The Src Family Kinase Hck Interacts with Bcr-Abl by a Kinase-independent Mechanism and Phosphorylates the Grb2-binding Site of Bcr
1997; Elsevier BV; Volume: 272; Issue: 52 Linguagem: Inglês
10.1074/jbc.272.52.33260
ISSN1083-351X
AutoresMarkus Warmuth, Michael Bergmann, Andrea Prieß, Kathrin Häuslmann, Bertold Emmerich, Michael Hallek,
Tópico(s)Eosinophilic Disorders and Syndromes
Resumobcr-abl, the oncogene causing chronic myeloid leukemia, encodes a fusion protein with constitutively active tyrosine kinase and transforming capacity in hematopoietic cells. Various intracellular signaling intermediates become activated and/or associate by/with Bcr-Abl, including the Src family kinase Hck. To elucidate some of the structural requirements and functional consequences of the association of Bcr-Abl with Hck, their interaction was investigated in transiently transfected COS7 cells. Neither the complex formation of Hck kinase with Bcr-Abl nor the activation of Hck by Bcr-Abl was dependent on the Abl kinase activity. Both inactivating point mutations of Hck and dephosphorylation of Hck enhanced its complex formation with Bcr-Abl, indicating that their physical interaction was negatively regulated by Hck (auto)phosphorylation. Finally, experiments with a series of kinase negative Bcr-Abl mutants showed that Hck phosphorylated Bcr-Abl and induced the binding of Grb2 to Tyr177 of Bcr-Abl. Taken together, our results suggest that Bcr-Abl preferentially binds inactive forms of Hck by an Abl kinase-independent mechanism. This physical interaction stimulates the Hck tyrosine kinase, which may then phosphorylate the Grb2-binding site in Bcr-Abl. bcr-abl, the oncogene causing chronic myeloid leukemia, encodes a fusion protein with constitutively active tyrosine kinase and transforming capacity in hematopoietic cells. Various intracellular signaling intermediates become activated and/or associate by/with Bcr-Abl, including the Src family kinase Hck. To elucidate some of the structural requirements and functional consequences of the association of Bcr-Abl with Hck, their interaction was investigated in transiently transfected COS7 cells. Neither the complex formation of Hck kinase with Bcr-Abl nor the activation of Hck by Bcr-Abl was dependent on the Abl kinase activity. Both inactivating point mutations of Hck and dephosphorylation of Hck enhanced its complex formation with Bcr-Abl, indicating that their physical interaction was negatively regulated by Hck (auto)phosphorylation. Finally, experiments with a series of kinase negative Bcr-Abl mutants showed that Hck phosphorylated Bcr-Abl and induced the binding of Grb2 to Tyr177 of Bcr-Abl. Taken together, our results suggest that Bcr-Abl preferentially binds inactive forms of Hck by an Abl kinase-independent mechanism. This physical interaction stimulates the Hck tyrosine kinase, which may then phosphorylate the Grb2-binding site in Bcr-Abl. Bcr-Abl (p210 bcr-abl ), the transforming agent in chronic myeloid leukemia, is the gene product of the bcr-abl hybrid gene, which results from the Philadelphia translocation t(9;22) by fusing parts of the c-abl gene, normally located on chromosome 9, to the bcr gene on chromosome 22 (1Ben-Neriah Y. Daley G.Q. Mes-Masson A.M. Witte O.N. Baltimore D. Science. 1986; 233: 212-214Crossref PubMed Scopus (655) Google Scholar, 2Groffen J. Stephenson J.R. Heisterkamp N. de Klein A. Bartram C.R. Grosveld G. Cell. 1984; 36: 93-99Abstract Full Text PDF PubMed Scopus (1255) Google Scholar). Previous studies have demonstrated that Bcr-Abl is a constitutively active tyrosine kinase (3Konopka J.B. Watanabe S.M. Witte O.N. Cell. 1984; 37: 1035-1042Abstract Full Text PDF PubMed Scopus (679) Google Scholar) that has transforming capacity in fibroblasts and hematopoietic cells (4Lugo T.G. Witte O.N. Mol. Cell. Biol. 1989; 9: 1263-1270Crossref PubMed Google Scholar, 5Daley G.Q. Baltimore D. Proc. Natl. 