The BCR/ABL oncogene alters interaction of the adapter ... - Nature

Leukemia (1997) 11, 376–385  1997 Stockton Press All rights reserved 0887-6924/97 $12.00

The BCR/ABL oncogene alters interaction of the adapter proteins CRKL and CRK with cellular proteins N Uemura, R Salgia, J-L Li, E Pisick, M Sattler and JD Griffin Division of Hematologic Malignancies, Dana-Farber Cancer Institute and Harvard Medical School, 44 Binney Street, Boston, MA 02115, USA

The Philadelphia chromosome translocation generates a chimeric oncogene, BCR/ABL, which causes chronic myelogenous leukemia (CML). In primary leukemic neutrophils from patients with CML, the major tyrosine phosphorylated protein is CRKL, an SH2-SH3-SH3 adapter protein which has an overall homology of 60% to CRK, the human homologue of the v-crk oncogene. In cell lines transformed by BCR/ABL, CRKL was tyrosine phosphorylated, while CRK was not. We looked for changes in CRK- and CRKL-binding proteins in Ba/F3 hematopoietic cell lines which were transformed by BCR/ABL. Anti-CRK II or antiCRKL immunoprecipitates were probed by far Western blotting with CRK II- or CRKL-GST fusion proteins to display CRK- and CRKL-coprecipitating proteins. There was a striking qualitative difference in the proteins coprecipitating with CRKL and CRK II. In untransformed cells, three major proteins coprecipitated with CRKL, identified as C3G, SOS and c-ABL. Each of these proteins was found to interact with the CRKL-SH3 domains, but not the SH2 domain. After BCR/ABL transformation, the CRKL SH3-domain binding proteins did not change, with the exception that BCR/ABL now coprecipitated with CRKL. Compared to CRKL, very few proteins coprecipitated with CRK II in untransformed, quiescent cells. After BCR/ABL transformation, both the CRKL- and CRK-SH2 domains bound to a new complex of proteins of approximate molecular weight 105–120 kDa. The major protein in this complex was identified as p120CBL. Thus, in these hematopoietic cell lines, CRKL is involved to a greater extent than CRK II in normal signaling pathways that involve c-ABL, C3G and SOS. In BCR/ABL-transformed cells, CRKL but not CRK II, appears to form complexes which potentially link BCR/ABL, c-ABL, C3G, and SOS to the protooncoprotein, p120 CBL. Keywords: chronic myelogenous leukemia; BCR/ABL; signal transduction; adapter proteins; CRKL; CRK

Introduction The t(9;22) Philadelphia chromosome translocation generates a chimeric oncogene, BCR/ABL, which causes chronic myelogenous leukemia (CML). The BCR/ABL oncogene generates a fusion protein, p210BCR/ABL, which is translocated to the cytoskeleton and activated as a tyrosine kinase.1–3 In cell lines either derived from patients with advanced phase CML or generated by transfection of the BCR/ABL oncogene, there are many cellular proteins which are constitutively tyrosine phosphorylated by p210BCR/ABL, directly or indirectly, but the importance of these phosphoproteins for transformation is largely unknown. By contrast, in the early (stable) phase of the leukemia, there are only a few proteins which either interact with BCR/ABL or are phosphorylated by BCR/ABL. In earlier studies, we and others identified a 39 kDa tyrosine phosphoprotein complexed with BCR/ABL in CML stable phase neutrophils to be the adapter protein, CRKL.4–7. The CRKL protein has an overall amino acid homology of 60% to CRK II, one of the two products generated through Correspondence: JD Griffin Received 29 October 1996; accepted 4 December 1996

