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

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 chim- alternative splicing of the human CRK proto-oncogene.8,9 eric oncogene, BCR/ABL, which causes chronic myelogenous CRKL has the same overall organization as CRK II, consisting leukemia (CML). In primary leukemic neutrophils from patients of one N-terminal SH2 domain followed by two SH3 domains, with CML, the major tyrosine phosphorylated protein is CRKL, 9 an SH2-SH3-SH3 adapter protein which has an overall hom- and does not contain other known functional motifs. CRK I ology of 60% to CRK, the human homologue of the v-crk onco- lacks the C-terminal SH3 domain of CRK II and CRKL, and gene. In cell lines transformed by BCR/ABL, CRKL was tyrosine overexpression of CRK I, but not CRK II, leads to transform- phosphorylated, while CRK was not. We looked for changes in ation of mammalian fibroblasts.8 v-crk is the oncogene in the CRK- and CRKL-binding proteins in Ba/F3 hematopoietic cell avian retrovirus CT10, and relative to CRK, v-Crk has a lines which were transformed by BCR/ABL. Anti-CRK II or anti- deletion of the C-terminal SH3 domain and the major tyrosine CRKL immunoprecipitates were probed by far Western blotting 221 10 with CRK II- or CRKL-GST fusion proteins to display CRK- and phosphorylation site at tyr . Tyrosine phosphorylation on CRKL-coprecipitating proteins. There was a striking qualitative CRK tyr221 creates an intramolecular binding site for the CRK difference in the proteins coprecipitating with CRKL and CRK SH2 domain, possibly inhibiting its binding to other pro- II. In untransformed cells, three major proteins coprecipitated teins.11,12 The functions of CRK and CRKL in normal signaling with CRKL, identified as C3G, SOS and c-ABL. Each of these are unknown although recent studies have linked CRK to sig- proteins was found to interact with the CRKL-SH3 domains, but 13 not the SH2 domain. After BCR/ABL transformation, the CRKL naling in normal T cells, and we have recently observed that SH3-domain binding proteins did not change, with the excep- CRKL is involved in signaling pathways activated by inte- 14 tion that BCR/ABL now coprecipitated with CRKL. Compared grins. Both CRK I and v-Crk have been shown to bind to to CRKL, very few proteins coprecipitated with CRK II in specific proline-rich sequences in c-Abl through the CRK SH3 untransformed, quiescent cells. After BCR/ABL transformation, domain.11,15 Several CRK or v-Crk binding proteins have been both the CRKL- and CRK-SH2 domains bound to a new com- identified, including C3G, EPS15, CBL, ABL, SOS and paxil- plex of proteins of approximate molecular weight 105–120 kDa. 5,11,15–19 CBL lin. Proteins which bind to bacterially expressed CRK The major protein in this complex was identified as p120 . 20 Thus, in these hematopoietic cell lines, CRKL is involved to and CRKL in vitro have been examined by Feller et al and a greater extent than CRK II in normal signaling pathways shown to be similar, including C3G and SOS. that involve c-ABL, C3G and SOS. In BCR/ABL-transformed Interestingly, despite the structural similarities of CRK II and cells, CRKL but not CRK II, appears to form complexes which CRKL, and the known interaction of c-CRK II with c-Abl, our potentially link BCR/ABL, c-ABL, C3G, and SOS to the proto- earlier studies indicated that CRKL is phosphorylated and oncoprotein, p120CBL. bound to p210BCR/ABL in CML cells, while CRKI and CRK II Keywords: chronic myelogenous leukemia; BCR/ABL; signal trans- 5,6 duction; adapter proteins; CRKL; CRK are not. 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 Introduction showed that this interaction is specifically induced by the BCR/ABL oncogene.5 Also, de Jong et al21 recently demon- CBL The t(9;22) Philadelphia chromosome translocation generates strated an interaction between CRKL and p120 in a chimeric oncogene, BCR/ABL, which causes chronic myel- BCR/ABL-positive cell lines, suggesting that CRKL could be ogenous leukemia (CML). The BCR/ABL oncogene generates involved in linking BCR/ABL to cellular signaling pathways. a fusion protein, p210BCR/ABL, which is translocated to the In contrast to CRK, there is still little known about the cytoskeleton and activated as