Expression-cloning method for identifying target proteins for eukaryotic tyrosine kinases and novel target proteins

BACKGROUND OF THE INVENTION
1. Field of the Invention
The invention, in the field of molecular and cell biology, relates to a novel method, based on direct expression cloning, for identifying target proteins capable of binding to and/or serving as substrates for receptor or cytoplasmic tyrosine kinases. The invention also relates to novel proteins identified using this method.
2. Description of the Background Art
A variety of polypeptide growth factors and hormones mediate their cellular effects by interacting with cell surface receptors and soluble or cytoplasmic polypeptide containing molecules having tyrosine kinase enzymatic activity (for review, see Williams, L. T. et al., Science 243:1564-1570 (1989); Ullrich, A. et al., Cell 61:203-212 (1990); Carpenter, G. et al. J. Biol. Chem. 265:7709-7712 (1990)). The interaction of these ligands with their receptors induces a series of events which include receptor dimerization and stimulation of protein tyrosine kinase activity. For the epidermal growth factor receptor (EGFR) as well as other receptors with tyrosine kinase activity, such as the platelet-derived growth factor receptor (PDGFR), kinase activation and receptor autophosphorylation result in the physical association of the receptor with several cytoplasmic substrates (Ullrich et al., supra).
Two substrates for the EGFR kinase have now been definitively identified in living cells: (a) the phosphatidylinositol specific phospholipase C-.gamma. (PLC-.gamma.) and (b) the GTPase activating protein (GAP), a protein which may be in the effector loop of the ras protein (Margolis, B. et al. Cell 57:1101-1107 (1989b); Meisenhelder, J. et al. Cell 57:1109-1122 (1989); Molloy, C. J. et al. Nature 342:711-714 (1989); Wahl, M. I. et al. J. Biol. Chem. 265:3944-3948 (1990); Ellis, C. et al. Nature 343:377-381 (1990); Kaplan, D. R. et al. Cell 61 121-133 (1990)).
Similarly, activated PDGFR was shown to tyrosine phosphorylate, and to become associated with PLC-.gamma., GAP, and cellular tyrosine kinases such as pp60.sup.src (Gould, K. L. et al., Molec. Cell. Biol. 8:3345-3356 (1988); Meisenhelder, J. et al., Cell 57:1109-1122 (1989); Molloy, C. J. et al., Nature 342:711-714 (1989); Kaplan, D. R. et al., Cell 61:121-133 (1990); Kazlauskas, A. et al., Science 247:1578-1581 (1990); Krypta, R. M. et al., Cell 62:481-492 (1990); Margolis, B. et al,, Science 248:607-610 (1990)). While the exact sites responsible for the association of EGFR with either PLC-.gamma. or GAP have not been completely clarified, recent work has begun to identify regions on both the substrate and receptor which contribute to the association.
SH2 (src homology 2) domains appear to be the regions responsible for the association of several tyrosine kinase substrates with activated growth factor receptorse SH2 domains are conserved sequences of about 100 amino acids found in cytoplasmic non-receptor tyrosine kinases such as pp60src, PLC-.gamma., GAP and v-crk (Mayer, B. J. et al., Nature 332:272-275 (1988); Pawson, T. Oncogene 3:491-495 (1988)). While having distinct catalytic domains, all these molecules share conserved SH2 and SH3 (src homology 3) domains and the ability to associate with receptors with tyrosine kinase activity (Anderson, D. et al., Science 250:979-982 (1990)).
Tyrosine kinase activation and receptor autophosphorylation are prerequisites for the association between growth factor receptors and SH2 domain-containing proteins (Margolis, B. et al., Mol. Cell. Biol. 10:435-441 (1990); Kumjian et al., Proc. Natl. Acad. Sci. USA 86:8232-8239 (1989); Kazlauskas, A. et al,, Science 247:1578-1581 (1990)). In particular, the carboxy-terminal (C-terminal) fragment of the EGFR, which contains all the known autophosphorylation sites, binds specifically to the SH2 domains of GAP and PLC-.gamma. (see below). Hence, a major site of association exists between the SH2 domain of these substrate proteins and the tyrosine phosphorylated C-terminal tail of the EGFR.
With the recognition that binding to the activated tyrosine kinase receptor is conserved among several substrate proteins, efforts to identify additional substrates which share these properties have been undertaken. Target proteins which bind to activated receptors have been identified by analysis of proteins that co-immunoprecipitate with growth factor receptors, or that bind to receptors attached to immobilized matrices (Morrison, D. K. et al., Cell 58:649-657 (1989); Kazlauskas, A. et al., EMBO J. 9:3279-3286 (1990)). While the identity of some of these proteins is known, several others detected utilizing these approaches have not been fully characterized. Moreover, it is possible that rare target molecules which interact with activated receptors have not been detected due to the limited sensitivity of these techniques; the actual stoichiometry of binding may be low, and the detergent solution necessary to solubilize proteins may disrupt binding.
Conventional approaches to isolate and clone these proteins have been arduous, requiring the use of large quantities of tissue or cells line to purify sufficient amounts of protein for microsequence analysis and subsequent conventional cDNA cloning. Therefore, a need for new approaches for the cloning and subsequent isolation and identification of these proteins is recognized in the art.
SUMMARY OF THE INVENTION
It is an object of the present invention to overcome the deficiencies of the related art.
It is also an object of the present invention to understand and gain control over the regulation of cell growth and oncogenesis by providing the ability to identify target proteins for tyrosine kinases, including both receptor and cytoplasmic tyrosine kinases in eukaryotic organisms.
It is a further object of the present invention to provide a novel expression/cloning system for the rapid cloning of target proteins which bind tyrosine kinase proteins which are present intracellularly and in cell receptors of eukaryotes. The cloning method is based on the ability of a certain class of substrates to bind specifically to the tyrosine-phosphorylated carboxy-terminus (C-terminus) of the proteins having tyrosine kinase activity. Non-limiting examples include proteins that bind at least one of cytoplasmic and receptor tyrosine kinases, such as a receptor tyrosine kinase found in epidermal growth factor receptor (EGFR) (see Example VI, below).
Another object of the present invention is to provide a method of cloning tyrosine kinase target proteins, which method has important advantages over conventional cloning methods, including avoidance of the laborious and costly task of purifying potential target proteins for microsequencing analysis.
Another object of the present invention is to provide a method for identifying receptor target molecules having tyrosine kinase activity whose association with activation receptors could not otherwise be detected using conventional techniques.
Another object of the present invention is to provide for the identification of structurally or functionally related proteins which, though only weakly homologous at the nucleic acid level, are similar in their property of binding to activated receptors with tyrosine kinase activity, which latter ability is important since conventional screening methods used to identify related genes are typically based on low stringency nucleic acid hybridization. Conventional hybridization-based screening would not have been successful in cloning and identifying such tyrosine kinase target proteins of the present invention, exemplified as non limiting examples as GRB-1, GRB-2, GRB-3, GRB-4 or GRB-7, because of their lack of similarity at the DNA level.
