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• W2013859462 abstract "The hematopoietic cells from patients with Bcr-Abl-positive chronic myelogenous leukemia exhibit multiple abnormalities of cytoskeletal function. The molecular events leading to these abnormalities are not fully understood. Previously we showed that Bcr-Abl elicits ubiquitin-dependent degradation of Abl interactor proteins. Because recent studies have suggested a role of Abl interactor proteins in the pathway that regulates cytoskeletal function, we investigated whether mutations in Bcr-Abl that interfere with the signaling to Abl interactor proteins affect its leukemogenic activity. We report here that the Src homology 3 domain and C-terminal proline-rich sequences of Bcr-Abl are required for its binding to Abl interactor 2 as well as for the induction of Abl interactor 2 degradation. Although the deletion of these regions did not affect the ability of the mutant Bcr-Abl to transform hematopoietic cells to growth factor independence, it abrogated its ability to stimulate spontaneous cell migration on fibronectin-coated surfaces. Furthermore, the mutant Bcr-Abl, defective in binding to Abl interactor 2 and inducing its degradation, failed to induce chronic myelogenous leukemia-like disease in mouse. These results are consistent with a role of Abl interactor proteins in the regulation of cytoskeletal function as well as in the pathogenesis of Bcr-Abl-induced leukemogenesis. The hematopoietic cells from patients with Bcr-Abl-positive chronic myelogenous leukemia exhibit multiple abnormalities of cytoskeletal function. The molecular events leading to these abnormalities are not fully understood. Previously we showed that Bcr-Abl elicits ubiquitin-dependent degradation of Abl interactor proteins. Because recent studies have suggested a role of Abl interactor proteins in the pathway that regulates cytoskeletal function, we investigated whether mutations in Bcr-Abl that interfere with the signaling to Abl interactor proteins affect its leukemogenic activity. We report here that the Src homology 3 domain and C-terminal proline-rich sequences of Bcr-Abl are required for its binding to Abl interactor 2 as well as for the induction of Abl interactor 2 degradation. Although the deletion of these regions did not affect the ability of the mutant Bcr-Abl to transform hematopoietic cells to growth factor independence, it abrogated its ability to stimulate spontaneous cell migration on fibronectin-coated surfaces. Furthermore, the mutant Bcr-Abl, defective in binding to Abl interactor 2 and inducing its degradation, failed to induce chronic myelogenous leukemia-like disease in mouse. These results are consistent with a role of Abl interactor proteins in the regulation of cytoskeletal function as well as in the pathogenesis of Bcr-Abl-induced leukemogenesis. chronic myelogenous leukemia Abl interactor bone marrow bone marrow transplantation glutathione S-transferase Src homology white blood cell interleukin wild type Bcr-Abl is generated by a reciprocal t(9;22)(q34;q11) chromosome translocation that fuses varying amounts of the breakpoint cluster region (bcr) gene on chromosome 22 with sequences upstream of the second exon of c-abl on chromosome 9. Depending on the amount of bcr sequences fused, three different Bcr-Abl fusion proteins with molecular masses of 185 kDa (p185Bcr-Abl), 210 kDa (p210Bcr-Abl), and 230 kDa (p230Bcr-Abl) may be produced (1Rosenberg N. Witte O.N. Adv. Virus Res. 1988; 35: 39-81Crossref PubMed Scopus (127) Google Scholar, 2Gotoh A. Broxmeyer H.E. Curr. Opin. Hematol. 