CA2113494A1 - Method for assaying for substances which affect bcr-abl mediated transformation - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE
A method for assaying for a substance that affects BCR-ABL
mediated transformation which comprises reacting the substance with a first compound containing Grb2, or a portion thereof, and a second compound containing a Grb2 binding site on BCR-ABL, Sos or Shc under conditions where the first and second compounds are capable of forming a complex, and comparing with a control. The invention also relates to a method of using the substance to regulate BCR-ABL mediated transformation; and a pharmaceutical composition comprising a compound containing Grb2 or a portion thereof, the Grb2 binding site on BCR-ABL, Sos or Shc, or mimetics thereof, and a pharmaceutically acceptable carrier. Methods of treating patients suffering from CML or ALL are also contemplated.

Description

Title: ~ETHOD POR ASSA~ING FOR SUBSTANOES W~IC~ AFFECT
BCR--ABL I~DIATED TRANSFORMATION

FIELD OF TIIE INVENq~ION
The invention relates to a method for assaying for a substance that affects BCR-ABh mediated transformation; a method of using the substance to regulate BCR-ABL mediated transformation; a pharmaceutical composition comprising a compound containing Grb2 or a portion thereof, preferably a Src homology 2 ~SH2) domain, a Grb2 Src homology 3 ~SH3) domain, a Grb2 binding site on BCR-ABL, Sos or Shc, or mimetics thereof, and a pharmaceutically acceptable carrier; and methods of treatment using the compositions.
BACKGROUND OF q~HE INVENTION
Many of the extracellular signals that normally regulate the proliferation and differentiation of hematopoietic cells activate transmembrane or receptor-associated cytoplasmic protein-tyrosine kinases, which in turn transiently activate intracellular signalling pathways (Veillette et al., 1989, Cell 55, 301-308;
Burkhardt et al., 1991, Proc. Natl. Acad. Sci.UsA 88, 7410-7414.; Niura et al., 1991, Mol. Cell. Biol. 11, 4895-4902; Rottapel et al., l991, ~ol. Cell. Biol. ll, 3043-3051; Scherr, l991, Trends in Genetics 7, 398-402;
Grinstein and Furuya, 1992, J. Biol. Chem. 267, 18122-18125; Torigoe et al., 1992, Blood 80, 617-624; Wang et al., 1992, EMBO J. 11, 4899-4908; Hanazono et al., 1993, EMBO J. 12, 1641-1646; Kolanus et al., 1993, Cell 74, 171-183). Gain-of-function mutations within genes encoding tyrosine kinases can lead to constitutive activation of their products, and thereby induce aberrant proliferation and neoplastic transformation. A well-characterized example in the hematopoietic system involves the rearrangements of the BCR and ABL genes in Philadelphia ~ 1 1 3 1 .~

chromosome positive (Ph+) chronic myelogeneous leukemia (CML), acute lymphocytic leukemia (ALL), and in rare cases Ph+ acute myelogeneous leukemia (AML) resulting in the formation of chimeric BCR-ABL oncogenes that are strongly implicated in the etiology of these leukemias ~Konopka et al., 1984, CELL 37, 1035-10422; Shtivelman et al., 1985, Nature 315, 550-554; Mes-Masson et al., 1986, Proc. Natl.
Acad. Sci. VSA 83, 9768-9772; Clark et al., 1987, Science 235, 85-88; Daley et al., 1987, Science 237, 532-535;
Hermans et al., 1987, Cell 51, 33-40; Elefanty et al., 1990, EMBO J. 9, 1068-1078; Heisterkamp et al., 1990, Nature 334, 251-253; Kelliher et al., Proc. Natl. Acad.
Sci. USA 87, 6649-6653, 1990; Gishizky et al., 1993, Proc.
Natl. Acad. Sci. USA 90, 3755-3759). Depending on the precise breakpoint within the BCR gene, fusion proteins of 210 kDa (P210) or 185 kDa (P185) are produced, which localize to the cytoplasm, and possess deregulated ABL
tyrosine kinase activity (Konopka et al., 1984, CELL 37, 1035-10422; Kloetzer et al. 1985, Virology 140:230-238;
Mes-Masson et al., 1986, Proc. Natl. Acad. Sci. USA 83, 9768-9772; Clark et al., 1987, Science 235, 85-88; Hermans et al., 1987, Cell 51, 33-40; Lugo et al., 1987, Science 237, 532-535; Dhut et al., 1991, Oncogene 6, 1459-1464;
McWhirter and Wang, 1991, Mol. Cell. Biol. 11, 1553-1565).
P210 and P185 BCR-ABL contain identical ABL-derived sequences but vary in the number of BCR-encoded amino acid residues, containing the N-terminal 927 ~or 902) or 426 amino acids of BCR respectively. The tyrosine kinase activity of the BCR-ABL proteins strongly correlates with their transforming potential in tissue culture, and is higher for the 185 kDa form which is characteristically associated with acute leukemias (Lugo et al., 1987, Science 237, 532-535 ; McLaughlin et al., 1989, Mol. Cell.
Biol. 9, 1866-1889; Kelliher et al., 1991, Proc. Natl.
Acad. Sci. USA 87, 6649-6653).
Understanding the mechanisms by which BCR-ABL
proteins elicit their biological effects, requires the 2 :~ ~ 3 ~

identification of their intracellular targets. A large number of tyrosine kinase targets are characterized by conserved non-catalytic Src Homology 2 (SH2) domains, sequences of ~95 amino acids that bind with high affinity to specific phosphotyrosine-containing sites such as those found on activated receptor tyrosine kinases, and thereby direct protein-protein interactions that regulate cytoplasmic signal transduction pathways (reviewed in Cantley et al., 1991, Cell 64, 281-302 ; Xoch et al., 1991, Science 252, 668-674; Pawson and Schlessinger, 1993, J. Current Biol. 3, 434-4423. SH2 domains are often located adjacent to a distinct non-catalytic motif, the Src Homology 3 (SH3) domain, which also functions in protein-protein interactions (reviewed in Musacchio et al., 1992, FEBS 307, 55-61; Pawson and Gish, 1992, Cell 71, 359-362; Pawson and Schlessinger, 1993, J. Current Biol. 3, 434-442). 5Ome of these SH2/SH3-containing proteins also possess intrinsic catalytic domains which may be directly regulated by tyrosine phosphorylation while others lack obvious catalytic sequences and function as adaptor molecules to create multi-protein signalling complexes. In the latter category are two recently described proteins encoded by the grbZ and shc genes (Lowenstein et al., 1992, Cell 70, 431-442; Pelicci et al., 1992, Cell 70, 93-104).
Grb2, the mammalian homolog of Caenorhabditis elegans Sem-5 and Drosophila Drk, is comprised entirely of a single SH2 domain flanked by two SH3 domains (Lowenstein et al., 1992, Cell 70, 431-442). Genetic evidence from C.
elegans and Drosophila indicates that Sem-5 and Drk are required for activation of the Ras pathway by receptor tyrosine kinases (Clark et al., 1992, Science 235, 85-88;
Simon et al., 1993, Cell 73, 169-177; Olivier et al., 1993, Cell 73, 179-191). In mammalian cells, Grb2 directly couples a subset of phosphorylated receptor tyrosine kinases, which it binds with its SH2 domain (Lowenstein et al., 1992, Cell 70, 431-442; Rozakis-Adcock 2113~

et al., 1992, Nature 360, 689-692; Buday and Downward, 1993, Cell 73, 611-620), to mSosl, a Ras guanine nucleotide releasing protein (GNRP) (Bowtell et al., 1992, Proc. Natl. Acad. Sci. USA 89, 6511-1515; Rozakis-Adcock et al., 1992, Nature 360, 689-692; Buday and Downward, 1993, Cell 73, 611-620; Chardin et al., 1993, Science 260, 1338-1343; Egan et al., 1993, Nature 363, 45-51; Rozakis-Adcock et al., 1993, Nat~re 363, 83-85; Li et al., Nature 363, 85-88 1993). The SH3 domains of Grb2 associate with proline-rich motifs in the C-terminal tail of mSosl (chardin et al., 1993, Science 260, 1338-1343; Li et al., 1993, Nature 363, 85-88; Rozakis-Adcock et al., 1993, Nature 363, 83-85) allowing the association of mSosl and Ras-GNRP activity with autophosphorylated epidermal growth factor (EGF) receptors (Gale et al., 1993, Nature 363, 88-92). These interactions apparently stimulate conversion of inactive GDP-bound Ras to the active GTP-bound form (Gale et al., 1993, Nature 363, 88-92).
Although Grb2 associates with activated tyrosine kinases, it is not an efficient substrate for tyrosine phosphorylation in either Drosophila or mammalian cells (Lowenstein et al., 1992, Cell 70, 431-442; Rozakis-Adcock et al., 1992, Nature 360, 689-692 ; Olivier et al., 1993, Cell 73, 179-191 ). Hence, Grb2 function is apparently regulated by the inducible binding of its SH2 domain to phosphotyrosine sites.
The mammalian shc gene encodes three overlapping proteins of 46, 52 and 66 kDa which possess a C-terminal SH2 domain, a more central glycine/proline-rich region and variable amounts of N-terminal sequence (Pelicci et al., 1992, Cell 70, 93-104). p465hC and p525hC arise by differential use of translation initiation sites within a 3.4 kb shc mRNA, and are expressed in all mammalian cells that have been analyzed. A minor and variably expressed 66 kDa protein is encoded by a distinct shc transcript.
Overexpression of p465hC and p525hC induces transformation of mouse fibroblasts (Pelicci et al., 1992, Cell 70, 93-104) .`, ' ,' . `' '-2~13~

and activates the Ras pathway in PC12 cells (Rozakis~
Adcock et al., 1992, Nature 360, 689-692 ), suggesting that Shc proteins might normally be involved in mitogenic signalling. Shc proteins are phosphorylated on tyrosine in response to growth factors such as EGF and insulin (pelicci et al., 1992, Cell 70, 93-104; Pronk et al., 1993, J. Biol. Chem. 268, 5748-5753), and in cells transformed by the v-Src or v-Fps tyrosine kinases (McGlade et al., 1992, Proc. Natl. Acad. Sci. USA 89, 8869-8873), and concomitantly bind the SH2 domain of the Grb2 protein. Grb2 and Shc are thus implicated in the control of Ras by tyrosine kinases in a number of different biological systems.
The activation of p21raS appears critical for the induction of DNA synthesis and morphological transformation by oncogenic tyrosine kinases (Mulcahy et al., 1985, Nature 313, 241-248; Smith et al., 1986, Gene 67, 31-40 ; Feig and Cooper, 1988, Nol. Cell. Biol. 8, 3235-3243).
SUNNARY OF THE INVENTION
The present inventors have significantly found that proteins that participate in Ras activation mediate the effects of BCR-ABL. In screening for proteins that associate with BCR-ABL, the present inventors found that BCR-ABL tyrosine kinases in both human leukemic cells and transformed rodent fibroblasts, are bound to Grb2, a polypeptide with the structure SH3-SH2-SH3 (Lowenstein et al., 1992). Grb2 can function as a molecular adaptor by linking a subset of receptor tyrosine kinases and other phosphotyrosine-containing proteins to mSosl, a Ras-GNRP
(Buday and Downward, 1993, supra; Chardin et al., 1993, Science 260, 1338-1343 ; Egan et al., 1993, Nature 363, 45-51; Li et al., 1993, supra; Rozakis-Adcock et al., 1993, supra; Gale et al., 1993, Nature 363, 88-92; Skolnik et al., 1993, E~BO J. 12, 1929-1936). Genetic data from C. elegans and Drosophila suggest that the invertebrate homologues of Grb2 and mSosl are crucial in coupling 2 ~
:

receptor tyrosine kinases to activation of the Ras pathway (Simon et al., 1991; Clark et al., 1992; Olivier et al., 1993; Simon et al., 1993).
The present inventors found that BCR-ABL
complexed with an activator of Ras guanine nucleotide exchange, mSosl, in BCR-ABL-transformed cells. These findings suggest that Grb2 binds directly to BCR-ABL
oncoproteins through its SH2 domain, thereby recruiting mSosl, which is constitutively associated with the Grb2 SH3 domains, into a heterotrimeric BCR-ABL-Grb2-mSosl complex. The association of Grb2 with receptor tyrosine kinases is normally transiently induced by receptor auto-phosphorylation, which creates a specific binding site for the Grb2 SH2 domain (Lowenstein et al., 1992, supra; Buday and Downward, 1993, supra; Rozakis-Adcock et al., 1993, supra; Pawson and Schlessinger, 1993, supra). However, Grb2 only binds to a subset of receptor tyrosine kinases upon growth factor stimulation (Suen et al., 1993, Mol.
Cell. Biol. 13, 5500-5512), and has not been previously observed to bind stably to cytoplasmic tyrosine kinases.
The present inventors therefore investigated the mechanism by which Grb2 and BCR-ABL might stably associate in ~CR-ABL-transformed cells.
In vitro, the Grb2 SH2 domain specifically bound to BCR-ABL proteins. The Sem-5 SH2 domain binds preferentially to phosphotyrosine followed at the +2 position by an Asn (Songyang et al., 1993, Cell 72, 767-778). Examination of the sequences of P185 and P210 BCR-ABL revealed a single potential binding site for the Grb2 SH2 domain (Tyr-Val-Asn-Val), centered on Tyr~77within the common N-terminal BCR-encoded sequence of BCR-ABL
oncoproteins. The present inventors demonstrated that Tyrl77 undergoes autophosphorylation, and that the interaction of BCR-ABL with the Grb2 SH2 domain is specifically inhibited by a soluble phosphotyrosine-containing peptide representing the Tyr177 phosphorylation site. The ability of this peptide to bind with high 2 ~

affinity to the Grb2 SH2 domain, and thereby block the interaction with BCR-ABL or the immobilized BCR peptide, is dependent on Asn~79 at the +2 position relative to phosphotyrosine.
The ability of BCR-ABL oncoproteins to bind stably to Grb2, and hence to mSosl, therefore appears to result from autophosphorylation at Tyr~77 which creates a specific binding site for the Grb2 SH2 domain. The N-terminal BCR sequence is implicated in activation of the ABL tyrosine kinase domain (Muller et al., 1991, Mol.
Cell . Biol . 11, 1785-1792) and enhances the F-actin binding function of ABL (NcWhirter and Nang, 1991, Mol.
Cell . Biol . 11, 1553-1565). The present inventors' work indicates that BCR sequences also contribute directly to interactions of BCR-ABL proteins with their targets by providing autophosphorylation sites which allow BCR-ABL to couple directly to SH2-containing signalling proteins.
Thus, inhibiting the interactions of Grb2 with the phosphorylated Tyr177site of BCR-ABL represents an approach to reversing the transformed phenotype of Ph+ leukemic cells.
Neither Grb2 nor mSosl are significantly phosphorylated on tyrosine in BCR-ABL-transformed cells, suggesting that their function is regulated by their physical association with phosphotyrosine-containing proteins. Since a fraction of BCR-ABL localizes to F-actin filaments (McWhirter and Wang, 1991, supra;
NcWhirter and Wang, 1993, E~O J. 12, 1533-1546), binding of Grb2-mSosl to BCR-ABL may relocalize Ras-GNRP activity to the cortical cytoskeleton, providing access to Ras.
Alternatively, mSosl activity may be stimulated by interaction of Grb2-mSosl with BCR-ABL. P160 BCR
complexes with BCR-ABL in vivo (Campbell et al., 1990, oncogene 5, 773-776; Liu et al., 1993, Oncogene 8, 35 101-109) and undergoes transphosphorylation on Tyr177 in vitro, indicating that BCR itself, which is expressed by the normal chromosome 22 in Ph+ leukemic cells, may also -: . .:

