Association of Bcr-Abl with the Proto-oncogene Vav Is Implicated in Activation of the Rac-1 Pathway
2002; Elsevier BV; Volume: 277; Issue: 14 Linguagem: Inglês
10.1074/jbc.m112397200
ISSN1083-351X
AutoresFlorian Bassermann, Thomas Jähn, Cornelius Miething, Petra Seipel, Ren-Yuan Bai, Sunita Coutinho, Christian Peschel, Justus Duyster, Victor L. J. Tybulewicz,
Tópico(s)Chronic Lymphocytic Leukemia Research
ResumoVav is a guanine nucleotide exchange factor for the Rho/Rac family predominantly expressed in hematopoietic cells and implicated in cell proliferation and cytoskeletal organization. The oncogenic tyrosine kinase Bcr-Abl has been shown to activate Rac-1, which is important for Bcr-Abl induced leukemogenesis. Previous studies by Matsuguchi et al. (Matsuguchi, T., Inhorn, R. C., Carlesso, N., Xu, G., Druker, B., and Griffin, J. D. (1995) EMBO J. 14, 257–265) describe enhanced phosphorylation of Vav in Bcr-Abl-expressing Mo7e cells yet fail to demonstrate association of the two proteins. Here, we report the identification of a direct complex between Vav and Bcr-Abl in yeast, in vitro and in vivo. Furthermore, we show tyrosine phosphorylation of Vav by Bcr-Abl. Mutational analysis revealed that the SH2 domain and the C-terminal SH3 domain as well as a tetraproline motif directly adjacent to the N-terminal SH3 domain of Vav are important for establishing this phosphotyrosine dependent interaction. Activation of Rac-1 by Bcr-Abl was abrogated by co-expression of the Vav C terminus encoding the SH3-SH2-SH3 domains as a dominantnegative construct. Bcr-Abl transduced primary bone marrow from Vav knock-out mice showed reduced proliferation in a culture cell transformation assay compared with wild-type bone marrow. These results suggest, that Bcr-Abl utilizes Vav as a guanine nucleotide exchange factor to activate Rac-1 in a process that involves a folding mechanism of the Vav C terminus. Given the importance of Rac-1 activation for Bcr-Abl-mediated leukemogenesis, this mechanism may be crucial for the molecular pathogenesis of chronic myeloid leukemia and of importance for other signal transduction pathways leading to the activation of Rac-1. Vav is a guanine nucleotide exchange factor for the Rho/Rac family predominantly expressed in hematopoietic cells and implicated in cell proliferation and cytoskeletal organization. The oncogenic tyrosine kinase Bcr-Abl has been shown to activate Rac-1, which is important for Bcr-Abl induced leukemogenesis. Previous studies by Matsuguchi et al. (Matsuguchi, T., Inhorn, R. C., Carlesso, N., Xu, G., Druker, B., and Griffin, J. D. (1995) EMBO J. 14, 257–265) describe enhanced phosphorylation of Vav in Bcr-Abl-expressing Mo7e cells yet fail to demonstrate association of the two proteins. Here, we report the identification of a direct complex between Vav and Bcr-Abl in yeast, in vitro and in vivo. Furthermore, we show tyrosine phosphorylation of Vav by Bcr-Abl. Mutational analysis revealed that the SH2 domain and the C-terminal SH3 domain as well as a tetraproline motif directly adjacent to the N-terminal SH3 domain of Vav are important for establishing this phosphotyrosine dependent interaction. Activation of Rac-1 by Bcr-Abl was abrogated by co-expression of the Vav C terminus encoding the SH3-SH2-SH3 domains as a dominantnegative construct. Bcr-Abl transduced primary bone marrow from Vav knock-out mice showed reduced proliferation in a culture cell transformation assay compared with wild-type bone marrow. These results suggest, that Bcr-Abl utilizes Vav as a guanine nucleotide exchange factor to activate Rac-1 in a process that involves a folding mechanism of the Vav C terminus. Given the importance of Rac-1 activation for Bcr-Abl-mediated leukemogenesis, this mechanism may be crucial for the molecular pathogenesis of chronic myeloid leukemia and of importance for other signal transduction pathways leading to the activation of Rac-1. The proto-oncogene p95 Vav-1 is expressed predominantly in hematopoietic cells (1.Katzav S. Packham G. Sutherland M. Aroca P. Santos E. Cleveland J.L. Oncogene. 