1997; 4: 3-11Crossref PubMed Scopus (64) Google Scholar, 3Raitano A.B. Whang Y.E. Sawyers C.L. Biochim. Biophys. Acta. 1997; 1333: F201-F216PubMed Google Scholar). Expression of Bcr-Abl is associated with greater than 95% of human chronic myelogenous leukemia (CML)1and ∼20% of acute lymphocytic leukemia cases (1Rosenberg N. Witte O.N. Adv. Virus Res. 1988; 35: 39-81Crossref PubMed Scopus (127) Google Scholar, 2Gotoh A. Broxmeyer H.E. Curr. Opin. Hematol. 1997; 4: 3-11Crossref PubMed Scopus (64) Google Scholar, 3Raitano A.B. Whang Y.E. Sawyers C.L. Biochim. Biophys. Acta. 1997; 1333: F201-F216PubMed Google Scholar). Mice transgenic for Bcr-Abl (4Heisterkamp N. Jenster G. ten Hoeve J. Zovich D. Pattengale P.K. Groffen J. Nature. 1990; 344: 251-253Crossref PubMed Scopus (594) Google Scholar) or reconstituted withBcr-Abl-transduced bone marrow (BM) cells (5Daley G.Q. Van Etten R.A. Baltimore D. Science. 1990; 247: 824-830Crossref PubMed Scopus (1929) Google Scholar, 6Pear W.S. Miller J.P. Xu L. Pui J.C. Soffer B. Quackenbush R.C. Pendergast A.M. Bronson R. Aster J.C. Scott M.L. Baltimore D. Blood. 1998; 92: 3780-3792Crossref PubMed Google Scholar, 7Zhang X. Ren R. Blood. 1998; 92: 3829-3840Crossref PubMed Google Scholar, 8Li S. Ilaria Jr., R.L. Million R.P. Daley G.Q. Van Etten R.A. J. Exp. Med. 1999; 189: 1399-1412Crossref PubMed Scopus (421) Google Scholar) developed leukemia that recapitulated many aspects of human CML. The inducible expression of Bcr-Abl in transgenic mice demonstrated that Bcr-Abl is required for both induction and maintenance of the leukemia (9Huettner C.S. Zhang P. Van Etten R.A. Tenen D.G. Nat. Genet. 2000; 24: 57-60Crossref PubMed Scopus (341) Google Scholar). Although these data provide strong evidence to support a direct role of Bcr-Abl in leukemogenesis, it remains unclear how the oncogenic activation of a single proto-oncogene induces malignancy in vivo with comprehensive changes in hematopoietic cell growth, differentiation, and homing. In vitro studies of Bcr-Abl-transformed cells suggest that the expression of Bcr-Abl promotes cell proliferation, enhances cell survival, and alters cell adhesion and migration (2Gotoh A. Broxmeyer H.E. Curr. Opin. Hematol. 1997; 4: 3-11Crossref PubMed Scopus (64) Google Scholar, 3Raitano A.B. Whang Y.E. Sawyers C.L. Biochim. Biophys. Acta. 1997; 1333: F201-F216PubMed Google Scholar, 10Clarkson B.D. Strife A. Wisniewski D. Lambek C. Carpino N. Leukemia. 1997; 11: 1404-1428Crossref PubMed Scopus (69) Google Scholar, 11Salgia R. Li J.L. Ewaniuk D.S. Pear W. Pisick E. Burky S.A. Ernst T. Sattler M. Chen L.B. Griffin J.D. J. Clin. Invest. 1997; 100: 46-57Crossref PubMed Scopus (149) Google Scholar, 12Salgia R. Quackenbush E. Lin J. Souchkova N. Sattler M. Ewaniuk D.S. Klucher K.M. Daley G.Q. Kraeft S.K. Sackstein R. Alyea E.P. von Andrian U.H. Chen L.B. Gutierrez-Ramos J.C. Pendergast A.M. Griffin J.D. Blood. 1999; 94: 4233-4246PubMed Google Scholar). How the changes in these fundamental cellular processes in Bcr-Abl-expressing cells lead to the clinical phenotype of CML remains largely unknown. Bcr-Abl proteins contain multiple domains important in interactions with other cellular proteins involved in the regulation of mitogenic and apoptotic pathways (2Gotoh A. Broxmeyer H.E. Curr. Opin. Hematol. 1997; 4: 3-11Crossref PubMed Scopus (64) Google Scholar, 3Raitano A.B. Whang Y.E. Sawyers C.L. Biochim. Biophys. Acta. 1997; 1333: F201-F216PubMed Google Scholar). They also contain domains and motifs capable of binding to cytoskeleton proteins as well as the proteins involved in regulation of cell adhesion and migration (13McWhirter J.R. Wang J.Y. EMBO J. 1993; 12: 1533-1546Crossref PubMed Scopus (286) Google Scholar, 14Salgia R. Uemura N. Okuda K. Li J.L. Pisick E. Sattler M. de Jong R. Druker B. Heisterkamp N. Chen L.B. Groffen J. Griffin J.D. J. Biol. Chem. 1995; 270: 29145-29150Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar, 15Bhat A. Kolibaba K. Oda T. Ohno-Jones S. Heaney C. Druker B.J. J. Biol. Chem. 1997; 272: 16170-16175Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 16Sattler M. Salgia R. Leukemia. 