The methods of the present invention take advantage of the discovery that the C-terminus of the EGFR protein in which the tyrosine residues are phosphorylated can bind substrates as described herein. By creating a labelled polypeptide which substantially corresponds to at least a portion of phosphorylation domain of a tyrosine kinase, a probe is provided having at least one phosphorylated tyrosine. Such a probe can be used to detect, identify and/or purify target proteins from solutions or as part of screening of cDNA expression libraries from eukaryotic cells or tissues. Such tyrosine kinase target proteins, discovered according to the present invention, are termed "GRB" (for growth factor Receptor Bound) for the initial receptor tyrosine kinases used, but which target proteins are not limited to growth factor receptors. Accordingly, GRBs of the present invention include target proteins for any eukaryotic tyrosine kinase which are provided according to the present invention.
The novel cloning methodology of the present invention has been designated, "CORT" (for Cloning Of Receptor Targets), and may also be applied to detecting, identifying, cloning or purifying target proteins for any tyrosine kinase, such as a soluble, cytoplasmic or receptor tyrosine kinase.
The method of the present invention is proposed as a novel approach having both generality and rapidity for the identification and cloning of target molecules for tyrosine kinases.
The present invention is thus directed to a method for detecting a target protein in solution, which is a target of a receptor or cytoplasmic tyrosine kinase, the target protein being capable of binding to at least a portion of a tyrosine-phosphorylated polypeptide of the receptor or cytoplasmic tyrosine kinase, the method comprising:
(a) contacting the solution (as a cell, an extract thereof, a lysate thereof, or a supernatant thereof) with a solid phase carrier, causing the binding of the protein to the carrier to provide a carrier-bound target protein;
(b) incubating the carrier-bound target protein with the tyrosine-phosphorylated polypeptide, which has been detectably labeled, allowing the polypeptide to bind to the carrier-bound protein;
(c) removing materials not bound to the carrier-bound target protein;
(d) detecting the presence or measuring the amount of the tyrosine-phosphorylated polypeptide bound to the carrier,
thereby quantitatively or qualitatively detecting the target protein in said solution.
In one embodiment, the receptor or cytoplasmic tyrosine kinase is any eukaryotic tyrosine kinase, such as epidermal growth factor receptor, the platelet-derived growth factor receptor, or the fibroblast growth factor receptor, pp60.sup.v-src, pp160.sup.gag-abl, pp130.sup.gag-fps, pp59.sup.c-fyn, PDGF receptor B, CSF-1 receptor pp150.sup.c-fms, pp150.sup.v-fms, EGF receptor, Insulin Receptor, IGF-1 receptor, pp68.sup.gag-ros, PLC-.gamma., middle t-pp60.sup.c-src middle t-pp62.sup.c-yes, and the consensus sequences EEEEEY(PO.sub.4)MPMXX (SEQ ID NO:11), EEEEEY(PO.sub.4)VPMXX (SEQ ID NO:12), DDDDDY(PO.sub.4)MPMXX (SEQ ID NO:13), and DDDDDY(PO.sub.4)VPMXX (SEQ ID NO:14) or a phosphorylatable fragment thereof, preferably a polypeptide of about 10 to 250 amino acid residues, more preferably 10 to 40 or 15 to 50 residues, wherein the polypeptide is produced recombinantly, synthetically or by enzymatic digestion of a purified tyrosine kinase molecule.
This method is preferably performed using a prokaryotic cell, most preferably a bacterial cell such as E. Coli. The cell may also be eukaryotic, such as a yeast or a mammalian cell.
Preferably, the phosphorylated polypeptide is detectably labeled.
The solid phase carrier can be any material which can be used to bind a target protein for a tyrosine kinase. The carrier may preferably be a nitrocellulose membrane, such as to which are transferred proteins released for lysed bacterial cells when a library is being screened.
The present invention also provides a method for mapping to a eukaryotic, such a mammalian, human, murine, or other eukaryotic chromosome a gene encoding a protein which is capable of binding to a tyrosine-phosphorylated polypeptide portion of a receptor or cytoplasmic tyrosine kinase molecule, the method comprising:
(a) infecting a host or host cells which a eukaryotic gene expression library;
(b) detecting a clone expressing the protein using a method according to claim 1;
(c) sequencing the DNA of the clone; and
(d) mapping the sequence to a eukaryotic chromosome.
The present invention is also directed to a polypeptide probe useful in the detection of the expression of a protein capable of binding to a tyrosine-phosphorylated polypeptide portion of a receptor or cytoplasmic tyrosine kinase. The probe comprises an amino acid sequence derived from the tyrosine-phosphorylated portion of the receptor or cytoplasmic molecule, or a functional derivative thereof, lacks the tyrosine kinase domain, and the sequence must contain at least one phosphotyrosine residue, such as 1,2,3,4,5,6,7,8,9,10 or 11 phosphotyrosines. The probe should be detectably labeled with known labels.
A preferred probe has between about 10 and 250 amino acid residues, preferably 10-35, 16-30, 21-35, 15-35, or 20-40 residues.
The probe of the present invention is useful for detecting target proteins for receptor or cytoplasmic tyrosine kinases including but not limited to, epidermal growth factor receptor (EGFR), platelet-derived growth factor receptor (PDGFR), fibroblast growth factor receptor (FGFR), colony stimulating factor-1, (CSF-1), insulin receptor, phospholipase C-.gamma. (PLC-.gamma.) and insulin like growth factor-1, (IGF 1), pp60.sup.v-src, pp160.sup.gag-abl, pp130.sup.gag-fps, pp59.sup.c-fyn, PDGF receptor B, CSF-1 receptor, pp150.sup.c-fms, pp150.sup.v-fms, EGF receptor, insulin receptor, IGF-1 receptor, pp68.sup.gag-ros, PLC, middle t-pp60.sup.c-arc, middle t-62.sup.c-yes, and the consensus sequence EEEEEY(PO4)MPMXX (SEQ ID NO:11), EEEEY (PO4) VPMXX (SEQ ID NO:12), DDDDY (PO4) MPMMXX (SEQ ID NO:13), and DDDDDY(PO4) VPMXX (SEQ ID NO:14) or a phosphorylatable fragment thereof, e.g., as described Cantley et al., Cell. 64:281-302 (1991) or Ulrich and Schlessinger Cell 61:203-312 (1990), which references are entirely herein incorporated by reference.