a tyrosine kinase.1–3 In cell lines involvement of CRKL in signaling pathways of either normal either derived from patients with advanced phase CML or gen- or malignant cells. In an effort to compare in a more general erated by transfection of the BCR/ABL oncogene, there are manner the interactions of CRKL and CRK II in normal and many cellular proteins which are constitutively tyrosine phos- BCR/ABL transformed cells, we have generated monoclonal phorylated by p210BCR/ABL, directly or indirectly, but the antibodies to CRKL, and used them to compare CRKL and CRK importance of these phosphoproteins for transformation is lar- binding proteins in untransformed and BCR/ABL-transformed gely unknown. By contrast, in the early (stable) phase of the hematopoietic cell lines. 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 phospho- Materials and methods protein complexed with BCR/ABL in CML stable phase neutrophils to be the adapter protein, CRKL.4–7. Cell lines and culture The CRKL protein has an overall amino acid homology of 60% to CRK II, one of the two products generated through 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% Correspondence: JD Griffin fetal calf serum (FCS) and 10% WEHI-3B cell-conditioned Received 29 October 1996; accepted 4 December 1996 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 377 ing p210BCR/ABL were generated using previously described depleted cell lysates and immune complexes were then methods.22 NIH3T3, CTLL, U937, HL-60, Daudi, Ramos, Jur- washed five times with lysis buffer and dissolved in sample kat, H9, K562, BV173, Hela, HepG2 and Cos cells were buffer by boiling for 5 min. Immunodepleted cell lysates were obtained from the ATCC (Rockville, MD, USA) and were cul- subjected to immunoblotting after being concentrated by tured in RPMI 1640 with 10% FCS. 32Dcl3 cells were Microcon 10 (Amicon, Beverly, MA, USA). Protein samples obtained from Dr Joel Greenberger (University of Pittsburgh), dissolved in Laemmli’s sample buffer were separated under and were cultured as for Ba/F3 cells.23 Mo7e cells24 were reducing conditions by SDS-polyacrylamide gel electro- obtained from Dr Steven Clark (Genetics Institute, Cambridge, phoresis (5–10% gradient gels) and electrophoretically trans- MA) and cultured in Dulbecco’s modified Eagle’s medium ferred to Immobilon PVDF membranes (Millipore, Bedford, with 20% FCS and rhGM-CSF at 10 ng/ml. TF-1 cells were MA, USA). The membranes were blocked with 5% non-fat dry obtained from the ATCC and cultured in RPMI 1640 with 10% milk in TBS (10 mM Tris-HCl, pH 8.0, 150 mM NaCl) and pro- FCS and rhGM-CSF at 10 ng/ml. bed 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 Antibodies and Reagents chemiluminescence system (Amersham).

GST fusion proteins containing full length CRKL (GST-CRKL)- CRKL-SH2, GST-CRKL-SH2-SH3(N) and GST-CRKL-SH3(N)- Far Western blotting SH3(C), were obtained from Dr John Groffen (Children’s Hos- pital, Los Angeles, CA) and generated as described pre- Ba/F3 cells were incubated with or without rmIL-3 at 10 ng/ml viously.5 GST-CRK fusion proteins were obtained from Dr for 5 min after starvation in RPMI 1640 with 0.5% bovine Bruce Mayer (Children’s Hospital, Boston, MA, USA). To gen- serum albumin without WEHI-CM for 16 h. Unstimulated and erate anti-CRKL monoclonal antibodies, female Balb/c mice IL-3-stimulated Ba/F3 and Ba/F3-p210 cells were lysed as were immunized with a series of five biweekly subcutaneous described above and the lysates were subjected to immuno- injections of purified GST-CRKL fusion protein (10 ␮g per precipitation using anti-CRKL mAb 5-6, anti-CRK antibody, or injection). Spleen cells of immunized mice and NS-1 myel- preimmune mouse serum. Immunoprecipitates were then sub- oma cells were fused with polyethylene glycol, and jected to SDS-PAGE and transferred to Immobilon PVDF hybridomas screened by enzyme-linked immunosorbent assay membranes as described above. The membranes were (ELISA) and immunoblot. Two monoclonal antibodies recog- blocked with 5% non-fat dry milk in PBS-T (0.1% Tween 20 nizing different epitopes were further characterized (mAb 2-2 in PBS, pH 7.4) and probed with GST-CRKL, GST-CRKL-SH2, and mAb 5-6). Isotypes of antibodies were determined by