1998; 12: 637-644Crossref PubMed Scopus (96) Google Scholar). Previously we and others identified Abl interactor (Abi) proteins that bind to both the Src homology 3 (SH3) domain and the C-terminal proline-rich regions of Abl kinase through dual SH3-PXXP interactions (17Dai Z. Pendergast A.M. Genes Dev. 1995; 9: 2569-2582Crossref PubMed Scopus (241) Google Scholar, 18Shi Y. Alin K. Goff S.P. Genes Dev. 1995; 9: 2583-2597Crossref PubMed Scopus (218) Google Scholar). Two highly related genes, abi-1 and abi-2, were cloned. The corresponding protein products share overall 69% identity with the greatest homology observed in the N-terminal homeobox-like domain, proline-rich sequences, and the C-terminal SH3 domain (17Dai Z. Pendergast A.M. Genes Dev. 1995; 9: 2569-2582Crossref PubMed Scopus (241) Google Scholar, 18Shi Y. Alin K. Goff S.P. Genes Dev. 1995; 9: 2583-2597Crossref PubMed Scopus (218) Google Scholar). In addition to the interaction with Abl kinase, Abi proteins also interact with other signaling molecules such as the Abl-related gene product Arg (19Wang B. Mysliwiec T. Krainc D. Jensen R.A. Sonoda G. Testa J.R. Golemis E.A. Kruh G.D. Oncogene. 1996; 12: 1921-1929PubMed Google Scholar), epidermal growth factor receptor substrate Eps8 (20Biesova Z. Piccoli C. Wong W.T. Oncogene. 1997; 14: 233-241Crossref PubMed Scopus (90) Google Scholar), the cytoskeleton protein spectrin (21Ziemnicka-Kotula D. Xu J. Gu H. Potempska A. Kim K.S. Jenkins E.C. Trenkner E. Kotula L. J. Biol. Chem. 1998; 273: 13681-13692Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar), and the guanine nucleotide exchange factor Sos (22Scita G. Nordstrom J. Carbone R. Tenca P. Giardina G. Gutkind S. Bjarnegard M. Betsholtz C. Di Fiore P.P. Nature. 1999; 401: 290-293Crossref PubMed Scopus (286) Google Scholar, 23Fan P.D. Goff S.P. Mol. Cell. Biol. 2000; 20: 7591-7601Crossref PubMed Scopus (53) Google Scholar). Although little is known about the function of Abi, recent studies suggested a role of Abi in the regulation of signal transduction mediated by small GTP-binding proteins. Abi-1 (also known as e3b1) forms a tricomplex with Eps8 and Sos-1 in vivo and regulates Rac-specific guanine nucleotide exchange factor activities in vitro (22Scita G. Nordstrom J. Carbone R. Tenca P. Giardina G. Gutkind S. Bjarnegard M. Betsholtz C. Di Fiore P.P. Nature. 1999; 401: 290-293Crossref PubMed Scopus (286) Google Scholar). It was therefore proposed that Abi-1 is an important player in the transduction of signals from Ras to Rac, a pathway important in the regulation of cytoskeletal function. Consistent with this proposal, microinjection of fibroblasts with anti-Abi-1 antibodies resulted in abrogation of Rac-dependent membrane ruffling in response to platelet-derived growth factor stimulation (22Scita G. Nordstrom J. Carbone R. Tenca P. Giardina G. Gutkind S. Bjarnegard M. Betsholtz C. Di Fiore P.P. Nature. 1999; 401: 290-293Crossref PubMed Scopus (286) Google Scholar). The involvement of Abi proteins in signaling mediated by the wild type and oncogenic forms of Abl kinases is suggested by several lines of evidence. Abi proteins bind to c-Abl and are substrates of the Abl kinases (17Dai Z. Pendergast A.M. Genes Dev. 1995; 9: 2569-2582Crossref PubMed Scopus (241) Google Scholar, 18Shi Y. Alin K. Goff S.P. Genes Dev. 1995; 9: 2583-2597Crossref PubMed Scopus (218) Google Scholar). Overexpression of Abi-1 potently suppresses the transforming activity of viral Abl (v-Abl) in NIH3T3 