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21~3~

activate Grb2-mSosl.
The shc gene products may be involved in Ras signalling, through an inducible interaction with Grb2 (Rozakis-Adcock et al ., 1992, supra). The investigations described herein identify the Grb2-binding site in Shc as pTyr-Val-Asn-Val which is also present at the autophosphorylation site located at Tyrl77 within the BCR
region of the oncogenic tyrosine kinase BCR-ABL as described above. Investigations described herein also show a correlation between Grb2-binding with Shc and Shc transforming activity.
The present inventors found that a minor fraction of Shc co-precipitated with BCR-ABL proteins.
This physical association of Shc polypeptides with BCR-ABL
may serve to facilitate Shc phosphorylation. In BCR-ABL-transformed cells, tyrosine phosphorylated Shc proteins associated with Grb2. The multiple Grb2 complexes that are formed may act synergistically to enhance Ras activation, and to enable BCR-ABL oncoproteins to transform hematopoietic cells.
The finding that BCR-ABL oncoproteins complex with Grb2 and Sos, and that phosphorylated Shc proteins associated with Grb2 in BCR-ABL transformed cells allows the identification of substances which affect BCR-ABL
mediated transformation and which accordingly may be used in the treatment of conditions such as chronic myelogenous leukemia and acute lymphoblastic leukemia. Furthermore, the finding of a correlation between Grb2-binding with Shc and Shc transforming activity allows the identification of substances which regulate the expression of Shc and which may be useful in the treatment of conditions such as malignancles.
Therefore, the present invention relates to a method for assaying for a substance that affects BCR-ABL
mediated transformation, preferably an antagonist of BCR-ABL mediated transformation, which comprises reacting a first compound containing Grb2 or a portion thereof, ~, i 3 4 9 i~

g _ , ' ' ~

preferably an SH2 or SH3 domain of Grb2, and a second compound containing a Grb2 binding site on BCR-ABL, Sos or Shc, or mimetics thereof, in the presence of a substance which is suspected of affecting BCR-ABL mediated transformation, under conditions where the first and second compounds are capable of forming a complex, and comparing with a control.
In one embodiment of the invention, a method is provided for assaying for a substance that affects BCR-ABL
mediated transformation comprising reacting a first compound containing the SH2-domain of Grb2, with a second compound containing the Grb2 binding site on BCR-ABL or Shc, or mimetics thereof, in the presence of a substance which is suspected of affecting BCR-ABL mediated transformation, under conditions where the first compound and the second compound are capable of forming a complex assaying for complexes, or free first and second compounds, and comparing with a control.
In another embodiment of the invention, a method is provided for assaying a medium for an antagonist of BCR-ABL mediated transformation comprising reacting a first compound containing an SH2 domain of Grb2, with a second compound containing the Grb2 binding site on BCR-ABL, or mimetics thereof, in the presence of a suspected antagonist of BCR-ABL mediated transformation, under conditions where the first compound and the second compound are capable of forming a complex, assaying for the complex, or free first and second compounds, and comparing with a control.
In another embodiment of the invention, a method i9 provided for assaying for the presence of a substance that affects BCR-ABL mediated transformation comprising reacting a first compound containing an SH3-domain of Grb2, and a second compound containing the ~rb2 binding site on SOS, in the presence of a substance which is suspected of affecting BCR-ABL mediated transformation, under conditions where the first compound and the second ~3~L~s compound are capable of forming a complex, assaying for complexes or free first and second peptides, and comparing with a control.
Still further the invention contemplates a method for assaying for a substance that affects Shc mediated transformation which comprises reacting a first compound containing an SH3 domain of Grb2, and a second compound containing a Grb2 binding site on Shc, or a mimetic thereof, in the presence of a substance which is suspected of affecting Shc mediated transformation under conditions where the first and second compounds are capable of forming a complex, and comparing with a control.
The invention further provides a composition comprising a compound containing Grb2 or a portion thereof, preferably an SH2 domain or SH3 domain of Grb2, the Grb2 binding site on BCR-ABL, Sos or Shc, or mimetics thereof, and a pharmaceutically acceptable carrier. A
composition comprising an antisense sequence constructed by inverting the nucleotide sequences of Grb2, Sos or Shc is also contemplated. The compositions may be used as an antagonist of one of the interactions of BCR-ABL and Grb2, and Grb2 and Sos or Shc.
The invention also relates to a method of using a compound containing Grb2 or a portion thereof, preferably an SH2 domain or SH3 domain of Grb2 , or the Grb2 binding site on BCR-ABL, Sos or Shc, or mimetics thereof, to regulate the interaction of Grb2 with BCR-ABL, Sos, or Shc.
The invention also contemplates a method of treating a patient suffering from CNL, ANL or ALL
comprising administering an effective amount of a compound containing Grb2 or a portion thereof preferably, an SH2 domain or an SH3 domain of Grb2, or a Grb2 binding site on BCR, Sos or Shc, or mimetics thereof, or a substance identified by the methods of the invention.
These and other aspects of the present invention ' `' ~' r ~

-2~3~9~ :
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will become evident upon reference to the following detailed description and attached drawings. In addition, -~
reference is made herein to various publications, which are hereby incorporated by reference in their entirety.
DESCRIPTION OF THE DRAWINGS
The invention will now be described in rela~ion to the drawings in which~
Figure 1 shows the amino acid sequence of :~
-.--. :::
Grb2;
Figure 2 shows the nucleotide sequence of Grb2;
Figure 3 shows the amino acid sequence of Shc; -~
Figure 4 shows the nucleotide sequence of Shc;
Figure 5 shows the amino acid sequence of mSosl;
Figure 6 shows the nucleotide sequence of mSosl;
Figure 7 shows the amino acid sequence of mSos2; ~ -E~
Figure 8 shows the nucleotide sequence of mSos2;
Figure 9 illustrates that Grb2 associates with multiple phosphoproteins in BCR-ABL-transformed cells;
Figure 10 illustrates that BCR-ABL oncoproteins form complexes with Grb2 in vivo;
Figure 11 illustrates that the SH2 domain of Grb2 binds BCR-ABL oncoproteins in vitro; ~ :~
Figure 12 illustrates that the Grb2 SH2 domain directly recognizes BCR-ABL proteins but not c-Abl;
Figure 13 illustrates that Tyr177 in P210 BCR-ABL
is an autophosphorylation site recognized by the Grb2 SH2 :~
domain, and undergoes transphosphorylation in P160 BCR;
Figure 14 illustrates that the Grb2 SH2 domain specifically interacts with the BCR-encoded sequence :
30 p.Tyr177-Val-Asn-Val; ::
Figure 15 illustrates that ~CR-ABL oncoproteins complex with mSosl in vivo;
Figure 16 illustrates that SHC proteins are tyrosine phosphorylated and associated with Grb2 in Ph+ ~
35 leukemic cells; and :
Figure 17 illustrates that the tyrosine ~ -phosphorylation of Shc proteins and their association with ~

. . , .. - - - -, . . . . . . .

Grb2 in Rat-1 fibroblasts is induced by expression of BCR-- ~ . ,- ~.
ABL oncoproteins.
DETAILED DESCRIPTION OF THE INVENTION
As hereinbefore mentioned, the present invention relates to a method for assaying for the presence of a substance that affects BCR-ABL mediated transformation.
The term "BCR-ABL mediated transformation" used herein refers to the interactions of Grb2 or portions thereof, with its binding sites on BCR-ABL, Sos or Shc, or mimetics thereof, and includes the binding of the SH2 domain of Grb2 to BCR or to Shc and the binding of the SH3 domains of Grb2 to Sos, or any modifications to the SH2 domain or SH3 domain or to the binding sites on BCR, Sos or Shc associated therewith, to form complexes thereby activating the Ras pathway which induces DNA synthesis and morphological transformation.
The invention also relates to a method for assaying for the presence of a substance that affects Shc mediated transformation. The term "Shc mediated transformation" used herein refers to the interactions of Grb2 or portions thereof, with its binding sites on Shc, or mimetics thereof, and includes the binding of the SH2 domain of Grb2 to Shc, or any modifications to the SH2 domain or to the binding sites on Shc associated therewith, to form complexes thereby activating the Ras pathway which induces DNA synthesis and morphological transformation.
The term "mimetics" referred to herein refers to functional equivalents of Grb2 or its SH2 and SH3 domains, and of SH2 or SH3 binding sites on BCR-ABL, Sos or Shc.
The selection of mimetics may be done using conventional methods such as described in P.S. Farmer, in Drug Design, E.J. Ariens, Ed. (Academic Press, New York, 1980) vol. 10, ppll9-143; J.B. Ball, P.F. Alewood, J. Mol. Recognition 3, 55 (1990); B.A. Morgan and J.A. Gainor, Annu. Rep. Med.
Chem. 24, 243 (1989); R.M. Freidinger, Trends Pharmacol.
Sci. 10, 270 (1989) and as illustrated in G.L. James et : : :-; -:

.

al, Science, 260, 1937, 1993 and N.E. Kohl et al, Science 260,1934, 1993, which are all incorporated herein by reference.
In a method of the invention a substance suspected of affecting BCR-ABL mediated transformation is reacted with a first compound. The first compound may be a polypeptide or a peptide containing Grb2 or a portion thereof, preferably SH2 or SH3 domains of Grb2. Grb2 is comprised entirely of a single SH2 domain flanked by two SH3 domains (Lowenstein et al., 1992, Cell 70, 431-442).
The amino acid and nucleotide sequences of Grb2 are shown in Figures 1 and 2 and in the Sequence Listing as SEQ. ID.
NOS. 1 and 2, respectively. The N-terminal SH3 domain is at amino acid residues 5-55; the SH2 domain is at amino acid residues 60-157; and, the C-terminal SH3 domain is at amino acid residues 163-214 as shown in Figure 1 and in SEQ. ID. NO.l.
The second compound used in the methods of the invention is selected so that it binds with the first compound to form a complex. The complex is preferably capable of activating the Ras pathway. The second compound may be selected by reacting a candidate compound which has been expressed and phosphorylated in a host cell with Grb2 or portions thereof, and determining binding activity. In a preferred method a tyrosine phosphorylated second compound which is a polypeptide or peptide may be identified by expressing a sequence encoding residues of the compound as a fusion protein, preferably a TrpE fusion protein, in a host cell, preferably a microorganism, most preferably Escherichia coli; inducing tyrosine phosphorylation of the fusion protein by infecting the host cell with a vector encoding the cytoplasmic domain of a protein tyrosine kinase, preferably the Agtll bacteriophage encoding the cytoplasmic domain of the Elk tyrosine kina~e (ABl-Elk); isolating host cells capable of expressing the fusion protein and the vector as a lysogen;
labelling the host cell preferably with [32P]orthophosphate; sequentially inducing the fusion protein and the tyrosine kinase; immunoprecipitating labelled lysates with antibodies to the fusion protein, preferably anti-TrpE antibodies (tYTrpE), separating the 5 immune complexes and subjecting to autoradiography and phosphoamino acid analysis. Once tyrosine phosphorylation of the candidate second compound has been identified, it can be assayed for its binding to Grb2 or portions thereof, isolated for example as a GST fusion protein .
10 Using this method complementary first and second compounds may be identified.
The second compound may contain a Grb2 binding site on BCR-ABL proteins preferably P210 or P185, or a mimetic thereof. The amino acid and nucleotide sequence of 15 P210 and P185 are described in Mes-Masson, A.M. et al.
Proc. Natl. Acad. Sci. USA 83, 9768-9772, 1986; Fainstein E. et al. Nature 330, 386-388, 1987; and Hermans, A. et al, Cell 51, 33-40, 1987. Preferably, the second compound is a peptide containing the sequence Tyr-Val-Asn-Val 20 phosphorylated on Tyr. Examples of suitable second compounds are the phosphopeptides DAEKPFpY177VNVEFK which corresponds to amino acids 171-182 of human BCR-ABL, with a carboxy terminal lysine added, GHQQPGADAEKPFpY177VNVE
which corresponds to amino acids 166-181 of human BCR-ABL, 25 and KGHQQPGADAEKPFpY177VNVE, which corresponds to amino acids 165-181 of human BCR-ABL. (Note that Table 1 sets out the meanir.g of the single letter amino acid designations used herein).
The second compound may also be a polypeptide or 30 peptide which contains a Grb2 binding site on Shc, or Sos, or mimetics thereof. The amino acid and nucleotide sequences of the 46 kDa and 52 kDa Shc proteins are shown in Figures 3 and 4 and in the Sequence Listing as SEQ ID
NOS. 2 and 3. More particularly, the 52 kDa peptide starts 35 at amino acid residue 1 and the 46 kDa peptide starts at amino acid residue 51 as shown in Figure 3 and SEQ ID. NO.
2. The core of the Grb2 binding site on the Shc protein ~ ~ ~ 3 ~ 9 ~

- 15 ~
. -has been found to be pY317VNV tamino acid residues 312-315) (See Example 7 herein). The second compound may accordingly contain the sequence Tyr-Val-Asn-Val phosphorylated on Tyr. An example of a second phosphorylated peptide that contains the Grb2 binding site on Shc which may be used in the methods of the invention is ELFDDPSpY317VNVQNI,DK.
The amino acid and nucleotide sequences of mSosl and mSos2 are shown in Figures 5 to 8 and in the Sequence Listing as SEQ ID NOS. 4, 5, 6, and 7. The SH3 domains of Grb2 associate with proline-rich motifs in the C-terminal tail of mSos (amino acids 1149-1161, 1178-1190, 1210-1222 and 1288-1300 of mSosl as shown in Figure 5 and amino acids 1107-1119, 1133-1145, 1164-1176 and 1178-1189 in Figure 6). Thus, sequences modelled on the tail of Sos may be contained in the second compound. A synthetic peptide containing the sequences PPPVPPR, PPPLPPR, PPAIPPR, PPLLPPR, PPPPPPR, PPPVPLR, GPPVPPR, and APPVPPR which has been shown to block the binding of Grb2 to Sos are preferably used. Examples of such peptides are EVPVPPPVPPRRR, EPLIPPPLPPRKK, HLDSPPAIPPRQP, SDDDPPAIPPRQP, PPESPPLLPPREP, PVPSPPPPPPRDP, LPDTPPPVPLRPP, HSIAGPPVPPRQS, and AHLPAPPVPPRQS.
The first and second compounds may be synthetically constructed. They may also be expressed in a host cell, preferably a bacterial host cell as a fusion protein, for example a glutathione-S-transferase (GST) fusion protein as discussed above. Grb2, Grb2 SH2, and N-or C-terminal Grb2 SH3 domain fusion proteins may be prepared as described in Lowenstein et al., 1992, Cell 70, 431-442. The second compound may be phosphorylated using the methods described above and in N. Reedijk et al., EMBO
Journal 11(4):1365-1372, 1992.
Conditions which permit the formation of complexes between the first and second compounds may be selected having regard to factors such as the nature and amounts of the substance to be tested and the first and ';

2 ~

second compounds. Generally a known concentration of the first and/or second compounds are used in the methods of the invention.
It will be appreciated that the selection of the first and second compounds in the methods of the invention will depend on the nature of the substance to be assayed.
It will also be appreciated that the selection of a specific complementary first and second peptide in the method of the invention will allow for the identification of a specific substance that affects a specific interaction. Thus, the identification of the specific site of binding of the Grb2 SH2 domain to BCR-ABL and of Grb2 SH3 domain to Shc as described herein allows the identification of specific substances that affect the interaction of BCR-ABh and Grb2, and Shc and Grb2.
The complexes, and free first and second compounds in the methods of the invention may be isolated by conventional isolation techniques, for example, salting out, chromatography, electrophoresis, gel filtration, fractionation, absorption, polyacrylamide gel electrophoresis, agglutination, or combinations thereof.
The assaying for complexes or free first and second compounds in the methods of the invention may be carried out using known methods. To facilitate the assay of the components, antibody against the first or second compound which may be labelled, or a labelled first or second compound may be utilized.
The first or second compounds may be used to prepare antibodies. Within the context of the present invention, antibodies are understood to include monoclonal antibodies! polyclonal antibodies, antibody fragments (e.g~ Fab, and F~ab') 2 and recombinantly produced binding partners. Polyclonal antibodies may be readily generated by one of ordinary skill in the art from a variety of warm-blooded animals such as horses, cows, various fowl, rabbits, mice, or rats. Monoclonal antibodies may also be readily generated using conventional techniques (see U.S.

