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. constructs, a sequence coding for the influenza virus hemag- The mass population of cells expressing the AN-Grb2 mutant glutinin epitope (HA) was fused in-frame at the N terminus of protein grew as a monolayer in liquid culture and exhibited the mutant and wild-type Grb2 genes. Epitope-tagged Grb2 contact inhibition upon reaching confluence (data not shown). genes were cloned into the mammalian expression vector, pCGn, which contains the hygromycin drug-resistance gene Table 1. Grb2 SH3-deletion mutant proteins inhibit the growth of (14). BCR-ABL-transformed cells in soft agar A quantitative assay was single-step reverse-transformation no. per 104 cells used to directly test the effect of SH3-deletion mutant and Colonies, wild-type Grb2 proteins on BCR-ABL-induced oncogenesis. Rat-1/ Rat-1/ Rat-1 fibroblasts expressing the BCR-ABL protein exhibit a Grb2 construct Rat-1 BCR-ABL Raf' K562 transformed phenotype as evidenced by their ability to grow as Empty vector Mock 1 566 620 187 foci in liquid culture and form colonies in soft agar (13). Several p210 BCR-ABL-expressing Rat-1 cell lines (Rat-1/ WT 2 734 665 209 BCR-ABL) were established that exhibited these transformed characteristics and grew as tumors in nude mice. These Rat- SH3 AN 0 14 653 13 1/BCR-ABL fibroblasts were transfected with the Grb2 con- structs and cultured for 9-14 days in the presence of hygro- AC 1 119 598 86 mycin to select for expression of the transduced construct. After drug selection, the levels of BCR-ABL and Grb2 pro- Data are mean number of colonies observed in three to five independent experiments. Mass populations of drug-selected cells teins were evaluated (Fig. 1). Approximately equal amounts of were seeded at densities ranging between 1 x 103 and 5 x 104 cells per wild-type and mutant forms of Grb2 were expressed. Similar 6-cm2 dish, depending on the cloning efficiency of each cell type. levels of the BCR-ABL protein were expressed in all of the cell Sarpples were plated in duplicate in medium containing 20% fetal calf populations tested (Fig. 1). To eliminate the possible variation serum. Macroscopic colonies (>0.4 mm) were counted after 14 days. that results from studying only clonal cell lines, mass popula- Colony numbers were adjusted for i04 cells total. WT, wild type. Downloaded by guest on September 29, 2021 Medical Sciences: Gishizky et al Proc. Natl. Acad. Sci. USA 92 (1995) 10891 We observed >95% reduction in the number of colonies after the mutant proteins. The ability of tyrosine kinase oncogenes expression of the AN-Grb2 protein (Table 1). Cells expressing to transform cells correlates with their ability to activate the AC-Grb2 protein grew as monolayers in liquid culture but transcription from promoters containing Ras-responsive ele- did not become quiescent when confluent.' Growth of AC- ments such as the ets/AP-1 motif (18). We utilized a chlor- GRB2-expressing Rat-1/BCR-ABL fibroblasts in soft agar amphenicol acetyltransferase (CAT) reporter gene construct was also suppressed, albeit to a lesser extent than that observed under the control of a ,B-globin promoter containing four for the AN-Grb2 mutant (Table 1). Rat-1/BCR-ABL cell lines tandem Ras-responsive elements (pB4X-CAT). Rat-l/BCR- expressing either the AN- or AC-Grb2 mutant protein did not ABL fibroblasts expressing the AN-Grb2 mutant protein ex- grow as tumors in nude mice (M.L.G., D.C., and A.M.P., hibited 20% of the Ras activity of mock-transfected controls unpublished data). Thus, Grb2 SH3-deletion mutant proteins (Fig. 3 Upper). Suppression of BCR-ABL-induced Ras acti- can interfere with BCR-ABL-induced transformation when vation was also observed in AC-Grb2-expressing