Proc. Natl. Acad. Sci. USA Vol. 92, pp. 10889-10893, November 1995 Medical Sciences Mutant forms of growth factor-binding protein-2 reverse BCR-ABL-induced transformation (adaptor protein/leukemia/Ras/protein-tyrosine kinase/signaling) MIKHAIL L. GISHIZKY*t, DAVID CORTEZt, AND ANN MARIE PENDERGASTt *Department of Hematology/Oncology, SUGEN, Inc., 515 Galveston Drive, Redwood City, CA 94063; and tDepartment of Pharmacology, Duke University Medical Center, Durham, NC 27710 Communicated by H. Hanafusa, Rockefeller Institute, New York NY July 11, 1995 (received for review November 9, 1994) ABSTRACT Growth factor-binding protein 2 (Grb2) is an BCR-ABL is a chimeric oncogene implicated in the patho- adaptor protein that links tyrosine kinases to Ras. BCR-ABL genesis of Philadelphia chromosome (Ph')-positive human is a tyrosine kinase oncoprotein that is implicated in the leukemias. The BCR-ABL oncogene is formed by the recip- pathogenesis of Philadelphia chromosome (Ph')-positive leu- rocal translocation of sequences from the ABL gene on kemias. Grb2 forms a complex with BCR-ABL and the nu- chromosome 9 into BCR sequences on chromosome 22 (6). cleotide exchange factor Sos that leads to the activation of the This translocation can generate two chimeric proteins, p185 Ras protooncogene. In this report we demonstrate that Grb2 and p210, that exhibit increased tyrosine kinase activity com- mutant proteins lacking amino- or carboxyl-terminal src homol- pared with the normal ABL protein (7, 8). A strong correlation ogy SH3 domains suppress BCR-ABL-induced Ras activation exists between the oncogenic potency and tyrosine kinase and reverse the oncogenic phenotype. The Grb2 SB13-deletion activity of BCR-ABL proteins and their ability to activate Ras mutant proteins bind to BCR-ABL and do not impair tyrosine (9, 10). Previously, we and others demonstrated that Grb2 kinase activity. Expression of the Grb2 SH3-deletion mutant forms a complex with BCR-ABL in vivo (9, 11). This associ- proteins in BCR-ABLtransformed Rat-i fibroblasts and in the ation is mediated by direct binding of the Grb2 SH2 domain to human Ph'-positive leukemic cell line K562 inhibits their ability Tyr-177 in the BCR portion of BCR-ABL. Mutant BCR-ABL to grow as foci in soft agar and form tumors in nude mice. that carries the Tyr-177 -> Phe change no longer binds Grb2 Furthermore, expression of the Grb2 SH3-deletion mutants in (9) and is impaired in its ability to activate Ras (9) and to K562 cells induced their differentiation. Because Ras plays an induce cellular transformation in Rat-1 cells (9, 12). This important role in signaling by receptor and nonreceptor tyrosine implicates Grb2 as a potential regulator of BCR-ABL-induced kinases, the use of interfering mutant Grb2 proteins may be oncogenesis. applied to block the proliferation ofother cancers that depend in We report that Grb2 proteins lacking either the amino- or part on activated tyrosine kinases for growth. carboxyl-terminal SH3 domain can inhibit BCR-ABL-induced transformation. The ability of the Grb2 mutants to reverse the A central issue in the study of oncogenesis is to identify the key transformed phenotype correlates with their ability to inhibit effectors responsible for transmitting the mitogenic stimulus Ras activation. Expression of Grb2 mutant proteins in the induced by activated protein tyrosine kinases. Tyrosine kinases human Ph1-positive chronic myelogenous leukemia (CML) associate with a variety of cytoplasmic proteins through phos- cell line K562 induced the cells to differentiate. These data photyrosine-dependent and -independent protein-binding do- suggest that BCR-ABL transforms hematopoietic cells in part mains, the best characterized of which are the src homology by blocking differentiation and inducing proliferation in a domains SH2 and SH3 (1, 2). An increasingly important group Ras-dependent manner. of SH2/SH3-containing proteins are the "adaptor" proteins such as growth factor-binding protein 2 (Grb2)/sem-5, the P85 subunit of phosphatidylinositol 3-kinase (PI3-K), Nck, Crkl, MATERIALS AND METHODS and Crk (2). These molecules consist almost exclusively of Cells and Viruses. The Ph'-positive K562 cell line was protein-binding domains and appear to function as "connec- maintained in RPMI medium with 10% (vol/vol) fetal calf tors" integrating mitogenic and other extracellular signals serum. Rat-1 fibroblasts were grown in Dulbecco's modified among multiple intracellular signaling pathways. The strongest Eagle's medium (DMEM) with 5% fetal calf serum. BCR- evidence implicating adaptor proteins as critical components ABL transformed Rat-1 fibroblasts were established as de- in signal transduction cascades has been obtained from studies scribed (13). of Grb2 (3). Helper-free retroviral stocks were prepared by transient Grb2 is the human homolog of the Caenorhabditis elegans hyperexpression in COS cells as described (8). gene sem-5 and functions by directly coupling tyrosine kinases Antibodies. Anti-ABL rabbit