2 1 :i 3 ~
.
- 17 ~

Patent Nos. RE 32,011, 4,902,614, 4,543,439, and 4,411,993 which are incorporated herein by reference; see also Nonoclonal Antibodies, Hybridomas: A New Dimension in Biological Analyses, Plenum Press, Kennett, McKearn, and Bechtol (eds.), 1980, and Antibodies: A Laboratory Manual, Harlow and Lane (eds.), Cold Spring Harbor Laboratory Press, 1988, which are also incorporated herein by reference). Binding partners may also be constructed utilizing recombinant DNA techniques to incorporate the variable regions of a gene which encodes a specifically binding antibody (See Bird et al., Science 242:423-426, 1988).
The antibodies against the first or second compounds, and/or the first and second antibodies may be labelled with various enzymes, fluorescent materials, luminescent materials and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, biotin, alkaline phosphatase, ~-galacto6idase, or acetylcholinesterase; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; and examples of suitable radioactive material include radioactive phosphorous 32p, iodine I125, I131 or tritium. Antibodies or the first and second compounds may also be coupled to electron dense substances, such as ferritin or colloidal gold, which are readily visualised by electron microscopy.
Radioactive Iabelled materials may be prepared by radiolabeling with 12sI by the chloramine-T method (Greenwood et al, Biochem. J. 89:114, 1963), the lactoperoxidase method (Marchalonis et al, Biochem. J.
124:921, 1971), the Bolton-Hunter method (Bolton and Hunter, Biochem. J. 133:529, 1973 and Bolton Review 18, ~mersham International Limited, Buckinghamshire, England, 1977), the iodogen method (Fraker and Speck, Biochem.
Biophys. Res. Commun. 80:849, 1978), the Iodo-beads method .~::: .. :

2 i ~ 3 '~
- 18 ~

(Markwell Anal. Biochem. 125:427, 1982) or with tritium by reductive methylation (Tack et al., J. Biol. Chem.
255:8842, 1980).
Known coupling methods (for example Wilson and Nakane, in "Immunofluorescence and Related Staining Techniques~, W. Knapp et al, eds, p. 215, Elsevier/North-Holland, Amsterdam & New York, 1978; P. Tijssen and E.
Kurstak, Anal. Biochem. 136:451, 1984) may be used to prepare enzyme labelled materials. Fluorescent labelled materials may be prepared by reacting the material with umbelliferone, fluorescein, fluorescein isothiocyanate, dichlorotriazinylamine fluorescein, dansyl chloride, derivatives of rhodamine such as tetramethyl rhodamine isothiocyanate, or phycoerythrin.
The first or second compounds used in the method of the invention may be insolubilized. For example, the first or second compounds may be bound to a suitable carrier. Examples of suitable carriers are agarose, cellulose, dextran, Sephadex, Sepharose, carboxymethyl cellulose polystyrene, filter paper, ion-exchange resin, plastic film, plastic tube, glass beads, polyamine-methyl vinyl-ether-maleic acid copolymer, amino acid copolymer, ethylene-maleic acid copolymer, nylon, silk, etc. The carrier may be in the shape of, for example, a tube, test plate, beads, disc, sphere etc.
The insolubilized firs~ and second compounds may be prepared by reacting the material with a suitable insoluble carrier using known chemical or physical methods, for example, cyanogen bromide coupling.
The methods of the invention may be used to assay for a substance that affects BCR-ABL mediated transformation, preferably a suspected antagonist.
Generally an antagonist may constitute any molecule with similar binding activity to the natural ligand but incapable of propagating the biological response nGrmally induced by the ligand. For example, an antagonist may constitute a molecule containing the Grb2 SH2 or SH3 :; .: ~:: ~:.

3~ 1 binding sites on Shc, Sos or BCR-ABL. The antagonist may be an endogenous physiological substance or it may be a natural or synthetic drug.
It will be understood that the substances that can be assayed using the methods of the invention may act on one or more of the Grb2 binding site on BCR-ABL, Sos, or Shc, or the binding site on Grb2 or the SH2 or SH3 domains thereof, including agonist binding sites, competitive antagonist binding sites, non-competitive antagonist binding sites or allosteric sites.
The methods of the present invention enable the screening of substances for use in the treatment of conditions such as CML, AML, and ALL. Such substances may be tested in animal models such as the rat c-myc cell transformation system described by T. Lugo and O.N. Witte, 1989, Nol. Cell. Biol. 9:1263-1270; the BaF3 cell system described by J.W. Whirter and J.Y.J. Wang, EMBO, 12, 1533~
1546; the mouse transgenic bcr/abl pl90 mice described by N. Heisterkamp et al. Nature 344:251, 1990; and, the Daley model of bone marrow transplantation (Science 247:824-830, 1990).
The methods of the present invention enable the screening of substances for use in the treatment of malignancies including malignancies induced by oncogenically activated tyrosine kinases for example mutations of the ret and trk genes result in the activation of their encoded receptor tyrosine kinases in papillary thyroid carcinomas and multiple endocrine neoplasia type 2A, and amplification and overexpression of the genes encoding Neu, the epidermal growth factor receptor and platelet-derived growth factor receptor are detected in breast carcinoma or glioblastomas.
The present invention also provides for experimental model systems for studying BCR-ABL mediated transformation. For example experimental models could be constructed where there are mutations in Grb2, Sos or Shc and the animals do not succumb to BCR-ABL transformation.

~ ~ i 3 ~

The invention also relates to a pharmaceutical composition comprising a compound containing Grb2 or a portion thereof, preferably an SH2 domain or an SH3 domain, a Grb2 binding site on BCR-ABL, Sos or Shc, or mimetics thereof. The pharmaceutical compositions may also contain substances identified using the methods described herein. The pharmaceutical compositions may be used as an antagonist of one of the interactions of BCR-ABL and G-b2 or Grb2 and Sos or Shc. Accordingly, the pharmaceutical compositions may be used to treat conditions such as ALL, CML, and malignancies as described above.
The pharmaceutical compositions of the invention contain Grb2 or portions thereof, or a Grb2 binding site on BCR-ABL, Sosl or Shc, or mimetics thereof, alone or together with other active substances. Such pharmaceutical compositions can be for oral, topical, rectal, parenteral, local, inhalant or intracerebral use. They are therefore in solid or semisolid form, for example pills, tablets, creams, gelatin capsules, capsules, suppositories, soft gelatin capsules, gels, membranes, tubelets. The compositions of the invention may also be conjugated to transport molecules such as transferrin to facilitate transport of the composition across the blood brain barrier.
The pharmaceutical compositions of the invention can be intended for administration to humans or animals.
Dosages to be administered depend on individual needs, on the desired effect and on the chosen route of administration.
The pharmaceutical compositions can be prepared by per se known methods for the preparation of pharmaceutically acceptable compositions which can be administered to patients, and such that an effective quantity of the active substance is combined in a mixture with a pharmaceutically acceptable vehicle. Suitable vehicles are described, for example, in Remington's Pharmaceutical Sciences (Remington's Pharmaceutical . - .

..... . . .. .

2 ~ ~ 3 ~1 n, ~

.
Sciences, Mack Publishing Company, Easton, Pa., ~SA 1985).
On this basis, the pharmaceutical compositions include, albeit not exclusively, Gxb2 or a portion thereof, preferably and SH2 or SH3 domain, or a Grb2 binding site on BCR, Sos or Shc, or mimetics thereof, in association with one or more pharmaceutically acceptable vehicles or diluents, and contained in buffered solutions with a suitable pH and iso-osmotic with the physiological fluids.
The present invention also provides for methods in which a patient suffering from a disorder, such as CML, AML, and ALL, iS treated with an effective amount of Grb2 or a portion thereof, preferably an SH2 domain or SH3 domain, or a Grb2 binding site on BCR, Sos or Shc, or mimetics thereof. Therapeutic methods comprising administering substances identified with the methods of the present invention are also within the scope of the present invention.
The invention also contemplates a method of using the substances identified using the methods of the invention and the pharmaceutical compositions of the invention for treating autologous transplants.
Transplantation of hematopoietic cells from peripheral blood and/or bone marrow is increasingly used in combination with high-dose chemo- and/or radiotherapy for the treatment of CML and ALL. The success of an autologous transplant to a large degree depends on the elimination of cells in the transplant that pose a risk to the transplant recipient such as tumour cells in autologous transplants that may cause recurrence of the malignant growth. The present invention provides a method of treating autologous transplants with the substances identified using the methods of the invention and the pharmaceutical compositions of the invention to regulate BCR-ABL mediated transformation in tumor cells in the transplant that may cause recurrence of malignant growth.
The invention contemplates pharmaceutical . . : .
compositions containing an antisense sequence which is constructed by inverting the nucleotide sequences of Grb2, Sos, or Shc as shown in Figures 2, 4, 6, and 8 and in SEQ
ID NOS 2,4, 6, and 8 relative to their normal presentation for transcription. The antisense sequences may be constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art. The antisense sequences may also be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed with the mRNA or gene e.g.
phosphorothioate derivatives and acridine substituted nucleotides. The antisense sequences may be produced biologically using an expression vector introduced into cells in the form of a recombinant plasmid, phagemid or attenuated virus in which antisense mRNA sequences are produced under the control of a high efficiency regulatory region, the activity of which may be determined by the cell type into which the vector is introduced.
For a discussion of the regulation of gene expression using anti-sense genes see Weintraub, H. et al., Antisense RNA as a molecular tool for genetic analysis, Reviews - Trends in Genetics, Vol. 1(1) 1986.
The compositions of the invention containing antisense sequences may be used in gene therapy to prevent BCR-ABL mediated transformation and accordingly to treat CML and ALL. The pharmaceutical composition may contain recombinant molecules comprising an antisense sequence and it may be directly introduced into hematopoietic cells for example in bone marrow or peripheral blood using in vivo delivery vehicles such as retroviral vectors, adenoviral vectors and DNA virus vectors. They may also be introduced into such cells in vivo using physical techniques such as microinjection and electroporation or chemical methods such as coprecipitation and incorporation of DNA into liposomes.

-~ 2~3~n~ ' , The pharmaceutical compositions may also be delivered in the form of an aerosol or by lavage.
The following non-limiting examples are illustrative of the present invention~
EXAMPLES
The following materials and methods were used in Examples 1 to 6~
NATERIALS AND METHODS
Cell culture The K562 cell line was obtained from the American Tissue Type Collection. The Ph+ve ALL cell line SUPB15 was generously provided by S. Smith (University of Chicago). K562 and SUPB15 cells were maintained in RPMI
medium supplemented with 10% fetal bovine serum ~FBS).
Rat-1 fibroblasts were grown in Dulbecco modified Eagle medium (DME) containing 5% FBS. Rat-1 cells stably expressing P185 or P210 BCR-ABL (Lugo et al., 1987, Science 237, 532-535) were maintained in DME supplemented with 5% FBS and 50 ~g ml~1 of G418.
Immunoprecipitations and Western blotting Immunoprecipitations were carried out as previously described (Moran et al., 1990, Proc. Natl.
Acad. Sci. USA, 87 8622-8626). All cells were lysed in 1 ml of PhC lysis buffer (50 mM of N-2 hydroxyethyl piperazine-N'-2 ethane sulfonic acid (HEPES, pH 7.5), 150 mM NaCl, 10% (vol/vol) glycerol, 1% (vol/vol) Triton X-100, 1.5 mN NgCl2, 1 mN EGTA, 10 ~g ml~1 of leupeptin, 10 ~g ml~1 of aprotinin, 1 mN sodium orthovanadate, 10 mN
sodium pyrophosphate, 100 mM sodium fluoride and 1 mM
phenylmethanesulfonyl fluoride). Cell lysates were centrifuged for 15 minutes at 10,000 g and the supernatant collected. In some experiments, protein concentrations of the clarified supernatants were determined and similar quantities of protein immunoprecipitated. Cell lysates were incubated and gently rocked at 4~C for 90 minutes with anti-ABL pEx5 antibody (Ronopka et al., 1984, CELL 37, 1035-10422), anti-Grb2 50 antibody (Lowenstein et al., ',' 2 ~. ~ 3 ~

1992, Cell 70, 431-442), anti-SHC antibody tPelicci et al., 1992, Cell 70, 93-104) or mSosl antibody (sowtell et al ., 1992, Proc. Natl . Acad . Sci . VSA 89, 6511-1515) and 100 ~1 of 10% protein A sepharose. The immmune complexes were washed 3 times with 20 mM HEPES (pH 7.5), 10%
glycerol, 0.1% Triton X-100, 150 mM NaCl and 1 mM sodium orthovanadate (HNTG), and heated in sodium dodecyl sulfate sample buffer prior to gel electrophoresis and transferred to nitrocellulose with a semi-dry transfer apparatus at 0.8 mA.cm~2 for 60 minutes. For immunoblotting with ABL, SHC and Grb2 antibodies, filters were blocked in Tris buffered saline (TBS) (20 mM Tris.HCl) (pH 7.5), 150 mM
NaCl) with 5~ Carnation skim milk powder. Antibody incubations were carried out in TBS with 3% milk powder and 0.1% Tween. To detect ABL proteins, a mouse monoclonal antibody directed against the carboxy terminal region of c-ABL was used at a concentration of 1 ~g ml~l (Schiff-Maker et al., 1986, ~. Virol 57, 1182-1186). Grb2 55 antibodies raised against the entire Grb2 molecule were used for Western blotting at a 1/200 dilution (Lowenstein et al., 1992, Cell 70, 431-442). mSosl immunoblotting was performed as previously described (Rozakis-Adcock et al., 1993, Nature363, 83-85). For anti-phosphotyrosine immunoblotting, filters were blocked in TBS containing 5%
bovine serum albumin and 1% ovalbumin and incubated with affinity purified anti-phosphotyrosine antiserum (Letwin et al., 1988, Oncogene 3, 621-627). Blots were then probed with [125I]-labelled protein A (35~Ci/ml, Amersham) in blocking solution and exposed to Kodak XAR-5 film. For anti-ABL blots, a secondary incubation was carried out with horseradish peroxidase conjugated anti-immunoglobulin antiserum (sigma) prior to development with chemiluminescent reagents (Amersham) and exposure to Kodak XRP-5 film.
Bacterial fusion proteins Regions of Grb2 and the ABL SH3 domain were isolated by the polymerase chain reaction (PCR) and : ~` ~ : ~ `':

~' " ~' . :
~., ~ . ..