GST-CRKL-SH3(N)-SH3(C), GST-CRK, GST-CRK-SH2, or GST- class-specific anti-mouse immunoglobulin antibodies CRK-SH3(N) fusion proteins or GST protein (as a negative (Amersham, Arlington Heights, IL, USA). Epitopes of anti- control) in binding buffer (1% non-fat dry milk, 25 mM Na bodies were mapped by immunoblotting on GST-CRKL fusion PO4, pH 7.2, 150 mM NaCl, 0.1% Tween 20, 2.5 mM EDTA, proteins. Anti-CRK monoclonal antibody was obtained from 20 mM NaF, 1 mM dithiothreitol) with aprotinin (10 ␮g/ml) Transduction Laboratories (Lexington, KY, USA). Anti-phos- and leupeptin (10 ␮g/ml) for 2 h. After washing with PBS-T, photyrosine mAb, 4G1025 was kindly provided by Dr Brian membranes were probed with anti-GST mAb (Santa Cruz Druker (University of Oregon Health Sciences Center, Port- Biotechnology, 1:500) for 1 h, washed again and further land, OR, USA). Anti-Abl mAb (Ab-3 clone) was purchased probed with HRP-coupled anti-mouse IgG antibody for 1 h, from Oncogene Science (Manhasset, NY, USA). Anti-Sos-1 washed again, and then subjected to the ECL chemilumi- antibody for immunoblotting was purchased from Santa Cruz nescence system. Biotechnology (Santa Cruz, CA, USA), anti-Sos-1 antibody for immunoprecipitation was purchased from Transduction Lab- oratories, anti-C3G polyclonal Ab, and anti-p120CBL poly- Results clonal antibody were purchased from Santa Cruz Biotechnol- ogy. Normal mouse serum was used as a negative control Generation and characterization of specific antibody for immunoprecipitation experiments. monoclonal antibodies against CRKL

Two monoclonal antibodies against CRKL, 2-2 (IgG1) and 5- Immunoblotting and immunoprecipitations 6 (IgG2a) were generated by immunizing mice with a GST- CRKL fusion protein. Partial epitope mapping using deletion Cells were washed with ice cold phosphate-buffered saline, mutants of CRKL fusion proteins revealed that mAb 2-2 recog- and were lysed in buffer containing 50 mM Tris (pH 8.0), 150 nizes an epitope in the N-terminal SH3 and mAb 5-6 mM NaCl, 1% NP-40 (w/v), 0.5% deoxycholic acid (w/v), recognizes an epitope in the C-terminal SH3 domain (data not 100 mM NaF, 1 mM phenylmethylsulfonyl ﬂuoride, 20 ␮g/ml shown). CRKL expression was examined in various cell lines aprotinin, 1 mM sodium orthovanadate, and 40 ␮g/ml leupep- by immunoblot (Figure 1 shows results using mAb 5-6, and tin at 108 cells/ml. After incubation on ice for 20 min, cell the same result was obtained using mAb 2-2 (data not lysates were centrifuged at 12 000 g for 15 min. The resulting shown)). CRKL was detected in all cell lines examined includ- supernatants were subjected to immunoprecipitation or ing nonhematopoietic cell lines (NIH3T3, Hepa, HepG2 and immunoblotting directly as whole cell lysates. For immuno- Cos), T cell lines (CTLL, Jurkat and H9), B cell lines (Ba/F3, precipitation, cell lysates were incubated with anti-CRKL anti- Daudi and Ramos), a monocytic line (U937) and myeloid cell bodies (2-2 or 5-6), anti-CRK antibody or preimmune mouse lines (32D, HL-60, MO7e, TF-1, K562 and BV173). In other serum and protein A sepharose (for mAb 5-6 or preimmune studies, both mAbs were found to recognize human, murine mouse serum) or protein G sepharose (for mAb 2-2) for 3 h at and monkey CRKL, but did not react with CRK II proteins from 4°C. After incubation, supernatants were saved as immuno- these species (data not shown). CRKL binding proteins in transformed hematopoietic cell lines N Uemura et al 378 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 Ͼ75% 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 mar- kers are indicated in kilodaltons (kDa). CRK and CRKL bind to similar proteins in a direct binding assay