The present invention also includes a method for preparing the above probe, comprising
(a) providing the receptor or cytoplasmic tyrosine kinase, or a recombinantly, enzymatically or synthetically produced fragment thereof, wherein the receptor or cytoplasmic tyrosine kinase, or fragment thereof, has both a tyrosine kinase domain and a tyrosine-phosphorylated domain, the tyrosine-phosphorylated domain including at least one tyrosine residue capable of being phosphorylated by the tyrosine kinase;
(b) incubating the receptor or cytoplasmic tyrosine kinase, or fragment, with detectably labeled adenosine triphosphate under conditions permitting phosphorylation of the tyrosine residue, causing phosphorylation of the tyrosine residue thereby producing the probe. In a preferred embodiment, the method includes the step of;
(c) additionally treating the phosphorylated receptor or cytoplasmic tyrosine kinase molecule with an agent capable of cleaving the molecule between the tyrosine kinase domain and the tyrosine-phosphorylated domain.
A preferred cleaving agent is cyanogen bromide.
In another embodiment, the above method involves a genetically engineered receptor-like derivative which is a polypeptide encoded by a DNA molecule comprising a DNA sequence encoding tyrosine kinase, linked to a DNA sequence encoding a selective enzymatic cleavage site, linked to a DNA sequence encoding the tyrosine-phosphorylated domain, and wherein the agent is an enzyme capable of cleaving at this cleavage site. Preferred enzymes are Factor Xa and thrombin.
Also provided is a method for purifying from a complex mixture a protein which is capable of binding to a tyrosine-phosphorylated polypeptide portion of a receptor or cytoplasmic tyrosine kinase molecule, the method comprising:
(a) contacting the complex mixture with a solid phase carrier to which a probe is bound, allowing the protein to bind to the probe;
(b) removing materials not bound to the carrier; and
(c) eluting the bound protein from the carrier,
thereby purifying the protein.
The present invention is also directed to a protein, GRB-1, having the amino acid sequence shown in FIG. 4A-4I (SEQ ID NO:5). The invention also includes polypeptides having an amino acid sequence substantially corresponding to the amino acid sequence of a protein, GRB-2, which includes the amino acid sequence shown in FIG. 26A-26C ((SEQ ID NO:6). The invention also includes polypeptides having an amino acid sequence substantially corresponding to the amino acid sequence of a protein, GRB-3, which includes the amino acid sequence shown in FIG. 34A-34C (SEQ ID NO:8). The invention also includes polypeptides having an amino acid sequence substantially corresponding to the amino acid sequence of a protein, GRB-4, which includes the amino acid sequence shown in FIG. 35A-35B (SEQ ID NO:9). The invention also includes polypeptides having an amino acid sequence substantially corresponding to the amino acid sequence of a protein, GRB-7, which includes the amino acid sequence shown in FIG. 36A-36G (SEQ ID NO:10}.
The invention is also directed to a DNA molecule encoding a polypeptide having a amino acid sequence substantially corresponding to the amino acid sequence of at least one of GRB-1, GRB-2, GRB-3, GRB-4 or GRB-7 proteins. Included are DNA molecules encoding functional derivatives of these proteins. When the DNA molecule naturally occurs, it is substantially free of the nucleotide sequences with which it is natively associated. The DNA molecules of this invention may be expression vehicles, such as plasmids.
Also provided is a host transformed with each of the above DNA molecules.
The present invention also includes a process for preparing a target protein substantially corresponding to the amino acid sequence GRB-1, GRB-2, GRB-3, GRB-4 or GRB-7 protein, comprising:
(a) culturing a host comprising a recombinant nucleic acid having a nucleotide sequence encoding the target protein under culturing conditions such that the target protein is expressed in recoverable amounts; and
(b) recovering the protein from the culture.

BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a filter blot pattern showing that the carboxy-terminus of the EGFR interacts with GAP-SH2 immobilized on nitrocellulose filters. Bacterially-expressed trpE/GAP-SH2 fusion protein or trpE as a control was spotted at various concentrations onto nitrocellulose filters. The filters were hybridized overnight with (.sup.32 P)-labelled C-terminal domain of the EGFR. Autoradiography was for 2 hours.
FIG. 2 is a schematic diagram depicting the method of cloning of receptor or cytoplasmic tyrosine kinase targets (CORT). C-terminal domain of the EGFR is phosphorylated with radiolabelled phosphorous. Lambda gt11 library was plated at a density of 4.times.10.sup.4 plaques per 150 mm plate. The plaques were overlaid with IPTG-impregnated nitrocellulose filters for 12 hours, after which the plaques were transferred to nitrocellulose and incubated with the labelled probe. Positive colonies are then selected for further analysis.
FIG. 3A-3B shows autoradiograms of phage expressing GRB-1 protein. 3A) Primary screen demonstrating one positive signal (arrow) out of 40,000 phage plated. 3B) Plaque purification of phage expressing GRB-1. All plaques bound to the (.sup.32 P)-labelled C-terminal domain of the EGFR.
FIG. 4A-4I shows the DNA sequence and predicted amino acid sequence of GRB-1 (SEQ ID NO:1). The protein has 724 amino acid residues.
FIG. 5 compares the sequences of the SH2 domains of GRB-1 with other proteins with similar motifs. 7op) SH2 domains of GRB-1, c-src, v-abl, bovine PLC-.gamma., GAP, and V-crk. N and C refer to N-Terminal and C-terminal
SH2 domains respectively. Conservation amino acid substitutions are as defined by Shwartz and Dayhoff: (A,G,P,S,T); (L,I,V,M); (D,E,N,Q); (K,R,H); (F,Y,W); and C. Bold letters identify those positions where the same or a conservative amino acid substitution is present at 5 or more positions. Boxes identify conserved motifs. Bottom) A similar comparison of the SH3 domain of GRB-1.
FIG. 6 is a schematic diagram comparing the structural organization of the SH2 and SH3 domains. The scheme includes known proteins containing SH2 and SH3 domains, such as c-src, v-crk, PLC-.gamma., GAP1 and GRB-1.
FIG. 7 is a Northern blot of monkey mRNA with GRB-1 probe. 5 .mu.g of poly (A)+MRNA, obtained from various monkey tissue, was electrophoresed on 1.2%/2.2M agarose-formaldehyde gel. The blot was hybridized with a (32P)-nick translated DNA probe corresponding to the insert from clone ki4.
FIG. 8 is a gel pattern showing that antibodies to GRB-1 immunoprecipitate a protein of 85 kDa from biosynthetically labelled cells.degree. Cells were metabolically labelled with (.sup.35 S)methionine, after which lysates were prepared and immunoprecipitated with either immune (I) or preimmune (P) serum. The immunoprecipitated protein was separated on a 8% SDS/PAGE. Autoradiography was performed overnight. Cell lines used include human glioblastoma cell line, U1242, rat bladder carcinoma cell line, NBT-II and NIH-3T3 cells.
FIG. 9A-9B depicts several wild-type and mutant proteins used in the studies. In both figures, the portion of the of "TM" represents the extracellular domain of the protein (excluding the cleaved signal sequence at the amino terminus) and the portion of the diagram to the right of "TM" represents the cytoplasmic domain of the protein. (A) EGF receptor constructs with their known or predicted autophosphorylation sites. Wild-type (W.T.), Kinase negative (K721A), and carboxy-terminal deletion (CD126), were immunoprecipitated from previously described transfected NIH373 cells expressing -300,000 EGF receptors. EGFR-C represents a deletion mutant containing the cytoplasmic domain of the EGF receptor produced by baculovirus-infected SF9 cells. (B) Structure of PLC-.gamma. and trpE/GAP SH2 proteins indicating location of the SH2 and SH3 domains and PLC-.gamma. tyrosine phosphorylation sites.