~ 1 1 3 ~

- ~ . .

subcloned into pGex expression vectors (Smith and Johnson, 1991, Gene 67, 31-40). Full length Grb2 and Grb2 SH2 fusion proteins have been described (Lowenstein et al., 1992, Cell 70, 431-442). The N- and C-terminal Grb2 SH3 fusion proteins contain portions of Grb2 corresponding to amino acid residues 1-58 and 159-217 respectively. The ABL-SH3 fusion protein corresponds to amino acids 929-1020 of P210 BCR-ABL. For the Abl SH2 fusion protein, the N~
terminal region of type IV murine c-Abl was isolated by PCR, beginning with the Stul restriction site and terminating at the 3l end of the SH2 domain. The Abl SH3 domain was subsequently deleted by PCR, and the fragment subcloned into a pGex vector. Bacterial cultures expressing the pGex vectors were grown in LB and ampicillin 100 ~g ml~1, and induced with 1 mM isopropyl thiogalactopyranoside (IPTG) for 4 hours at 30 or 37C.
The induced bacteria were lysed by sonication in PLC LB, and the glutathione-S-transferase (GST) fusion proteins recovered from clarified lysates by glutathione-agarose (Pharmacia). The amount of each GST fusion protein was determined by comparison of bands on a Coomassie Blue stained gel to a dilution series of bovine serum albumin;
similar quantities of fusion proteins were incubated with cell lysates for 90 minutes at 4C. SH2 binding proteins were recovered by centrifugation, washed 3 times in HNTG
and resuspended in sodium dodecyl sulfate sample buffer.
Two-dimensional trypsin/V8 protease maps of BCR-ABL and BCR proteins.
For analysis of in vitro autophosphorylated sites, P~10 and P185 BCR-ABL were immunoprecipitated from K562 and SUPB15 cells respectively, by ABL monoclonal p6D
antibody raised against a synthetic peptide corresponding to amino acids 51-64 of human c-ABL (Li et al., 1988, Oncogene 2, 559-566). Proteins were labelled in an in vitro kinase assay using y-32P-ATP, separated by SDS-PAGE
and extracted from the gel as described (Liu et al., 1993, Oncogene 8, 101-109). P160 BCR was immunoprecipitated 2 ~ i 3~lt~

from K562 cells by antibody raised against a synthetic peptide corresponding to amino acids 1256-1271 of the C-terminal portion of BCR, and transphosphorylated by P210 BCR-ABL in vitro as previously described (Liu et al., 1993, Oncogene 8, 101-109). Performic acid oxidized proteins were first treated with N-tosyl-L-phenylalanyl chloromethyl ketone (TPCK)-treated trypsin for 22 hours and then after boiling for 5 minutes treated with V8 protease (Glu-C) for 22 hours under conditions that favor cleavage after glu (pH 7. 8 ammonium bicarbonate) (Liu et al., 1993, Oncogene 8, 101-109). Peptides were separated by two-dimensional electrophoresis and chromatography, sequenced by manual Edman degradation, and the elution position of 32P-labelled phosphotyrosine monitored (Sullivan and Wong, l991, Anal. Biochem. 197, 65-68; Roach and Wang, 1991, Nethods in Enzymology 201:200-226). For analysis of peptides that bound to GST-Grb2 SH2, a trypsin/V8 protease digest of in vitro labelled P210 BCR-ABL was mixed with immobilized GST-Grb2 SH2 or GST-Abl SH2 20 fusion proteins for 90 minutes at 4C. Bound peptides were removed by centrifugation, and the remaining peptides analyzed by two-dimensional electrophoresis and chromatography.
BIAcore analysis of phosphopeptides interacting with Grb2 25 SH2 or full length Grb2.
Phosphopeptides were synthesized by 9-fluorenyl methoxy carbonyl (Fmoc) chemistry according to published methods (Piccione et al., 1993, J. Biol. Chem. 268, 3197~
3202). The sequences of these synthetic phosphopeptides 30 were: BCR, DAEKPFp.Y177VNVEFK, corresponding to amino acids 171-182 of human BCR, with a carboxy terminal lysine added; Asn~Ala BCR, identical to the BCR peptide except for substitution of alanine for asparagine at the +2 residue carboxy terminal to Tyr1n; SHC, 35 ELFDDPSp.Y317VNVQNLDK corresponding to the Grb2 recognition site on the SHC protein (Pelicci et al., 1992, Cell 70, 93-104); PDGFR 771, SSNp.Y771MAPYDNYK, corresponding to the ` " ' ~ ` `, ` ' :

3 ~

GAP recognition site of the PDGF receptor (Kashishian et al., 1992, EMBO J. 11, 1373-1382); PDGFR 1009, SSVLp.Yl~WTAVQP corresponding to the Syp recognition site on the PDGF receptor (Kazlauskas et al., 1993, Proc. Natl.
Acad. Sci. USA 90:6939-6942). For peptide immobilization to the sensor chip surface, a 0.5 mN solution of BCR
phosphopeptide in 50 mN HEPES (pH 7.5) and 2M NaCl was used following conditions previously described (Payne et al., 1993, Proc. Natl. Acad. Sci. USA 90, 4902-4906). GST
fusion proteins containing full length human Grb2 or the SH2 domain were resuspended in 50 mM HEPES (pH 7.5), 150 mN NaCl and 3.0 mN EDTA, and concentrated using a Centricon 10 microconcentrator device (Amicon). Solutions (100 ~1) containing 1 ~N GST-Grb2 or 1 ~M GST-Grb2 SH2 and the indicated concentration of soluble peptide in 50 mN
HEPES, pH 7.5, 150 mM NaCl and 3 mM EDTA, was injected across the surface containing immobilized BCR
phosphotyrosine peptide. The amount of GST-Grb2 or GST-Grb2 SH2 protein bound was estimated from the steady-state surface plasmon resonance signal at a fixed time following the end of the injection and the percentage bound relative to in~ection of GST-Grb2 SH2 alone calculated (Ponzetto et al., 1993, Mol. Cell. Biol. 13, 4600-4608). After in~ection the peptide surface was regenerated using 2N
GuHCl.

Grb2 forms a stable complex with BCR-ABL oncoproteins in v vo Fibroblasts transformed by P185 or P210 BCR-ABL
possess a variety of phosphotyrosine-containing proteins (Lugo et al ., 1987, Science 237, 532-535) which might potentially associate with the Grb2 SH2 domain and thereby stimulate the Ras pathway. To test whether Grb2 interacts with phosphoproteins in BCR-ABL-transformed cells, anti-Grb2 immunoprecipitates from Rat-l (Rl) fibroblasts transformed by expression of P185 BCR-ABL (Rl-P185) were immunoblotted with antibodies to phosphotyrosine.

Multiple phosphoproteins were detected in association with Grb2 (Figure 9). In Figure 9 the positions of P185 BCR-ABL
and p525hC are indicated by arrows, and the mobilities of size markers shown. One prominent phosphotyrosine-containing protein that co-precipitated with Grb2 had the same mobility as P185 BCR-ABL, suggesting that Grb2 and BCR-ABL proteins might form a stable complex in vivo. To investigate this possibility, anti-Grb2 immunoprecipitates from lysates of Rl-P185 cells, or R1 fibroblasts expressing P210 BCR-ABL (R1-P210), were probed with anti-ABL antibody. Both forms of BCR-ABL were directly identified as co-precipitating with Grb2 ~Figure 10a). In the reciprocal experiment, Grb2 was found to be associated with BCR-ABL, precipitated with anti-ABL antibodies (Figure 10b). Duplicate samples were subsequently immunoblotted with anti-ABL antibodies (a) or anti-Grb2 antibodies (b). Similar experiments using the CNL K562 and the Ph+ ALL SUPB15 cell lines, which express P210 and P185 BCR-ABL respectively (Lozzio and Lozzio, 1975, Blood 45, 321-334; Naumovski et al., 1988, Cancer Res. 48, 2876-2879), also demonstrated the co-precipitation of Grb2 and BCR-ABL (data not shown).
The association of Grb2 with the EGF receptor is strictly dependent on receptor autophosphorylation (Lowenstein et al., 1992, Cell 70, 431-442; Buday and Downward, 1993, Cell 73, 611-620; Rozakis-Adcock et al., 1993, Nature 363, 83-85). To as~ess the potential role of BCR-ABL autophosphorylation in the interaction with Grb2, anti-Grb2 immunoprecipitates from R1-P185 cells were treated with potato acid phosphatase, prior to immunoblotting with anti-ABL antiserum. Phosphatase treatment diminished the association of BCR-ABL with Grb2, indicating that recognition of a BCR-ABL phosphorylation site by Grb2 is likely to contribute to this interaction (data not shown).

The Grb2-SH2 domain is sufficient to bind BCR-ABL
, . - -~i~3~n;~ ;

oncoproteins in vitro The region of Grb2 responsible for its association with P210 and P185 BCR-ABL was further analyzed by reconstitution of these interactions in vitro.
Full length Grb2 was expressed in bacteria as a glutathione-S-transferase (GST) fusion protein, and isolated by chromatography on glutathione-sepharose beads.
Immobilized GST-Grb2 bound specifically to P210 BCR-ABL
(Figure 11) or P185 BCR-ABL (data not shown) when incubated with lysates of Rl-P210 or Rl-P185 cells. More particularly, bacterially expressed GST fusion proteins containing full length Grb2, the Grb2 SH2 domain alone or either the N- or C-terminal Grb2 SH3 domains alone, were immobilized and incubated with lysates of Rl-P210 cells.
GST was included as a negative control. Proteins bound to the immobilized fusion proteins were recovered and immunoblotted with anti-ABL antibodies. An anti-ABL
immmunoprecipitate was included, and the position of P210 BCR-ABL is depicted by an arrow in Figure 11. By utilizing a GST fusion protein containing the isolated SH2 domain of Grb2, the Grb2 SH2 domain alone was found to be sufficient to form a stable complex with BCR-ABL (Figure 11).
Co-precipitation of BCR-ABL with Grb2 may be the consequence of a direct interaction between the two proteins, or may require an intermediary molecule. To determine whether Grb2 SH2 domains are capable of directly recognizing BCR-ABL proteins, a filter binding assay was performed (Nayer et al., 1991, Proc. Natl. Acad. Sci. USA
88, 627-631). BCR-ABL and endogeneous c-Abl proteins were immunoprecipitated from R1, Rl-P185 and Rl-P210 cells, subjected to SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose. Replica filters were incubated either with a bacterial lysate containing the GST-Grb2 SH2 fusion protein or GST alone. Binding of the GST proteins to the immobilized Abl proteins was detected by incubation of the filters with anti-GST antibodies :::: .

Ll followed by 125I-labelled protein A. In this assay, GST-Grb2 SH2 bound to P210 and P185 BCR-ABL but not c-Abl, whereas GST alone was incapable of binding to any of the Abl proteins (Figure 12). Nore particularly, Abl proteins were immunoprecipitated from Rl, Rl-P185 and Rl-P210 cells, separated by gel electrophoresis and transferred to nitrocellulose. Replica filters were incubated with bacterial lysates containing GST or GST-Grb2 SH2 fusion proteins as indicated. The filters were subsequently incubated with anti-GST antibodies and probed with [12sI]-labelled protein A. An identical filter was also blotted with anti-ABL antibodies to indicate the amount of c-Abl or BCR-ABL protein in each sample. The positions of pl45 8bl, P185 BCR-ABL and P210 BCR-ABL are indicated.
These results indicate that the SH2 domain of Grb2 directly binds to BCR-ABL proteins under denaturing conditions.

Tyrl77 in BCR-ABL is an autophosphorylation site recoanized by the Grb2-SH2 domain SH2 domains bind with high affinity to tyrosine phosphorylated ligands (Cantley et al., 1991, Cell 64, 281-302; Escobedo et al., 1991, ~ol. Cell. Biol. 11, 1125-1132; Songyang et al., 1993, Cell 72, 767-778). The specificity of ligand binding is largely dictated by the three residues immediately C-terminal to the phosphotyrosine. These residues, together with the phosphorylated tyrosine, can make direct contact with the SH2 domain (Eck et al., 1993, Nature 362, 87-91; Waksman et al., 1993, Cell 72, 779-790. ). When employed to screen a degenerate phosphopeptide library, the Grb2 SH2 domain showed a strong preference for Asn at the +2 position, two residues C-terminal to phosphotyrosine (Songyang et al., 1993, Cell 72, 767-778). Indeed the three known Grb2 SH2-binding sites, located on SHC, the EGF-receptor and IRS-l, all have Asn at the +2 position (Buday and Downward, 1993, Cell 73, 611-620 ; Skolnik et 2; 1 ~

al., 1993, ÆNBO J. 12, 1929-1936 ; Pawson and Schlessinger, 1993, J. Current Biol. 3, 434-442; See Example 6). BCR-ABL proteins contain a single potential Grb2-binding site, Tyr177-Val~Asn-Val, identical to that found in the SHC protein. Remarkably, this site is located within the BCR-encoded sequence that is common to both BCR-ABL proteins. BCR-ABL -has been shown to autophosphorylate in vitro and in vivo on tyrosine residues within the N-terminal BCR sequence (Liu et al., 1993, Oncogene 8, 101-109; Lu et al., 1993, Blood 82, 1257-1263). To test which BCR-ABL phosphopeptides are recognized by Grb2, P210 BCR-ABL was immunoprecipitated from K562 cells, autophosphorylated in an in vitro kinase assay using y-32P-ATP, and digested with trypsin followed by V8 protease. The proteolytic digest was then incubated with immobilized GST-Grb2 SH2 or Abl SH2 fusion proteins.
The immobilized fusion proteins and any associated phosphopeptides were removed by centrifugation, and the remaining peptides were analyzed by two-dimensional electrophoresis and chromatography. Two phosphorylated peptides, H and L, were specifically depleted from the BCR-ABL trypsin/V8 protea~e digest by incubation with immobilized GST-Grb2 SH2 fusion proteins ~Figure 13a and 13b) but not by GST-Abl SH2 domains (data not shown~.
Phosphoamino acid analysis of peptides H and L indicated that peptides H and L contained phosphotyrosine, but not phosphothreonine or phosphoserine (data not shown).
Manual Edman degradation of peptides H and L (partial V8) placed the phosphotyrosine at cycle numbers 14 and 15 respectively (Figure 13e and 13f).
Nore particularly, Figure 13 shows that Tyr177 in P210 BCR-ABL is an autophosphorylation site recognized by the Grb2 SH2 domain, undergoes transphosphorylation in P160 BCR, and is also an autophophorylation site in P185 BCR-ABL. Figure 13a shows a two-dimensional tryptic/V8 protease map of P210 BCR-ABL. P210 BCR ABL was immunoprecipitated from X562 cells, labelled in an in ' vitro kinase assay using ~-32-ATP, and digested with trypsin followed by V8 protease. Peptides were separated by two-dimensional electrophoresis and chromatography.
Previously reported tryptic phosphopeptides are identified by number (Liu et al., 1993, Oncogene 8, 101-109), and peptides produced by the action of both trypsin and V8 protease are identified by capit 1 letters. The previously reported tryptic peptide #10 ~Liu et al., 1993, oncogene 8, 101-109) likely contains peptide L. Figure 13b shows the depletion of peptides H and L by immobilized Grb2 SH2 fusion proteins. The trypsin/V8 double digest of P210 BCR-ABL shown in (a) was mixed with immobilized GST
fusion proteins containing the Grb2 SH2 domain, and peptides remaining in the supernatant separated by two-dimensional electrophoresis and chromatography.Comparison with (a) indicates selective depletion of peptides H and L. In conjunction with the mobilities of the peptides in two-dimensional electrophoresis and chromatography, these results suggested the following peptide sequences: peptide H, GHGQPGADAEKPFpY177VNVE;
peptide L, KGHGQPGADAEKPFpY177VNVE. The underlined Glu (E) appeared resistant to cleavage by V8 protease. These results indicate that Tyr177 is indeed phosphorylated, and that peptides containing Tyr177 are specifically recognized by the Grb2 SH2 domain. Figure 13c shows the two-dimensional tryptic~V8 protease map of P160 BCR
transphosphorylated by P210 BCR-ABL. P160 BCR
immunoprecipitated from K562 cells was transphosphorylated in vitro, digested with trypsin and V8 protease, and the peptides resolved by two-dimensional electrophoresis and chromatography. Tyr177 is also transphosphorylated in P160 BCR (Figure 13c) which forms stable complexes with BCR-ABL
in cells ((Liu et al., 1993, Oncogene 8, 101-109; Lu et al., 1993, Blood 82, 1257-1263; Campbell et al., 1990, oncogene 5, 773-776). Figure 13d show the two-dimensional tryptic/V8 protease digest of P185 BCR-ABL. P185 BCR-ABL
was immunoprecipitated from SUPB15 cells, and analyzed as 2 ~ ~ 3 ~

outlined in (a). P185 BCR-ABL also utilizes Tyr177 as an in vitro autophosphorylation site (Figure 13d). Figures 13e and f show the elution positions of 32P-labelled phosphotyrosine by manual Edman degradation of peptides H
5 (e) and L (f). The counts corresponding to each cycle number are the cpm remaining after the cycle.