alternative splicing of the human CRK proto-oncogene.8,9 CRKL has the same overall organization as CRK II, consisting of one N-terminal SH2 domain followed by two SH3 domains, and does not contain other known functional motifs.9 CRK I lacks the C-terminal SH3 domain of CRK II and CRKL, and overexpression of CRK I, but not CRK II, leads to transformation of mammalian fibroblasts.8 v-crk is the oncogene in the avian retrovirus CT10, and relative to CRK, v-Crk has a deletion of the C-terminal SH3 domain and the major tyrosine phosphorylation site at tyr221.10 Tyrosine phosphorylation on CRK tyr221 creates an intramolecular binding site for the CRK SH2 domain, possibly inhibiting its binding to other proteins.11,12 The functions of CRK and CRKL in normal signaling are unknown although recent studies have linked CRK to signaling in normal T cells,13 and we have recently observed that CRKL is involved in signaling pathways activated by integrins.14 Both CRK I and v-Crk have been shown to bind to specific proline-rich sequences in c-Abl through the CRK SH3 domain.11,15 Several CRK or v-Crk binding proteins have been identified, including C3G, EPS15, CBL, ABL, SOS and paxillin.5,11,15–19 Proteins which bind to bacterially expressed CRK and CRKL in vitro have been examined by Feller et al20 and shown to be similar, including C3G and SOS. Interestingly, despite the structural similarities of CRK II and CRKL, and the known interaction of c-CRK II with c-Abl, our earlier studies indicated that CRKL is phosphorylated and bound to p210BCR/ABL in CML cells, while CRKI and CRK II are not.5,6 In other preliminary studies, we also identified one protein, the focal adhesion protein paxillin, which is bound to CRKL, but not to CRKI or CRK II, in CML cells, and further showed that this interaction is specifically induced by the BCR/ABL oncogene.5 Also, de Jong et al21 recently demonstrated an interaction between CRKL and p120CBL in BCR/ABL-positive cell lines, suggesting that CRKL could be involved in linking BCR/ABL to cellular signaling pathways. In contrast to CRK, there is still little known about the involvement of CRKL in signaling pathways of either normal or malignant cells. In an effort to compare in a more general manner the interactions of CRKL and CRK II in normal and BCR/ABL transformed cells, we have generated monoclonal antibodies to CRKL, and used them to compare CRKL and CRK binding proteins in untransformed and BCR/ABL-transformed hematopoietic cell lines. Materials and methods

Cell lines and culture The murine IL-3-dependent pro-B cell line, Ba/F3, was obtained from Dr Alan D’Andrea (Dana-Farber Cancer Institute) and cultured in RPMI 1640 medium containing 10% fetal calf serum (FCS) and 10% WEHI-3B cell-conditioned medium (WEHI-CM; as a source of IL-3). Ba/F3 cells express-

CRKL binding proteins in transformed hematopoietic cell lines N Uemura et al

ing p210BCR/ABL were generated using previously described methods.22 NIH3T3, CTLL, U937, HL-60, Daudi, Ramos, Jurkat, H9, K562, BV173, Hela, HepG2 and Cos cells were obtained from the ATCC (Rockville, MD, USA) and were cultured in RPMI 1640 with 10% FCS. 32Dcl3 cells were obtained from Dr Joel Greenberger (University of Pittsburgh), and were cultured as for Ba/F3 cells.23 Mo7e cells24 were obtained from Dr Steven Clark (Genetics Institute, Cambridge, MA) and cultured in Dulbecco’s modified Eagle’s medium with 20% FCS and rhGM-CSF at 10 ng/ml. TF-1 cells were obtained from the ATCC and cultured in RPMI 1640 with 10% FCS and rhGM-CSF at 10 ng/ml.

Antibodies and Reagents GST fusion proteins containing full length CRKL (GST-CRKL)CRKL-SH2, GST-CRKL-SH2-SH3(N) and GST-CRKL-SH3(N)SH3(C), were obtained from Dr John Groffen (Children’s Hospital, Los Angeles, CA) and generated as described previously.5 GST-CRK fusion proteins were obtained from Dr Bruce Mayer (Children’s Hospital, Boston, MA, USA). To generate anti-CRKL monoclonal antibodies, female Balb/c mice were immunized with a series of five biweekly subcutaneous injections of purified GST-CRKL fusion protein (10 mg per injection). Spleen cells of immunized mice and NS-1 myeloma cells were fused with polyethylene glycol, and hybridomas screened by enzyme-linked immunosorbent assay (ELISA) and immunoblot. Two monoclonal antibodies recognizing different epitopes were further characterized (mAb 2-2 and mAb 5-6). Isotypes of antibodies were determined by class-specific anti-mouse immunoglobulin antibodies (Amersham, Arlington Heights, IL, USA). Epitopes of antibodies were mapped by immunoblotting on GST-CRKL fusion proteins. Anti-CRK monoclonal antibody was obtained from Transduction Laboratories (Lexington, KY, USA). Anti-phosphotyrosine mAb, 4G1025 was kindly provided by Dr Brian Druker (University of Oregon Health Sciences Center, Portland, OR, USA). Anti-Abl mAb (Ab-3 clone) was purchased from Oncogene Science (Manhasset, NY, USA). Anti-Sos-1 antibody for immunoblotting was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA), anti-Sos-1 antibody for immunoprecipitation was purchased from Transduction Laboratories, anti-C3G polyclonal Ab, and anti-p120CBL polyclonal antibody were purchased from Santa Cruz Biotechnology. Normal mouse serum was used as a negative control antibody for immunoprecipitation experiments.