The antibodies identified two bands of CRKL protein, migrating at approximately 37 and 39 kDa, in the BCR/ABL- Since CRKL and CRK are ‘adapter proteins’ and could poten- transformed cell line K562 (Figure 2b). Both antibodies were tially transduce signals by linking proteins through the SH2 capable of precipitating more than 90% of CRKL from K562 and SH3 domains, it is important to identify proteins which cells (Figure 2c). However, immune complexes with antibody bind to each domain of CRKL and CRK directly in vivo.To 5-6 contained reproducibly more coprecipitating tyrosine compare the binding of CRKL and CRK to cellular proteins, phosphoproteins than did immune complexes formed with we first performed far Western blotting with GST-CRK II and antibody 2-2 for unknown reasons (Figure 2a), and antibody GST-CRKL fusion proteins as probes on whole cell lysates 5-6 was used in future experiments. In studies not shown, from Ba/F3 and Ba/F3-p210 cells (Figure 3). Remarkably simi- tyrosine phosphorylation of CRKL was examined in three lar patterns were observed except for proteins of 60–70 kDa, hematopoietic cell lines before and after transformation with indicating that both CRKL and CRK have the potential to inter- BCR/ABL (32Dcl3, Ba/F3 and MO7e) and in two cell lines act with a highly overlapping population of cellular proteins derived from patients with CML (K562 and BV173). Tyrosine in these cell lines. Both GST-CRK and GST-CRKL bound to phosphorylated CRKL was found to be restricted to cell lines major bands at approximate molecular weights of 190 and a transformed by BCR/ABL. In K562 cells, the major tyrosine series of proteins at 140–170 kDa in untransformed cells, and phosphoproteins in anti-CRKL immune complexes were 210, to a similar set of proteins in BCR/ABL-transformed Ba/F3 cells 190, 140, 120 and 85–90 kDa, in addition to p39 CRKL itself, plus a 210 kDa protein and proteins at 105–120 kDa (Figure and confirm earlier studies in K562 cells by ten Hoeve et al.18 3). The 70 kDa protein bound more to CRKL than CRK, while We also observed that virtually no tyrosine phosphoproteins the 60 kDa protein bound more to CRK than CRKL. The coprecipitated with CRKL under the conditions employed here 70 kDa protein bound through an SH3 domain interaction, in cell lines not transformed by BCR/ABL or in untransformed, while the 60 kDa protein bound through an SH2 domain quiescent, cells. CRK II has previously been shown to be widely expressed.26 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 binding proteins in transformed hematopoietic cell lines N Uemura et al 379 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 for- ming 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

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 Figure 3 Direct binding of GST-CRK and GST-CRKL fusion protein cells, no significant bands were detected as CRKL-SH2-bind- in vitro to proteins from untransformed and BCR/ABL-transformed ing proteins by probing with a GST-CRKL-SH2 fusion protein, × 6 Ba/F3 cells. Whole cell lysates (0.8 10 cells) from unstimulated although a small amount of a 120 kDa protein was observed (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 with longer development times (data not shown). Using GST- fusion proteins. CRK-SH2 as a probe, a 120 kDa protein was detected in anti- CRK immunoprecipitates in BCR/ABL transformed cells, but not in quiescent untransformed cells (Figure 6). CRK and CRKL coprecipitate with different amounts of cellular proteins in both untransformed and BCR/ABL- transformed Ba/F3 cells The major CRKL SH3-binding proteins are SOS, C3G, c-ABL and p210BCR/ABL The experiment shown in Figure 3 indicates that both CRK and CRKL can potentially bind effectively to an overlapping Previous studies have identified c-ABL, C3G and SOS as cellu- set of cellular proteins. However, in vitro binding does not lar proteins which can interact with c-CRK and possibly, necessarily reflect in vivo binding. To assess in vivo interac- CRKL.5,11,15–19,22 Since the molecular weights of the proteins tions of CRK and CRKL, we performed far Western blotting in identified by far Western blot as CRKL binding proteins were which anti-CRKL (mAb 5-6) or anti-CRK immunoprecipitates consistent with SOS, C3G and ABL, these proteins were exam- were subjected to direct detection by GST-CRKL or GST-CRK ined directly in anti-CRKL immune complexes with specific II fusion proteins, respectively (as described in Materials and antibodies (Figure 7). SOS, C3G and c-ABL proteins are con- methods). This technique has the advantage that it is semi- tained within the 140–170 protein complex detected by quantitative and that it only detects proteins which both CRKL-SH3. The p210 band was shown by anti-ABL blotting coprecipitate with CRKL or CRK and also bind directly to to be p210BCR/ABL. Interestingly, the