The Grb2 SH2 domain specifically interacts with the BCR-encoded sequence p.Tyr177-Val-Asn-Val The specificity of Grb2 binding to the Tyr177 autophosphorylation site on BCR-ABL was investigated using Biosensor technology (Felder et al., 1993, Mol. Cell.
Biol . 13, 1449-1455; Panayatou et al ., 1993, Mol . Cell .
Biol . 13, 3567-3576; Payne et al ., 1993, Proc. Natl .
15 Acad. Sci. USA 90, 4902-4906). A synthetic phosphopeptide (designated BCR) corresponding to the BCR-encoded residues 171-182 and containing phosphotyrosine at position 177, was immobilized to the sensor chip of a Pharmacia BIAcore instrument; soluble full length Grb2 or Grb2 SH2 fusion 20 proteins bound to this immobilized phosphopeptide, as measured by surface plasmon resonance. Soluble GST-Grb2 or GST-Grb2 SH2 were mixed with varying concentrations of the phosphopeptides under test, and injected across the sensor chip surface containing the immobilized BCR
25 peptide. The affinities of various phosphotyrosine~
containing peptides for GST-Grb2 or GST-Grb2 SH2 were then assayed by their abilities to compete with this interaction. Soluble GST-Grb2 or GST-Grb2 SH2 were mixed with varying concentrations of the phosphopeptides under 30 test, and injected across the sensor chip surface containing the immobilized BCR peptide. Addition of the soluble BCR phosphopeptide, or a phosphopeptide corresponding to the Grb2 binding site on SHC (Pelicci et al., 1992, Cell 70, 93-104), specifically inhibited the 35 interaction between the immobilized BCR phosphopeptide and the Grb2 fusion proteins, whereas phosphopeptides corresponding to the GAP or the Syp recognition sites on . :-:..' :::
.- -:: - . ~.

2~3~ ' the platelet-derived growth factor (PDGF) receptor (Kashishian et al., 1993, EMBO J. 11, 1373-1382;
Kazlauskas et al., 1993, Proc. Natl. Acad. Sci. USA, 90:6939-6942) did not (Figure 14). Substitution of Ala 5 for Asn at the ~2 position in the BCR peptide abolished the inhibition observed with the wildtype BCR
phosphopeptide. The wildtype BCR peptide, but not the Asn)Ala BCR peptide, also inhibited the binding of P210 BCR-ABL from Rl-P210 cell lysates to an immobilized Grb2 10 SH2 fusion protein (data not shown).
More particularly, a BCR phosphotyrosine-containing synthetic peptide corresponding to residues 171-182 of human BCR, was immobilized to the sensor chip surface of a Pharmacia BIAcore instrument, and binding of 15 GST-Grb2 fusion proteins to this peptide was monitored by surface plasmon resonance (Felder et al., 1993, Mol. Cell.
Biol. 13, 1449-1455; Panayatou et al., 1993, Nol. Cell.
Biol. 13, 3567-3576 ; Payne et al., 1993, Proc. Natl.
Acad. Sci. USA 90, 4902-4906). The affinities of various 20 phosphotyrosine-containing peptides for GST-Grb2 (solid symbols) or GST-Grb2 SH2 (open symbols) were assayed by their abilities to compete with this interaction. Soluble GST-Grb2 or GST-Grb2 SH2 proteins were mixed with varying concentrations of the phosphotyrosine-containing peptides 25 BCR (O), SHC (v), Asn~Ala BCR (~), PDGFR 771 (Il), or PDGFR
1009 (o) and injected across the sensor chip surface containing the immobilized BCR peptide. The sequences of these synthetic phosphopeptides were: BCR, DAEKPFp.Y177VNVEFK, corresponding to amino acids 171-182 of 30 human BCR, with a carboxy terminal lysine added; Asn~Ala BCR, identical to the BCR peptide except for substitution of alanine for asparagine at the +2 residue carboxy terminal to ~rr177; SHC, ELFDDPSp.Y317VNVQNLDK corresponding to the Grb2 recognition site on the SHC protein (Pelicci 35 et al., 1992, Cell 70, 93-104); PDGFR 771, SSNp.Y771NAPYDNYK, corresponding to the GAP recognition site of the PDGF receptor (Kashishian et al., 1992, EMBO J. 11, : ~: '"-~:, 1373-1382); PDGFR 1009, SSVLp.Y1~TAVQP corresponding to the Syp recognition site on the PDGF receptor (Kazlauskas et al., 1993l Proc. Natl. Acad. Sci. USA 90:6939-6942).
Taken together, these results indicate that one function of the BCR moiety of BCR-ABL oncoproteins is to provide a site for tyrosine phosphorylation that can directly couple BCR-ABL to Grb2 and hence to the Ras pathway, and that Asn at the +2 position is also critical for this interaction.

Recruitment of mSosl into a complex with BCR-ABL
The SH3 domains of Grb2 bind constitutively to proline-rich motifs within the C-terminal tail of mSosl (Rozakis-Adcock et al., 1993, Nature 363, 83-85; Li et al., 1993, Nature 363, 85-88). Upon stimulation with EGF, the mSosl-Grb2 complex binds to the activated EGF receptor at the inner plasma membrane, an event that may enable Sos to stimulate the dissociation of GDP from Ras (Buday and Downward, 1993, Cell 73, 611-620). Since mSosl is widely expressed in hematopoietic cells (Bowtell et al., 1992, Proc. Natl. Acad. Sci. USA 89, 6511-1515), and thus may participate in Ras activation in leukemic cells, the inventors investigated whether the association of Grb2 with BCR-ABL proteins was accompanied by recruitment of mSosl into this complex.
Figure 15 shows that BCR-ABL oncoproteins complex with mSosl in vivo. In particular, Figure 15a shows an immunoblot of lysates of Rl or R1-P210 cells immunoprecipitated with anti-ABL or anti-mSosl antibodies, and the immunoprecipitated proteins subsequently immunoblotted with anti-ABL antibodies. Normal rabbit serum (C) was included as a negative control. Figure 15b shows an immunoblot of Rl-P185 cell lysates, immuno~
precipitated with anti-mSosl or anti-Grb2 antibody, and immunoblotted with anti-ABL antibody. Figure 15c shows an immunoblot of lysates of R1 or Rl-P210 cells immuno-precipitated with anti-mSosl antibody or anti-Grb2 '.: '' ~', i 3 '~

- 36 - :~
: :,:
antibody, and immunoblotted with anti-mSosl antibody.
P210 BCR-ABL but not endogeneous c-abl, co-precipitated with mSosl. Similar results were obtained with P185 BCR-ABL which co-precipitated with mSosl from Rl-P185 cell lysates (Figure 15b). The association of mSosl with BCR-ABL likely occurs through the Grb2 adaptor protein since mSosl co-precipitated with Grb2 from lysates of both ~he parental R1 cells and the BCR-ABL-transformed cells (Figure 15c). The ABL SH3 domain, which also binds proline rich motifs (Ren et al., 1993, Science 259, 1157-1161) and might potentially associate directly with mSosl, did not appear to bind efficiently to mSosl in vitro (data not shown). Like Grb2, mSosl was not significantly phosphorylated on tyrosine in BCR-ABL-transformed cells (data not shown).

Shc proteins are phosphorylated on tyrosine and associated with Grb2 in cells transformed by P210 or P185 BCR-ABL
Analysis of phosphotyrosine-containing proteins associated with Grb2 in BCR-ABL-transformed cells suggests that Grb2 might bind phosphorylated ligands in addition to BCR-ABL (Figure 9). A number of tyrosine kinases phosphorylate the SH2-containing Shc proteins, which apparently stimulate the Ras pathway through inducible association with Grb2-mSosl (McGlade et al., 1992, Proc.
Natl. Acad. Sci. USA 89, 8869-8873; Pelicci et al., 1992, Cell 70, 93-104; Rozakis-Adcock et al., 1992, Nature 360, 689-692; Pronk et al., 1993, J. Biol. Chem. 268, 5748-5753; Rozakis-Adcock et al., 1993, Nature 363, 83-85).
K562 cells express three SHC isoforms, of 46, 52 and 66 kDa, of which p525hC is the predominant species (Figure 16).
Figure 16 illustrates that SHC proteins are tyrosine phosphorylated and associated with Grb2 in Ph+
leukemic cells. Lysates of the K562 cell line were immunoprecipitated with anti-SHC or anti-ABL antibodies, and the precipitated proteins immunoblotted with anti-phosphotyrosine, anti-Shc or anti-Grb2 antibodies as : ', :- . ~:~::

~: : ~

-- 2 ~

indicated. Normal rabbit serum was included as a negative control (C). The positions of P210 BCR-ABL, the 66, 52 and 46 kDa SHC proteins and Grb2 are indicated on Figure 16.
Prolonged exposure of this autoradiogram revealed a minor level of phosphotyrosine on p665hC~ and co-precipitation of p465h', as well as p525hC~ with BCR-ABL (data not shown).
Figure 17 illustrates that the tyrosine phosphorylation of Shc proteins and their association with Grb2 in Rat-l fibroblasts is induced by expression of BCR-ABL oncoproteins. Figure 17a is an immunoblot of R1, R1-- .. :
P185 and R1-P210 cell lysates immunoprecipitated with anti-SHC antibodies and the immunoprecipitates immunoblotted with anti-SHC or anti-phosphotyrosine antibodies as indicated. The positions of the 66, 52 and 46 kDa Shc proteins are indicated. Figure 17b is an immunoblot of lysates of R1 and R1-P185 cells containing similar quantities of total protein, immunoprecipitated with anti-Grb2, anti-SHC or normal rabbit serum (C), and the immune complexes blotted with anti-SHC antibodies.
In ~ummary, p525hC and p465hC were phosphorylated on tyrosine in K562 cells, and a minor fraction of these proteins was associated with BCR-ABL (Figure 16). Shc proteins were also phosphorylated on tyrosine in the Ph+
ALL line SUPB15 (data not shown), and in Rl-P185 and R1-P210 cells, but not in the parental R1 cells (Figure 17).
Both isoforms of BCR-ABL therefore elicit the phosphorylation of Shc proteins on tyrosine in vivo. To determine whether Grb2 binds to Shc proteins in cells expressing BCR-ABL, anti-Grb2 immunoprecipitates were immunoblotted with anti-SHC antibodies. No Shc proteins co-precipitated with Grb2 from parental R1 cells.
However, anti-SHC antibodies recognized proteins of 46 and 52 kDa in anti-Grb2 immunoprecipitates from lysates of Rl-P185 cells (Figure 17). Furthermore, phosphotyrosine-containing proteins with these mobilities were detected in anti-Grb2 immunoprecipitates from R1-P185 cells (Figure 2113~n~ :~

9). In the reciprocal experiment, co-precipitation of Grb2 by SHC antibodies was also observed tdata not shown).
The association of Shc and Grb2 was also detected in the Ph+ leukemic cell lines, and in Rl-P210 cells (Figure 16 and data not shown). These results indicate that BCR-ABL
proteins induce the formation of a complex between tyrosine phosphorylated Shc and Grb2.

This investigation addressed whether the interaction between Shc and Grb2 proteins is both necessary and sufficient for induction of mitogenesis by overexpression of Shc proteins. The Grb2-binding site on Shc proteins has been mapped and the transforming potential of Shc mutants defective for Grb2-binding has been investigated.
Phosphorylation of Shc Tyr317 creates a Grb2-binding site.
Analysis of the primary Shc protein sequence revealed a putative phosphorylation site at Tyr317, located amino-terminal to the SH2 domain within the CH
region. The specificity with which SH2 domains bind to a phosphorylated site is primarily dictated by the three residues immediately C-terminal to the phosphotyrosine (in the +1, +2 and +3 positions). Screening of a degenerate phosphopeptide library has established that the Grb2 SH2 domain binds preferentially to peptides with the sequence pY-X-N-X, with a particularly strong selectivity for the Asn at the +2 position. Similarly, the C.elegans Sem-5 SH2 domain preferentially selects pY-L/V-N-V/P. The Shc Y317 site contains the sequence YVNV, which corresponds well to the consensus Grb2 SH2-binding motif predicted from the phosphopeptide library ~creen. Hence, phosphorylation of Tyr317 may be responsible for creating the high affinity binding site for the Grb2 SH2 domain.
To test this hypothesis, a GST-fusion protein containing amino acids 280 to 473 of the p52SHC (GST-SHC+), was used in an in vitro assay to test binding to 2~13 the Tyr317 site. The GST-SHC+ protein was inducibly phosphorylated on tyrosine in bacteria by co-expression with a ~-galactosidase fusion polypeptide containing the catalytic domain of the Elk protein-tyrosine kinase (Reedijk et al., ENBO Journal 11 (4J :1365-1372, 1992. ) . The GST-SHC+ protein contained no detectable phosphotyrosine prior to induction of the Elk tyrosine kinase, but became tyrosine phosphorylated following expression of the Elk fusion protein. A mutant version of the GST-SHC+ protein, GST-SHC+(F317), in which Tyr317 was changed to phenylalanine, was not phosphorylated under similar conditions suggesting that Tyr317 is the only site in this protein which is phosphorylated by Elk. Similar results were obtained when GST-SHC+ or GST-SHC+(F317) were purified and used as the substrates for the purified EGF-receptor kinase domain, or the v-Src tyrosine kinase (data not shown). Furthermore, when different Shc polypeptide fragments were expressed as GST fusion proteins, and incubated with purified v-Src tyrosine kinase, only polypeptides containing the region surrounding Tyr317 were substrates for in vitro tyrosine phosphorylation.
To test Grb2-binding, tyrosine phosphorylated GST-SHC+ was immobilized, incubated with a lysate of Rat-2 fibroblasts, and the resulting complexes immunoblotted with anti-Grb2 antibodies. Using this approach, Grb2 was found to bind specifically to the phosphotyrosine-containing form of GST-SHC+. In contrast the GST-SHC+(F317) variant, that lacks the Tyr317 phosphorylation site, did not bind detectably to Grb2 under the same circumstances. To investigate the involvement of the Tyr317 site in binding of Grb2 to wild type Shc in more detail, a 15 amino acid phosphopeptide corresponding to the Shc sequence encomp~ssing phosphorylated Tyr317, was tested for its ability to compete for the binding of '' ~:~','`-,' tyrosine phosphorylated GST-SHC+ to Grb2. Addition of the Tyr317 phosphopeptide to a concentration of 200 nM
inhibited the binding of phosphorylated GST-SHC+ to Grb2 in a Rat-2 cell lysate (IC50). In contrast an unrelated phosphotyrosine-containing peptide (pY771) corresponding to the binding site for Ras GTPase activating protein on the bPDGF-receptor did not inhibit Grb2 binding by phosphorylated GST-SHC+.
$hese results strongly suggest that Shc phosphorylation at Tyr317 creates a binding site for the Grb2 SH2 domain. Since high affinity SH2-binding apparently requires specific amino acids C-terminal to the phosphorylated tyrosine, substitution of residues at the +1 to +3 positions relative to Tyr317 might be anticipated to modulate Grb2-binding. In particular the Asn at the +2 site (Asn319) is characteristic of Grb2-binding sites. To test this prediction the codon for Asn319 of p52 SHC was mutated to encode alanine, and introduced into the GST-SHC+ vector. This mutant was efficiently phosphorylated both by the Elk tyrosine kinase in bacteria, and by the EGF-receptor kinase domain in vitro. Despite its efficient phosphorylation on tyrosine, the Ala319 Shc mutant did not bind to Grb2 in a Rat-2 cell lysate as efficiently as wt GST-SHC+. This result suggests that the high affinity binding of Grb2 to Shc proteins requires phosphorylation of Shc at Tyr317, which lies within a high affinity binding motif for the Grb2 SH2 domain, pYVNV, of which the Asn at the +2 position is a crucial component.
Shc contains another motif that might possibly represent a Grb2-binding site, YYND.
Shc tyrosihe 317 is required for GRB-2 association in vivo.
Having established that phosphorylation of Shc Tyr317 is sufficient to promote Grb2-binding in vitro, the .. __.. ... . . . ... . .. . . . . . .