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Hematol. 1996; 94: 5-16Crossref PubMed Scopus (42) Google Scholar) may also play pivotal roles in the pathogenesis of chronic myeloid leukemia. However, the precise mechanisms of transformation by p210 bcr-abl are unknown, and some characteristics of chronic myeloid leukemia, like induction of blast crisis after chronic phase or prolonged viability of chronic myeloid leukemia cells under serum starvation, are still unexplained. Some critical domains of Bcr-Abl that are necessary for transformation and induction of leukemia have been identified. The coiled-coil oligomerization domain, localized at the N terminus of Bcr-Abl, seems to induce tetramerization of Bcr-Abl, which is in turn necessary for the constitutive activation of the tyrosine kinase of Bcr-Abl, as well as for the complex formation with other Src homology (SH) 1The abbreviations used are: SH, Src homology; k.n., kinase negative; Ab, antibody; PCR, polymerase chain reaction; wt, wild type; IP, immunoprecipitation; DOTAP,N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methylsulfate. 2-containing proteins (22Tauchi T. Miyazawa K. Feng G.-S. Broxmeyer H.E. Toyama K. J. Biol. Chem. 1997; 272: 1389-1394Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, 23McWhriter J.R. Galasso D.L. Wang J.Y.J. Mol. Cell. Biol. 1993; 13: 7587-7595Crossref PubMed Scopus (376) Google Scholar). Further important residues or domains within Bcr are the tyrosine at position 177, which is a binding site for the Ras adaptor protein Grb2 (24Pendergast A.M. Quilliam L.A. 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The putative function of these domains is to direct the subcellular localization of Bcr-Abl to compartments where it interacts with specific proteins via specific binding motifs (28Pawson T. Nature. 1995; 373: 573-580Crossref PubMed Scopus (2234) Google Scholar). The optimal binding motif for SH3 domains is polyproline (PXXP) (29Ren R. Mayer B.J. Cicchetti P. Baltimore D. Science. 1993; 259: 1157-1161Crossref PubMed Scopus (1022) Google Scholar), whereas SH2 domains predominantly bind to phosphorylated tyrosine residues in a specific amino acid context (30Songyang Z. Cantley L.C. Trends Biochem. Sci. 1995; 20: 470-475Abstract Full Text PDF PubMed Scopus (333) Google Scholar). In addition, the SH3 and SH2 domains of Bcr-Abl seem to regulate the tyrosine kinase activity as well as the transforming capacity of Abl proteins in vivo (26Pendergast A.M. Muller A.J. Havlik M.H. Maru Y. Witte O.N. Cell. 1991; 66: 161-171Abstract Full Text PDF PubMed Scopus (309) Google Scholar, 31Mayer B. 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Science. 1992; 256: 382-385Crossref PubMed Scopus (177) Google Scholar), and an actin binding site allowing interaction with the cytoskeleton (37McWhriter J.R. Wang J.Y.J. EMBO J. 1993; 12: 1533-1546Crossref PubMed Scopus (286) Google Scholar). We have recently described the activation and association of two members of the Src family of tyrosine kinases, p53/56 lyn and p59 hck, with Bcr-Abl (38Danhauser-Riedl S. Warmuth M. Druker B.J. Emmerich B. Hallek M. Cancer Res. 1996; 56: 3589-3596PubMed Google Scholar). Src kinases are composed of a N-terminal unique domain, a PXXP-binding SH3 domain, a phosphotyrosine-binding SH2 domain, a tyrosine kinase domain, and a C-terminal tail, which is closely involved in negative regulation of the kinase activity (39Brown M. Cooper J. Biochim. Biophys. Acta. 1996; 1287: 121-149Crossref PubMed Scopus (1086) Google Scholar). One of the common features of Src family kinases seems to be their mechanism of autoregulation. Two cooperative mechanisms negatively regulate the activity of Src family kinases (40Sicheri F. Moarefi I. Kuriyan J. Nature. 