Immunoblotting and immunoprecipitations Cells were washed with ice cold phosphate-buffered saline, and were lysed in buffer containing 50 mM Tris (pH 8.0), 150 mM NaCl, 1% NP-40 (w/v), 0.5% deoxycholic acid (w/v), 100 mM NaF, 1 mM phenylmethylsulfonyl fluoride, 20 mg/ml aprotinin, 1 mM sodium orthovanadate, and 40 mg/ml leupeptin at 108 cells/ml. After incubation on ice for 20 min, cell lysates were centrifuged at 12 000 g for 15 min. The resulting supernatants were subjected to immunoprecipitation or immunoblotting directly as whole cell lysates. For immunoprecipitation, cell lysates were incubated with anti-CRKL antibodies (2-2 or 5-6), anti-CRK antibody or preimmune mouse serum and protein A sepharose (for mAb 5-6 or preimmune mouse serum) or protein G sepharose (for mAb 2-2) for 3 h at 4°C. After incubation, supernatants were saved as immuno-

depleted cell lysates and immune complexes were then washed five times with lysis buffer and dissolved in sample buffer by boiling for 5 min. Immunodepleted cell lysates were subjected to immunoblotting after being concentrated by Microcon 10 (Amicon, Beverly, MA, USA). Protein samples dissolved in Laemmli’s sample buffer were separated under reducing conditions by SDS-polyacrylamide gel electrophoresis (5–10% gradient gels) and electrophoretically transferred to Immobilon PVDF membranes (Millipore, Bedford, MA, USA). The membranes were blocked with 5% non-fat dry milk in TBS (10 mM Tris-HCl, pH 8.0, 150 mM NaCl) and probed with primary antibodies for 2 h. After washing, membranes were further probed with HRP-coupled secondary antibodies for 1 h, washed again and subjected to the ECL chemiluminescence system (Amersham).

Far Western blotting Ba/F3 cells were incubated with or without rmIL-3 at 10 ng/ml for 5 min after starvation in RPMI 1640 with 0.5% bovine serum albumin without WEHI-CM for 16 h. Unstimulated and IL-3-stimulated Ba/F3 and Ba/F3-p210 cells were lysed as described above and the lysates were subjected to immunoprecipitation using anti-CRKL mAb 5-6, anti-CRK antibody, or preimmune mouse serum. Immunoprecipitates were then subjected to SDS-PAGE and transferred to Immobilon PVDF membranes as described above. The membranes were blocked with 5% non-fat dry milk in PBS-T (0.1% Tween 20 in PBS, pH 7.4) and probed with GST-CRKL, GST-CRKL-SH2, GST-CRKL-SH3(N)-SH3(C), GST-CRK, GST-CRK-SH2, or GSTCRK-SH3(N) fusion proteins or GST protein (as a negative control) in binding buffer (1% non-fat dry milk, 25 mM Na PO4, pH 7.2, 150 mM NaCl, 0.1% Tween 20, 2.5 mM EDTA, 20 mM NaF, 1 mM dithiothreitol) with aprotinin (10 mg/ml) and leupeptin (10 mg/ml) for 2 h. After washing with PBS-T, membranes were probed with anti-GST mAb (Santa Cruz Biotechnology, 1:500) for 1 h, washed again and further probed with HRP-coupled anti-mouse IgG antibody for 1 h, washed again, and then subjected to the ECL chemiluminescence system. Results

Generation and characterization of specific monoclonal antibodies against CRKL Two monoclonal antibodies against CRKL, 2-2 (IgG1) and 56 (IgG2a) were generated by immunizing mice with a GSTCRKL fusion protein. Partial epitope mapping using deletion mutants of CRKL fusion proteins revealed that mAb 2-2 recognizes an epitope in the N-terminal SH3 and mAb 5-6 recognizes an epitope in the C-terminal SH3 domain (data not shown). CRKL expression was examined in various cell lines by immunoblot (Figure 1 shows results using mAb 5-6, and the same result was obtained using mAb 2-2 (data not shown)). CRKL was detected in all cell lines examined including nonhematopoietic cell lines (NIH3T3, Hepa, HepG2 and Cos), T cell lines (CTLL, Jurkat and H9), B cell lines (Ba/F3, Daudi and Ramos), a monocytic line (U937) and myeloid cell lines (32D, HL-60, MO7e, TF-1, K562 and BV173). In other studies, both mAbs were found to recognize human, murine and monkey CRKL, but did not react with CRK II proteins from these species (data not shown).