cell lines, present in amounts greater than or equal to endogenous albeit to a lesser extent than that observed in cells expressing wild-type Grb2 in cells (Fig. 1). the AN-Grb2 mutant (Fig. 3 Upper). The greater potency of the Binding of Grb2 SH3-Deletion Mutants to BCR-ABL Does AN-Grb2 mutant to suppress BCR-ABL-induced Ras activa- Not Inhibit BCR-ABL Tyrosine Kinase Activity. Direct bind- tion and inhibit transformation is consistent with the observed ing of Grb2 to BCR-ABL in vivo requires only interaction preference of Sos for binding the N-terminal SH3 domain of between the Grb2 SH2 domain and phosphorylated Tyr-177 in BCR-ABL (9, 11). To confirm that Grb2 SH3-deletion mutant proteins bind to BCR-ABL in vivo, we examined whether the two proteins coimmunoprecipitate. Cell lysates prepared from 100- Rat-1/BCR-ABL cells expressing the HA epitope-tagged Grb2 proteins were subjected to immunoprecipitation with anti-ABL or anti-HA epitope antibodies. The immunoprecipi- 0 tates were incubated in the presence of radioactive ATP under - 50- conditions that promote autophosphorylation of the BCR- C ABL kinase. Comparable levels of BCR-ABL protein were -F precipitated from cells expressing wild-type or mutant Grb2 proteins with anti-ABL or anti-HA epitope antibody (Fig. 2). i No BCR-ABL protein was detected in lysates from cells transfected with the empty vector subjected to the immuno- precipitation procedure with the anti-HA epitope antibody. These experiments demonstrate that the Grb2 SH3-deletion mutants form a stable complex with BCR-ABL in vivo and that x the mutants do not inhibit BCR-ABL tyrosine kinase activity. I-i I'q Grb2 Mutants Inhibit BCR-ABL-Induced Ras Activation. To determine whether the growth inhibitory effect of the Grb2 mutant proteins was due to their ability to block BCR-ABL- Ratl Ratl/BCR-ABL induced activation of the Ras signaling pathway, we evaluated the level of Ras activity in Rat-1/BCR-ABL cells expressing + vector + AN -Grb2 + AC -Grb2

CN cm ge. A|-GDP

Anti-abi ~~~cm Z%_ 00 Anti-abl Antibody -GTP Bcr/AbI -_ 3 - 205 _*~~~~~~~~~~~~~~~. .. *** * .*b.* anti-RAS _ + - 116 +. _ + _ + Antibody % GTP Bound 17% 35% 20% 21% Anti-HA Antibody i e1III FIG. 3. Grb2 SH3 deletion mutants inhibit BCR-ABL-stimulated Ras activation. (Upper) Measurement of Ras-dependent transcrip- Bcr/AbI -_0- 205 tional Clonal lines of Rat-1 and Rat-1/BCR-ABL cells, ;f. activation. expressing AN- or AC-Grb2 proteins or vector alone as indicated, were transfected with 1 ,ug of pB4X-CAT reporter plasmid. Forty-eight 116 hours after transfection, cells were harvested and'assayed for CAT activity. Samples represent lysates from normal Rat-1 cells, Rat-1/ BCR-ABL + empty vector, Rat-1/BCR-ABL + AN-Grb2, Rat-1/ BCR-ABL + AC-Grb2. Data were recorded in arbitrary units; the FIG. 2. Grb2 SH3-deletion mutant proteins bind to BCR-ABL in amount of CAT activity detected in the Rat-1/BCR-ABL cells ex- vivo. Samples correspond to mass populations of Rat-l/BCR-ABL pressing the vector alone was set at 100%. Results are means ± SD' of cells transfected with the indicated construct at the top of each lane. data from four independent experiments. (Lower) The parental Rat-1 Cell lysates were prepared and treated with anti-ABL pex5 antibodies cells and Rat-1/BC'R-ABL cell lines expressing the Grb2 mutants were (Upper) or anti-HA epitope antibody 12CA5 (Lower). The immuno- incubated overnight in the presence of [32P]orthophosphate. After the precipitates were extensively washed and subjected to an in vitro incubation, the amount 'of [32P]GTP and [32P]GDP bound to Ras was autokinase assay (9). Samples were analyzed by SDS/PAGE in 8% gel determined for each cell line. The percent GTP bound was calculated and visualized by autoradiography. Sizes are in kDa. as GTP/(GTP + GDP) x 100. Downloaded by guest on September 29, 2021 10892 Medical Sciences: Gishizky et al. Proc. Natl. Acad. Sci. USA 92 (1995)