1997; 385: 602-609Crossref PubMed Scopus (1047) Google Scholar): the interaction of the SH3 domain with a polyproline type II helix located between the SH2 domain and the kinase domain and an interaction of the tyrosine phosphorylated C-terminal tail (Tyr501 in Hck) with the SH2 domain. On the contrary, phosphorylation of a conserved autophosphorylation site within the activation loop of the kinase domain (Tyr390 in Hck) positively regulates the kinase activity (41Cooper J. MacAuley A. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 4232-4236Crossref PubMed Scopus (137) Google Scholar). Autophosphorylated Src retains significant activity even if phosphorylated at the negative regulatory tyrosine in the C-terminal tail (42Boerner R. Kassel D. Barker S. Ellis B. DeLacy P. Knight W. Biochemistry. 1996; 35: 9519-9525Crossref PubMed Scopus (84) Google Scholar). This study presents experiments on structural and functional requirements for the interaction of Bcr-Abl with Src kinases. Using several mutants of Bcr-Abl and Hck we were able to demonstrate that the mechanism of interaction of Bcr-Abl with Src-Kinases is independent of Src and Abl kinase activity and is not mediated by any of the known Bcr-Abl-binding motifs. Moreover, the experiments showed that Tyr177 is phosphorylated by Hck, because coexpression of Hck induced the binding of Grb2 to k.n. Bcr-Abl; this effect was abrogated by a Y177F point mutation. Reagents for cell lysis were purchased from Sigma Chemicals (Deisenhofen, Germany). SDS-polyacrylamide gel electrophoresis was performed with chemicals provided by Bio-Rad (München, Germany) with the exception of acrylamide/bisacrylamide, which was purchased from Boehringer Bioproducts (Ingelheim, Germany). The polyclonal antibodies (Abs) against Hck (N-30), Lyn (44Druker B.J. Okuda K. Matulonis U. Salgia R. Roberts T. Griffin J.D. Blood. 1992; 79: 2215-2220Crossref PubMed Google Scholar), Bcr (N-20 and 7C6), Abl (K-12), and Grb2 (c-23) and the anti-phosphotyrosine Ab PY20 as well as the corresponding blocking petides were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The monoclonal anti-Abl Ab Ab3 was purchased from Oncogene Sciences (Uniondale, NJ). For immunoblotting, all primary Abs were used at 1:1000 dilutions, with the exception of PY20 and anti-Abl Ab3, which were used in a 1:500 dilution. Secondary Abs were either purchased from Bio-Rad (coupled with alkaline phosphatase) or Amersham (coupled with horseradish peroxidase; ECL detection system) and used in dilutions from 1:2000 to 1:5000. The wild type cDNA clone of bcr-abl was provided by Dr. George Daley (Massachusetts General Hospital, Boston, MA) (43Daley G.Q. Van Etten R.A. Baltimore D. Science. 1990; 247: 824-830Crossref PubMed Scopus (1929) Google Scholar). The point mutations Y177F, R1053L, and Y1294F were introduced by site-directed mutagenesis using the Bio-Rad mutaphage kit. For the introduction of the Y177F mutation, a bcr-abl subfragment containing the bcr sequences up to the AccI restriction site was used. This fragment was subsequently recloned into full-length bcr-abl using StuI/NsiI digests. The R1053L and Y1294F mutations were introduced into a KpnI/EcoRI fragment. Thereafter, a KpnI/KpnI fragment from pUCbcr-abl (one KpnI site form pUC, the other at position 3082 in bcr-abl) containing bcr and the abl 5′ sequences up to the KpnI site was added to reconstitute full-length bcr-abl. All mutated fragments