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In studies not shown, CRK II expression was readily detected, as expected, by Western blot in all the cell lines described above using a commercially available monoclonal antibody. Also, both anti-CRKL and anti-CRK antibodies were found to precipitate .75% of the cellular CRKL or CRK, respectively, in a single precipitation. Figure 1 Expression of CRKL in cell lines. Two hundred micrograms of whole cell lysate protein from the indicated cell lines were separated by SDS-PAGE (10%), transferred to PVDF membrane, and immunoblotted with anti-CRKL mAb (5-6). Molecular weight markers are indicated in kilodaltons (kDa).

The antibodies identified two bands of CRKL protein, migrating at approximately 37 and 39 kDa, in the BCR/ABLtransformed cell line K562 (Figure 2b). Both antibodies were capable of precipitating more than 90% of CRKL from K562 cells (Figure 2c). However, immune complexes with antibody 5-6 contained reproducibly more coprecipitating tyrosine phosphoproteins than did immune complexes formed with antibody 2-2 for unknown reasons (Figure 2a), and antibody 5-6 was used in future experiments. In studies not shown, tyrosine phosphorylation of CRKL was examined in three hematopoietic cell lines before and after transformation with BCR/ABL (32Dcl3, Ba/F3 and MO7e) and in two cell lines derived from patients with CML (K562 and BV173). Tyrosine phosphorylated CRKL was found to be restricted to cell lines transformed by BCR/ABL. In K562 cells, the major tyrosine phosphoproteins in anti-CRKL immune complexes were 210, 190, 140, 120 and 85–90 kDa, in addition to p39 CRKL itself, and confirm earlier studies in K562 cells by ten Hoeve et al.18 We also observed that virtually no tyrosine phosphoproteins coprecipitated with CRKL under the conditions employed here in cell lines not transformed by BCR/ABL or in untransformed, quiescent, cells. CRK II has previously been shown to be widely expressed.26

CRK and CRKL bind to similar proteins in a direct binding assay Since CRKL and CRK are ‘adapter proteins’ and could potentially transduce signals by linking proteins through the SH2 and SH3 domains, it is important to identify proteins which bind to each domain of CRKL and CRK directly in vivo. To compare the binding of CRKL and CRK to cellular proteins, we first performed far Western blotting with GST-CRK II and GST-CRKL fusion proteins as probes on whole cell lysates from Ba/F3 and Ba/F3-p210 cells (Figure 3). Remarkably similar patterns were observed except for proteins of 60–70 kDa, indicating that both CRKL and CRK have the potential to interact with a highly overlapping population of cellular proteins in these cell lines. Both GST-CRK and GST-CRKL bound to major bands at approximate molecular weights of 190 and a series of proteins at 140–170 kDa in untransformed cells, and to a similar set of proteins in BCR/ABL-transformed Ba/F3 cells plus a 210 kDa protein and proteins at 105–120 kDa (Figure 3). The 70 kDa protein bound more to CRKL than CRK, while the 60 kDa protein bound more to CRK than CRKL. The 70 kDa protein bound through an SH3 domain interaction, while the 60 kDa protein bound through an SH2 domain interaction (data not shown).

Figure 2 Tyrosine phosphoproteins coimmunoprecipitated with two anti-CRKL monoclonal antibodies. Cell lysate from K562 cells (2 × 106 cells) was subjected to immunoprecipitation with anti-CRKL mAbs 2-2, 5-6 or normal mouse serum as a negative control (C). Immunoprecipitates (IP) and immunodepleted cell lysates were separated by SDS-PAGE (5–10% gradient gel) and transferred to PVDF membrane. (a) Anti-phosphotyrosine (4G10) blot. (b) Anti-CRKL (5-6) blot. (c) After the first immunoprecipitation, supernatants were then subjected to anti-CRKL (5-6) blotting. The heavy band at about 55 kDa represents the immunoglobulin heavy chain.


CRKL and CRK, respectively, as demonstrated by reprobing of the blots with CRKL and CRK monoclonal antibodies (Figure 4).

CRK- and CRKL-SH3 binding proteins To determine which of these proteins bind to the SH3 domains of CRKL, we performed far Western blotting using a GST-CRKL-SH3(N)-SH3(C) fusion protein as a direct probe. As shown in Figure 5, it was found that most of the proteins forming a broad 140–170 kDa band detected in both Ba/F3 and Ba/F3-p210 cells were CRKL SH3-binding proteins. In addition to these proteins, a single protein around 70 kDa detected only in untransformed Ba/F3 cells and a single protein around 210 kDa detected only in Ba/F-p210 cells were found to be CRKL-SH3-binding proteins. No proteins were detected with a CRK SH3 probe which could be identified by far Western blotting with GST-CRK-SH3 (Figure 5).