1995; 11: 1079-1088PubMed Google Scholar). The protein consists of several conserved domains, among them a calponin homology domain, a Dbl homology domain, a pleckstrin homology domain, and a cysteine-rich region, as well as an Src homology 2 (SH2) 1The abbreviations used are: SH2Src homology 2SH3Src homology 3GEFGDP/GTP exchange factorCMLchronic myeloid leukemiaWTwild typeFCSfetal calf serumSCFstem cell factorGSTglutathione S-transferaseEGFPenhanced green fluorescent proteinIMDMIscove's modified Dulbecco's mediumILinterleukinTStemperature-sensitive domain framed by two SH3 domains, suggesting a role for Vav in multiple signal transduction pathways (2.Adams J.M. Houston H. Allen J. Lints T. Harvey R. Oncogene. 1992; 7: 611-618PubMed Google Scholar, 3.Bustelo X.R. Mol. Cell. Biol. 2000; 20: 1461-1477Crossref PubMed Scopus (449) Google Scholar). Deletion of the N-terminal region encoding the calponin homology domain results in oncogenic activation of Vav (1.Katzav S. Packham G. Sutherland M. Aroca P. Santos E. Cleveland J.L. Oncogene. 1995; 11: 1079-1088PubMed Google Scholar). Vav has been shown to be important for the transduction of signals from the T-cell receptor; Vav knock-out animals have defects in T cell development, and their T-cells proliferate poorly and produce little IL-2 in response to T-cell receptor stimulation (4.Turner M. Mee P.J. Walters A.E. Quinn M.E. Mellor A.L. Zamoyska R. Tybulewicz V.L. Immunity. 1997; 7: 451-460Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar, 5.Fischer K.D. Kong Y.Y. Nishina H. Tedford K. Marengere L.E. Kozieradzki I. Sasaki T. Starr M. Chan G. Gardener S. Nghiem M.P. Bouchard D. Barbacid M. Bernstein A. Penninger J.M. Curr. 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Vav is phosphorylated in response to T-cell receptor stimulation and to cytokines and growth factors like IL-3, SCF, platelet-derived growth factor, or granulocyte/macrophage colony-stimulating factor, suggesting that it is also involved in the signal transduction pathways activated by these receptors (9.Margolis B. Hu P. Katzav S. Li W. Oliver J.M. Ullrich A. Weiss A. Schlessinger J. Nature. 1992; 356: 71-74Crossref PubMed Scopus (307) Google Scholar, 10.Bustelo X.R. Barbacid M. Science. 1992; 256: 1196-1199Crossref PubMed Scopus (170) Google Scholar, 11.Matsuguchi T. Inhorn R.C. Carlesso N. Xu G. Druker B. Griffin J.D. EMBO J. 1995; 14: 257-265Crossref PubMed Scopus (134) Google Scholar, 12.Alai M. Mui A.L. Cutler R.L. Bustelo X.R. Barbacid M. Krystal G. J. Biol. Chem. 1992; 267: 18021-18025Abstract Full Text PDF PubMed Google Scholar). Through this mechanism, the SH2 domain of Vav mediates association with the tyrosine kinase receptor itself or with cytoplasmic tyrosine kinases like Lyn, JAK, or ZAP-70 (3.Bustelo X.R. Mol. Cell. Biol. 2000; 20: 1461-1477Crossref PubMed Scopus (449) Google Scholar, 9.Margolis B. Hu P. Katzav S. Li W. Oliver J.M. Ullrich A. Weiss A. Schlessinger J. Nature. 1992; 356: 71-74Crossref PubMed Scopus (307) Google Scholar, 11.Matsuguchi T. Inhorn R.C. Carlesso N. Xu G. Druker B. Griffin J.D. EMBO J. 1995; 14: 257-265Crossref PubMed Scopus (134) Google Scholar, 13.Bustelo X.R. Ledbetter J.A. Barbacid M. Nature. 1992; 356: 68-71Crossref PubMed Scopus (245) Google Scholar). Vav has been shown to be a GDP/GTP exchange factor (GEF) for members of the Rho family of GTPases (14.Crespo P. Schuebel K.E. Ostrom A.A. Gutkind J.S. Bustelo X.R. Nature. 