Grb2 (refs. 19 and 20; M.L.G., D.C., and A.M.P., unpublished 40 data). *a5 To further evaluate the effect of the Grb2 mutants on I BCR-ABL-induced Ras activation, we directly measured the amount of GTP and GDP bound to Ras. Expression of the Grb2 2 30- mutant proteins in Rat-1/BCR-ABL cells decreased the amount U 0 of GTP bound to Ras (Fig. 3 Lower). The percentage of GTP 0) bound to Ras in the AN-Grb2- and AC-Grb2-expressing BCR- T. 20- ABL-transformed cell lines was similar to that observed in nontransformed Rat-1 cells. These measurements of GTP- and C-CD GDP-bound Ras confirm that Grb2 mutant proteins inhibit o10- BCR-ABL-induced Ras activation in vivo. 0 Grb2 Mutants Do Not Inhibit Growth of Raf Gene- E Transformed Rat-i Fibroblasts. If the Grb2 mutant proteins Ca I- function by blocking the signal transduction pathway prior to Q IV, A1- Ras, then transformation by genes whose products are known IC b' to function downstream of Ras in the signaling cascade should not be affected. One such gene encodes the Raf serine/ threonine kinase. Genetic, biochemical, and biological studies FIG. 4. Hemoglobin content in K562 cells expressing wild-type (WT) and mutant Grb2 proteins. Cell lysates were prepared, and indicate that Raf is positioned downstream of Ras in 'the hemoglobin content was determined by using a benzidine stain as signaling cascade (21, 38-40). Rat-1 cell lines transformed described (17). Triplicate samples were analyzed for each cell popu- with a gene encoding an activated form of Raf (Raf) (22) were lation. Data represent the amount of hemoglobin per 1 ,ug of total transfected with the Grb2 cDNA constructs and cultured in the cellular protein transfected with the indicated constructs. presence of hygromycin for 9 days. After drug selection, the levels of the Grb2 proteins were determined, and the mass evaluated whether the Grb2 SH3-deletion mutant proteins populations of transfected Rat-1/Raf' cells were evaluated for could reverse the BCR/ABL-induced oncogenic phenotype in their ability to grow in soft agar. We observed that Rat-1/Raf' hematopoietic cells. The Grb2 constructs were introduced into cells that expressed a >5-fold excess'of AN- or AC-Grb2 Phl-positive K562 cells, a p210 BCR-ABL-expressing cell line protein compared with endogenous wild-type Grb2 protein established from a CML patient in erythroid blast crisis (23). (data not shown) were not inhibited in their ability to grow in After transfection and 48 hr of culture in the presence of soft agar (Table 1). These data show that the Grb2 mutant hygromycin, the levels of endogenous and transfected Grb2 proteins inhibit the BCR-ABL mitogenic signal by uncoupling proteins were determined by immunoblot analysis (data not the signal transduction pathway upstream of Raf. shown). At the same time, cells were harvested from liquid Grb2 Mutants Do Not Inhibit the Growth of Rat-1 Fibro- culture, and equal numbers of viable cells were seeded in soft blasts. To determine whether mutant Grb2 proteins inhibit the agar. In each experiment the level of the transfected Grb2 growth of nontransformed cells, Rat-1 fibroblasts were trans- mutant proteins was at least 5 times greater than that of fected with expression constructs coding for wild-type, AN-, or endogenous Grb2 (data not shown). Cultures seeded with AC-Grb2 and then were cultured for 12-14 days in the K562 cells expressing the AN-GRB2 mutant protein showed presence of hygromycin. After drug selection, the number of 10% of the colonies in soft agar shown by mock-transfected foci in