CRK- and CRKL-SH2 binding proteins

Figure 3 Direct binding of GST-CRK and GST-CRKL fusion protein in vitro to proteins from untransformed and BCR/ABL-transformed Ba/F3 cells. Whole cell lysates (0.8 × 106 cells) from unstimulated (Ba/F3(−)) and IL-3-stimulated Ba/F3 (Ba/F3(+)) and Ba/F3-p210 cells were subjected to far Western blotting using GST-CRK or GST-CRKL fusion proteins.

CRK and CRKL coprecipitate with different amounts of cellular proteins in both untransformed and BCR/ABLtransformed Ba/F3 cells The experiment shown in Figure 3 indicates that both CRK and CRKL can potentially bind effectively to an overlapping set of cellular proteins. However, in vitro binding does not necessarily reflect in vivo binding. To assess in vivo interactions of CRK and CRKL, we performed far Western blotting in which anti-CRKL (mAb 5-6) or anti-CRK immunoprecipitates were subjected to direct detection by GST-CRKL or GST-CRK II fusion proteins, respectively (as described in Materials and methods). This technique has the advantage that it is semiquantitative and that it only detects proteins which both coprecipitate with CRKL or CRK and also bind directly to CRKL or CRK, respectively. Anti-CRKL immunoprecipitates were found to contain several cellular proteins in both untransformed and BCR/ABL-transformed Ba/F3 cells when probed with GST-CRKL, while a GST probe was negative (Figure 4). In untransformed cells, the most prominent CRKL binding proteins detected were p140–170 and p70. In BCR/ABL-transformed cells, the pattern was similar except that the p70 protein was diminished, and proteins migrating at approximately 210 kDa and 105–120 kDa were also detected. No prominent proteins which are smaller than 50 kDa were detected, suggesting that CRKL might not bind to itself, at least in an inter-molecular manner. In contrast, anti-CRK precipitates did not contain detectable CRK-binding proteins in untransformed cells, although a prominent 120 kDa protein was detected in BCR/ABL-transformed cells (Figure 4). Both anti-CRKL and anti-CRK antibodies effectively precipitated

The same procedure was performed using a GST-CRKL-SH2 fusion protein as a direct probe. Two proteins around 105– 120 kDa detected only in Ba/F3-p210 cells were identified as CRKL SH2-binding proteins (Figure 6). In untransformed Ba/F3 cells, no significant bands were detected as CRKL-SH2-binding proteins by probing with a GST-CRKL-SH2 fusion protein, although a small amount of a 120 kDa protein was observed with longer development times (data not shown). Using GSTCRK-SH2 as a probe, a 120 kDa protein was detected in antiCRK immunoprecipitates in BCR/ABL transformed cells, but not in quiescent untransformed cells (Figure 6).

The major CRKL SH3-binding proteins are SOS, C3G, c-ABL and p210 BCR/ABL Previous studies have identified c-ABL, C3G and SOS as cellular proteins which can interact with c-CRK and possibly, CRKL.5,11,15–19,22 Since the molecular weights of the proteins identified by far Western blot as CRKL binding proteins were consistent with SOS, C3G and ABL, these proteins were examined directly in anti-CRKL immune complexes with specific antibodies (Figure 7). SOS, C3G and c-ABL proteins are contained within the 140–170 protein complex detected by CRKL-SH3. The p210 band was shown by anti-ABL blotting to be p210BCR/ABL. Interestingly, the binding of SOS, C3G and c-ABL proteins with CRKL is constitutive and is not changed following BCR/ABL transformation. Also, after transformation, c-ABL is not displaced by BCR/ABL, as about the same amount of c-ABL and BCR/ABL are precipitated. Thus, considering also the data shown in Figure 3, and consistent with previous studies, CRK can apparently bind through its SH3 domains to c-ABL, C3G and SOS, the amount of these proteins actually coprecipitating with CRK is significantly less than that with CRKL in the cell lines examined. This also includes BCR/ABL, thus presumably explaining the observation that CRKL, but not CRK, is an in vivo substrate of BCR/ABL. To confirm the apparent quantitative difference between CRKL- and CRK-binding proteins in Ba/F3 cells, we performed the reverse immunoprecipitations, immunoprecipitations with anti-C3G, ABL or SOS; followed by immunoblotting with antiCRK or anti-CRKL. CRKL was detected in C3G, ABL, and SOS

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Figure 4 Detection of proteins coprecipitating with CRK and CRKL in untransformed and BCR/ABL-transformed Ba/F3 cells. Lysates from 15 × 106 unstimulated Ba/F3 cells (Ba/F3(−)), Ba/F3 cells stimulated with IL-3 (Ba/F3(+)), or Ba/F3-p210 cells were immunoprecipitated with antiCRKL mAb (5-6), anti-CRK mAb, or preimmune mouse serum as a negative control (C). Immunoprecipitates were then separated by SDS-PAGE and transferred to PVDF membranes. Proteins on the membranes were probed with GST-CRK, GST-CRKL fusion proteins or GST alone as a control. The heavy band at about 55 kDa represents the immunoglobulin heavy chain of the antibody used for immunoprecipitation.