1997; 385: 169-172Crossref PubMed Scopus (682) Google Scholar). It has been demonstrated that tyrosine phosphorylation of Vav in vitro and in vivo by the tyrosine kinase Lck can activate its Rac GEF activity (14.Crespo P. Schuebel K.E. Ostrom A.A. Gutkind J.S. Bustelo X.R. Nature. 1997; 385: 169-172Crossref PubMed Scopus (682) Google Scholar). The GEF activity of Vav is encoded by the Dbl homology domain. In addition to tyrosine phosphorylation, products of phosphatidylinositol 3′-kinase activation have been proposed to contribute to the GEF activation of Vav via the PH domain (15.Han J. Luby-Phelps K. Das B. Shu X. Xia Y. Mosteller R.D. Krishna U.M. Falck J.R. White M.A. Broek D. Science. 1998; 279: 558-560Crossref PubMed Scopus (710) Google Scholar). Rac activation leads to a wide array of cellular responses including membrane ruffling, JNK (c-Jun N-terminal kinase) activation, and transcriptional activation from Rho/Rac-responsive elements such as NF-AT and NF-κB. Src homology 2 Src homology 3 GDP/GTP exchange factor chronic myeloid leukemia wild type fetal calf serum stem cell factor glutathione S-transferase enhanced green fluorescent protein Iscove's modified Dulbecco's medium interleukin temperature-sensitive Both the SH2 and SH3 domains of Vav have been shown to bind to a whole array of signaling molecules. Among them, as mentioned above, are tyrosine kinases such as Lck, JAK-2, and ZAP-70, which bind to the SH2 domain, and adapter molecules like Cbl, Grb2, and SLB70, binding to the SH3 domain. In addition, heterogeneous ribonucleoprotein (hnRNP), the focal adhesion protein zyxin, Ku-70, hnRNP-c, and hSiah2 have been identified as binding partners of Vav (reviewed in Ref. 3.Bustelo X.R. Mol. Cell. Biol. 2000; 20: 1461-1477Crossref PubMed Scopus (449) Google Scholar). The chimeric oncogene Bcr-Abl is crucial for the pathogenesis of chronic myeloid leukemia (CML) and a subset of acute lymphoblastic leukemia (16.Sawyers C.L. N. Engl. J. 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One of the best studied and defined interactions is the binding of Grb2 to tyrosine 177 in the Bcr part of Bcr-Abl, which links Bcr-Abl to Sos/Ras pathway (21.Puil L. Liu J. Gish G. Mbamalu G. Bowtell D. Pelicci P.G. Arlinghaus R. Pawson T. EMBO J. 1994; 13: 764-773Crossref PubMed Scopus (401) Google Scholar, 22.Pendergast A.M. Quilliam L.A. Cripe L.D. Bassing C.H. Dai Z. Li N. Batzer A. Rabun K.M. Der C.J. Schlessinger J. Gishizky M.L. Cell. 1993; 75: 175-185Abstract Full Text PDF PubMed Scopus (593) Google Scholar). In addition, a multitude of other adapter proteins and substrates of Bcr-Abl have been identified such as Grb10, Grb4, SHC, Crk, Crkl, phosphatidylinositol 3-kinase, and STAT (signal transducers and activators of transcription), which are thought to be involved in the molecular pathogenesis of CML (23.Bai R. Jahn T. Schrem S. Munzert G. Weidner K. JYJ W. Duyster J. Oncogene. 1998; 17: 941-948Crossref PubMed Scopus (44) Google Scholar, 24.Coutinho S. Jahn T. Lewitzky M. 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Instead it was reported that Vav associates and is phosphorylated by JAK-2 in these cells (11.Matsuguchi T. Inhorn R.C. Carlesso N. Xu G. Druker B. Griffin J.D. EMBO J. 1995; 14: 257-265Crossref PubMed Scopus (134) Google Scholar). In this paper, in contrast to the previous report, we demonstrate that Vav interacts directly with Bcr-Abl. This interaction is dependent on the tyrosine kinase activity of Bcr-Abl and an unusual binding mechanism involving a proline-rich region and the two SH3 domains framing the SH2 domain of Vav. We present evidence that complex formation of Bcr-Abl and Vav is necessary for activation of the Rac pathway by Bcr-Abl, which has been shown to be crucial for Bcr-Abl-mediated leukemogenesis (34.Skorski T. Wlodarski P. Daheron L. Salomoni P. Nieborowska-Skorska M. Majewski M. Wasik M. Calabretta B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 11858-11862Crossref PubMed Scopus (84) Google Scholar). The DNA for Bcr-AblΔSal was cloned into the yeast expression vector BTM116 (35.Weidner K.M. Di C.S. Sachs M. Brinkmann V. Behrens J. Birchmeier W. Nature. 