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 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 neutro- phils 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, mem- branes were further probed with HRP-coupled secondary anti- bodies 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 mg 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).