binding of SOS, C3G and CRKL or CRK, respectively. Anti-CRKL immunoprecipitates c-ABL proteins with CRKL is constitutive and is not changed were found to contain several cellular proteins in both following BCR/ABL transformation. Also, after transformation, untransformed and BCR/ABL-transformed Ba/F3 cells when c-ABL is not displaced by BCR/ABL, as about the same amount probed with GST-CRKL, while a GST probe was negative of c-ABL and BCR/ABL are precipitated. Thus, considering (Figure 4). In untransformed cells, the most prominent CRKL also the data shown in Figure 3, and consistent with previous binding proteins detected were p140–170 and p70. In studies, CRK can apparently bind through its SH3 domains to BCR/ABL-transformed cells, the pattern was similar except that c-ABL, C3G and SOS, the amount of these proteins actually the p70 protein was diminished, and proteins migrating at coprecipitating with CRK is significantly less than that with approximately 210 kDa and 105–120 kDa were also detected. CRKL in the cell lines examined. This also includes BCR/ABL, No prominent proteins which are smaller than 50 kDa were thus presumably explaining the observation that CRKL, but not detected, suggesting that CRKL might not bind to itself, at least CRK, is an in vivo substrate of BCR/ABL. in an inter-molecular manner. In contrast, anti-CRK precipi- To confirm the apparent quantitative difference between tates did not contain detectable CRK-binding proteins in CRKL- and CRK-binding proteins in Ba/F3 cells, we performed untransformed cells, although a prominent 120 kDa protein the reverse immunoprecipitations, immunoprecipitations with was detected in BCR/ABL-transformed cells (Figure 4). Both anti-C3G, ABL or SOS; followed by immunoblotting with anti- anti-CRKL and anti-CRK antibodies effectively precipitated CRK or anti-CRKL. CRKL was detected in C3G, ABL, and SOS CRKL binding proteins in transformed hematopoietic cell lines N Uemura et al 380

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 anti- CRKL 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 Figure 6 Detection of CRK- and CRKL-binding proteins using used were prepared as described in the legend for Figure 4 using anti- CRK-SH2 and CRKL-SH2 fusion proteins as probes. The membranes CRK and anti-CRKL immune complexes. Proteins on the membranes used were prepared as described in the legend for Figure 4. The pro- were probed with GST-CRK-SH3(N) or GST-CRKL-SH3(N)-SH3(C) teins on the membranes were probed with GST-CRK-SH2 or GST- fusion proteins. The heavy band at about 55 kDa represents the CRKL-SH2 fusion proteins. The heavy band at about 55 kDa rep- immunoglobulin heavy chain of the antibody used for immunoprecip- resents the immunoglobulin heavy chain of the antibody used for itation. immunoprecipitation. CRKL binding proteins in transformed hematopoietic cell lines N Uemura et al 381

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 pres- p120CBL antibody (Figure 9), consistent with the observations ence of BCR/ABL did not affect the amount of CRKL detected, of de Jong21, Ribon27 and our previous studies.22 This result except for anti-ABL immunoprecipitates. These results are suggests that CRKL could form complexes in vivo that link c- consistent with the notion that CRKL is a more abundant bind- ABL, BCR/ABL, or guanine nucleotide exchange factors, such ing partner than CRK for C3G, c-ABL and SOS. as C3G and SOS, with a protooncoprotein, p120CBL,in It is interesting that a 70 kDa CRKL-SH3-binding protein BCR/ABL-transformed cells. The effects of this complex on was detected only in untransformed Ba/F3 cells, and disap- transformation will be interesting to study. CRK may also form pears after BCR/ABL transformation. A 70 kDa protein also such complexes,27 but our data suggest that CRKL is quantitat- binds to CRKL-SH3 in another murine hematopoietic cell line, ively more important. Overall, these observations suggest a 32D, and is again diminished dramatically in BCR/ABL-trans- mechanism to explain the observations by our group and formed 32D cells (data not shown). The identity of this protein others that p120CBL is a prominent target of BCR/ABL both in is currently unknown. 