1996; 384: 173-176Crossref PubMed Scopus (507) Google Scholar). A cDNA library derived from K562 cells was used (CLONTECH, Heidelberg, Germany) as the prey. Various Bcr-Abl mutants were used in the screen as described previously (23.Bai R. Jahn T. Schrem S. Munzert G. Weidner K. JYJ W. Duyster J. Oncogene. 1998; 17: 941-948Crossref PubMed Scopus (44) Google Scholar, 24.Coutinho S. Jahn T. Lewitzky M. Feller S. Hutzler P. Peschel C. Duyster J. Blood. 2000; 96: 618-624Crossref PubMed Google Scholar). For in vitro translation and expression in 293 cells, the cDNAs of Bcr-Abl WT, Bcr-Abl mutants, F46Wt, and the F46 mutants were cloned in pcDNA 3.1, a mammalian expression vector containing the cytomegalovirus promoter and the SV40 origin of replication (Invitrogen). Cloning of the corresponding human F46/Vav mutant into the full-length murine Vav c-DNA generated VavR696L and VavAAΔCSH3. Expression of these chimerical constructs was performed using the pCMV Tag II vector (Invitrogen). Tyrosine to phenylalanine mutants Bcr-Abl/115F, Bcr/177F-Abl, Bcr/246F-Abl, Bcr/246F-Abl/449F, Bcr/246F-Abl/342F, 449F, Bcr/246F-Abl/353F, 449F, and Bcr/177F-Abl/393 were generated by site-directed mutagenesis using overlapping oligonucleotides and Pfu DNA polymerase (Stratagene, Heidelberg, Germany). The numbers correspond to the human Bcr and murine c-Abl sequence. The Bcr-Abl temperature-sensitive mutant (TSBcr-Abl) was generated by subcloning a temperature-sensitive v-Abl mutant (DP) into Bcr-Abl (23.Bai R. Jahn T. Schrem S. Munzert G. Weidner K. JYJ W. Duyster J. Oncogene. 1998; 17: 941-948Crossref PubMed Scopus (44) Google Scholar). The Bcr/1–509-Abl, Bcr/1–242-Abl, and Bcr/1–63-Abl constructs have been described previously (4.Turner M. Mee P.J. Walters A.E. Quinn M.E. Mellor A.L. Zamoyska R. Tybulewicz V.L. Immunity. 1997; 7: 451-460Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar). Point mutations of the F46/Vav cDNA were conducted using the QuikChange mutagenesis kit following the instructions of the supplier (Stratagene), and deletion mutants were made by PCR using oligonucleotides containing EcoRI and XhoI restriction sites and standard cloning procedures. All constructs derived from site-directed mutagenesis and PCR were verified by automatic sequence analysis. 293 cells were maintained in Dulbecco's modified Eagle's medium (Invitrogen) containing 10% FCS and penicillin/streptomycin (200 units of penicillin/ml and 200 μg of streptomycin/ml). K562, Jurkat, Mo7e, and Mo7e/p210 cells were maintained in RPMI 1640 medium (Invitrogen) with 10% fetal calf serum (Seromed, Berlin, Germany) and penicillin/streptomycin. The medium for Mo7e was supplemented with granulocyte/macrophage colony-stimulating factor (R&D Systems DPC Bierman GmbH, Wiesbaden, Germany). SCF stimulation of Mo7e cells was performed at a final concentration of 200 ng/ml SCF at 37 °C for 5 min and subsequent resuspension in ice-cold phosphate-buffered saline. Transfections were performed with the DOTAP transfection reagent (Roche Molecular Biochemicals). The cDNAs of F46/Vav and F46/Vav mutants were cloned in-frame into the vector pGEX KG to make GST (glutathione S-transferase) fusion proteins. WT and mutant Bcr-Abl proteins were in vitro translated and 35S-radiolabeled using the TNT system (Promega, Heidelberg, Germany). The translation mix was diluted to a final concentration of 25 mm Tris·HCl (pH 7.4), 10 mm MgCl2, 1 mm dithiothreitol, and 100 μm cold ATP and incubated for 30 min at 4 °C to allow for autophosphorylation of the translated proteins. Reactions were stopped by dilution to a final concentration of 10 