fibroblasts by inhibiting the v-Abl-stimulated extracellular signal-regulated kinase pathway (18Shi Y. Alin K. Goff S.P. Genes Dev. 1995; 9: 2583-2597Crossref PubMed Scopus (218) Google Scholar, 23Fan P.D. Goff S.P. Mol. Cell. Biol. 2000; 20: 7591-7601Crossref PubMed Scopus (53) Google Scholar). More recently we reported that Bcr-Abl elicits the ubiquitin-dependent degradation of Abi-2 (24Dai Z. Quackenbush R.C. Courtney K.D. Grove M.G. Cortez D. Reuther G.W. Pendergast A.M. Genes Dev. 1998; 12: 1415-1424Crossref PubMed Scopus (103) Google Scholar). Significantly, the expression of Abi-2 is lost in cell lines and bone marrow cells isolated from patients with aggressive Bcr-Abl-positive leukemia (24Dai Z. Quackenbush R.C. Courtney K.D. Grove M.G. Cortez D. Reuther G.W. Pendergast A.M. Genes Dev. 1998; 12: 1415-1424Crossref PubMed Scopus (103) Google Scholar). These data are consistent with a hypothesis that the loss of Abi-2 may play a role in the progression of Bcr-Abl-positive leukemias. To test this hypothesis, we mapped the sequences in Bcr-Abl that are required for its interaction with Abi-2. This enabled the generation of mutant forms of Bcr-Abl defective in binding to Abi-2 and therefore allowed the evaluation of the effects of the mutations on Bcr-Abl-induced cellular transformation and leukemogenesis. In this report we show that the deletion of the SH3 domain and C-terminal proline-rich sequences in Bcr-Abl not only abrogates its interaction with Abi-2 but also prevents Abi-2 degradation, prevents spontaneous cell migration on a fibronectin-coated surface, and impairs leukemogenesis. BaF3 cells and 32D cells were grown in RPMI medium containing 10% fetal bovine serum and 10% WEHI-3-conditioned medium as a source of IL-3. The primary bone marrow cells obtained from control and diseased BMT mice were cultured in minimum essential medium α medium containing 20% fetal bovine serum with or without added growth factors for 5 days before being subjected to Western blot analysis. The retroviral vectors MIGR1 and MSCV were kindly provided by Dr. W. S. Pear (University of Pennsylvania, Philadelphia, PA) and Dr. R. G. Hawley (University of Toronto, Toronto, Canada), respectively. To construct retroviral vectors expressing p185wt and p185ΔSH3, the cDNA fragments encoding p185wt and p185ΔSH3 (deletion of amino acids 414–519) were released from pGEMp185Bcr-Abl and pGEMp185Δ414–519 (25Pendergast A.M. Muller A.J. Havlik M.H. Clark R. McCormick F. Witte O.N. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 5927-5931Crossref PubMed Scopus (123) Google Scholar), respectively, by restriction digestion with EcoRI. The purified cDNA fragments were then ligated to MIGR1 or MSCV at the EcoRI site. To generate retroviral vectors expressing p185ΔC and p185ΔSH3ΔC, pGEMp185Bcr-Abl and pGEMp185Δ414–519 were digested with AatII, which released 5′ cDNA sequences encoding amino acids 1–819 of p185Bcr-Abl or amino acids 1–714 of p185Δ414–519, respectively. The purified cDNA fragments were ligated to pGEMAblΔSH3Δ544–637 (17Dai Z. Pendergast A.M. Genes Dev. 1995; 9: 2569-2582Crossref PubMed Scopus (241) Google Scholar) at theAatII site to replace 5′ sequences of Abl. The resulting plasmids, designated pGEMp185ΔC and pGEMp185ΔSH3ΔC, respectively, were digested withEcoRI, which released the cDNA fragments encoding p185ΔC and p185ΔSH3ΔC. The released cDNA fragments were purified and ligated to MIGR1 and MSCV at theEcoRI site. The retroviral packaging cell lines GP+E-86 and Bosc 23, kindly provided by Drs. A. Bank (Columbia University, New York, NY) and W. S. Pear (University of Pennsylvania), respectively, were used to generate retroviral stocks as described previously (26Pear W.S. Nolan G.P. Scott M.L. Baltimore D. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8392-8396Crossref PubMed Scopus (2306) Google Scholar). The retroviral stocks were stored at −80 °C until use. To determine the titers of retroviral supernatants, a serial dilution was made and used to transduce NIH3T3 cells as described previously (27Yan X.Q. Lacey D. Fletcher F. Hartley C. McElroy P. Sun Y. Xia M. Mu S. Saris C. Hill D. Hawley R.G. McNiece I.K. Blood. 1995; 86: 4025-4033Crossref PubMed Google Scholar). After 10 days of selection with G418, the stable transfectants were counted, and titers were calculated. Stable mass populations of BaF3 cells expressing wild type and mutant forms of Bcr-Abl were generated by retroviral transduction as described previously (17Dai Z. Pendergast A.M. Genes Dev. 1995; 9: 2569-2582Crossref PubMed Scopus (241) Google Scholar). Retroviral transduction of mouse bone marrow cells was performed as described previously (27Yan X.Q. Lacey D. Fletcher F. Hartley C. McElroy P. Sun Y. Xia M. Mu S. Saris C. Hill D. Hawley R.G. McNiece I.K. Blood. 1995; 86: 4025-4033Crossref PubMed Google Scholar) with minor modifications. Briefly, 8–12-week-old male (C57BL/6J × DBA/2J) F1 (BDF1) mice (Charles River Laboratories, Wilmington, MA) were treated with 5-fluorouracil (150 mg/kg of body weight) 4 days prior to marrow harvest. Marrow cells were incubated with retroviral supernatants (6 × 105 cells/ml) containing 15% fetal bovine serum, 6 µg/ml Polybrene, 0.1% bovine serum albumin, 2.5 ng/ml IL-3 (PeproTech, Inc., Rocky Hill, NJ), 100 ng/ml IL-6, 100 ng/ml IL-11, and 100 ng/ml recombinant rat stem cell factor at 37 °C in 5% CO2 for 3 days. The retroviral supernatants were replaced daily with fresh supernatants. The transduced BM cells were washed with phosphate-buffered saline and were injected into lethally irradiated (9.5 grays) syngeneic female mice (6–8 weeks old) through the tail vein at 4 × 105 cells/mouse. Double layer agar cultures in 35-mm dishes were established as described previously (28Bradley T. Hodgson G. Rosendaal M. J. Cell. Physiol. 1978; 94: 517-522Crossref Scopus (186) Google Scholar). The minimum essential medium α medium supplemented with 20% fetal bovine serum was used for all cultures. Cultures were incubated at 37 °C in 5% CO2 for 14 days after which colonies containing greater than 50 cells were scored using a dissecting microscope. Two weeks after bone marrow transplantation, mice were monitored for disease development. Mouse peripheral blood samples were obtained under anesthesia by retro-orbital puncture. A differential count of white blood cells (WBCs) was performed using the Advia 120 Hematology System (Bayer Inc., Tarrytown, NY) with veterinary software for analysis of mouse blood. Disease development was judged by elevated WBCs as well as symptoms such as abnormal gait and labored breathing. Moribund animals were sacrificed by CO2 asphyxiation and were examined for tumors or other visible abnormalities. Collection of spleens, livers, and bone marrow cells was performed immediately after sacrifice. All protocols used were approved by institutional review committees at the University of Colorado Health Sciences Center. As described previously (17Dai Z. Pendergast A.M. Genes Dev. 1995; 9: 2569-2582Crossref PubMed Scopus (241) Google Scholar), anti-Abi-2 antibody was raised to a recombinant glutathioneS-transferase (GST)-Abi-2Δ1–100 fusion protein. The antibody was affinity-purified by a standard technique (29Harlow E. Lane D. Using Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1999: 74-76Google Scholar). Immunoblots were performed as described previously (17Dai Z. Pendergast A.M. Genes Dev. 1995; 9: 2569-2582Crossref PubMed Scopus (241) Google Scholar) using anti-Abi-2 