Figure 5 Detection of CRK- and CRKL-binding proteins using CRK-SH3 and CRKL-SH3 fusion proteins as probes. The membranes used were prepared as described in the legend for Figure 4 using antiCRK and anti-CRKL immune complexes. Proteins on the membranes were probed with GST-CRK-SH3(N) or GST-CRKL-SH3(N)-SH3(C) fusion proteins. The heavy band at about 55 kDa represents the immunoglobulin heavy chain of the antibody used for immunoprecipitation.

Figure 6 Detection of CRK- and CRKL-binding proteins using CRK-SH2 and CRKL-SH2 fusion proteins as probes. The membranes used were prepared as described in the legend for Figure 4. The proteins on the membranes were probed with GST-CRK-SH2 or GSTCRKL-SH2 fusion proteins. The heavy band at about 55 kDa represents the immunoglobulin heavy chain of the antibody used for immunoprecipitation.


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Figure 7 Identification of major CRKL-SH3-binding proteins. The membranes used for far Western blotting were stripped and reprobed with specific antibodies against c-ABL, SOS or C3G. The heavy band at about 55 kDa represents the immunoglobulin heavy chain of the antibody used for immunoprecipitation.

immunoprecipitates, while CRK was not (Figure 8). The presence of BCR/ABL did not affect the amount of CRKL detected, except for anti-ABL immunoprecipitates. These results are consistent with the notion that CRKL is a more abundant binding partner than CRK for C3G, c-ABL and SOS. It is interesting that a 70 kDa CRKL-SH3-binding protein was detected only in untransformed Ba/F3 cells, and disappears after BCR/ABL transformation. A 70 kDa protein also binds to CRKL-SH3 in another murine hematopoietic cell line, 32D, and is again diminished dramatically in BCR/ABL-transformed 32D cells (data not shown). The identity of this protein is currently unknown.

p120CBL antibody (Figure 9), consistent with the observations of de Jong21, Ribon27 and our previous studies.22 This result suggests that CRKL could form complexes in vivo that link cABL, BCR/ABL, or guanine nucleotide exchange factors, such as C3G and SOS, with a protooncoprotein, p120CBL, in BCR/ABL-transformed cells. The effects of this complex on transformation will be interesting to study. CRK may also form such complexes,27 but our data suggest that CRKL is quantitatively more important. Overall, these observations suggest a mechanism to explain the observations by our group and others that p120CBL is a prominent target of BCR/ABL both in cell lines and in primary leukemic cells from patients with CML.

The major CRK-SH2 and CRKL-SH2-binding protein is p120CBL

Discussion

One of the 120 kDa proteins detected by CRKL-SH2 and CRKSH2 in BCR/ABL-transformed cells was identified as p120CBL, by probing anti-CRKL immunoprecipitates with an anti-

BCR/ABL is a tyrosine kinase oncogene which transforms hematopoietic cells in vivo and in vitro, but does not effectively transform many non-hematopoietic cell lines.28 The

Figure 8 CRKL is more abundant than CRK in ABL, SOS or C3G immunoprecipitates. The cells used are Ba/F3 and Ba/F3-p210 cells. Whole cell lysate (WCL; 1 × 106 cells) or immunoprecipitates of cell lysate (70 × 106 cells) with anti-Abl, anti-Sos, anti-C3G or preimmune mouse serum (as a negative control; C) antibodies were processed as described. Membranes were immunoblotted with anti-CRK or anti-CRKL (56) antibodies.


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Figure 9 Identification of the major CRKL-SH2-binding protein. The membranes used for far Western blotting were stripped and reprobed with a specific antibody against p120CBL .