mmTris·HCl (pH 7.4), 5 mm EDTA, 130 mm NaCl, 1% Triton, 1 mm phenylmethylsulfonyl fluoride, and 10 μg/ml each phenantroline, aprotinin, leupeptin, and pepstatin (lysis buffer). GST fusion proteins were added and incubated for 1 h at 4 °C. Protein complexes were collected on glutathione-agarose beads (Amersham Biosciences, Inc.), washed thoroughly with NETN buffer (100 mm NaCl, 1 mm EDTA, 50 mmTris·HCl (pH 7.4), 0.5% Nonidet P-40, 1 mmphenylmethylsulfonyl fluoride, 5 mm benzamidine), and subjected to SDS-PAGE. In vitro translated proteins were visualized by autoradiography. For binding experiments with cell extracts, 1 × 107 cells were solubilized in lysis buffer, precleared with glutathione-beads, and incubated with the GST fusion protein for 3 h at 4 °C. Protein complexes were collected on glutathione-beads, washed thoroughly with lysis buffer or NETN buffer, and subjected to SDS-PAGE. Immunoblotting was performed with the antibodies indicated. Bcr-Abl and Abl was detected by immunoblotting using an Abl-specific antibody 8E9 (BD PharMingen, Hamburg, Germany). Tyrosine phosphorylation was detected with the monoclonal anti-phosphotyrosine antibody, 4G10 (Upstate Biotechnology, Lake Placid, NY), and PY20 (Transduction Laboratories, Lexington, KY). Polyclonal anti-Vav and anti-Rac-1 antibodies came from Santa Cruz Biotechnology, Heidelberg, Germany. Monoclonal anti-Xpress® and anti-Flag antibodies were from Invitrogen. Immunoprecipitation was done as described previously (36.Duyster J. Baskaran R. Wang J.Y.J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 1555-1559Crossref PubMed Scopus (106) Google Scholar). Briefly, 1 × 107 cells were solubilized in lysis buffer containing 10 mm Tris·HCl (pH 7.4), 5 mm EDTA, 130 mm NaCl, 1% Triton, 1 mm phenylmethylsulfonyl fluoride, 1 mm Na3VO4, and 10 μg/ml each phenantroline, aprotinin, leupeptin, and pepstatin. After clarification by centrifugation, antibody-protein complexes were brought down with 30 μl of protein A-Sepharose (Amersham Biosciences, Inc.). Immunoprecipitations were analyzed by SDS-PAGE followed by immunoblotting with anti-Abl (8E9), monoclonal anti-phosphotyrosine antibody 4G10 (Upstate Biotechnology), PY20 (Transduction Laboratories), anti-Flag (Invitrogen), and polyclonal anti-Vav antibodies (Santa Cruz) as described previously (36.Duyster J. Baskaran R. Wang J.Y.J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 1555-1559Crossref PubMed Scopus (106) Google Scholar). Bands were visualized using the ECL system (Amersham Biosciences, Inc.). Bcr-Abl and kinase-defective Bcr-Abl were in vitro translated using the TNT system (Promega). The translation mix was diluted to a final concentration of 10 mm Tris·HCl (pH 7.4), 5 mm EDTA, 130 mm NaCl, 1% Triton, 1 mm phenylmethylsulfonyl fluoride, 1 mm Na3VO4, and 10 μg/ml each phenantroline, aprotinin, leupeptin, and pepstatin. Immunoprecipitation was performed with 2 μg of anti-Abl (Ab-3). The immune complexes were collected on protein A-Sepharose beads, washed twice each with lysis buffer containing 500 mm NaCl, lysis buffer plus 100 mm NaCl, and lysis buffer alone and twice with kinase buffer containing 25 mm Tris· HCl (pH 7.4), 10 mm MgCl2, and 1 mmdithiothreitol. The pellet was resuspended in 20 μl of kinase buffer with 1 μg of GST-F46/Vav as a substrate. Reactions were started by the addition of 15 μm cold ATP and 10 μCi of [γ-32P]ATP and incubated for 30 min at 30 °C. Reactions were stopped by the addition of Laemmli buffer, heated at 95 °C for 10 min, and resolved by SDS-PAGE. 293 cells (3 × 105) were cotransfected in 60-mm dishes using the DOTAP transfection reagent (Roche Molecular Biochemicals) with 3 μg of plasmid encoding Rac-1, 2.5 μg of the plasmid expressing WT-Bcr-Abl, KD-Bcr-Abl, or the control vector, and 5 μg of the dominant-negative construct Vav/F46 or Vav/F46AAΔCSH3 or the control vector. 