FIG. 10A-10B is a gel pattern showing association of PLC-.gamma. with EGFR mutants. Wild-type (HER14), carboxy-terminal deletion (DC126), or kinase-negative (K721A) EGFR were immunoprecipitated with anti-EGFR mAb108. Receptors were autophosphorylated with (.gamma.-.sup.32 P-ATP. Concomitantly EGFR-C was added to protein A-Sepharose beads alone or to immunoprecipitated K721A receptors either with or without ATP. After further washes to remove ATP, lysate from --15.times.10 6 PLC-.gamma. overexpressing 3T-P1 cells was added and mixed for 90 min at 4 C. After washing to remove unbound PLC-.gamma., proteins were separated on a 6% SDS-gel and transferred to nitrocellulose for immunoblotting. One eighth of the sample was utilized for anti-PTyr blotting (FIG. 10A), the remainder for anti-PLC-.gamma. blotting (FIG. 10B) (exposure time 14 h).
FIG. 11 is a gel pattern showing that phosphorylation of PLC-.gamma. reduces its binding to the EGF receptor. Full length EGFR was immunoprecipitated with mAb108, and allowed to autophosphorylate. Lysate from PLC-.gamma. overexpressing 3T-P1 cells was added and mixed for 90 min at 4.degree. C. After binding, ATP was added to one half of the samples allowing the PLC-.gamma. molecules to be phosphorylated by the EGF receptor. SDS-PAGE sample buffer was then added to one half of the EGFR-PLC-.gamma. complexes (NO WASH, left panel) and directly loaded onto the 6% gel. The other half was washed three times with HNTG and then loaded on the gel (WASH, right panel. After running duplicate samples on SDS-PAGE, the proteins were transferred to nitrocellulose and probed with anti-PLC-.gamma. and (.sup.125 I)protein A. The bands were subsequently cut from the nitrocellulose and quantitated in a .gamma. counter. After three washes with HNTG, 50.+-.5% (Mean.+-.SEM, n=4) of the non-phosphorylated PLC-.gamma. remained bound to the EGFR while only 22, 4% of the phosphorylated PLC-.gamma. remained (exposure time: 12 h).
FIGS. 12A and 12B are representations of a gel pattern showing binding of EGFR-C to trpE proteins. In FIG. 12A, EGFR-C (0.5 .mu.g) was immunoprecipitated with antibody C and washed. MnCl.sub.2 alone or MnCl.sub.2 and ATP were then added to facilitate autophosphorylation of TrpE or trpE/GAP SH2 (approximately 2 .mu.g). The immunoprecipitates were separated on a 10% SDS-gel, transferred to nitrocellulose and immunoblotting was performed with anti-trpE. For comparison, about 0.1 .mu.g of trpE or trpE/GAP SH2 lysate was loaded directly on to the gel (right panel of A). In FIG. 12B, trpE or trpE/GAP SH2 was immunoprecipitated with anti-trpE antibodies and washed. Phosphorylated or non-phosphorylated EGFR-C (0.5 .mu.g) was then added and allowed to bind as above. After washing, samples were separated on a 10% gels transferred to nitrocellulose and probed with antibody C. The two samples on the right represent 0.5 .mu.g of phosphorylated and non-phosphorylated kinase loaded directly onto the gel (exposure time: 2 h).
FIGS. 13A and 13B are representations of a gel pattern showing binding of trpE/GAP SH2 to wild-type and mutant EGFR. In FIG. 13A, wild-type receptor (HER14) or the carboxy-terminal deletion CD126 receptor were immunoprecipitated with mAb 108. MnCl.sub.2 alone or MnCl.sub.2 and ATP were then added to the autophosphorylated half of the receptor-containing samples. One set of CD126 was also cross-phosphorylated with 0.5 .mu.g of EGFRC. TrpE/GAP SH2 was then added for 90 min at 4.degree. C. and, after three more washes, loaded onto SDS-PAGE. After transfer to nitrocellulose, blots were probed with anti-trpE (left panel), anti-EGFR RK2 (center panel), or anti-PTyr (right panel). RK2 and anti-PTyr are both 1/8 of the total sample and were separated on 7% SDS-PAGE. The remaining sample was loaded on a 10% gel for the anti-trpE blot (exposure time 14 h).
In FIG. 13B, lysates from NIH3T3 2.2 cells containing no EGFR (3T3) or from cells with kinase-negative receptors (K21A) were immunoprecipitated with mAb108. To all immunoprecipitates, 0.5 .mu.g of EGFR-C was added and then MnCl.sub.2 alone or MnCl.sub.2 and ATP. trpE/GAP SH2 was added and samples prepared and immunoblotted as in (A) (exposure time 19 h).
FIG. 14 is a gel pattern showing binding of PLC-.gamma. and trpE/GAP SH2 to the CNBr cleaved C-terminal fragment of EGFR. EGFR-C (10 .mu.g) was incubated in a Centricon 30 in 20 mM HEPES, pH 7.5 with 100 .mu.g BSA as a carrier protein. The phosphorylated and non-phosphorylated EGFR-C were then each divided in two with one half being stored in buffer while the other half was cleaved with CNBr. The four samples either with or without ATP, and with or without CNBr were then each brought up in 500 .mu.l 1% Triton X-100 lysis buffer, split in two, and immunoprecipitated with anti-C antibody. After washing the immunoprecipitates, lysates containing PLC-.gamma. or trpE/GAP SH2 were added. Immunoblotting was then performed on the samples as above with anti-trpE or anti-PLC-7. For the right panel, a fraction of the cleaved and uncleaved EGFR-C (0.1 .mu.g) was loaded directly on the gel without immunoprecipitation and immunoblotted with RK2 (exposure time 14 h). The dark band seen in all lines of the anti-trpE blot runs at about 40 kDa (also seen in FIG. 13) and represents (.sup.125 I)protein A binding to the heavy chain of the immunoprecipitating antibody.
FIG. 15 is a gel pattern showing binding of the tyrosine phosphorylated C-terminal EGFR fragment to trpE/GAP SH2 but not to trpE. EGFR-C (5 .mu.g) was autophosphorylated by the addition of (.gamma.-32P)ATP. The phosphorylated EGFR-C was concentrated in a Centricon 30, and then cleaved with CNBr in 70% formic acid. One half of the sample (350,000 c.p.m.) was allowed to bind to trpE or trpE/GAP SH2 as in FIG. 12B, washed and run on a 10% SDS-gel. (A) Binding of phosphorylated CNBr cleaved EGFR-C to trpE (B) Binding of phosphorylated CNBr cleaved EGFR-C to trpE GAP SH2 (C) 3000 c.p.m. of CNBr-cleaved EGFR-C (D) for comparison 3000 c.p.m. of cleaved EGFR-C (exposure time 20 h). EGFR 984/1186 indicates the sequence of the tyrosine autophosphorylated fragment generated by CNBr.