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 CRK- BCR/ABL is a tyrosine kinase oncogene which transforms SH2 in BCR/ABL-transformed cells was identiﬁed as p120CBL, hematopoietic cells in vivo and in vitro, but does not effec- by probing anti-CRKL immunoprecipitates with an anti- tively 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 (5- 6) antibodies. CRKL binding proteins in transformed hematopoietic cell lines N Uemura et al 382 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 GST- CRKL-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 anti- CRK 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 c- 11,13,15,20,27,34–38 Figure 9 Identification of the major CRKL-SH2-binding protein. ABL has been readily demonstrated. None the The membranes used for far Western blotting were stripped and re- less, the available data suggest that there is at least a quantitat- probed with a specific antibody against p120CBL. ive 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 chimeric oncoprotein p210BCR/ABL is known to bind the actin domain binds intramolecularly to the ptyr-X-X-pro motif con- cytoskeleton through a conserved C-terminal domain in c-ABL taining the major tyrosine phosphorylation site at tyr221.11 This and this interaction is believed to be important for transform- would block interaction of the SH2 domain with cellular pro- ation.2,3 It has been suggested that p210BCR/ABL recruits sig- teins and could also interfere with access to the SH3 domains. naling proteins which ultimately affect adhesion, proliferation However, in both the untransformed and BCR/ABL-trans- and viability.29–33 In the stable phase of the disease, the tyro- formed cell lines tested here, CRK was not detectably tyrosine sine kinase activity of BCR/ABL is apparently lower than dur- phosphorylated, and it is therefore not likely that intramolecu- ing the blast phase, and increased tyrosine phosphorylation is lar binding of CRK SH2 is the explanation for the relatively detectable on only a few cellular proteins. One of these pro- reduced amount of CRK SH3 binding proteins. The possibility teins, CRKL, is phosphorylated in leukemic cells from patients that the CRKL SH2 can undergo any type of intramolecular both early and late in their course, and has been shown to binding has not been assessed. We did not observe direct physically interact with BCR/ABL through its SH3 binding in vitro of CRKL-SH2 to phosphoCRKL when anti- domains.4,6,7,21 CRKL immune complexes were probed with a GST-CRKL-SH2 The SH2 and SH3 domains of CRKL are related to CRK II, fusion protein in a far Western blot, although this does not and in vitro binding studies suggest that the two adapter pro- prove that intramolecular binding does not occur in vivo. teins bind to a highly overlapping set of proteins. However, The immunoprecipitation/far Western assay used is semi- in vivo, CRK II and CRKL may or may not have related func- quantitative since the fusion protein probes are used in excess tions. In the studies reported here, we have carefully com- during the direct binding assay. However, if there are multiple pared the protein interactions of CRKL and CRK in a hemato- SH2-binding sites on a protein, the assay would overestimate poietic cell line, both untransformed and transformed by the abundance of that protein relative to others in the immune BCR/ABL. complex. It is likely that a fraction of the CRKL molecules are Specifically, we used a combination of immunoprecipit- involved in a small number of protein complexes in quiescent, ation and far Western blotting to examine CRKL- and CRK- untransformed cells, and that several novel protein complexes associated proteins. When anti-CRKL monoclonal antibody are formed after transformation. In untransformed cells, CRKL CRKL binding proteins in transformed hematopoietic cell lines N Uemura et al 383 is complexed through its SH3 domains to a group of proteins phorylated on tyrosine residues in cells transformed by acti- which include C3G, SOS and c-ABL.11,20,36 It is not possible vated ABL oncogenes, and also that p120CBL and BCR/ABL to compare precisely the relative amounts of these three pro- form a complex. Furthermore, de Jong et al have reported that teins, although preliminary serial depletion studies suggest p120CBL