65 h post-transfection, cells were starved in FCS-free media and harvested 12 h later. Cell lysates were precleared with glutathione-beads and incubated with equal amounts of GST-PAK for 1 h at 4 °C. Samples were separated by SDS-PAGE, and proteins were detected with a Rac-1 specific antibody (Santa Cruz). To normalize transfection efficiencies, lysates were analyzed by immunoblotting with anti-Rac-1 (Santa Cruz), anti-Abl 8E9 (BD PharMingen), and anti-Xpress® (Invitrogen). Retroviral supernatant was produced by transiently transfecting the Phoenix ecotropic producer line (Gary Nolan, Stanford, CA) with the Mig-p210 or Mig vectors (W. Pear, Philadelphia, PA) as described previously (37.Bai R.Y. Ouyang T. Miething C. Morris S.W. Peschel C. Duyster J. Blood. 2000; 96: 4319-4327Crossref PubMed Google Scholar). Medium was changed at 36 h after transfection and collected after 48 h. Titers of ∼1 × 105-1 × 106 helper virus-free viral particles were obtained as measured by fluorescence-activated cell sorter titer of EGFP-positive infected Rat-1 cells. 2 × 104 cells were seeded onto each 60-mm culture dish in IMDM containing 10% FCS and 0.1% agarose on a base of bottom agar (IMDM, 10% FCS, 0.6% agar). The growth layer was allowed to harden and was subsequently overlaid with a top agar layer (IMDM, 10% FCS, 0.1% agar). Colonies were analyzed ∼2 weeks after plating. Wild-type and Vav−/− mice (4.Turner M. Mee P.J. Walters A.E. Quinn M.E. Mellor A.L. Zamoyska R. Tybulewicz V.L. Immunity. 1997; 7: 451-460Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar) were treated with 150 mg/kg 5-fluorouracil 4 days prior to bone marrow harvest. BM cells flushed from tibia and femur were preincubated for 1 day in bone marrow medium (IMDM, Invitrogen) supplemented with 30% FCS (Seromed), 1% bovine serum albumin, 1% glutamine, 0,5% penicillin-streptomycin, 100 μm2-mercaptoethanol, and 1 μm hydrocortisone) 50 ng/ml mouse SCF, 8 ng/ml mouse IL-3, and 12 ng/ml human IL-6 (all from R&D Systems). After 1 day of preincubation, 1 × 106 bone marrow cells were coincubated with 3 ml of viral supernatant and 1 ml of new medium in the presence of the above mentioned growth factors and 4 μg/ml polybrene. New supernatant was added after 24 h. 3 days after infection, cells were washed in phosphate-buffered saline. Transformation of bone marrow cells in liquid culture was determined as described previously (39.Afar D.E. Han L. McLaughlin J. Wong S. Dhaka A. Parmar K. Rosenberg N. Witte O.N. Colicelli J. Immunity. 1997; 6: 773-782Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). Briefly, 1 × 105 infected cells were plated in bone marrow medium in 12-well plates in a volume of 2 ml. Nonadherent cells were counted after 9 days. To isolate signaling intermediates important for the oncogenic signal transduction of Bcr-Abl, a yeast two-hybrid screen was performed using Bcr-(1–509)-Abl/Lex A as a bait and a cDNA library obtained from a cell line established from a CML patient in blast crisis of the disease (K562). An identical 800-bp clone was isolated four times, which encoded the C-terminal SH3-SH2-SH3 region of p95 Vav (F46/Vav, amino acids 560–845) (Table I). Co-expression of kinase-active Bcr-Abl and F46/Vav resulted in histidine autotrophy and growth of the yeast cells, indicating interaction of Bcr-Abl with F46/Vav (Fig. 1A). The interaction of Vav/F46 and Bcr-Abl was specific, as the control protein lamin showed no binding (Fig. 1A). Yeast cells co-expressing F46/Vav and a tyrosine kinase-defective mutant of Bcr-Abl (KDBcr-Abl) failed to grow, indicating that Bcr-Abl autophosphorylation is required for this interaction (Fig. 1A). Binding of F46/Vav to Bcr-Abl in yeast suggested a direct interaction between Vav and Bcr-Abl. To confirm a direct association of