were completely sequenced to confirm the mutations and to exclude additional mutations. Double and triple mutants of these residues were accomplished by substitution of the DraIII/DraIII fragment of the R1053L mutant with a corresponding fragment containing the Y1294F mutation and/or by substitution of the HindIII/HindIII fragment (first HindIII site from pUC19 and second HindIII site at position 2609 in bcr-abl) of the R1053L and Y1294F single mutants and of the R1053L/Y1294F double mutant with a corresponding fragment containing the Y177F mutation. The A-/B-box deletion (ΔA-/B-box) was introduced by digesting a BsiEI/SacI subfragment of Bcr-Abl cloned into pUC19 with EcoNI and BglII. Following religation the fragment was sequenced to ensure that the reading frame had been saved. Finally, the mutation was repackaged into full-length bcr-abl using BsaAI/NheI digests. The double mutants bcr-abl k.n./Y177F and bcr-ablk.n./ΔA-/B-box were obtained using the same strategy as used for the generation of the Y177F/R1053L or Y177F/Y1294F double mutants. Deletion of amino acids 1–223 of bcr-abl was accomplished by PCR mutagenesis using the following primers: 5′-ggcgaattcatgggggatgcatccaggcccccttac-3′ and 5′-ccggaattctcattttgaactctgcttaaatccagt-3′. The PCR fragment was subcloned into pUC containing an EcoRI/HindIII fragment of wild type bcr-abl using EcoRI/NheI digests. This fragment was converted to full-length by ligating the deleted subfragment with a HindIII fragment containing the missing bcr and abl sequences. For deletion of the noncatalytical C-terminal portion of Bcr-Abl (amino acids 1426–2031), a similar strategy was used. The primer sequences were 5′-cacgccagtcaacagtctggag-3′ and 5′-gggcaggaattctcactgcagcaaggtactcacaga-3′. The PCR fragment was subcloned into the KpnI/EcoRI sites of pUC19. This fragment was converted to full-length by ligation of the subfragment with a KpnI fragment containing the residual bcr-abl sequences. All PCR reactions were run with Vent polymerase (New England Biolabs, Beverly, MA). Fragments were sequenced to ensure that no mutations had been introduced during the PCR reactions. All bcr-abl mutants were cloned into pcDNA3 (Invitrogen, Leek, Netherlands) for expression in COS7 cells. The wild type (wt) cDNA clone of hck was purchased from ATCC (Rockville, Maryland). Tyr501 was mutated to phenylalanin by PCR modification using the following primers: 5′-gaatgtgaattcatggggtgcatgaagtccaag-3′ and 5′-tggatagaattctcazggctgctgttgaaactggctctc-3′. The resulting full-length hck fragment was subcloned into pUC19 and sequenced subsequently. The mutations K269R and Y390F were accomplished using a two-fragment PCR strategy; wt hck was cloned into a pUC vector that had been modified by deleting one PvuI site. The resulting vector, pUCΔNdeI/XbaI was used as a template for two PCR reactions producing PCR fragments overlapping at the remaining PvuI site within in the ampicillin resistance (AmpR) gene of pUC and meeting within the hck insert near the triplet to be mutated. Thus, the primers binding in hckallowed us to obtain PCR fragments that could be blunt end-ligated without introducing a deletion; one of these primers contained a mutagenic triplet. Finally, the PCR fragments were PvuI-digested and ligated. For mutation of Lys269 the following primers were used: 5′-gtggcagtgcggacgatgaagccaggg-3′ and 5′-cttggtgtgcttgttgtaggtggc-3′. Sequences for primers used for the mutation of Tyr390 were 5′-gacaacgagtttacggctcgggaaggg-3′ and 5′-gtcaatgacccgggccaggccaaagtc-3′. The sequences for the primers binding in pUC were 5′-ccagccagccggaagggccgagcg-3′ and 5′-ctcttactgtcatgccatccg-3′. All hck constructs as well as a wt lyn cDNA were cloned into the EcoRI site of the expression vector pApuro (vector and lyn cDNA provided by Dr. Seth Corey, Children's Hospital of Pittsburgh, PA). 