chimeric oncoprotein p210BCR/ABL is known to bind the actin cytoskeleton through a conserved C-terminal domain in c-ABL and this interaction is believed to be important for transformation.2,3 It has been suggested that p210BCR/ABL recruits signaling proteins which ultimately affect adhesion, proliferation and viability.29–33 In the stable phase of the disease, the tyrosine kinase activity of BCR/ABL is apparently lower than during the blast phase, and increased tyrosine phosphorylation is detectable on only a few cellular proteins. One of these proteins, CRKL, is phosphorylated in leukemic cells from patients both early and late in their course, and has been shown to physically interact with BCR/ABL through its SH3 domains.4,6,7,21 The SH2 and SH3 domains of CRKL are related to CRK II, and in vitro binding studies suggest that the two adapter proteins bind to a highly overlapping set of proteins. However, in vivo, CRK II and CRKL may or may not have related functions. In the studies reported here, we have carefully compared the protein interactions of CRKL and CRK in a hematopoietic cell line, both untransformed and transformed by BCR/ABL. Specifically, we used a combination of immunoprecipitation and far Western blotting to examine CRKL- and CRKassociated proteins. When anti-CRKL monoclonal antibody

immunoprecipitates were probed with GST-CRKL (full length), a complex of CRKL-coprecipitating proteins from 140– 170 kDa were the major proteins detected in non-transformed hematopoietic cells. In the same cell line transformed by BCR/ABL, the 140–170 kDa complex was detected as well as a 210 kDa protein and proteins at 105–120 kDa. Using GSTCRKL-SH2 and -SH3 domain fusion proteins as probes, the 140–170 kDa series of proteins in both untransformed and transformed cells and the 210 kDa protein were found to bind directly to the SH3 domain of CRKL, but not the CRKL SH2 domain. In contrast, the 105–120 kDa proteins observed only in BCR/ABL-transformed cells bound only to the CRKL SH2 domain. These data suggest that in untransformed cells, CRKL is in a complex with several proteins through its SH3 domain(s), but that its SH2 domain is largely uncomplexed in quiescent cells. It will be of interest to determine if the CRKL SH2 domain can be induced to bind to cellular proteins following activation by integrin crosslinking, adhesion, or stimulation by other growth factors or cytokines. Interestingly, when these studies were repeated with antiCRK monoclonal antibodies, CRKL and CRK II were found to be quantitatively different in terms of association with cellular proteins in both untransformed and BCR/ABL-transformed Ba/F3 cells, although quite similar when tested by direct far Western analysis of total cellular proteins. While proteins coprecipitating with CRKL through interactions mediated by the CRKL SH3 domains were readily identified, proteins were difficult to detect in anti-CRK immunoprecipitates. This was not due to failure to adequately immunoprecipitate CRK II, failure of the GST-CRK II fusion proteins to function as probes in far Western blots, or competition between the anti-CRK antibody and cellular proteins for binding to the same site on CRK II. It is quite possible that small amounts of proteins are bound to the CRK II SH3 domains, below the limit of detection of the technique used here. In fact, in other cell systems, binding of CRK II to cellular proteins such as C3G, SOS and cABL has been readily demonstrated. 11,13,15,20,27,34–38 None the less, the available data suggest that there is at least a quantitative difference between CRKL and CRK II in binding to proteins through the SH3 domains in these hematopoietic cell lines. Previous studies with CRK II have suggested that the SH2 domain binds intramolecularly to the ptyr-X-X-pro motif containing the major tyrosine phosphorylation site at tyr221.11 This would block interaction of the SH2 domain with cellular proteins and could also interfere with access to the SH3 domains. However, in both the untransformed and BCR/ABL-transformed cell lines tested here, CRK was not detectably tyrosine phosphorylated, and it is therefore not likely that intramolecular binding of CRK SH2 is the explanation for the relatively reduced amount of CRK SH3 binding proteins. The possibility that the CRKL SH2 can undergo any type of intramolecular binding has not been assessed. We did not observe direct binding in vitro of CRKL-SH2 to phosphoCRKL when antiCRKL immune complexes were probed with a GST-CRKL-SH2 fusion protein in a far Western blot, although this does not prove that intramolecular binding does not occur in vivo. The immunoprecipitation/far Western assay used is semiquantitative since the fusion protein probes are used in excess during the direct binding assay. However, if there are multiple SH2-binding sites on a protein, the assay would overestimate the abundance of that protein relative to others in the immune complex. It is likely that a fraction of the CRKL molecules are involved in a small number of protein complexes in quiescent, untransformed cells, and that several novel protein complexes are formed after transformation. In untransformed cells, CRKL