antibody, 8E9 anti-abl monoclonal antibody (PharMingen, San Diego, CA), and mixtures of anti-phosphotyrosine antibodies PY20 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), PY69 (ICN Biomedicals, Inc.), and 4G10 (Upstate Biotechnology, Inc., Waltham, MA) as indicated in the text. Bosc 23 cells were transiently transfected with MSCV, MSCVp185wt, MSCVp185ΔSH3, MSCVp185ΔC, and MSCVp185ΔSH3ΔC. Two days after transfection, cells were lysed and incubated with GST or GST-Abi-2 fusion protein bound to glutathione-Sepharose beads as described previously (17Dai Z. Pendergast A.M. Genes Dev. 1995; 9: 2569-2582Crossref PubMed Scopus (241) Google Scholar). Bound proteins were separated by SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose, probed with anti-Abl antibody, and visualized by the enhanced chemiluminescence detection system. The spontaneous cell migration assay was performed as described previously (30Uemura N. Griffin J.D. J. Biol. Chem. 1999; 274: 37525-37532Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). The inserts of Transwell plates (0.33-cm2 growth area, 8-µm pores; Corning Costar Corp., Cambridge, MA) were coated with human fibronectin (Sigma Chemical Co.). The bottom chambers of the Transwell plates contained 600 µl of RPMI medium plus 1% bovine serum albumin. The BaF3 cells transduced with MIGR1 vector or MIGR1 containing cDNAs for wild type and mutant forms of Bcr-Abl were starved in RPMI medium containing 1% bovine serum albumin for 6 h. The cells were resuspended in RPMI medium containing 1% bovine serum albumin at a concentration of 1 × 106 cells/ml. One hundred microliters of cells were added into the inserts and allowed to migrate into the bottom chambers for 6–8 h. c-Abl binds to Abi proteins through dual SH3-PXXP interactions (17Dai Z. Pendergast A.M. Genes Dev. 1995; 9: 2569-2582Crossref PubMed Scopus (241) Google Scholar, 18Shi Y. Alin K. Goff S.P. Genes Dev. 1995; 9: 2583-2597Crossref PubMed Scopus (218) Google Scholar). To determine whether the direct interaction is required for Bcr-Abl to induce Abi-2 degradation, retroviral vectors containing cDNAs for wild type (p185wt) and mutant forms of p185Bcr-Abl with deletions in the SH3 domain (p185ΔSH3), C-terminal proline-rich sequences (p185ΔC), or both SH3 and C-terminal proline-rich regions (p185ΔSH3ΔC) were generated (Fig.1 A). The abilities of these wild type and mutant forms of p185Bcr-Abl to bind to Abi-2 were tested by an in vitro binding assay. The p185wt bound to Abi-2 because it was detected in GST-Abi-2 precipitates by anti-Abl Western blot analysis (Fig. 1 B,lane 6). In addition to full-length p185wt, proteins with faster mobility were also detected by the anti-Abl antibody in GST-Abi-2 precipitates. These are likely the truncated forms of Bcr-Abl that underwent degradation during incubation because they were also detectable in immunoprecipitates by anti-Abl antibody (data not shown). The deletion of the SH3 domain did not affect binding of p185ΔSH3 to Abi-2 (Fig. 1 B, lane 9). The deletion of the C-terminal proline-rich sequences reduced, but did not abrogate, binding of p185ΔC to Abi-2 (Fig.1 B, lane 12). In contrast, deletion of both the SH3 domain and C-terminal proline-rich sequences completely abolished the interaction between p185ΔSH3ΔC and Abi-2 (Fig.1 B, lane 15). These wild type and mutant forms of p185Bcr-Ablwere then introduced into the murine hematopoietic cell line BaF3 by retroviral transduction and tested for their abilities to induce Abi-2 degradation. BaF3 cells express Abi-2 that migrated on SDS-polyacrylamide gels as a major doublet and minor bands with apparent molecular masses of 60, 65, and 70 kDa, respectively (Fig.1 C, lane 1). The two bands with slower mobility are a reflection of the phosphorylation (31Courtney K.D. Grove M. Vandongen H. Vandongen A. LaMantia A.S. Pendergast A.M. Mol. Cell. Neurosci. 