FIG. 16A-16C shows the partial nucleotide sequence and predicted amino acid sequence of GRB-2.
FIG. 17 is a comparison of sequence homology of avian crk to GRB-3 with dots indicating homologous amino acids.
FIG. 18 is a protein sequence of nck compared to that of GRB-4 for amino acid sequence homology.
FIG. 19 is a GRB-7 (SEQ ID NO:1) protein sequence.
FIG. 20 is a schematic representation of GRB-7 to include the proline rich, P2B2, rasGAP and SH2 domain homology.
FIG. 21 is a comparison of a GRB-7 amino acid sequences with SH2 domains from avian c-src, human PLC-.GAMMA.1, GRB-1/p85, mouse fyn, GRB-3 and GRB-4.
FIG. 22 is a comparison of a GRB-7 amino acid sequence with rasGAP.
FIG. 23 is a comparison of a GRB-7 amino acid sequence with P2B2.
FIG. 24 is a representation of a Northern blot analysis of GRB-7 mRNA.
FIG. 25A-25C is a comparison of binding of the phosphorylated EGFR carboxy-terminus to PLC-g fragments expressed in a .lambda.gt11 or T7 polymerase based library.
FIG. 26A-26C include a cDNA (SEQ ID NO:2) and protein sequence (SEQ ID NO:2) of GRB2 clone 10-53, with '5 and '3 untranslated flanking sequences; SH2 (thick line) and SH3 (thin lines) domains are indicated.
FIG. 26F is a schematic representation of the overall domain structure of GRB2.
FIGS. 26D and 26E are sequence alignments of GRB2 SH2 and SH3 domains, respectively, with other proteins. N and C refer to N-terminal and C-terminal domains, respectively. The one letter code is used to indicate amino acid residues. Bold letters identify those positions where the same or a conservative amino acid substitution is present at that position. Compared are PLC.gamma.1, GAP, v-src, v-abl, v-crk and p85. The SH2 domain of GRB2 is most similar to the SH2 domain of v-fgr (43% similarity) and the N-terminal SH3 domain is most similar to the SH3 domain of human vav (48% similarity).
FIGS. 27A-27B show the analysis of expression of GRB2 in various murine tissues and cell lines. 27A shows a Northern analysis in murine tissues, with tissue of origin as indicated, with 20 .mu.g total RNA loaded per lane. The sizes of the GRB2 transcripts (relative to BRL size markers indicated) are 3.8 kb and 1.5 kb.
FIG. 27B shows immunoprecipitation of GRB2 from (.sup.35 S)methionine labeled HER14 lysates with preimmune (lane 1) and immune GRB2 antiserum (Ab50) (lane 2). Immunoblot analysis of GRB2 from lysates of HER14 cells with Ab86 (lane 3). Molecular weight markers (sized in kDa) are indicated. Arrow indicates band corresponding to GRB2 protein. Exposure times are 24 hours.
FIG. 28 shows the association of endogenous GRB2 with EGFR in HER14 cells. HER14 cells mock treated lanes 1, 3, 5) or EGF treated (lanes 2, 4, 6) were lysed and immunoprecipitated with anti-EGF receptor antibodies (mAb 108), subjected to SDS-PAGE, and after transfer to nitrocellulose, blotted with polyclonal anti-EGFR antibodies (Anti-C) lanes 1 and 2), anti-phosphotyrosine antibodies (lanes 3 and 4), or anti-GRB2 antibodies (Ab86) (lanes 5 and 6). The immunoblots were labeled with .sup.125 I-protein A followed by autoradiography at -70.degree. C. Anti-GRB2 blot were exposed for 24 hrs. Anti-EGFR and antiP-tyr blots were exposed for 16 hrs. The positions of molecular weight markers (sized in kDa) are indicated.
FIG. 29 is a schematic representation of GRB2-GST fusion proteins. Gluthatione-S-transferase fusion proteins of full size GRB2 and various regions of GRB2 were generated and purified by affinity chromatography utilizing glutathione agarose beads, as described in methods. Shown are the SH2 domain of GRB2 (SH2), the amino terminal SH3 (N-SH3), carboxy terminal SH3 (C-SH3), the amino terminal SH3 and SH2 domains (N-SH3 SH2), and the SH domain with the carboxy terminal SH3 domain (SH2 C-SH3). GST region of fusion proteins is not shown.
FIG. 30 represents the binding of GST-GRB2 fusion proteins to activated growth factor receptors in vitro.degree. Binding of fusion proteins to the tyrosine phosphorylated proteins (lanes i through 6) and EGFR (lanes 7 through 10) in control and EGF stimulated HER14 cell lysates, and tyrosine phosphorylated proteins in control and PDGF stimulated lysates (lanes 11 through 14). Lysates were incubated with equal amounts of fusion proteins immobilized on glutathione-agarose beads. Bound proteins were washed, subjected to SDS-PAGE and immunoblotted with antiphosphotyrosine (lanes 1 through 6, 11 through 14)) or anti EGF-receptor (lanes 7 through 10) antibodies. The immunoblots were labelled with .sup.125 I-protein. A followed by autoradiography at -70.degree. C. exposure time 16 hrs. The positions of the molecular weight markers are indicated (sizes in kDA).
FIG. 31 shows data representing the lack of significant phosphorylation of GRB2 in HER14 cells following stimulation with EGF. (.sup.32 P)orthophosphate (lanes 1 through 4) or (.sup.35 S) methionine (lanes 5 through 8) metabolically labeled HER14 cells were lysed following mocked EGF treatment. The precleared lysates were immunoprecipitated with either preimmune or anti-GRB2 antibodies (Ab50), and subjected to SDS-PAGE and autoradiography. Two hour (32P) and two day (.sup.35 S) exposure times are shown. The position of GRB2 and the co-immunoprecipitating 55 kDa phosphoprotein are marked with arrows.
FIG. 32 presents the alignment of amino acid sequences of GRB2 and sem-5 (single letter code). Boxes surround the SH2 and SH3B domains as indicated. Bold capital letters indicate identical amino acids, capital letters indicate conservative substitutions.
FIG. 33 is a representation showing a model for the interaction between EGF receptor and GRB2 and their C. elegans counterparts. Tyrosine autophosphorylated EGFR (or let-23) binds to the SH domain of GRB2 (or sem-5). Ras (or let-60) acts downstream leading to either cell proliferation or vulval development.
FIG. 34A-34C is a cDNA (SEQ ID NO:3) and protein sequence (SEQ ID NO:8) of GRB-3.