21~ 349 !~

- 41 ~
' . ~ ' ' involvement of this site in the formation of a Shc-Grb2 complex was investigated in cells. To investigate whether Tyr317 is a Grb2-binding site in vivo, mammalian expression vectors were constructed with a wild type human SHC cDNA, encoding the p52 and p46 Shc proteins, or a mutant SHC cDNA in which the codon for Tyr317 was replaced with Phe (TM). In addition both the SHC and TM cDNAs were tagqed with a foreign epitope, to allow the ectopically expressed Shc proteins to be immunologically distinguished from endogenous Shc polypeptides, which are ubiquitously expressed. To this end, the SHC and TM cDNAs were fused in-frame with a 162bp fragment of the PML cDNA, which encodes a 54 AA PML peptide (6,7 kDa), to obtain the SHC-TAG and TM-TAG cDNAs (note that two proline residues were introduced as a flexible linker between the Shc and PML
sequences). The two tagged cDNAs, therefore, have the potential to encode two PML-tagged SHC proteins of 53 and 58 KDa (p535HC-TAG and p585HC-T~G) containing wild type Shc sequences, and p53T~TAG and p58TMTAG with a Phe317 substitution.
To test the tyrosine phosporylation and Grb2-binding properties of these Shc proteins, the SHC-TAG and TM-TAG cDNAs were introduced into rodent fibroblasts that overexpress the EGFR (SAA cells). Nestern blot analysis of the transduced SAA cells (SAA-TM-TAG and SAA-SHC-TAG) with anti-SHC antibodies revealed the endogenous Shc proteills (p46shc p52shc and p665hC) and two PML-tagged exogenous Shc proteins of approximately 53 and 58 kDa (p53sHc-TAG and p5gsHG-TAG or p53TM-TAG and p58TM-T~G
respectively). To ensure that the tagging procedure did not interfere with antibody recognition of either the Shc or PML epitopes, SAA-TM-TAG and SAA-SHC-TAG cell lysates were immunoprecipitated with anti-PML antibodies and western blotted with anti-SHC antibodies. The anti-SHC

.. . ~ ' ' ' 2iL 3~

antibody recognized two proteins (53 and 58 XDa approximately) in the anti-PML immunoprecipitates, that comigrated with the p535HC-TAG and p58SHC-TAG detected b western blotting of the SAA-SHC-TAG whole cell lysate with anti-SHC antibody. To ensure that SAA-TM-TAG and SAA-SHC-TAG cells expressed comparable amounts of EGFR, EGF-stimulated cells were western blotted with anti-Ptyr antibodies.
The tyrosine phosphorylation of the wild type and TM Shc proteins, and their potential to bind Grb2, were then examined in EGF-stimulated SAA-TM-TAG and SAA-SHC-TAG cells. Lysates prepared from serum-starved cells, or from cells stimulated for 5 minutes with EGF, were immunoprecipitated with anti-PML antibodies and the immunoprecipitates were immunoblotted with anti-SHC, anti-phosphotyrosine (pTyr) or anti-Grb2 antibodies. EGF
stimulation induced a marked retardation of the electrophoretic mobility of the p53sHc-TAs and p58SHC-TAG
proteins, which contain wild type Shc sequences; such a reduction in the mobility of Shc proteins has been previously attributed to their tyrosine phosphorylation following growth factor stimulation. Indeed, the phsphOtyrosine content of p535Hc-TAG and p585HC-TAG increased considerably upon EGF stimulation. In addition, phosphorylated p535HCTAG and p58sHcTAG formed stable complexes with Grb2 in EGF-stimulated cells.
In contrast, both the reduction in electrophoretic mobility and the increase in tyrosine phosphorylation were far more limited for the p53TM-TAG and p58TM-TAG proteins after EGF stimulation, and no binding of these mutant proteins to Grb2 was detected. However, it should be noted that the degree of basal phosphorylation of p53TM-T~G and p58TM-T~G was never truly zero and it slightly increased upon EGF stimulation.
.

2 1 :~ 3 Overall these results provide strong evidence that, in vivo, Tyr317 is the major site for Shc phosphorylation by the EGFR, and that it is the sole Shc high affinity binding site for Grb2.
Mutation of the Shc Grb2-binding site abrogates Shc transformin~ potential.
To ascertain whether phosphorylation of Tyr317 and consequent binding of Grb2 are essential for the transforming activity of Shc proteins, the effects of the wild type Shc and TM proteins on the growth properties of cultured fibroblasts were investigated. For these purposes SHC and TN cDNAs were expressed in NIH-3T3 cells, and clones overexpressing the SHC or TM proteins (NIH-SHC, NIH-TM clone) were analyzed for their cell cycle properties and capacity to form colonies in soft agar.
Three clones that overexpressed the wild type Shc proteins, and three that overexpressed the TN proteins, were selected on the basis that they expressed similar levels of Shc proteins; three clones transfected with the expression vector only (C-NIH) were used as controls.
The effects of wild type or TM Shc proteins on cell cycle indices were investigated by analyzing the DNA
content of propidium iodine (PI)-stained nuclei isolated from three SHC-NIH, TM-NIH and C-NIH cell lines in flow cytometry studies (Dean, 1980; Fried et al., 1978). The data obtained from three experiments were separately summed and statistically compared. The cells were cultured in 10~ serum until they were almost confluent and were then serum-deprived for 24 hours. The cell-cycle phase distribution was similar in .the exponentially growing NIH-C, NIH-SHC and NIH-TM cell lines. In contrast, there was a significantly (p < 0.007) larger proportion of cycling cells in the serum-free NIH-SHC
cells than in the serum-free C-NIH or TM-NIH cell lines.

: " ' ' ' S ~ 3.~ n ~ ~:

- 44 ~

NIH-SHC, NIH-T~ and NIH-C cell lines were plated in triplicate at varying cell concentrations in soft agar medium, supplemented with 20% serum, and colonies were scored after 14 days~ Whereas the NIH-S~C clones formed colonies at a frequency comparable with that previously reported, the NIH-TM ard NIH-C clones did not form colonies. `~
These data indicate that the integrity of the Tyr317 site is required for Shc transformation of cultured -~-fibroblasts. They also suggest that formation of the Shc~
Grb2 complex is an obligatory step in the chain of events by which overexpression of Shc proteins leads to neoplastic transformation. The correlation between Grb2-binding and Shc-induced transformation supports the notion that transformation by Shc involves activation of the Ras signalling pathway.
Deletion of the Shc amino-terminus does not affect the Shc transformina potential.
To assess whether the interaction with Grb2 is, by itself, sufficient for the transformation of NIH-3T3 cells by Shc, or whether other regions of Shc might be involved in this activity, a Shc mutant (D5') was expressed that lacked the amino-terminal XX amino acids, but retained the CH domain and Tyr317 site as well as the SH2 domain (D5' cDNA). The D5' cDNA, which encodes a 26 kDa protein, was expressed in NIH-3T3 cells (NIH-D5'). In addition, this cDNA was tagged with the PML epitope (to give D5'-TAG), and the resulting 33 kDa product was expressed in SAA cells (SAA-D5'-TAG). The p33D5-TAG protein was phosphorylated following stimulation of the SAA-D5'-TAG cells ~ith EGF, and that it bound efficiently to Grb2.
Flow cytometry analysis of the DNA content of PI-stained nuclei from serum-starved NIH-D5' cells revealed a high proportion of cycling cells, comparable to that seen with : -~ `~ ,' `':';

:: . ~ . .
--.::: --: ~:

2~3~

NIH-SHC cells. Agar plating of NIH-D5~ cells demonstrated that all clones examined were capable of forming colonies, although the size and frequency of these colonies were slightly inferior to those of the NIH-SHC cells.
It, therefore, appears that the 200 amino -terminal amino-acids of the Shc protein are not essential to the transforming potential of SHC and that interaction with Grb2 and, by inference, activation of Ras, may be sufficient to induce cellular transformation. - - -Having illustrated and described the principles of the invention in a preferred embodiment, it should be appreciated to those skilled in the art that the invention can be modified in arrangement and detail without departure from such principles. We claim all modifications coming within the scope of the following claims.

~ ; :; ' ';

.:~

2 ~ t~

- 4 6 - - : : ~

TABLE 1 ;~

AM~JO A~:ID DESlGNA'IlONS
i. , ~
A Ala :~

E alu :~
F Phe G c~
H His Ile -: ~
K Lys ::
L Lcu :
M Mct N Asn Q Gln R ~rg ~ :
S Scr T Thr :
V Val . ' W Trp :
Y Tyr :~
, . .. ~

.

:
''.'` .' ~' "~':' . .... . :~

:-:: :- i.
:::

- 47 ~

SEQUENCE LISTING

(1) GENERAL INFORNATION~
ti) APPLICANT: PAWSON, TONY
ARLINGHAUS, RALPH
PUIL, LORRI
GISH, GERALD
(ii) TITLE OF INVENTION: NETHOD FOR ASSAYING FOR SUBSTANCES WHICH
AFFECT BCR-ABL MEDIATED TRANSFORNATION :~
(iii) NUMBER OF SEQUENCES: 8 (iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Bereskin & Parr (B) STREET: 40 King Street Uest (C) CITY: Toronto .
(D) STATE: Ontario (E) COUNTRY: Canada :~
(F) ZIP: M5H 3Y2 (v) COMPUTER READABLE FORM:
(A) MEDIVM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTUARE: PatentIn Release ~1.0, Yersion ~1.25 (vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: US
(B) FILING DATE: ~
(C) CLASSIFICATION: : ~ ::
(viii) ATTORNEY/AGENT INFORMATION: .~ : ~
(A) NAME: Kurdydyk, Linda M. : :~ :-(B) REGISTRATION NUMBER: 34,971 ~ ... :
(C) REFERENCE/DOCKET NUMBER: 3153-089 ~ :
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (416) 364-7311 .~
(B) TELEFAX: (416) 361-1398 -~ .
(C) TELEX: 06-23115 ~ :
:.
(2) INFORMATION FOR SEQ ID NO:l:
(i) SEQUENCE CHARACTERISTICS: :::
(A) LENGTH~ 217 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single :: : :~
(D) TOPOLOGY: linear - ~:.
(ii) MOLECULE TYPE: peptide ' :':

2 i ~ 3 .~ ~ ~

(vi) ORIGINAL SOURCE: ~: ~
(A) ORGANISM: Homo sspiens (vii) IMMEDIATE SOURCE:
(A) LIBRARY: gtll human brainstem ,~
(B) CLONE: Grb2 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:
Met Glu Ala Ile Ala Lys Tyr Asp Phe Lys Ala Thr Ala Asp Asp Glu Leu Ser Phe Lys Arg Gly Asp Ile Leu Lys Val Leu Asn Glu Glu Cys 20 25 30 ~ -Asp Gln Asn Trp Tyr Lys Ala Glu Leu Asn Gly Lys Asp Gly Phe Ile ~ . .

Pro Lys Asn Tyr Ile Glu Met Lys Pro His Pro Trp Phe Phe Gly Lys 50 55 60 . .
Ile Pro Arg Ala Lys Ala Glu Glu Met Leu Ser Lys Gln Arg His Asp : -65 70 75 80 :~ -Gly Ala Phe Leu Ile Arg Glu Ser Glu Ser Ala Pro Gly Asp Phe Ser Leu Ser Val Lys Phe Gly Asn Asp Val Gln His Phe Lys Val Leu Ar~
100 105 110 '~
Asp Gly Ala Gly Lys Tyr Phe Leu Trp Val Val Lys Phe Asn Ser Leu : :~
115 120 125 -~ --Asn Glu Leu Val Asp Tyr His Arg Ser Thr Ser Val Ser Arg Asn Gln 130 135 140 :~
Gln Ile Phe Leu Arg Asp Ile Glu Gln Val Pro Gln Gln Pro Thr Tyr Val Gln Ala Leu Phe Asp Phe Asp Pro Gln Glu Asp Gly Glu Leu Gly Phe Arg Arg Gly Asp Phe Ile His Val Met Asp Asn Ser Asp Pro Asn Trp Trp Lys Gly Ala Cys His Gly Gln Thr Gly Met Phe Pro Arg Asn Tyr Val Thr Pro Val Asn Arg Asn Val (2) INFORMATION FOR SEQ ID NO:2:
(i) SEQ~ENCE CHARACTERISTICS: ;~

: ~ ~
~``` 2t ~3'~
. ..
- 49 - :

(A) LENGTH: 1109 base pairs : ~
(B) TYPE: nucleic acid ~: i ( C ) STRANDEDNESS: s ingle -:
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) ~:
'~
(vi) ORIGINAL SOURCE: :
(A) ORGANISM: Homo sapiens (vii) IMMEDIATE SOURCE:
(A) LIBRARY: gtll ~uman brainstem ( B ) CLONE: Grb2 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
GCCAGTGAAT TCGGGGGCTC AGCCCTCCTC CCTCCCTTCC CCCTGCTTCA GGCTGCTGAG 60 ;

GACGAGCTGA GCTTCMMG GGGGGACATC CTCAAGGTTT TGAACGMGA ATGTGATCAG 180 .

AGCAMCAGC GGCACGATGG GGCCTTTCTT ATCCGAGAGA GTGAGAGCGC T5CTGGGGAC 360 ~ -GCCGGGAAGT ACTTCCTCTG GGTGGTGMG TTCAATTCTT TGAATGAGCT GGTGGATTAT 480 :

MAGGAGCTT GCCACGGGCA GACCGGCATG TTTCCCCGCA ATTATGTCAC CCCCGTGMC 720 :

GTTGGATTTA MMTGCCM MCTTACCTA TMMTTMGA AGAGTTTTTA TTACMMTTT 960 :- ~
TCACTGCTGC TCCTCTTTCC CCTCCTTTGT CTTTTTTTTC ATCCTTTTTT CTCTTCTGTC 1020 :::~ ~:

'' 2 ~ 4 n ~

(2) INFORMATION FOR SEQ ID NO:3: -(i) SEQUENCE CHARACTERISTICS: : :
(A) LENGTH: 473 amino acids : :
tB) TYPE: amino acid (C) STRANDEDNESS: single - :
(D) TOPOLOGY: linear . .
(ii) MOLECVLE TYPE: peptide .
(vi) ORIGINAL SOURCE: .
(A) ORGANISM: Homo sapiens (vii) IMMEDIATE SOURCE~
(A) LIBRARY: Human (B) CLONE: SHC Protein ~:

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
Met Asn Lys Leu Ser Gly Gly Gly Gly Arg Arg Thr Arg Val Glu Gly Gly Gln Leu Gly Gly Glu Glu Trp Thr Arg His Gly Ser Phe Val Asn -~-Lys Pro Thr Arg Gly Trp Leu His Pro Asn Asp Lys Val Met Gly Pro Gly Val Ser Tyr Leu Val Arg Tyr Met Gly Cys Val Glu Val Leu Gln `~
50 55 60 .. :~ ~ ;
Ser Met Arg Ala Leu Asp Phe Asn Thr Arg Thr Gln Val Thr Arg Glu 65 70 75 80 .
Ala Ile Ser Leu Val Cys Glu Ala Val Pro Gly Ala Lys Gly Ala Thr .