2000; 16: 244-257Crossref PubMed Scopus (54) Google Scholar). As reported previously (24Dai Z. Quackenbush R.C. Courtney K.D. Grove M.G. Cortez D. Reuther G.W. Pendergast A.M. Genes Dev. 1998; 12: 1415-1424Crossref PubMed Scopus (103) Google Scholar), expression of p185wt in BaF3 cells induced degradation of Abi-2 (Fig. 1 C, lane 2). In correlation with their binding capacities, p185ΔCexhibited reduced ability to induce Abi-2 degradation (Fig.1 C, lane 4), whereas p18ΔSH3ΔCwas completely deficient in inducing Abi-2 degradation (Fig.2 C, lane 5). Similar results were observed in another murine hematopoietic cell line 32D (data not shown). The deletion of the SH3 domain and C-terminal proline-rich sequences does not affect the tyrosine kinase activity of the mutant forms of Bcr-Abl. This was demonstrated by the assessment of the protein tyrosine phosphorylation in BaF3 cells expressing the wild type and mutant forms of p185Bcr-Abl. As would be expected, the wild type and mutant forms of p185Bcr-Abl were all tyrosine-phosphorylated and were able to stimulate protein tyrosine phosphorylation in BaF3 cells (Fig. 2 A). The C-terminal proline-rich sequences deleted in p185ΔCand p185ΔSH3ΔC also contain a binding site for the adapter proteins Crk and Crkl (16Sattler M. Salgia R. Leukemia. 1998; 12: 637-644Crossref PubMed Scopus (96) Google Scholar, 32Ren R. Ye Z.S. Baltimore D. Genes Dev. 1994; 8: 783-795Crossref PubMed Scopus (291) Google Scholar). In particular, Crkl is tyrosine-phosphorylated upon Bcr-Abl transformation and is believed to link Bcr-Abl to multiple downstream molecules (16Sattler M. Salgia R. Leukemia. 1998; 12: 637-644Crossref PubMed Scopus (96) Google Scholar). One of the molecules that associates with Crkl and Bcr-Abl is c-Cbl, a major tyrosine-phosphorylated protein in Bcr-Abl-expressing cells (15Bhat A. Kolibaba K. Oda T. Ohno-Jones S. Heaney C. Druker B.J. J. Biol. Chem. 1997; 272: 16170-16175Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 16Sattler M. Salgia R. Leukemia. 1998; 12: 637-644Crossref PubMed Scopus (96) Google Scholar). Therefore, we examined whether the deletion of the C-terminal proline-rich sequences in Bcr-Abl affects the tyrosine phosphorylation of Crkl and c-Cbl. Crkl and c-Cbl were not tyrosine-phosphorylated in control BaF3 cells (Fig. 2 B, lane 1). In contrast, the tyrosine phosphorylation of Crkl and c-Cbl was readily detected in BaF3 cells transformed by wild type as well as mutant forms of p185Bcr-Abl (Fig. 2 B, lanes 2–5). To determine whether the SH3 domain and C-terminal proline-rich sequences are required for Bcr-Abl to stimulate cell proliferation and survival, the mutant forms of p185Bcr-Abl were tested for their abilities to transform BaF3 cells. Normal BaF3 cells require IL-3 for their proliferation and survival (17Dai Z. Pendergast A.M. Genes Dev. 1995; 9: 2569-2582Crossref PubMed Scopus (241) Google Scholar, 33Cortez D. Stoica G. Pierce J.H. Pendergast A.M. Oncogene. 1996; 13: 2589-2594PubMed Google Scholar), whereas cells expressing p185wt were transformed and were able to proliferate in the absence of IL-3 (Fig. 3 A). The deletions of the SH3 domain and C-terminal proline-rich sequences in p185Bcr-Abl did not affect its ability to stimulate cell proliferation and survival because all three mutants, p185ΔSH3, p185ΔC, and p185ΔSH3ΔC, were capable of transforming BaF3 cells to IL-3 independence (Fig. 3 A). Because hematopoietic stem cells are thought to be primary natural targets of Bcr-Abl, we tested if the deletions of the SH3 domain and proline-rich regions in Bcr-Abl affect its ability to transform mouse BM cells enriched for hematopoietic stem cells by 5-fluorouracil treatment. An in vitro soft agar assay (28Bradley T. Hodgson G. Rosendaal M. J. Cell. Physiol. 