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Bcr Phosphorylated on Tyrosine 177 Binds Grb2

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Bcr Phosphorylated on Tyrosine 177 Binds Grb2 Oncogene (1997) 14, 2367 ± 2372 1997 Stockton Press All rights reserved 0950 ± 9232/97 $12.00 SHORT REPORT Bcr phosphorylated on tyrosine 177 binds Grb2 Guozhen Ma, Dai Lu, Yun Wu, Jiaxin Liu and Ralph B Arlinghaus Department of Molecular Pathology, The University of Texas, M.D. Anderson Cancer Centre, Houston, Texas 77030, USA We and others have shown that the Bcr-Abl oncoprotein (Chuang et al., 1995). Patients acquiring the Ph fuse 5' binds activators of the Ras pathway such as Grb2 and sequences of BCR to the bulk of the ABL gene Shc. Grb2 binding is mediated through a phosphorylated (Shtivelman et al., 1986; Hermans et al., 1987). The tyrosine residue (Y177) located within a consensus Grb2 resulting Bcr-Abl oncoprotein contains either 426 binding site encoded by the ®rst exon of the BCR gene. amino acids of Bcr or 902/926 amino acids of Bcr Our results indicate that P160 BCR is tyrosine fused to the Abl protein lacking the ®rst exon coding phosphorylated at the same site by Bcr-Abl in kinase sequences (Shtivelman et al., 1987). The resulting Bcr- assays (Puil et al., 1994). We performed experiments to Abl proteins are termed P185 BCR-ABL or P210 determine whether Bcr, which was tyrosine phosphory- BCR-ABL. Unlike the normal Abl protein, both Bcr- lated within cells by activated c-Abl, could also bind Abl proteins possess an activated tyrosine protein Grb2, and whether phosphotyrosine 177 was the major kinase activity (Konopka et al., 1984; Kloetzer et al., binding site. Complexes between Bcr and Abl were 1985; Kurzrock et al., 1987). detected in a hemopoietic cell line lacking Bcr-Abl and in The BCR-ABL gene is a major factor in causing and COS1 cells coexpressing both Bcr and Abl proteins. maintaining Ph-positive leukemia. Mice transplanted P160 BCR was tyrosine phosphorylated in COS1 cells with marrow cells expressing the Bcr-Abl oncoprotein coexpressing Abl and Bcr proteins. Similarly, various develop a number of leukemias, some of which deletion mutants of Bcr including BCRN553, BCRN413 resemble chronic myelogenous leukemia (CML) and BCRN221 were tyrosine phosphorylated by acti- (Daley et al., 1990). Transgenic mice expressing the vated c-Abl whereas BCRN159 was not. Wild-type Bcr P185 BCR-ABL protein develop acute lymphocytic and Bcr Y177F were examined under these conditions for leukemia (Heisterkamp et al., 1990). their ability to co-precipitate with Grb2. The results Bcr-Abl proteins form complexes with several showed that while wild-type tyrosine phosphorylated Bcr proteins including p120 Ras Gap (Druker et al., eciently bound Grb2, tyrosine phosphorylated Bcr 1992), P160 BCR (Campbell et al., 1990), 14-3-3 Y177F had greatly reduced Grb2-binding ability. Studies (Reuther et al., 1994), Crkl (ten Hoeve et al., 1994), with GST-SH2 (Grb2) revealed that tyrosine phosphory- Grb2 (Pendergast et al., 1993), Shc and Grb2 (Pull et lated Bcr was able to bind to GST SH2 (Grb2) but al., 1994; Tauchi et al., 1994) and an identi®ed protein tyrosine phosphorylated Bcr Y177F was de®cient in termed ph-P53 (Li et al., 1988). More recently, BCR- binding. These results indicate that the Bcr protein when Abl was reported to induce multimeric complexes of phosphorylated at tyrosine 177 binds Grb2, thereby signaling proteins that include p120 CBL, Pl-3 kinase, implicating Bcr as a potantial activator of the Ras c-Crk or Crkl, c-Abl and Bcr-Abl (Sattler et al., 1996). pathway. Crkl and Grb2 are members of the SH2/SH3 contain- ing proteins, which are part of an ever expanding Keywords: Bcr; Bcr-Abl; tyrosine phosphorylation; group of proteins involved in signal transduction. Grb2; Ras pathway; Philadelphia chromosome Because of past results in a number of systems, it is clear that Grb2 is an activator of the Ras pathway. Grb2 binds to the consensus sequence YVNV when the tyrosine residue is phosphorylated (Zhou et al., 1993). The BCR gene was ®rst discovered because of its This sequence is found in the ®rst exon coding region association with Philadelphia chromosome (Ph)- of BCR. We ®rst demonstrated that Bcr sequences positive leukemias (Nowell et al., 1960; Priest et al., contain several tyrosine residues that are major targets 1980; Champlin and Golde, 1985). The BCR gene for autophosphorylation by Bcr-Abl (Liu et al., 1993). encodes a 160 kDa protein that contains a novel These same sites are also phosphorylated in trans on serine/threonine kinase (Stam et al., 1987; Li et al., P160 BCR found in complexes with Bcr-Abl oncopro- 1989; Maru and Witte, 1991) and a Rac Gap function teins (Liu et al., 1993). (Diekmann et al., 1991). In addition, the central We showed that Bcr ®rst exon peptide sequences region of Bcr has homology to the DBL gene, known containing phosphotyrosine 177 labeled in the kinase to be associated with a guanine-nucleotide exchange reaction