is complexed through its SH3 domains to a group of proteins which include C3G, SOS and c-ABL.11,20,36 It is not possible to compare precisely the relative amounts of these three proteins, although preliminary serial depletion studies suggest that C3G is the most abundant and c-ABL the least abundant. We do not know if both SH3 domains of one CRKL molecule can be involved in separate binding interactions. If not, which seems likely, then there may be separate complexes containing CRKL and C3G, SOS, and c-ABL. It would be of interest to compare CRK II and CRKL binding proteins in other cell types, and the approach used here would facilitate such a comparison. C3G and SOS were examined in this study because of the observed molecular weights of the CRKL binding proteins and because of previous studies identifying possible CRK binding proteins. C3G was originally identified as a protein which could bind to the SH3 domain of CRK, and the binding site for the first CRK II SH3 domain was identified as a proline-rich sequence in C3G containing an important lysine residue.38,39 Sequence analysis of C3G suggested it was a guanine nucleotide exchange factor, and subsequent studies confirmed that C3G had exchange factor activity in vitro for the small GTP binding protein, Rap1, but not for p21ras.40 SOS is a well known guanine nucleotide exchange factor with in vitro activity for p21ras. SOS has been linked to the control of p21ras function in signaling pathways activated by several growth factor receptors, typically through binding to the two SH3 domains of GRB2. It is not yet known if the binding sites for the SH3 domain of CRKL overlap with the binding sites for the SH3 domains of GRB2. It is possible, however, that SOS can be linked to different cellular proteins through GRB2 and CRKL. It is also possible that individual SOS molecules bind both GRB2 and CRKL and that the resulting complexes contain other proteins depending on the pattern of tyrosine phosphorylation. There is abundant evidence that BCR/ABL can activate the p21ras pathway,25,41–45 and several investigators feel that this is an important pathway for transformation. Thus, the data presented here suggest that in addition to a direct link to SOS through binding of GRB2 to tyr177 of BCR/ABL,46 binding of CRKL to SOS could also affect SOS function. In contrast to GRB2, there are no data suggesting that CRKL directly links BCR/ABL to SOS, since both BCR/ABL and SOS interact with the CRKL SH3 domains. The interaction of CRKL with c-ABL in both untransformed and transformed cells is of considerable interest. Our data suggest that this association is constitutive, and not further increased in response to mitogens. The function of c-ABL remains largely unknown, although recent studies have linked ABL prominently to cell cycle control in the nucleus, and as part of the response to DNA damage induced by radiation.47 c-ABL is thought to be predominantly nuclear, while our studies indicate that CRKL is predominantly cytoplasmic (Uemura and Griffin, unpublished). We do not yet know if the c-ABL and CRKL interaction observed in these studies takes place in the nucleus, the cytoplasm or both. It is of interest that the major ABL-binding protein in hematopoietic cells is CRKL, while in non-hematopoietic cells, CRK II is also involved. In cells transformed by BCR/ABL, one novel CRKL binding protein was identified, p120CBL. p120CBL is the product of the widely expressed proto-oncogene c-p120CBL (for Casitas B lineage lymphoma).48 v-cbl is the oncogene in the CAS NS-1 retrovirus and was generated by a C-terminal truncation of cCbl.49,50 The CAS NS-1 retrovirus induces pre-B cell lymphomas and some myelogenous leukemias in mice. Recently, Andoniou and colleagues51 reported that p120CBL is phos-

phorylated on tyrosine residues in cells transformed by activated ABL oncogenes, and also that p120CBL and BCR/ABL form a complex. Furthermore, de Jong et al have reported that p120CBL binds CRKL in the K562 CML cell line. Thus, our data confirm that of de Jong et al, and suggests that CRKL could function to link BCR/ABL to p120CBL, and further suggests that p120CBL may be linked to other cellular proteins through CRKL, including C3G, SOS and c-ABL. p120Cbl may be of particular interest as a BCR/ABL substrate for several reasons. In normal cells, p120CBL is involved in signal transduction pathways in normal cells associated with proliferation or activation. For example, p120CBL is a common substrate of tyrosine kinases activated after cytokine receptor stimulation.52,53 Also, we have recently shown that p120CBL is tyrosine phosphorylated in hematopoietic cells in response to integrin crosslinking.54 The downstream signaling pathways are unknown, but p120CBL is physically associated with the p85 subunit of phosphoinositol-3-kinase in both activated normal cells55,56 and in BCR/ABL-transformed cells.22 The function of c-Cbl is unknown, however. A homologue of c-Cbl in C. elegans, Sli-1, is a negative regulator of tyrosine kinase signaling.57 The results presented here confirm that CRKL functions as a linker protein in CML cells.6,7,21 At the present time, the role of CRKL-containing complexes in transformation remains speculative. The fact that several of the proteins identified are known to play a role in integrin signaling is of interest, since significant adhesion defects in CML progenitor cells have been described by Verfaillie, Gordon, and our own groups.58–60 Further studies will be necessary to determine if these interactions lead to aberrant adhesive properties or other biological effects. Overall, we suggest that the interaction of BCR/ABL with p120CBL through CRKL, but not CRK, could contribute to the known signaling abnormalities which cause CML.

Acknowledgements This work was supported by NIH grants CA36167 (JDG) and CA60821 (RS).

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