Arg Arg Arg Lys Pro Cys Ser Arg Pro Leu Ser Ser Ile Leu Gly Arg 100 105 110 ~ :.
Ser Asn Leu Lys Phe Ala Gly Met Pro Ile Thr Leu Thr Val Ser Thr `~

Ser Ser Leu Asn Leu Met Ala Ala Asp Cys Lys Gln Ile Ile Ala Asn His His Met Gln Ser Ile Ser Phe Ala Ser Gly Gly Asp Pro Asp Thr 145 150 155 160 ~;~
Ala Glu Tyr Val Ala Tyr Val Ala Lys Asp Pro Val Asn Gln Arg Ala ::

Cys His Ile Leu Glu Cys Pro Glu Gly Leu Ala Gln Asp Val Ile Ser :~

~ ~ :L 3 ~ 9 -~

Thr Ile Gly Gln Ala Phe Glu Leu Arg Phe Lys Gln Tyr Leu Arg Asn Pro Pro Lys Leu Val Thr Pro His Asp Arg Met Ala Gly Phe Asp Gly 210 215 220 :-Ser Ala Trp Asp Glu Glu Glu Glu Glu Pro Pro Asp His Gln Tyr Tyr Asn Asp Phe Pro Gly Lys Glu Pro Pro Leu Gly Gly Val Val Asp Met Arg Leu Arg Glu Gly Ala Ala Pro Gly Ala Ala Arg Pro Thr Ala Pro :
260 265 270 ~ ~
Asn Ala Gln Thr Pro Ser His Leu Gly Ala Thr Leu Pro Val Gly Gln :- :
275 280 285 -.
Pro Val Gly Gly Asp Pro Glu Val Arg Lys Gln Met Pro Pro Pro Pro :
290 295 300 i:: ~
Pro Cys Pro Gly Arg Glu Leu Phe Asp Asp Pro Ser Tyr Val Asn Val .. :
305 310 315 320 : :~:
Gln Asn Leu Asp Lgs Ala Arg Gln Ala Val Gly Gly Ala Gly Pro Pro 325 330 335 ~ : :-.
Asn Pro Ala Ile Asn Gly Ser Ala Pro Arg Asp Leu Phe Asp Net Lys - :::.

Pro Phe Glu Asp Ala Leu Arg Val Pro Pro Pro Pro Gln Ser Val Ser : :

Met Ala Glu Gln Leu Arg Gly Glu Pro Trp Phe His Gly Lys Leu Ser Arg Arg Glu Ala Glu Ala Leu Leu Gln Leu Asn Gly Asp Phe Leu Val Arg Glu Ser Thr Thr Thr Pro Gly Gln Tyr Val Leu Thr Gly Leu Gln Ser Gly Gln Pro Lys His Leu Leu Leu Val Asp Pro Glu Gly Val Val Arg Thr Lys Asp His Arg Phe Glu Ser Val Ser His Leu Ile Ser Tyr 435 440 445 ::;~
His Met Asp Asn His Leu Pro Ile Ile Ser Ala Gly Ser Glu Leu Cys Leu Gln Gln Pro Val Glu Arg Lys Leu (I) INFOR~ATIO~ FOR SLQ ID NO:4:

~,"- ,'''':~:~- .: : ' . ': :

2113~

( i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 3031 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (vi) ORIGINAL SOURCE:
(A) ORGANISM: ~omo sapiens (vii) IMMEDIATE SOURCE:
(A) LIBRARY: Human (B) CLONE: SHC pr~tein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:

3 '~
_ s3 -... ~ .
:: . ~ .

~ ~ 3 '~ J ~ :

CCCCTCTTTT GGCCTTGTGG AT M GGGAGA GTTGACCGTT TTCATCCTGG CCTCCT~TTG 2940 (2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
tA) LENGTH: 1336 amino acids .
(B) TYPE: amino acid ::~
(C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide .: . .
(vi) ORIGINAL SOURCE: ~ :
(A) ORGANISM: Mus musculus ;~
(vii) IMMEDIATE SOURCE: : ~::
(A) LIBRARY: Mouse :~
(B) CLONE: Son of Sevenless 1 :

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
Met Leu Val Ser His Leu Ile Leu Pro Arg Lys Gln His Pro Ala Gly Thr Met Gln Ala Gln Gln Leu Pro Tyr Glu Phe Phe Ser Glu Glu Asn Ala Pro Lys Trp Arg Gly Leu Leu Val Pro Ala Leu Lys Lys Val Gln 35 40 45 : ::
Gly Gln Val His Pro Thr Leu Glu Ser Asn Asp Asp Ala Leu Gln Tyr - -Val Glu Glu Leu Ile Leu Gln Leu Leu Asn Met Leu Cys Gln Ala Gln Pro Arg Ser Ala Ser Asp Val Glu Glu Arg Val Gln Lys Ser Phe Pro :

His Pro Ile Asp Lys Trp Ala Ile Ala Asp Ala Gln Ser Ala Ile Glu :~
100 105 110 .
Lys Arg Lys Arg Arg Asn Pro Leu Ser Leu Pro Ala Glu Arg Ile His 115 120 125 - ~
His Leu Leu Arg Glu Val Leu Gly Tyr Lys Ile Asp His Gln Val Ser : ~ :

~' ' , ' ' ~:

- 21~ 3~n 1 Val Tyr Ile Val Ala Val Leu Glu Tyr Ile Ser ~la Asp Ile Leu Lys 145 150 155 160 . ::
Leu Val Gly Asn Tyr Val Arg Asn Ile Arg His Tyr Glu Ile Thr Lys :~:

Gln Asp Ile Lys Val Ala Met Cys Ala Asp Lys Val Leu Met Asp Met ~:
180 185 190 :
Phe His Gln Asp Val Glu Asp Ile Asn Ile Leu Ser Leu Thr Asp Glu ~ ~ ~
195 200 205 : :
Glu Pro Ser Thr Ser Gly Glu Gln Thr Tyr Tyr Asp Leu Val Lys Ala ~ ;
210 215 220 - ~
Phe Met Ala Glu Ile Arg Gln Tyr Ile Arg Glu Leu Asn Leu Ile Ile : ~:
225 230 235 240 : .
Lys Val Phe Arg Glu Pro Phe Val Ser Asn Ser Lys Leu Phe Ser Ser Asn Asp Val Glu Asn Ile Phe Ser Arg Ile Val Asp Ile His Glu Leu Ser Val Lys Leu Leu Gly His Ile Glu Asp Thr Val Glu Met Thr Asp Glu Gly Ser Pro His Pro Leu Val Gly Ser Cys Phe Glu Asp Leu Ala Glu Glu Leu Ala Phe Asp Pro Tyr Glu Ser Tyr Ala Arg Asp Ile Leu : :~

Arg Pro Gly Phe His Gly His Phe Leu Ser Gln Leu Ser Lys Pro Gly :~
325 330 335 : -:
:: - ~: - :,: . -::
~: . : : . ~: :
Ala Ala Leu Tyr Leu Gln Ser Ile Gly Glu Gly Phe Lys Glu Ala Val : :~

Gln Tyr Val Leu Pro Arg Leu Leu Leu Ala Pro Val Tyr His Cys Leu :~

His Tyr Phe Glu Leu Leu Lys Gln Leu Glu Glu Lys Ser Glu Asp Gln Glu Asp Lys Glu Cys Met Lys Gln Ala Ile Thr Ala Leu Leu Asn Val :~
385 390 395 400 :: ~: ~
Gln Ser Gly Met Glu Lys Ile Cys Ser Lys Ser Leu Ala Lys Arg Arg ~ : :-405 410 415 ~: :: :
Leu Ser Glu Ser Ala Cys Arg Phe Tyr Ser Gln Gln Met Lys Gly Lys Gln Leu Ala Ile Lys Lys Met Asn Glu Ile Gln Lys Asn Ile Asp Gly ~ "'~; ' 21~.3~
- 56 - :

435 440 445 .
: :.~:::. ~,; .
Trp Glu Gly Lys Asp Ile Gly Gln Cys Cys Asn Glu Phe Ile Met Glu Gly Thr Leu Thr Arg Val Gly Ala Lys His Glu Arg His Ile Phe Leu 465 470 475 480 ;~
Phe Asp Gly Leu Net Ile Cys Cys Lys Ser Asn His Gly Gln Pro Arg - ~ ~' - .
Leu Pro Gly Ala Ser Ser Ala Glu Tyr Arg Leu Lys Glu Lys Phe Phe : 500 505 510 Met Arg Lys Val Gln Ile Asn Asp Lys Asp Asp Thr Ser Glu Tyr Lys 515 520 525 ;~
His Ala Phe Glu Ile Ile Leu Lys Asp Gly Asn Ser Val Ile Phe Ser 530 535 540 ~ ~:
Ala Lys Ser Ala Glu Glu Lys Asn Asn Trp Met Ala Ala Leu Ile Ser eu Gln Tyr Arg Ser Thr Leu Glu Arg Met Leu Asp Val Thr Val Leu Gln Glu Glu Lys Glu Glu Gln Met Arg Leu Pro Ser Ala Glu Val Tyr . ~ H~

: ~ :
Arg Phe Ala Glu Pro Asp Ser Glu Glu Asn Ile Leu Phe Glu Glu Asn : ::
595 600 605 :
Val Gln Pro Lys Ala Gly Ile Pro Ile Ile Lys Ala Gly Thr Val Leu Lys Leu Ile Glu Arg Leu Thr Tyr His Met Tyr Ala Asp Pro Asn Phe :~
625 630 635 640 . ~::
Val Arg Thr Phe Leu Thr Thr Tyr Arg Ser Phe Cys Arg Pro Gln Glu Leu Leu Ser Leu Leu Ile Glu Arg Phe Glu Ile Pro Glu Pro Glu Pro Thr Glu Ala Asp Arg Ile Ala Ile Glu Asn Gly Asp Gln Pro Leu Ser Ala Glu Leu Lys Arg Phe Arg Lys Glu Tyr Ile Gln Pro Val Gln Leu 690 695 700 . . ~:
Arg Val Leu Asn Val Cys Arg His Trp Val Glu His His Phe Tyr Asp 705 710 715 720 : :~
Phe Glu Arg Asp Ala Asp Leu Leu Gln Arg Met Glu Glu Phe Ile Gly :~

.. ~:. ::.:

: :: ;~ . :::::
:: -:: -., :~: ....
:-::: :: ~- . :.::
.-: ~::: :. -.-:

~ 2 1 .~
::
- 57 _ '' '.

Thr Val Ar8 Gly Lys Ala Met Lys Lys Trp Val Glu Ser Ile Thr Lys : :

~: .
Ile Ile Gln Arg Lys Lys Ile Ala Arg Asp Asn Gly Pro Gly His Asn 755 760 765 :
Ile Thr Phe Gln Ser Ser Pro Pro Thr Val Glu Trp His Ile Ser Arg Pro Gly His Ile Glu Thr Phe Asp Leu Leu Thr Leu His Pro Ile Glu Ile Ala Arg Gln Leu Thr Leu Leu Glu Ser Asp Leu Tyr Arg Ala Val Gln Pro Ser Glu Leu Val Gly Ser Val Trp Thr Lys Glu Asp Lys Glu 820 825 830 :-:: -:-:::: -Ile Asn Ser Pro Asn Leu Leu Lys Met Ile Arg His Thr Thr Asn Leu Thr Leu Trp Phe Glu Lys Cys Ile Val Glu Thr Glu Asn Leu Glu Glu 850 855 860 : ;::. ::~
: . .-: :
Arg Val Ala Val Val Ser Arg Ile Ile Glu Ile Leu Gln Val Phe Gln Glu Leu Asn Asn Phe Asn Gly Val Leu Glu Val Val Ser Ala Met Asn 885 890 895 : : - : -:~
~ :~ '' :
Ser Ser Pro Val Tyr Arg Leu Asp His Thr Phe Glu Gln Ile Pro Ser :. . :

Arg Gln Lys Lys Ile Leu Glu Glu Ala His Glu Leu Ser Glu Asp His ~ :

:. ~ . - .:
Tyr Lys Lys Tyr Leu Ala Lys Leu Arg Ser Ile Asn Pro Pro Cys Val :: :

Pro Phe Phe Gly Ile Tyr Leu Thr Asn Ile Leu Lys Thr Glu Glu Gly 945 950 955 960 : .
Asn Pro Glu Val Leu Arg Arg His Gly Lys Glu Leu Ile Asn Phe Ser Lys Arg Arg Arg Val Ala Glu Ile Thr Gly Glu Ile Gln Gln Tyr Gln 980 985 990 :~
::
Asn Gln Pro Tyr-Cys Leu Arg Val Glu Pro Asp Ile Lys Arg Phe Phe Glu Asn Leu Asn Pro Net Gly Asn Ser Met Glu Lys Glu Phe Thr Asp 1010 1015 1020 :.:~

Tyr Leu Phe Asn Lys Ser Leu Glu Ile Glu Pro Arg His Pro Lys Pro ::~
-. - ::::
: :-;:`''` ~ ' .`.:
:: : :~,.: ' ' .` ' . ,' ~ ' .: ''~ ` :.

- 58 - ~:
.-, 1025 1030 1035 1040 ~;
Leu Pro Arg Phe Pro Lys Lys Tyr Ser Tyr Pro Leu Lys Ser Pro Gly ~::
1045 1050 1055 .
Val Arg Pro Ser Asn Pro Arg Pro Gly Thr Met Arg His Pro Thr Pro 1060 1065 1070 :
Leu Gln Gln Glu Pro Arg Lys Ile Ser Tyr Ser Arg Ile Pro Glu Ser Glu Thr Glu Ser Thr Ala Ser Ala Pro Asn Ser Pro Arg Thr Pro Leu - ::-Thr Pro Pro Pro Ala Ser Gly Thr Ser Ser Asn Thr Asp Val Cys Ser ~ :
llOS 1110 1115 1120 ::::~
Val Phe Asp Ser Asp His Ser Ala Ser Pro Phe His Ser Arg Ser Ala : .:-: -:
Ser Val Ser Ser Ile Ser Leu Ser Lys Gly Thr Asp Glu Val Pro Val :
1140 1145 1150 ~: -Pro Pro Pro Val Pro Pro Arg Arg Arg Pro Glu Ser Ala Pro Ala Glu : :~
1155 1160 1165 : :~
Ser Ser Pro Ser Lys Ile Met Ser Lys His Leu Asp Ser Pro Pro Ala :::
1170 1175 1180 ~ :
Ile Pro Pro Arg Gln Pro Thr Ser Lys Ala Tyr Ser Pro Arg Tyr Ser 1185 1190 1195 1200 ::~
le Ser Asp Arg Thr Ser Ile Ser Asp Pro Pro Glu Ser Pro Pro Leu eu Pro Pro Arg Glu Pro Val Arg Thr Pro Asp Val Phe Ser Ser Ser ro Leu His Leu Gln Pro Pro Pro Leu Gly Lys Lys Ser Asp His Gly 1235 1240 1245 : ~
Asn Ala Phe Phe Pro Asn Ser Pro Ser Pro Phe Thr Pro Pro Pro Pro -~ ~ ! ~.';
1250 1255 1260 . : :~
:, .. :., ~:
Gln Thr Pro Ser Pro His Gly Thr Arg Arg His Leu Pro Ser Pro Pro 1265 1270 1275 1280 ::~
Leu Thr Gln Glu Met Asp Leu His Ser Ile Ala Gly Pro Pro Val Pro : : - 1285 1290 1295 Pro Arg Gln Ser Thr Ser Gln Leu Ile Pro Lys Leu Pro Pro Lys Thr . ~ :
1300 1305 1310 :
Tyr Lys Arg Glu His Thr His Pro Ser Met His Arg Asp Gly Pro Pro :~

.:: ::: ,, , .-::
': :: -'''::.'. -':-: -:: ~ -:-- . - .: :
: ~ - :-:: :: ::

2 ~ 1 3 ~

. :: , Leu Leu Glu Asn Ala His Ser Ser (2) INFOR~5ATION FOR SEQ ID NO: 6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4783 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single :~: .-(D) TOPOLOGY: linear ( ii ) MOLECULE TYPE: DNA ( genomic ) ~ -(vi) ORIGINAL SOURCE:
(A) ORGANISM: Mus musculus : - - : ~
(vii) IMMEDIATE SOURCE:
(A) LIBRARY: Mouse :~
(B) CLONE: Son of Sevenless 1 :

(xi) SEQIIENCE DESCRIPTION: SEQ ID NO:6:
GTGTGTTCTA GTTGGGTGCA AGGTCTAGCC TAAGGCMTG CTTGTCTCCC ACCTCATCCT 60 ~ ~ :

CAGCGAGGAG MCGCGCCCA AGTGGCGGGG GCTGCTGGTG CCTGCGCTGA AAAAGGTTCA 180 -. :~
GGGGCAAGTT CACCCTACTC TTGAGTCTM TGATGATGCT CTTCAGTATG TTGMGAATT 240 .
MTTTTGCAA TTACTMMTA TGCTATGCCA AGCTCAGCCC CGGAGTGCTT CAGATGTGGA 300 ~ . -. ~:-: -: ,.