each culture was enumerated. In three separate exper- controls (Table 1). Analogous to what was observed in the iments, Rat-1 cells transfected with wild-type, AN-, or AC- Rat-l/BCR-ABL fibroblasts, the AC-Grb2 mutant also inhib- Grb2 plasmids gave rise to similar numbers of foci when ited K562 colony formation but to a lesser degree than did the compared to cells transfected with the empty vector (Table 2). AN-Grb2 (Table 1). These data strongly suggest that BCR- Consistent with other studies (15) Rat-1 cells transfected with ABL-induced Ras activation mediated by Grb2 is an essential plasmids expressing the dominant inhibitory mutant Ras pro- component in BCR-ABL-induced human malignancy. tein (Ras-Asn-17) showed a dramatic reduction in the number K562 cells retain their ability to differentiate and synthesize of foci formed. In addition, we have been able to establish hemoglobin (16, 17, 24). Previous studies have demonstrated Rat-1 cell lines that express the AN- or AC-Grb2 proteins at a that drugs that inhibit tyrosine kinase activity can be used to 2- to 5-fold higher level than endogenous Grb2. Expression of induce erythroid differentiation of K562 cells (16, 24). To the mutant Grb2 proteins has no apparent effect on the growth determine whether expression of the Grb2 mutants induced a of these cells in liquid culture or in soft agar (data not shown). similar differentiation process, a portion of the transfected These data suggest that mutant Grb2 proteins are not toxic to K562 cells used in the soft agar assays were lysed, and the nontransformed cells. amount of hemoglobin protein was determined for each Grb2 Mutants Inhibit Growth and Induce Differentiation of transfected cell population. We observed a dramatic increase the Human Ph'-Positive Leukemic Cell Line K562. Next we in the level of hemoglobin protein in cells expressing either the AN- or AC-Grb2 mutants (Fig. 4). In contrast, K562 cells Table 2. Growth of transfected Rat-1 cells in liquid culture expressing elevated levels ofwild-type Grb2 protein showed no Foci, no. per dish increase in hemoglobin when compared to mock-transfected Construct 1 2 3 controls. These data suggest that BCR-ABL may transform Exp. Exp. Exp. hematopoietic cells in part by blocking normal differentiation pCGn 61 ± 3 50 ± 4 63 ± 3 in a Ras-dependent manner. pCGn/Grb2WT 44 ± 3 39 ± 2 49 ± 2 pCGn/Grb2AN 52 ± 4 46 ± 1 55 ± 3 pCGn/Grb2AC 53 ± 2 49 ± 2 60 ± 4 DISCUSSION pZip 61±3 60±3 70±3 These studies demonstrate that mutant forms of adaptor pZip/Ras(Asnl7) 9 ± 2 8 ± 4 11 ± 2 proteins can functionally revert the oncogenic phenotype No vector 0 0 0 induced by a protein-tyrosine kinase. Expression of the Grb2 Data are means ± SD of foci in three separate cultures established SH3-deletion mutant proteins in normal and transformed cells with each transfected cell population. was not toxic. Rather, cells transformed by the BCR-ABL Downloaded by guest on September 29, 2021 Medical Sciences: Gishizky et al. Proc. Natl. Acad. Sci. USA 92 (1995) 10893 oncogene once again responded to external stimuli (such as pathways employed by specific tyrosine kinases in oncogenesis contact inhibition) and regained their ability to differentiate and in normal growth and development. (synthesize hemoglobin). This observation suggests that the level of endogenous wild-type Grb2 protein in these cells is We thank J. Schlessinger for his insight and encouragement sufficient to maintain normal signaling. Thus, the Grb2 mutant throughout these studies. We thank L. D. Cripe for preparation of the proteins may function by decreasing the amplitude of the Grb2 constructs; B. Joyce for technical assistance; M. Ostrowski for mitogenic signal initiated by the activated tyrosine