1978; 94: 517-522Crossref Scopus (186) Google Scholar) was performed to evaluate the colony-forming ability of the BM cells transduced with control retrovirus or retroviruses expressing the wild type and mutant forms of p185Bcr-Abl. In vitro growth of BM cells requires a mixture of growth factors for optimal colony formation. Normal BM cells and the BM cells transduced with control retrovirus did not form colonies in agar culture in the absence of growth factors. In contrast, the BM cells transduced with wild type and mutant forms of p185Bcr-Abl formed colonies in soft agar (Fig. 3 B). It appeared that the p185ΔC and p185ΔSH3ΔC were more potent in transforming BM cells because their expression in BM cells induced 3- and 2-fold increases, respectively, in colony formation compared with p185wt and p185ΔSH3. It was reported that the expression of Bcr-Abl in BaF3 cells stimulated spontaneous cell migration on fibronectin-coated surface (11Salgia R. Li J.L. Ewaniuk D.S. Pear W. Pisick E. Burky S.A. Ernst T. Sattler M. Chen L.B. Griffin J.D. J. Clin. Invest. 1997; 100: 46-57Crossref PubMed Scopus (149) Google Scholar). To determine whether the SH3 domain and C-terminal proline-rich regions of Bcr-Abl are required for this signaling pathway, we performed the Transwell cell migration assay (28Bradley T. Hodgson G. Rosendaal M. J. Cell. Physiol. 1978; 94: 517-522Crossref Scopus (186) Google Scholar). The spontaneous migration on fibronectin-coated membranes was examined in BaF3 cells expressing wild type and mutant forms of p185Bcr-Abl. Consistent with the previous report (11Salgia R. Li J.L. Ewaniuk D.S. Pear W. Pisick E. Burky S.A. Ernst T. Sattler M. Chen L.B. Griffin J.D. J. Clin. Invest. 1997; 100: 46-57Crossref PubMed Scopus (149) Google Scholar), a 3.6-fold increase in spontaneous migration on fibronectin-coated membrane was observed in cells expressing p185wt compared with that in control cells (Fig.4). The expression of p185ΔSH3 and p185ΔC also stimulated spontaneous migration but to a lesser extent compared with control cells. In contrast, no significant increase of spontaneous migration was observed in BaF3 cells expressing p185ΔSH3ΔC (Fig.4) compared with control cells. The findings that p185ΔSH3ΔC is defective in inducing Abi-2 degradation and stimulating spontaneous cell migration prompted us to ask if these defects affect its leukemogenic activity in vivo. This was tested by performing bone marrow transplantation studies in mice. BM cells from 5-fluorouracil-treated donor mice were transduced with either control retrovirus or retroviruses containing cDNAs for wild type and mutant forms of p185Bcr-Abl. To ensure that comparable transduction efficiencies were achieved with the different retroviruses, retroviral supernatants with approximately equivalent titers were used. The levels of wild type and mutant p185Bcr-Abl proteins expressed in transduced BM cells were determined by Western blot analysis (Fig.5 A, lanes 1–5). The transduced BM cells were then transplanted into lethally irradiated syngeneic recipient mice. Consistent with previous reports (6Pear W.S. Miller J.P. Xu L. Pui J.C. Soffer B. Quackenbush R.C. Pendergast A.M. Bronson R. Aster J.C. Scott M.L. Baltimore D. Blood. 1998; 92: 3780-3792Crossref PubMed Google Scholar, 7Zhang X. Ren R. Blood. 1998; 92: 3829-3840Crossref PubMed Google Scholar, 8Li S. Ilaria Jr., R.L. Million R.P. Daley G.Q. Van Etten R.A. J. Exp. Med. 1999; 189: 1399-1412Crossref PubMed Scopus (421) Google Scholar, 34Gross" @default.
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