteins with BCR-ABL. Assessment of the role of individual proteins FIG. 7. Binding of BCR-ABL proteins to bacterially expressed CRKL. Lysates from 32D, 32Dp210, or 32Dp210⌬P1,P2 cells were analyzed for binding to (A) GST-CRKL SH3n or (B) GST-CRKL SH2. Bound proteins were separated by SDS-PAGE and transferred to PVDF membranes. ABL proteins were detected using the 24 -21 ABL monoclonal antibody. The 32Dp210 and 32Dp210⌬P1,P2 lysates were normalized for BCR-ABL expression prior to this analysis. C, lysates from 32Dp210, 32Dp210⌬SH2, or 32Dp210⌬P1,P2 cells were analyzed for binding to the indicated GST-CRKL construct. The GST-CRKL SH3 contains both the N-and C-terminal SH3 domains of CRKL, and consistently less binding to this construct is seen as compared with the N-terminal SH3 domain alone. may require the use of cell lines generated from mice lacking some of these signaling proteins.