Bcr-Abl and Vav/F46, we tested the ability of GST-Vav/F46 to bind to in vitro translated Bcr-Abl. Fig. 1B shows that Vav/F46 is able to directly bind to Bcr-Abl in vitro. Moreover, we found two constructs carrying internal deletions in the Bcr part of Bcr-Abl (BCR/1–242-Abl and BCR/1–63-Abl) to bind to GST-Vav/F46 (data not shown). Bcr/1–63-Abl is missing the autophosphorylation site Tyr177 responsible for the binding of Bcr-Abl to Grb2, indicating that an Abl phosphorylation site other than Tyr177 is responsible for this phosphotyrosine-dependent interaction. These results together with the data obtained in yeast suggested that the Vav SH2 domain binds to an autophosphorylation site located in the Abl part of Bcr-Abl, which is different from Tyr177. Concordant with this hypothesis, complex formation of Vav could also be demonstrated with v-Abl (Fig. 2D). One tyrosine residue within the Abl portion of Bcr-Abl resembles, at least in part, the predicted consensus site for the Vav SH2 domain, YMEP, which is tyrosine 115 with the sequence -YITP- (40.Songyang Z. Shoelson S.E. McGlade J. Olivier P. Pawson T. Bustelo X.R. Barbacid M. Sabe H. Hanafusa H. Yi T. Mol. Cell. Biol. 1994; 14: 2777-2785Crossref PubMed Scopus (836) Google Scholar). In an attempt to identify the tyrosine residue in Abl that is responsible for complex formation with Vav, a series of tyrosine to phenylalanine mutations and combinations thereof were introduced into Bcr-Abl. These mutants, including Abl Tyr115, Abl Tyr393, the major Abl autophosphorylation site, and the Grb2 binding site, Bcr Tyr177, were tested for their ability to bind to the Vav/F46 fragment in a GST pull-down assay (Abl numbers refer to the Abl Ia isoform). All of the tested tyrosine-to-phenylalanine mutants retained the ability to bind Vav/F46 (data not shown). Thus, the complex between Vav and Bcr-Abl may involve multiple tyrosine residues or involve other binding motifs and intermediate proteins (for example, c-Cbl). Alternatively the binding may be dependent on a so-called "open" conformation that can only be adopted by kinase-active Bcr-Abl, as shown by the paper of Schindler et al. (41.Schindler T. Bornmann W. Pellicena P. Miller W.T. Clarkson B. Kuriyan J. Science. 2000; 289: 1938-1942Crossref PubMed Scopus (1629) Google Scholar).Table IBinding of Vav/F46 mutants to Bcr-AblEqual amounts of GST fusion proteins of the represented Vav/F46 mutants were tested for their binding affinity to Bcr-Abl from K562 lysates of 5 × 106 cells in a GST pull-down assay as described in Fig. 4. The amino acid numbers of the F46/Vav fragments are indicated. +++, strong binding; (+), very weak binding; −, no binding. R696L is an SH2 mutant defective in phosphotyrosine binding. AASH3-SH2-SH3 is a proline-to-alanine mutant of four prolines (amino acids 607–610). Dbl, Dbl homology; PH, pleckstrin homology; SH, Src homology; p-rich, proline-rich. Open table in a new tab Figure 2Vav interacts with Bcr-Abl and v-Abl in vivo. A, a temperature-sensitive mutant of Bcr-Abl (TS Bcr-Abl) was transiently transfected into 293 cells. TSBcr-Abl-transfected 293 cells were kept at 39 °C (restrictive temperature) or shifted to 32 °C (permissive temperature) for 4 h. Cell lysates were run on SDS-PAGE and immunoblotted with an anti-phosphotyrosine antibody (PY20) (upper panel) or an anti-Abl antibody (8E9) (lower panel). B, cell lysates from 1 × 107 TSBcr-Abl-transfected 293 cells kept at the restrictive (39 °C) or permissive (32 °C) temperature were immunoprecipitated with an anti-Vav antibody or a rabbit anti-mouse (RαM) antibody as control. Protein complexes were washed thoroughly and resolved by SDS-PAGE followed by immunoblotting with anti-Vav antibody (upper panel), anti-phosphotyrosine antibody (anti-ptyr, middle p
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