in vitro speci®cally bound to the SH2 domain activity (Ron et al., 1991). Recent studies indicate that of Grb2 (Puil et al., 1994). Both Bcr-Abl and Bcr the DBL homology domain within Bcr is functional contained this phosphotyrosine peptide sequence, indicating that Bcr if phosphorylated on tyrosine 177 will also bind to Grb2. In the experiments shown below we showed that Bcr and Abl formed stable Correspondence: R Arlinghaus complexes in cell lines and that full length Bcr and Received 20 August 1996; revised 21 January 1997; accepted amino terminal fragments of Bcr containing tyrosine 21 January 1997 177 were tyrosine phosphorylated by activated Abl. Tyr phosphorylated Bcr binds Grb2 GMaet al 2368 We demonstrated that tyrosine phosphorylated Bcr P6D, Li et al., 1988) immunoprecipitated P145 ABL coprecipitated with Grb2 whereas mutant Bcr (Y177F) (Figure 1b, lane 3) and coimmunoprecipitated P160 was defective in binding Grb2. BCR (lane 1). Similar experiments were performed in Previous results demonstrated that c-Abl and Bcr COS1 cells cotransfected with BCR and ABL genes when overexpressed in Baculovirus infected cells form with identical results. P5C anti-Bcr coprecipitated P145 complexes (Pendergast et al., 1991a). We performed ABL (Figure 1a, lane 7) whereas peptide blocked P5C experiments to determine whether Bcr and Abl form did not (lane 8), and P6D anti-Abl coprecipitated P160 complexes in a human pre-B cell line, SMS-SB (Smith BCR (Figure 1b, lane 2). These results establish that a et al., 1981). In these experiments, we screened either human hematopoietic cell line expressing both Bcr and anti-Bcr immunoprecipitates for the presence of P145 Abl contain stable complexes of Bcr and Abl and that ABL or anti-Abl immunoprecipitates for P160 BCR these complexes can also be detected in COS1 cells under conditions that allow detection of protein- coexpressing both proteins. protein complexes (Cambell et al., 1990; Liu et al., Our previous results have established that P160 BCR 1993). Immunoprecipitates were denatured, fractio- is tyrosine phosphorylated by Bcr-Abl (Lu et al., 1993). nated by SDS-polyacrylamide gel electrophoresis, and It was of interest to determine whether Bcr can be blotted onto Immobilon P ®lters (Millipore, Bedford, tyrosine phosphorylated by activated c-Abl. Therefore, MA) for Western blotting. Anti-Bcr (a peptide we examined COS1 cells overexpressing normal Abl in generated monoclonal antibody termed P5C, Li et al., the presence and absence of Bcr. In these experiments 1989) immunoprecipitated P160 BCR as detected by COS1 cells were transiently transfected with BCR-ABL Western blotting with a commercial anti Bcr (7C6, or ABL genes in the presence and absence of either full Santa Cruz Biotechnology, Santa Cruz, CA) (Figure length BCR or deletion mutants of BCR lacking 1a, lane 1). Peptide-blocked P5C had a greatly reduced varying lengths of the 3' end of the ®rst exon (Figure level of P160 BCR (lane 2). Screening the same blot 3). The methods used were as described by Lu et al. after stripping away the anti-Bcr antibody detected (1993). Brie¯y, BCR-ABL and ABL genes were P145 ABL in the P5C immunoprecipitate (Figure 1a, inserted into the pSG5 vector (Stratagene, La Jolla, lane 5) but had greatly reduced amounts of P145 ABL CA), which contains the early SV40 promoter to in the peptide-blocked sample (lane 6). Similarly, anti- enhance in vivo expression in cells expressing the large Abl (a peptide generated monoclonal antibody termed T antigen. COS1 cells were transfected, and extracts were analysed by Western blotting with either anti-Abl (8E9) monoclonal antibody (Guo et al., 1994), anti-Bcr monoclonal antibody (7C6, Santa Cruz Biotechnology, a Santa Cruz, CA) or rabbit anti-peptide antibodies 1234 made against Bcr 1 ± 16 (the N-terminus) or ¨ Bcr BCR(1256 ± 1271) (the C-terminus) (Campbell et al., 1990). Immunoprecipitates were denatured and fractio- nated by SDS gel electrophoresis, and blotted onto Immobilon ®lters for Western blotting. Simultaneous 5678 overexpression of both P145 ABL (1b) and full length Bcr (P160 BCR) induced tyrosine phosphorylation of ¨ Abl P160 BCR (Figure 2, lane 3), which was lacking in the anti-phosphotyrosine (Transduction Labs, Lexington, KY) blot of COS1 cells expressing Bcr alone (lanes 1 and 6) or Abl alone (lanes 2 and 4). That this tyrosine phosphorylated protein is P160 BCR, is indicated by its b presence only in COS1 cells coexpressing human P160 12 BCR and activated c-Abl (lanes 3, 5 and 7). The ¨ Bcr identity of the 175 kd protein phosphorylated on tyrosine residues (lane 3) present in cells coexpressing both BCR and ABL is unknown. Possibly, it is a hyperphosphorylated form of P160 BCR or another 34 form of BCR, which is present in trace amounts ¨ (compare lanes 3 and 7). Abl In order to investigate the in vivo tyrosine phosphorylation sites of Bcr, we expressed deletion Figure 1 Bcr forms stable complexes with Abl in cell lines.
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