FIG. 35A-35B is a cDNA (SEQ ID NO:4) and protein (SEQ ID NO:9) sequence of GRB-4.
FIG. 36A-36G is a cDNA (SEQ ID NO:7) and protein (SEQ ID NO:10) sequence of GRB-7.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
Methods, compounds and compositions have now been discovered to provide a means to understand and gain control over the regulation of cell growth and oncogenesis by providing the ability to identify target proteins for tyrosine kinases, including both receptor and cytoplasmic tyrosine kinases in eukaryotic organisms.
One embodiment of the present invention is to provide a novel expression/cloning system for the rapid cloning of target proteins which bind tyrosine kinase proteins which are present intracellularly and in cell receptors of eukaryotes. The cloning method is based on the discovery that certain class of substrates can bind specifically to the phosphorylated domain of proteins having tyrosine kinase activity.
According to another embodiment of the present invention, novel probes and methods using such probes for rapid expression cloning of DNA encoding proteins which have the characteristic of binding to the tyrosine-phosphorylated portion, such as the C-terminus, of a receptor tyrosine kinase molecule, which molecule is present in the cytoplasm or in cell receptors of eukaryotic receptors.
By the term "eukaryote" or "eurkaryotic" is intended any organism considered to have the attributes of a eukaryote, including a cell nucleus, mitochondria, chromosomes, etc., which are attributes which do not occur in bacteria, blue-green algae or viruses. Non-limiting examples of eukaryotes include yeast, fungi, insects, plants, mammals, birds, reptiles, amphibians. Mammals include, but are not limited to, humans, mice, rats, rabbits, cows, pigs, goats, sheep, horses, cats, dogs, etc.
Expression cloning is a method wherein the DNA being cloned encodes a protein which is expressed from a cloned library from a cell known or expected to have the desired protein. The desired DNA, typically in the form of a cDNA library, is detected by means of its expression and/or direct detection of the protein which it encodes. Expression cloning systems and library cloning are well-known in the art (see: Sambrook, J. et al. (Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989), and Ausubel et al, eds. (Current Protocols in Molecular Biology Wiley Interscience, N.Y. (1987, 1992)), which references are hereby entirely incorporated by reference).
According to the present invention, the protein is expressed according to known method steps from a library and the expressed protein, released from the cell it is expressed in is transferred to a solid carrier or support, such as a nitrocellulose filter as a non-limiting example, and detected using a detectable label for the expressed protein by known method steps.
One of the ways in which the polypeptide probe target protein can be detectably labeled is by providing peptide probes or anti-target protein antibodies and linking the peptide probes or antibodies to an enzyme for use in an enzyme immunoassay (EIA). This enzyme, in turn, when later exposed to an appropriate substrate, will react with the substrate in such a manner as to produce a chemical moiety which can be detected, for example, by spectrophotometric, fluorometric or by visual means. Enzymes which can be used to detectably label the antibody include, but are not limited to, malate dehydrogenase, staphylococcal nuclease, delta-5-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-6- phosphate dehydrogenase, glucoamylase and acetylcholinesterase. The detection can be accomplished by colorimetric methods which employ a chromogenic substrate for the enzyme. Detection may also be accomplished by visual comparison of the extent of enzymatic reaction of a substrate in comparison with similarly prepared standards.
Detection may additionally be accomplished using any of a variety of other immunoassays or detectably labeled peptide probes. For example, by radioactively labeling the peptide probes, anti-target protein antibodies or antibody fragments, such that the labeled target protein may also be detected through the use of a radioimmunoassay (RIA). A good description of RIA may be found in Laboratory Techniques and Biochemistry in Molecular Biology, by Work, T. S., et al., North Holland Publishing Company, New York (1978) with particular reference to the chapter entitled "An Introduction to Radioimmune Assay and Related Techniques" by T. Chard, incorporated by reference herein. A radioactive isotope ,such as .sup.32 p, .sup.35 S, .sup.12 C or .sup.3 H, can be detected by such means as the use of a gamma counter, a liquid scintillation counter or by autoradiography.
It is also possible to label the peptide probe or anti-target protein antibody with a fluorescent compound. When the fluorescently labeled peptide or antibody is exposed to light of the proper wave length, its presence can then be detected due to fluorescence. Among the most commonly used fluorescent labelling compounds are fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine. Suitable fluorescent probes are well known or commercially available, such as from Molecular Probes, Inc., Eugene Oreg.
The peptide probe or anti-target protein antibody can also be detectably labeled using fluorescence emitting metals such as .sup.152 Eu, or others of the lanthanide series. These metals can be attached to the peptide probe or anti-target protein antibody using such metal chelating groups as diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).
The peptide probe or anti-target protein antibody also can be detectably labeled by coupling it to a chemiluminescent compound. The presence of the chemiluminescent-tagged peptide probe or anti-target protein antibody is then determined by detecting the presence of luminescence that arises during the course of a chemical reaction. Examples of particularly useful chemiluminescent labeling compounds are luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester.
Likewise, a bioluminescent compound my be used to label the peptide probe or anti-target protein antibody of the present invention. Bioluminescence is a type of chemiluminescence found in biological systems in which a catalytic peptide probe or anti-target protein antibody increases the efficiency of the chemiluminescent reaction. The presence of a bioluminescent peptide probe or anti-target protein antibody is determined by detecting the presence of luminescence. Important bioluminescent compounds for purposes of labeling are luciferin, luciferase and aequorin,
The expression cloning method of the present invention for detecting and cloning a target protein for tyrosine kinase cytoplasmic or receptor protein my be used for detecting such target proteins from any eukaryotic cell source. For example, certain target molecules bind to the tyrosine phosphorylated portion of PDGFR and the colony stimulating factor-1 (CSF-1) (Coughlin, S. R. et al., Science 243:1191-1194 (1989); Kazlauskas, A. et al., Cell 58:1121-1133 (1989); Shurtleff, S. A. et al., EMBO J. 9:2415-2421 (1990); and Reedjik, M. et al., Mol. Cell. Biol. 10:5601-5608 (1990)). In these receptors, the tyrosine phosphorylation occurs in a kinase insert domain, rather than in the C-terminal domain as is the case with the EGFR. Therefore, specific polypeptide probes in the range of 10-250, such as 10-20, 20-30, 40-50, 70-100, or 100-200, amino acids utilizing the kinase insert domain, or a portion thereof as defined herein, and cytoplasmic or receptor or PDGFR or CSF-1 receptor can be similarly used for expression cloning. Similar probes can also be constructed for the fibroblast growth factor (FGF) receptor (which is tyrosine phosphorylated in the C-terminal domain) or the HER 2/neu receptor, both of which are also able to interact with SH2 containing proteins such as PLC-.gamma.. In other receptors, such as the insulin receptor, tyrosine phosphorylation occurs in the kinase domain itself.