32D, 32D k.n.bcr-abl (K1172R) and 32D wt bcr-abl (32Dp210) cells were obtained from Dr. Brian J. Druker (Oregon Health Sciences University, Portland, OR). 32D cells were transfected by electroporation as described (44Druker B.J. Okuda K. Matulonis U. Salgia R. Roberts T. Griffin J.D. Blood. 1992; 79: 2215-2220Crossref PubMed Google Scholar). 32D and 32D bcr-abl k.n. cells were grown in RPMI 1640 medium (Boehringer Bioproducts, Ingelheim, Germany) supplemented with 10% fetal calf serum (Boehringer Bioproducts) and 10% WEHI-3B conditioned medium to provide murine interleukin-3. 32Dp210 cells were cultured in RPMI 1640 supplemented with 10% fetal calf serum. COS7 cells were routinely grown in Dulbecco's modified Eagle's medium containing 4.5 g/liter glucose. For transient transfection, cells of one confluent 175-cm2 flask were diluted 1:3 and replated into 175-cm2 tissue culture flasks. 18–24 h thereafter, cells grown to >95% confluency were transiently transfected by lipofection using DOTAP (Boehringer, Mannheim, Germany) according to the guidelines of the manufacturer. Briefly, 50 μg of bcr-abl cDNA and/or 25 μg of hck cDNA were diluted to concentrations of 0.1 μg/μl and preincubated for 15 min with a 6-fold excess (in μg) of DOTAP. For transfection, Dulbecco's modified Eagle's medium containing 1.0 g/liter glucose, 10% fetal calf serum, and antibiotics was used. 24 h after transfection, cells were washed twice in ice-cold phosphate-buffered saline (Life Technologies, Inc., Eggersheim, Germany) and serum-deprived by incubation in Dulbecco's modified Eagle's medium containing 1.0 g/liter glucose and 0.5% fetal calf serum. Transfected cells were normally harvested 48 h after transfection by trypsinization. To protect cells from forming unresuspendible aggregates, 10 μg/ml aprotinin was added to the cells immediately after trypsinization. 32D cells were lysed in lysis buffer containing 1% Brij97 as described previously (38Danhauser-Riedl S. Warmuth M. Druker B.J. Emmerich B. Hallek M. Cancer Res. 1996; 56: 3589-3596PubMed Google Scholar). For lysis, COS7 cells were washed twice in ice-cold phosphate-buffered saline to remove remaining serum. Thereafter, cells were lysed in lysis buffer containing 1% Nonidet P-40, 20 mm Tris (pH 8.0), 50 mm NaCl, and 10 mm EDTA as well as 1 mmphenylmethylsulfonyl fluoride, 10 μg/ml aprotinin, 10 μg/ml leupeptin, and in most cases 2 mm sodium orthovanadate. In general, pelleted cells from one 175-cm2 tissue culture flask (about 5 × 107 cells) were resuspended in 500 μl of lysis buffer solution and incubated on ice for 25 min. Thereafter unsoluble material was removed by centrifugation at 15,000 × g. Afterward lysates were checked for protein concentrations using a Bio-Rad protein assay. For immunoprecipitation (IP), 150 μl of COS7 cell lysate was diluted by the addition of 450 μl of incubation buffer containing 20 mm Tris (pH 8.0), 50 mm NaCl, and 10 mm EDTA, 1 mm phenylmethylsulfonyl fluoride, 10 μg/ml aprotinin, 10 μg/ml leupeptin, and 2 mm sodium orthovanadate to inhibit phosphatase activity where desired. Lyn, Hck, and Bcr-Abl were precipitated by adding 5 μg of the appropriate Abs,i.e. anti-Lyn 44 for precipitation of Lyn, anti-Hck N-30 for precipitation of Hck, and either anti-Bcr 7C6 or anti-Abl K-12 for precipitation of Bcr-Abl. IP reactions were incubated overnight at 4 °C on a rotating plate. After 18 h of incubation, 125 μl of Sepharose A beads (Pharmacia Biotech Inc., Freiburg, Germany) diluted 1:1 in IP buffer (0, 1% Nonidet P-40, 20 mm Tris (pH 8.0), 50 mm NaCl, and 10 mm EDTA, 1 