kinase. the pCGn expression plasmid; U. Rapp for the activated Raf BXB The ability of BCR-ABL to transform fibroblasts and he- construct (referred to as Raf' in this manuscript); C. Der for the matopoietic cell lines pZipNeoSV and pZipRas-Asnl7 constructs; and G. Plowman, D. correlates with the ability to activate Ras Hayes, and A. Levitzki for critical reading and suggestions in the (9, 10, 25, 26). In the absence of Ras activation, BCR-ABL preparation of this manuscript. M.L.G. is supported by SUGEN, Inc.; does not induce a fully transformed phenotype (25-28). Our D.C. is a Howard Hughes Medical Institute Predoctoral fellow; and previous studies demonstrated that Grb2 can bind directly to A.M.P. is a Whitehead Scholar and a Scholar of the Leukemia Society BCR-ABL, resulting in the formation of a BCR-ABL-Grb2- of America. This work was supported in part by National Institutes of Sos complex in vivo, which leads to the activation of Ras (9). Health Grant CA61033 to A.M.P. The Grb2 SH2 domain can also bind to other phosphorylated proteins such as Shc (11) and the phosphotyrosine phos- 1. Pawson, T. & Schlessinger, J. (1993) Curr. Biol. 3, 434-442. phatase Syp (29). Both Shc and Syp can be in 2. Mayer, B. J. & Baltimore, D. (1993) Trends Cell Bio. 3, 8-13. phosphorylated 3. Lowenstein, E. J., Daly, R. J., Batzer, A. G., Li, W., Margolis, B., Lammers, different BCR-ABL-expressing cell types. The possibility ex- R., Ullrich, A., Skolnik, E. Y., Bar-Sagi, D. & Schlessinger, J. (1992) Cell ists that Grb2 may bind to phosphorylated Shc and Syp 70, 431-442. proteins, thus providing alternative pathways by which BCR- 4. Clark, S. G., Stern, M. J. & Horvitz, H. R. (1992) Nature (London) 356, ABL may be linked to Ras in different cell types. Regardless 340-344. 5. Schlessinger, J. (1993) Trends Biochem. Sci. 18, 273-275. of these other potential pathways, the ability of Grb2 SH3 6. Rosenberg, N. & Witte, 0. N. (1988) Adv. Virus Res. 35, 39-81. deletion mutant proteins to reverse the transformed phenotype 7. Konopka, J. B., Watanabe, S. M. & Witte, 0. N. (1984) Cell 37, 1035-1042. and induce differentiation of the Ph'-positive CML cell line K562 8. Muller, A. J., Young, J. C., Pendergast, A. M., Pondel, M., Littman, D. R. demonstrates the importance of this adaptor protein in the & Witte, 0. N. (1991) Mol. Cell. Biol. 11, 1785-1792. 9. Pendergast, A. M., Quilliam, L. A., Cripe, L. D., Bassing, C. H., Dai, Z., Li, maintenance of the BCR-ABL-induced oncogenic state. N., Batzer, A., Rabun, K. M., Der, C. J., Schlessinger, J. & Gishizky, M. L. Interaction of the Grb2 SH3 domain with Sos has been (1993) Cell 75, 175-185. validated as a critical component in the pathway by which 10. Mandanas, R. A., Leibowitz, D. S., Gharehbaghi, K., Tauchi, T., Burgess, tyrosine kinases induce Ras activation (5, 15). However, it is G. S., Miyazawa, K., Jayaram, H. N. & Boswell, H. S. (1993) Blood 82, 1838-1847. important to note that Grb2 SH3 domains can also bind other 11. Puil, L., Liu, J., Gish, G., Mbamalu, G., Bowtell, D., Pelicci, P. G., cellular proteins (19, 30-32). The biological relevapce of these Arlinghaus, R. & Pawson, T. (1994) EMBO J. 13, 764-773. other protein interactions is unknown. However, the ability of 12. Afar, D. E. H., Goga, A., McLaughlin, J., Witte, 0. N. & Sawyers, C. L. Grb2 to interact with dynamin leads one to speculate that this (1994) Science 264, 424-426. 13. Lugo, T. & Witte, 0. N. (1989) Mol. Cell. Biol. 9, 1263-1270. adaptor protein may also be involved in endocytosis and 14. Tanaka, M. & Herr, W. (1990) Cell 60, 376-386. membrane trafficking (30). In addition, a naturally occurring 15. Feig, L. A. & Cooper, G. M. (1988) Mol. Cell. 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