~ :~''` ~, .

`- ~113~

TCTTAGTCAG TTATCAMGC CTGGGGCAGC ACTTTATTTG CAGTCCATAG GCGMGGCTT lG80 CTGGGAGGGG MGGACATTG GACAGTGTTG CAATGAGTTC ATAATGGMG GMCTCTTAC ~ 440 ACGTGTAGGA GCCMMCACG AGAGACACAT AT``sTCTCTTC GATGGCTTM TGATTTGCTG lS00 .::,.'''' ",~ ,'~.,''' '~

AGAGACTTTT GACTTGCTCk CCTTACACCC AATAGAMTT GCTCGGCMC TCACTTTACT 2460 -- 2 ~ 13`~ ~

_61 . .: - ~ ::: -~:

:: ~ ':;

,.................................................................... .

, CCTTTGCTTT ACTCTTCTAC TTTAGAATAT TTTCGTAAAA GTTATTCAGA GGACTGTGAG 4500 ::

(2) INFORMATION FOR SEQ ID NO: 7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1297 amino acids ( B ) TYPE: amino ac id (C) STRANDEDNESS: single (D) TOPOLOGY: linear ( ii ) MOLECULE TYPE: peptide ( vi ) ORIGINAL SOURCE: :::
(A) ORGANISM: Mus musculus (vii) IMMEDIATE SOURCE: .
(A) LIBRARY: Mouse (B) CLONE: Son of Sevenless 2 (Yi) SEQUENCE DESCRIPTION: SEQ ID NO:7: . - -Pro Thr Leu Ser Ala Asn Glu Glu Ser Leu Tyr Tyr Ile Glu Glu Leu Ile Phe Gln Leu Leu Asn Lys Leu Cys Met Ala Gln Pro Arg Thr Val Gln Asp Val Glu Glu Arg Val Gln Lys Thr Phe Pro Elis Pro Ile Asp Lys Trp Ala Ile Ala Asp Ala Gln Ser Ala 60 Y

Arg Asn Pro Leu Leu Leu Pro Val Asp Lys Ile His Pro Ser Leu Lys ~ ; -Glu Val Leu Gly Tyr Lys Val Asp Tyr His Val Ser Leu Tyr Ile Val .: : - ::
go 95 Ala Val Leu Glu Tyr Ile Ser Ala Asp Ile Leu Lys Leu Ala Gly Asn ~ :

Tyr Val Phe Asn Ile Arg His Tyr Glu Ile Ser Gln Gln Asp Ile Lys ::::~
115 120 125 ::
Val Ser Net Cys Ala Asp Lys Val Leu Met Asp Met Phe Asp Gln Asp Asp Asp Ile Gly Leu Val Ser Leu Cys Glu Asp Glu Pro Cys Ser Ser Gly Glu Leu Asn Tyr Tyr Asp Leu Val Arg Thr Glu Ile Ala Glu Glu Arg Gln Tyr Leu Arg Glu Leu Asn Met Ile Ile Lys Val Phe Arg Glu Ala Phe Leu Leu Asp Arg Lys Leu Phe Lys Pro Ser Glu Ile Glu Lys Ile Phe Ser Asn Ile Ser Asp Ile His Glu Leu Thr Val Lys Leu Leu 210 215 220 ~ :
Gly Leu Ile Glu Asp Thr Val Glu Met Thr Asp Glu Ser Ser Pro His :: -~
225 230 235 240 ..
Pro Leu Ala Gly Ser Cys Phe Glu Asp Leu Ala Glu Glu Gln Ala Phe:~

: : ::- ..:--:. .-:
Asp Pro Tyr Glu Thr Leu Ser Gln Asp Ile Leu Ala Pro Glu Phe Asn Asp His Phe Ser Lys Leu Met Ala Arg Pro Ala Val Ala Leu His Phe ::

Gln Ser lle Ala Asp Gly Phe Lys Glu Ala Val Arg Tyr Val Leu Pro 290 295 300 .
Arg Leu Met Leu Val Pro Val Tyr His Cys Trp His Tyr Phe Glu Leu .

Leu Lys Leu Lys Ala Cys Ser Glu Glu Gln Glu Asp Lys Glu Cys Leu Asn Gln Ala Ile Thr Ala Leu Met Asn Leu Gln Gly Ser Met Asp Arg 340 345 350 :~
Ile Tyr Lys ~ln His Ser Pro Arg Arg Arg Pro Gly Asp Pro Val Cys Leu Phe Tyr Asn Arg Gln Leu Arg Ser Lys His Leu Ala Ile Lys Lys -~

Met Asn Glu Ile Gln Lys Asn Ile Asp Gly Trp Glu Gly Lys Asp Ile : .

,,., , ~ ~ . . - , ~ :

` `
2 :~ ~ 3 ~
::

Gly Gln Cys Cys Asn Glu Phe Ile Met Glu Gly Pro Leu Thr Arg Ile Gly Ala Lys His Glu Arg His Ile Phe Leu Phe Asp Gly Leu Met Ile Ser Cys Lys Pro Asn His Gly Gln Thr Arg Leu Pro Gly Tyr Ser Ser 435 440 445 : .:
Ala Glu Tyr Arg Leu Lys Glu Lys Phe Val Met Arg Lys Ile Gln Ile 450 455 460 : ::
Cys Asp Lys Glu Asp Ala Cy8 Glu Tyr Arg His Ala Phe Glu Leu Val :~

Ser Lys Asp Glu Asn Ser Val Ile Phe Ala Ala Lys Ser Ala Glu Glu 485 490 495 :
-: :. : -Lys Asn Asn Trp Met Ala Ala Leu Ile Ser Leu His Tyr Arg Ser Thr 500 505 510 ~: ;.
Leu Asp Arg Met Leu Asp Ser Val Leu Leu Lys Glu Glu Asn Glu Gln Pro Leu Arg Leu Pro Ser Pro Asp Met Tyr Arg Phe Val Val Thr Asp 530 535 540 - .
Ser Glu Glu Asn Ile Val Phe Glu Asp Asn Leu Gln Ser Arg Ser Gly : . ~
545 550 555 560 ~- .
. ::: , :: :. :::: :::, -, Ile Pro Ile Ile Lys Gly Gly Thr Val Val Lys Leu Ile Glu Arg Leu :~: .
565 570 575 : ::,:~
Thr Tyr His Met Tyr Ala Asp Pro Asn Phe Val Arg Thr Phe Leu Thr 580 585 590 - .-Thr Tyr Arg Ser Phe Cys Lys Pro Gln Glu Leu Leu Asn Leu Leu Ile ::

Glu Arg Phe Glu Ile Pro Glu Pro Glu Pro Thr Glu Ala Asp Lys Leu :.. ::~
610 615 620 . . :~
Ala Leu Glu Lys Gly Glu Gln Pro Ile Ser Ala Asp Leu Lys Arg Phe .: ,: ~. - ~ ,:
Arg Lys Glu Tyr Val Gln Pro Val Gln Leu Arg Val Leu Asn Val Phe Arg His Trp Val-Glu His His Tyr Tyr Asp Phe Glu Arg Asp Leu Glu 660 665 670 : ::
. . : :: .
Leu Leu Glu Arg Leu Glu Ser Phe Ile Ser Ser Val Arg Gly Lys Ala : :

Met Lys Lys Trp Val Glu Ser Ile Ala Lys Ile Ile Lys Arg Lys Lys : ~

~ ~ '', `, -'~ 1 1 c~ A r-690 695 700 .-Gln Ala Gln Ala Asn Gly Ile Ser His Asn Ile Thr Phe Glu Ser Ser Pro Pro Pro Val Glu Trp His Ile Ser Arg Thr Gly Gln Phe Glu Thr 725 730 73s he Asp Leu Met Thr Leu His Pro Ile Glu Ile Ala Arg Gln Leu Thr Leu Leu Glu Ser Asp Leu Tyr Arg Lys Val Gln Pro Ser Glu Leu Val - ,~:

Gly Ser Val Trp Thr Lys Glu Asp Lys Glu Ile Asn Ser Pro Asn Leu Leu Lys Met Ile Arg His Thr Thr Asn Leu Thr Leu Trp Phe Glu Lys Cys Ile Val Glu Ala Glu Asn Phe Glu Glu Arg Val Ala Val Leu Ser 805 810 815 -:-rg Ile Val Glu Ile Leu Gln Val Phe Gln Asp Leu Asn Asn Phe Asn ly Val Leu Glu Ile Val Ser Ala Val Asn Ser Val Ser Val Tyr Arg 835 840 845 :
.... -- ~ ... .~, Leu Asp His Thr Phe Glu Ala Leu Gln Glu Arg Lys Arg Arg Ile Leu Asp Asp Ala Val Glu Leu Ser Gln Asp His Phe Lys Lys Tyr Leu Val ~ ~ -Lys Leu Lys Ser Ile Asn Pro Pro Cys Val Pro Phe Phe Gly Ile Tyr Leu Thr Asn Ile Leu Lys Thr Glu Glu Gly Asn Ser Asp Phe Leu Lys Arg Lys Gly Lys Asp Leu Ile Asn Phe Ser Lys Arg Arg Lys Val Ala 915 920 925 :
Glu Ile Thr Gly Glu Ile Gln Gln Tyr Gln Asn Gln Pro Tyr Cys Leu :~
930 935 940 - :
Arg Thr Glu Pro Glu Met Arg Arg Phe Phe Glu Asn Leu Asn Pro Met 945 950 955 960 : : .
Gly Ile Leu Ser Glu Lys Glu Phe Thr Asp Tyr Leu Phe Asn Lys Ser 965 970 975 :
Leu Glu Ile Glu Pro Arg Asn Cys Lys Gln Pro Pro Arg Phe Pro Arg `` 21 ~

Lys Ser Thr Phe Ser Leu Lys Ser Pro Gly Ile Arg Pro Asn Ala Gly 995 1000 1005 ~: :
Arg His Gly Ser Thr Ser Gly Thr Leu Arg Gly His Pro Thr Pro Leu lO10 1015 1020 :~
Glu Arg Glu Pro Tyr Lys Ile Ser Phe Ser Arg Ile Ala Glu Thr Glu 1025 1030 1035 1040 :~
Leu Glu Ser Thr Val Ser Ala Pro Thr Ser Pro Asn Thr Pro Ser Thr Pro Pro Val Ser Ala Ser Ser Asp His Ser Val Phe Leu Asp Val Asp Leu Asn Ser Ser Cys Gly Ser Asn Thr Ile Phe Ala Pro Val Leu Leu ~ T::~

Pro His Ser Lys Thr Phe Phe Ser Ser Cys Gly Ser Leu His Lys Leu Ser Glu Glu Pro Leu Ile Pro Pro Pro Leu Pro Pro Arg Lys Lys Phe ::~

Asp ~is Asp Ala Leu Asn Ser Lys Gly Ala Val Lys Ser Asp Asp Asp ::

Pro Pro Ala Ile Pro Pro Arg Gln Pro Pro Pro Pro Lys Val Lys Pro Arg Val Pro Val Leu Met Gly Thr Phe Asp Gly Pro Val Pro Ser Pro Pro Pro Pro Pro Pro Arg Asp Pro Leu Pro Asp Thr Pro Pro Pro Val ::~

Pro Leu Arg Pro Pro Glu His Phe Ile Asn Cys Pro Phe Asn Leu Gln Pro Pro Pro Leu Gly His Pro His Arg Asp Pro Asp Trp Leu Arg Asp 1205 1210 1215 :~:
Val Ser Thr Cys Pro Asn Ser Pro Ser Thr Pro Pro Thr Thr Pro Ser :~

Pro Arg Ile Pro Arg Ser Cys His Leu Leu Ser Ser Ser His Ser Ser Leu Ala His Leu Pro Ala Pro Pro Val Pro Pro Arg Gln Asn Ser Ser Pro Leu Leu Pro Lys Leu Pro Pro Lys Thr Tyr Lys Ar8 Glu Leu Ser His Pro Pro Leu Tyr Arg Leu Pro Leu Leu Glu Asn Ala Glu Thr Pro .
.... ~ .

2 1 ~

Gln ( 2 ) INFORMATION FOR SEQ ID NO: 8: ;
ti) SEQUENCE CHARACTERISTICS~
(A) LENGTH: 5253 base psirs (B) TYPE: nucleic acid ~C) STRANDEDNESS: single ~M~
(D) TOPOLOGY: linear ( ii ) MOLECULE TYPE: DNA ( genomic) (vi) ORIGINAL SOURCE: : : x:::
(A) ORGANISM: Mus musculus (vii) IMMEDIATE SOURCE: .
(A) LIBRARY: Mouse (B) CLONE: Son of Sevenless 2 :: ~ .

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: -CCCACCCTCT CAGCTMTGA AGAGTCTCTC TATTATATTG MGMCTGAT TTTTCAGCTG 60 `
CTTMTMGC TATGCATGGC TCAACCMGG ACTGTTCMG ATGTTGAGGA ACGAGTTCM 120 :~

GMGTTTTGG GGTATMAGT GGACTACCAT GTGTCCCTCT ACATTGTGGC TGTATTGGAG 300 : :~

GGTGAGCTAA ACTATTATGA CCTCGTCAGG ACTGMMTTG CAGMGMMG ACAGTATCTA 540 ~ -TTCMGCCTT CTGMMTTGA AMGATTTTC AGTAACATTT CAGATATACA TGMTTGACT 660 - ~ -CCATTAGCTG GTAGCTGTTT TGMGATTTA GCAGAGGAGC MGCGTTTGA TCCCTATGM 780 . : ~ ~
ACATTATCAC AGGACATTCT TGCACCAGAG TTTAATGACC ACTTCAGCM GTTGATGGCC 840 . ~ -AGACCTGCAG TCGCTCTACA TTTTCAGTCC ATTGCTGACG GCTTTMGGA GGCTGTTCGT 900 ~
: : ':

2 ~ ~ 3 ~
::

' ~,' :::" ' t :: :

2 1 ~ 3 ~
~. ~

:~"`,"'`' `'' ;;. .'`

. 2 : , :: .~: . .
, . : -::: -.:-AAATTTTTGA MMGTTTTTC CAGATTAGTA ATATTTMMC AGAMATACT TTAAAAAGCT 5040 ` ~

TTATTAMTT TTTTAATCAG ACAGGATMM GCTTTGCCAT TTGGATACTA TCATTCMAG 5100 : :.~:
,~

' ' ~' ,.
- :: .'- ' ., `~.' '~'.

~' ~ ' ', :

''' ' '''"

.' ~

.... ,.. ... . .. .. : : `

Claims (6)

1. A method for assaying for a substance that affects BCR-ABL mediated transformation which comprises reacting the substance with a first compound containing Grb2 or an SH2 or SH3 domain thereof, and a second compound containing a Grb2 binding site on BCR-ABL, Sos or Shc, or mimetics thereof, under conditions where the first and second compounds are capable of forming a complex, and comparing with a control.

2. A method for assaying for a substance as claimed in claim 1 wherein the first compound contains the SH2-domain of Grb2, and the second compound contains the Grb2 binding site on BCR-ABL or Shc, or a mimetic thereof.

3. A method for assaying for a substance as claimed in claim 1 wherein the first compound contains an SH3-domain of Grb2, and the second compound contains the Grb binding site on Sos or a mimetic thereof.

4. A pharmaceutical composition comprising a compound containing Grb2 or a portion thereof, the Grb2 binding site on BCR, Sos or Shc, or mimetics thereof, and a pharmaceutically acceptable carrier.

5. A pharmaceutical composition as claimed in claim 4 comprising an SH2 domain or an SH3 domain of Grb2.

6. A method of treating a patient suffering from chronic myelogeneous leukemia, acute myelogenous leukemia or acute lymphocytic leukemia comprising administering an effective amount of a pharmaceutical composition as claimed in claim 4 or 5 or a substance identified by the method as claimed in claim 1.