polyclonal antibodies were to the Ras-activating nucleotide exchange factor Sos (3, 4). used for immunoprecipitation (7). A mouse anti-ABL mono- Mammalian Grb2 is a 26-kDa protein composed of a single clonal antibody (designated 21-63) was used for immunoblot- SH2 domain flanked by SH3 domains on the amino and ting (9). Grb2 proteins were detected by immunoblotting carboxyl termini (3). The Grb2 SH2 domain binds to phos- (Western blotting) of total cell lysates with an anti-Grb2 mouse phorylated tyrosine residues on tyrosine kinases and other monoclonal antibody (Transduction Laboratories, Lexington, proteins, while the SH3 domains bind to proline-rich se- KY). Epitope-tagged Grb2 proteins were also detected with quences of Sos (5). The ability of mammalian Grb2 gene to the 12CA5 mouse monoclonal antibody against influenza complement loss-of-function mutations in the C. elegans gene hemagglutinin [Babco (Emeryville, CA) or Boehringer Mann- sem-5 suggests that Grb2 is an essential element in the tyrosine heim]. kinase-stimulated mitogenic response (5). Abbreviations: SH3, src homology 3; SH2 src homology 2; CML The publication costs of this article were defrayed in part by page charge chronic myelogenous leukemia; Grb2, growth factor-binding protein 2; payment. This article must therefore be hereby marked "advertisement" in HA, hemagglutinin. accordance with 18 U.S.C. §1734 solely to indicate this fact. tTo whom reprint requests should be addressed. 10889 Downloaded by guest on September 29, 2021 10890 Medical Sciences: Gishizky et al. Proc. Natl. Acad. Sci. USA 92 (1995) Plasmid Constructions. Full-length human Grb2 cDNA or tcli c\ Grb2 mutants lacking the 5' or 3' SH3 domains were inserted CM O( c! o .0 in-frame into the BamHI site of a modified pCGn plasmid (D z c) 4 vector (14). The hygromycin-resistance gene was inserted CD < < > upstream of the simian virus 40 (SV40) origin of replication. o o 0 0 The constructs were verified by dideoxy chain-termination a ca sequence analysis. pZipNeoSV and pZipRas-Asnl7 have been Autokinase described (15). Transfections. Grb2 constructs were transfected into Rat-1, Bcr/Abl 0 Rat-1/BCR-ABL, and Rat-1/Raf' cells by the standard cal- - 205 cium phosphate transfection protocol (8, 9). K562 cells were transfected by electroporation with a Gene Pulser Electropo- rator (Bio-Rad). Transformation Assays. Soft agar assays were performed as - 116 described (9, 13). Liquid culture foci formation assays were performed by standard procedures. After transfection, cells - 80 were cultured for 24 hr, trypsinized, and replated at 1 X 105 cells per 10-cm2 dish in the presence of hygromycin at 250 ,ug/ml. Cells transfected with pZipNeoSV and pZipRas- Anti-Grb2 were cultured in the presence at (Asnl7) of G418 (Sigma) 0.5 Western - 49.5 mg/ml. The number of foci in each culture was determined 12-14 days later. Transcriptional Activation Assay. Transcriptional activation - 32.5 of expression from a Ras-responsive element (ets/AP-1) pro- moter was performed as described (9). endogenous oe Ras-Bound Guanine Nucleotide Ratio Determination. The Grb2 "- _ _ -27.5 procedure for assaying the percentage of GTP bound to Ras was essentially as described (16). The' amount of labeled GTP 41M and GDP in each sample was quantitated on a Betagen - 18.5 phosphor imager. The percent of GTP bound to Ras in each 1 2 34 sample was calculated as GTP/(GTP + GDP). FIG. 1. Expression of p210 BCR-ABL and Grb2 wild-type (WT) Binding Assays, in Vitro Autophosphorylation, and Immu- and mutant proteins in Rat-1 fibroblasts. Samples correspond to mass noblotting. Procedures for in vitro binding assays, in vitro populations of Rat-1/BCR-ABL cells transfected with the following labeling with [y-32P]ATP, and immunoblotting were carried plasmid DNAs: WT Grb2 (lane 1), AC-GRB2 (lane 2), AN-GRB2 out essentially as described (9). (lane 3), and an empty vector (lane 4). (Upper) Cells were lysed and Hemoglobin Protein Determinations. The amount of hemo- immunoprecipitated with anti-ABL pex4 antibodies, and the immu- globin present in mass populations of transfected K562 cells noprecipitates were subjected to an in vitro autokinase assay (9). was determined essentially as described (17). (Lower) Grb2 proteins were detected by Western blotting of total cell lysates with an anti-Grb2 mouse monoclonal antibody and visualized with the enhanced chemiluminescence detection system. The greater RESULTS size of the transfected wild-type Grb2 protein is due to the fusion of HA Grb2 Mutants Reverse the Oncogenic Phenotype of BCR- the epitope tag. ABL-Transformed Rat-i Fibroblasts. Mutant Grb2 genes tions of transfected Rat-1/BCR-ABL cells were evaluated for were constructed that deleted the SH3 domain on either the growth in liquid culture and in soft agar. amino (AN) or carboxyl (AC) terminus. To facilitate discrim- Expression of the AN-Grb2 mutant protein in Rat-1/BCR- ination of endogenous Grb2 protein from the transfected ABL cells caused reversion to a nontransformed phenotype.