mmphenylmethylsulfonyl fluoride, 10 μg/ml aprotinin, 10 μg/ml leupeptin, and 2 mm sodium orthovanadate) were added to each sample. Following an additional 2 h of incubation at 4 °C, the precipitates were washed three times with IP buffer and subsequently boiled in 2× sample buffer before loading on SDS gels. Peptide blocking experiments were performed as described previously (38Danhauser-Riedl S. Warmuth M. Druker B.J. Emmerich B. Hallek M. Cancer Res. 1996; 56: 3589-3596PubMed Google Scholar). For immune complex kinase assays of Src kinases precipitated from 32D cells, cell lysis, and the IP protocol were slightly modified; IP incubation periods were reduced to 3 h, and three times of washing with IP buffer were followed by washing the precipitates one time with kinase buffer (50 mm Tris (pH 7.4), 10 mmMnCl2). Kinase reaction and analysis of autophosphorylation was performed as described (38Danhauser-Riedl S. Warmuth M. Druker B.J. Emmerich B. Hallek M. Cancer Res. 1996; 56: 3589-3596PubMed Google Scholar). Gel electrophoresis and immunoblotting were performed using standard methods. Proteins were either transfered to polyvinylidene difluoride membranes (Millipore, Eschborn, Germany) or nitrocellulose (Schleicher & Schüll, Dassel, Germany). Immunoblots with PY20 were developed by using alkaline phosphatase-conjugated secondary Abs at a dilution of 1:2000 in Tris-buffered saline containing 5% bovine serum albumin when polyvinylidene difluoride membranes were probed. For detection of phosphorylated proteins transferred to nitrocellulose or other membranes, secondary horseradish peroxidase-conjugated Abs were used. The ECL detection system was used according to the guidelines of the manufacturer (Amersham, Braunschweig, Germany). Dephosphorylation was achieved by omitting orthovanadate, a potent phosphatase inhibitor, from the lysis buffer, followed by preincubation of cleared lysate at 4 °C for 24 h prior to IP. We have recently described the association and activation of two kinases of the Src family, p53/56 lyn and p59 hck, with or by Bcr-Abl (38Danhauser-Riedl S. Warmuth M. Druker B.J. Emmerich B. Hallek M. Cancer Res. 1996; 56: 3589-3596PubMed Google Scholar). The mechanism and function of this interaction is unknown. This led us to investigate the structural and functional requirements for the complex formation of Bcr-Abl with Hck kinase, a Src family member preferentially expressed in hematopoietic cells. To establish an expression system that would allow the rapid screening of Bcr-Abl and Hck mutants, wt full-length cDNAs of bcr-abland hck were cloned into appropriate mammalian expression vectors (pcDNA3 or pApuro) and prepared for transient transfection into COS7 cells. cDNAs were introduced into these cells using lipofection (see "Materials and Methods"). Fig.1 A demonstrates that considerable amounts of Bcr-Abl (lane 2) and Hck (lane 3) were expressed 48 h post-transfection when compared with lysates from cells transfected with control vector (lane 1). Cotransfection of bcr-abl and hck cDNAs (lane 4) lead to expression of Bcr-Abl and Hck similar to that of single cDNA transfections. To demonstrate that the expressed kinases were active in vivo, the blot was stripped and reblotted with an anti-phosphotyrosine Ab, PY20. Fig. 1 Bshows that both kinases were highly (auto)phosphorylated and that expression of these kinases resulted in an increased overall phosphotyrosine content in cellular proteins. Because both kinases, Bcr-Abl and Hck (lanes 2 and 3), seemed maximally activated, no synergism was detectable by anti-phosphotyrosine blotting when Bcr-Abl and Hck were coexpressed. We then wished to demonstrate that Bcr-Abl and Src kinases were found in