Accordingly, any tyrosine kinase protein or fragment thereof of 10-250 amino acids, e.g., as described in Cantley et al. Cell 64:281-302 (1991) (the entire contents of which are herein incorporated by reference), can be used to bind a target protein in solution which is contacted to the tyrosine kinase protein bound or associated with a carrier or support. The carrier or support can be any known material that associates with a tyrosine kinase or fragment thereof, such that, once the target protein is bound, the non-bound material can be removed from the carrier without dissociated the tyrosine kinase bound to the target protein.
Thus the tyrosine kinase protein is used as a protein probe to bind target proteins. Alternatively, a polypeptide of 10-250 amino acids, corresponding to at least a phosphorylation domain of the tyrosine kinase; or corresponding to a consensus sequence of a class or group of tyrosine kinases, can be used as the protein or polypeptide probe and may be detectably labeled.
Thus, while it will be appreciated that different sites are tyrosine-phosphorylated in different proteins, e.g., the C-terminal domain in the EGFR, the kinase domain in insulin receptor, and a kinase domain insert in PDGFR, the present invention recognizes the common features of all these structures, the presence of one or more phosphotyrosine residues, and the ability of certain cellular proteins to bind on the basis of affinity to a polypeptide containing one or more phosphotyrosines. While reference will generally be made below to a probe which is a C-terminal domain, with reference to the EGFR, this language is not intended to be limiting and is intended to include all of the other alternative tyrosine-phosphorylated domains discussed above.
The methods and approach of the present invention can be applied to the cloning and identification of all target molecules which are capable of interacting in a specific manner with tyrosine phosphorylated polypeptides, such as cytoplasmic tyrosine kinases or the activated phosphorylated receptors described herein. Additional proteins which bind to tyrosine-phosphorylated sequences, such as the tyrosine-specific phosphatases, e.g., R-PTPases (Sap, J. et al., Proc. Natl. Acad. Sci. USA 87:6112-6116 (1990); Kaplan, R. et al., Proc. Natl. Acad, Sci. USA 87:7000-7004 (1990) may also be used according to a method of the present invention. The methods are also applicable in the cloning and identification of proteins which bind to phosphorylated serine/threonine residues, as with serine/threonine-specific phosphatases as a non-limiting example.
Use of a polypeptide or protein probe of the present invention allows the rapid cloning of DNA and identification of the encoded proteins from eukaryotic DNA or RNA libraries, such as a gene expression library. The method is particularly useful with a bacteriophage lambda gt11 library or a T7 library. As a non-limiting example of a eukaryotic library, screening a human fetal brain lambda gt11 expression library has permitted the present inventors to clone several target protein genes and to characterize the proteins they encode. One, termed GRB-1, was fully, DNA sequenced (SEQ ID NO:1) and found to encode novel human protein with an amino acid sequence as shown in FIG. 4A-4I (SEQ ID NO:5) and a molecular weight of about 85 kDa which contained two SH2 domains and one SH3 domain (FIG. 4A-4I and FIG. 5). GRB-2 DNA (FIG. 26A-26C) (SEQ ID NO:2) also contains unique SH2 and SH3 domains in the amino acid sequence, (FIG. 26A-26C) (SEQ ID NO:6). GRB-3 DNA (SEQ ID NO:3) was also sequenced (FIG. 34A-34C) and the GRB-3 amino acid sequence (SEQ ID NO:8). GRB-4 DNA (SEQ ID NO:4) (FIG. 35A-35B) encoded a protein composed of three SH3 domains and one SH2 domain having the GRB-4 amino acid sequence (SEQ ID NO: 9) .
Several overlapping clones were identified which were used for DNA sequencing of GRB-7 (FIG. 36A-C) (SEQ ID NO:7) to obtain the full length GRB-7 amino acid sequence shown in FIG. 36A-36C (SEQ ID NO:10). A schematic representation of GRB-7 is displayed in FIG. 20 depicting the regions of similarity to known proteins. The GRB-7 protein is 535 amino acids in length (FIG. 36A-36G) (SEQ ID NO:7) and has one SH2 domain at its extreme carboxy-terminus. In FIG. 21, the SH2 domain of GRB-7 is compared to other SH2 domains including mouse fyn, human PLC-.gamma.1 and the crk and nck-like proteins of the present invention. Other protein motifs in GRB-7 were determined using Swissprot and GenEmbl databases, using software such as the University of Wisconsin Genetics Computer Group Sequence Analysis Software package (Devereaux et al Nucl. Acid Res. 12:387 (1984)). The Swissprot and GenEMBL database can be searched using known software, such as the FASTA and TFASTA respectively. Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85, 2444 (1988). Protein alignments can be performed using known software, such as BESTFIT, e.g., with conservative substitutions defined as a score of .gtoreq.0.8 using the symbol comparison table for proteins. Gribskov and Burgess, Nucleic Acid Research 14, 6745 (1984).
From such analysis, amino acids 242 to 339 of GRB-7 showed similarity to a sequence from the central region of ras GAP (21). Over this region of 91 amino acids from ras GAP, GRB-7 has 26% identity and 42% similarity allowing for conservative substitutions (FIG. 22). This region of ras GAP lies between the SH2/SH3 domains and the GTPase activating carboxy terminal region and has not been assigned a specific function (Martin et al Science 255:192 (1992)). The amino-terminal sequence of GRB-7 was found to be proline rich and thus has similarity to many other proline rich proteins. GRB-7 does have an extended region of limited similarity to the catalytic domain of protein phosphatase 2B (Guerini and Klee, Proc. Natl. Acad. Sci. USA 87:6112 (1990)) including this proline rich region (FIG. 23) but no significant similarity was found to other serine/threonine phosphatase such as protein phosphatase 1 or 2A.
A northern blot of GRB-7 in mouse tissues is presented in FIG. 25A-25C. Oligo dt selected mRNA was probed with GRB-7 cDNA using known methods. See Ausubel et al eds., Current Protocols in Molecular Biology, Wiley Interscience, New York., (1987, 1992) and Sap et al Proc. Natl. Acad. Sci. USA 87:6112 (1990), which are entirely incorporated herein by reference. The highest signal was detected in liver and kidney, but a signal was also detected in ovary and testes. On longer exposure, a weak signal was detectable in lung but not in heart, muscle, spleen or brain. The major transcript was seen at 2.4 kb which closely corresponds to the longest cDNA clone obtained.
GRB-7 represents another novel gene cloned using the CORT technology, according to the present invention. It belongs to a relatively rare group of proteins with SH2 domains but no SH3 domains including the fps tyrosine kinase, (I. Sadowski, J. C. Stone and T. Pawson, Mol. Cell. Biol. 6:4396 (1986)), protein tyrosine phosphatase 1C (Shen et al Nature (Lond.) 352:736 (1991)) and possibly tensin (Davis et al., Science 252:712 (1991}).