binds CRKL in the K562 CML cell line. Thus, our data that C3G is the most abundant and c-ABL the least abundant. confirm that of de Jong et al, and suggests that CRKL could We do not know if both SH3 domains of one CRKL molecule function to link BCR/ABL to p120CBL, and further suggests that can be involved in separate binding interactions. If not, which p120CBL may be linked to other cellular proteins through seems likely, then there may be separate complexes contain- CRKL, including C3G, SOS and c-ABL. p120Cbl may be of ing CRKL and C3G, SOS, and c-ABL. It would be of interest particular interest as a BCR/ABL substrate for several reasons. to compare CRK II and CRKL binding proteins in other cell In normal cells, p120CBL is involved in signal transduction types, and the approach used here would facilitate such a pathways in normal cells associated with proliferation or acti- comparison. vation. For example, p120CBL is a common substrate of tyro- C3G and SOS were examined in this study because of the sine kinases activated after cytokine receptor stimulation.52,53 observed molecular weights of the CRKL binding proteins and Also, we have recently shown that p120CBL is tyrosine phos- because of previous studies identifying possible CRK binding phorylated in hematopoietic cells in response to integrin proteins. C3G was originally identified as a protein which crosslinking.54 The downstream signaling pathways are could bind to the SH3 domain of CRK, and the binding site unknown, but p120CBL is physically associated with the p85 for the first CRK II SH3 domain was identified as a proline-rich subunit of phosphoinositol-3-kinase in both activated normal sequence in C3G containing an important lysine residue.38,39 cells55,56 and in BCR/ABL-transformed cells.22 The function of Sequence analysis of C3G suggested it was a guanine nucleo- c-Cbl is unknown, however. A homologue of c-Cbl in C. ele- tide exchange factor, and subsequent studies confirmed that gans, Sli-1, is a negative regulator of tyrosine kinase sig- C3G had exchange factor activity in vitro for the small GTP naling.57 binding protein, Rap1, but not for p21ras.40 SOS is a well The results presented here confirm that CRKL functions as known guanine nucleotide exchange factor with in vitro a linker protein in CML cells.6,7,21 At the present time, the activity for p21ras. SOS has been linked to the control of p21ras role of CRKL-containing complexes in transformation remains function in signaling pathways activated by several growth speculative. The fact that several of the proteins identified factor receptors, typically through binding to the two SH3 are known to play a role in integrin signaling is of interest, domains of GRB2. It is not yet known if the binding sites for since significant adhesion defects in CML progenitor cells the SH3 domain of CRKL overlap with the binding sites for have been described by Verfaillie, Gordon, and our own the SH3 domains of GRB2. It is possible, however, that SOS groups.58–60 Further studies will be necessary to determine if can be linked to different cellular proteins through GRB2 and these interactions lead to aberrant adhesive properties or other CRKL. It is also possible that individual SOS molecules bind biological effects. Overall, we suggest that the interaction both GRB2 and CRKL and that the resulting complexes con- of BCR/ABL with p120CBL through CRKL, but not CRK, could tain other proteins depending on the pattern of tyrosine phos- contribute to the known signaling abnormalities which phorylation. There is abundant evidence that BCR/ABL can cause CML. 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 Acknowledgements to SOS through binding of GRB2 to tyr177 of BCR/ABL,46 binding of CRKL to SOS could also affect SOS function. In contrast This work was supported by NIH grants CA36167 (JDG) and to GRB2, there are no data suggesting that CRKL directly links CA60821 (RS). 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 References and transformed cells is of considerable interest. Our data suggest that this association is constitutive, and not further 1 Muller AJ, Young JC, Pendergast AM et al. BCR first exon increased in response to mitogens. 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