a complex in COS7 cells, similar to our previous findings in myeloid cells (38Danhauser-Riedl S. Warmuth M. Druker B.J. Emmerich B. Hallek M. Cancer Res. 1996; 56: 3589-3596PubMed Google Scholar). For this purpose, lysates of transiently transfected COS7 cells coexpressing Bcr-Abl and Hck were subjected to immunoprecipitation with the polyclonal anti-Abl Ab K-12. Subsequent immunoblotting with the anti-Hck Ab N-30 demonstrated that Hck (Fig.1 C, lane 1) formed a complex with Bcr-Abl. Coprecipitation was completely blocked by the addition of corresponding blocking peptide, indicating that the coprecipitation of Hck with Bcr-Abl was not caused by unspecific binding (lane 2). Similar results were obtained when Bcr-Abl was precipitated using the polyclonal anti-Bcr Ab 7C6 (not shown). Moreover, we could also coprecipitate Bcr-Abl in anti-Hck IPs (not shown). Finally, similar results were obtained in cotransfection and coprecipitation experiments with Lyn and Bcr-Abl in COS7 cells (not shown). Several domains have been described as functionally relevant protein interaction modules of Bcr-Abl (Fig. 2 A) (8Goga A. McLaughlin J. Afar D.E.H. Saffran D.C. Witte O.N. Cell. 1995; 82: 981-988Abstract Full Text PDF PubMed Scopus (257) Google Scholar, 45Feller S.M. Ren R. Hanafusa H. Baltimore D. Trends Biochem. Sci. 1994; 19: 453-458Abstract Full Text PDF PubMed Scopus (182) Google Scholar, 46Cohen G.B. Ren R. Baltimore D. Cell. 1995; 80: 237-248Abstract Full Text PDF PubMed Scopus (926) Google Scholar). To investigate which of these regions were necessary for the interaction of Bcr-Abl with Src kinases, several mutations were introduced into bcr-abl cDNAs (Fig. 2 A). Surprisingly, none of these mutations introduced into bcr-abl alone or in combination was able to disrupt or diminish the formation of Bcr-Abl-Hck complexes in COS7 cells (Fig.2 D). Fig. 2 B shows a typical example of such an experiment. The hck gene was cotransfected into COS7 cells either in combination with a control vector (lane 1) or with various single, double, or triple bcr-abl mutants containing amino acid substitutions recently shown to be critical for Ras activation by Bcr-Abl (8Goga A. McLaughlin J. Afar D.E.H. Saffran D.C. Witte O.N. Cell. 1995; 82: 981-988Abstract Full Text PDF PubMed Scopus (257) Google Scholar): Tyr177, a binding site for the Grb2 adaptor protein; Arg1053, an amino acid embedded in the conserved FLVRESE motif of the Abl SH2 domain that is essential for SH2-directed binding to phosphorylated tyrosines; and Tyr1294, a major autophosphorylation site located in the Abl kinase domain. Lysates of transiently transfected cells were used for anti-Abl IPs. Subsequent immunoblot analysis demonstrated that considerable amounts of Hck were coprecipitated with wt Bcr-Abl (Fig.2 B, middle panel, lane 2). However, similar amounts of Hck were found in complexes with all single, double, and triple mutants (lanes 3–9). Similar results were obtained when Lyn was coexpressed with these mutants (not shown). Anti-Grb2 immunoblots showed that the association of Bcr-Abl with the Ras adaptor protein Grb2 was constantly disrupted by the Y177F mutation (Fig. 2 B, bottom panel, lanes 3,6, 7, and 9). Anti-Abl immunoblots demonstrated that equal amounts of Bcr-Abl were precipitated from Bcr-Abl-expressing cells (Fig. 2 B, top panel). Finally, control blots of whole cell lysates showed that comparable amounts of Hck and Grb2 were expressed in the different cells used for the experiment (Fig. 2 C). Longer exposure of films during chemoluminiscence detection also revealed that small amounts of Hck could be coprecipitated with endogenous c-Abl (fain
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