CORT methodology of the present invention provides proteins that interact with the EGFR and lie downstream of the EGFR signalling pathway. In general, in vitro associations between SH2 domain and tyrosine phosphorylated proteins correlate with interactions in living cells (McGlade et al., Mol. Cell. Biol. 12:991 (1992)). CORT methodology of the present invention is therefore expected to yield commercially important downstream signalling components of cytoplasmic tyrosine kinase target proteins, as well as growth factor receptors, as demonstrated by the finding that the C. elegans gene sem-5 is the homolog of human GRB-2. Sem-5 is crucial for vulval development, a process that requires the activity of let-23, an EGFR like tyrosine kinase. Accordingly, it is expected that sem-5 lies downstream of the activated let-23, and that GRB-2 serves a similar crucial function in EGFR signalling.
CORT methodology of the present invention can also be used to identify new SH2 proteins that interact with the EGFR. Seven different exemplary SH2 domain proteins are expected to have important signalling functions, With the use of the T7 polymerase based library, this methodology may be more easily applied, due to relatively higher levels of expressions which increase detectability, to any eukaryotic cytoplasmic or receptor tyrosine kinase proteins, such as growth factor receptor systems. Hence such a method of the present invention can also be used to clone other novel SH2 domain proteins using other growth factor receptor tyrosine kinases, including the use of T7 polymerase based libraries, by performing expression/cloning techniques involving protein-protein interactions and DNA binding proteins.
SH2 domains, such as in the GAP and PLC-.gamma. proteins, are responsible for the association of these proteins with the phosphorylated C-terminus of the EGFR (see Example VI, below). Thus, one function of SH2 domains is to juxtapose the intracellular portion of receptor tyrosine kinase molecules with their substrates to facilitate efficient tyrosine phosphorylation.
Detailed analysis of one of the cDNA clones of the present invention, GRB-1, identified using methods of the present invention, reveals a novel sequence containing two SH2 domains and one SH3 domain. This protein is expressed in various tissues and cell lines. Its predicted molecular weight, 85 kDa, is consistent with its migration on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).
By the term "cytoplasmic tyrosine kinase" is meant a soluble form of protein or polypeptide having tyrosine kinase which can be found in the intracellular portion of a cell. By the term "receptor tyrosine kinase" is intended a transmembrane protein having an extracellular receptor domain, and one or more intracellular domains, including at least one extracellular or intracellular domain having tyrosine kinase enzymatic activity. Additional intracellular domains may have sequence homology to SH2. These molecules are well known in the art (Williams, L. T. et al., Science 243:1564-1570 (1989); Ullrich, A. et al., Cell 61:203-212 (1990); Carpenter, G. et al., J. Biol. Chem. 265:7709-7712 (1990), which are entirely incorporated by reference).
The proteins which interact with, and which may be phosphorylated by, tyrosine kinases are referred to as "target" proteins for these kinases, as distinguished from the "ligands" for these receptors, which bind to the kinase.
According to the present invention, an expression cloning method is performed directly on a gene expression library, such as lambda gt11 or T7 expression library. In a preferred embodiment, the DNA is human cDNA. More preferably, the DNA is human fetal brain DNA. Using such a source as the starting material for the cloning of human genes has a great advantage over the alternative known means, in which a large amount of tissue is taken, and antibodies produced, or the protein purified and partially sequenced, and oligonucleotide probes are then prepared from this sequence and used to screen a genomic DNA or cDNA library. The advantage of bypassing these steps is of most relevance in the case of human genes, since tissue is generally not available in large quantities, with the exception of placenta.
The expression library may be screened in a single step. Preferably, the lambda plaques are blotted onto a solid carrier, preferably nitrocellulose, allowing the transfer of library DNA-encoded proteins which are expressed in the infected bacteria and transferred to the carrier. This carrier is then incubated with the probe of the present invention, as described herein. The probe is allowed to bind to proteins which have the capability of binding to the tyrosine-phosphorylated polypeptide. Based on the label used in the probe, such as an enzymatic, radioisotope or fluorescent label, an appropriate detection system is used to identify the plaques containing the protein of interest. The phage in these plaques are then selected, and the DNA inserts can then be re-cloned, excised and placed into other vectors, used for large scale expression of the protein, and the like, according to known method steps.
One of ordinary skill in the art will appreciate that the concentrations, times, temperatures can be varied depending on the precise nature of the system used, and will know how to vary the appropriate parameters without undue experimentation. Furthermore, general methods in this area are set forth in Sambrook et al. (supra).
Materials of which a solid phase carrier can be made include, but are not limited to, nitrocellulose, cellulose, paper, substituted polystyrenes, acrylonitriles, polycarbonate, polypetene, or silicone oxide.
The probe of the present invention is a tyrosine-phosphorylated polypeptide molecule derived from the C-terminal domain of a cytoplasmic or receptor tyrosine kinase. The polypeptide can have between about 10 and about 250 amino acids in length. The probe can be a phosphorylated native sequence or a functional derivative thereof (defined below).
Highly efficient phosphorylation is obtained by using the tyrosine kinase domain present on the tyrosine kinase molecule to autophosphorylate the C-terminal region at between 1 and 5 tyrosine residues. Known methods and conditions (described in detail in Example I) are used to phosphorylate the tyrosine residues. A preferred substrate is detectably labeled substrate such as (.gamma.-P.sup.32 -adenosine triphosphate). The source of tyrosine molecule used as the source material to make the probe can include molecules chemically purified from tissues or cells, or molecules produced by recombinant DNA methods.
When using recombinant techniques, a native cytoplasmic or receptor tyrosine kinase may be produced, or alternatively, a tyrosine kinase derivative may be produced. A preferred tyrosine kinase derivative includes the tyrosine kinase domain linked to the C-terminal domain. In another embodiment, the two domains may be produced as separate molecules, and mixed together to achieve tyrosine phosphorylation of the C-terminus-derived polypeptide.
The probe comprising a tyrosine-phosphorylated C-terminal portion of the tyrosine kinase, as described herein can be produced by recombinant means in the form of a fusion protein.
As used herein, a "fusion protein" may refer to a fused protein comprising a bacterial protein and a polypeptide of interest such as a protein having an SH2 domain. Alternatively, a fusion protein may also be an artificially constructed tyrosine kinase-like derivative, wherein a DNA sequence encoding the tyrosine kinase domain has been linked to a selective enzymatic cleavage site, which, in turn, is linked to a tyrosine kinase C-terminal domain having one or more tyrosine residues which can be phosphorylated by the kinase. Such a genetic construct encoding this type of "fusion protein" can be inserted into an expression vehicle and expressed in a bacterial or eukaryotic host. Once expressed, such a fusion protein can be allowed to autophosphorylate, wherein the kinase acts to phosphorylate the tyrosine residues in the C-terminal domain.degree. Following this phosphorylation, use of the appropriate enzyme will cleave at the selective cleavage site, thus separating the N-terminal kinase from the C-terminal phosphorylated polypeptide, which can now serve as a probe.
Expression of fusion proteins and modifications to increase yields and to provide cleavage sites, etc., are well known. See, e.g., Ausubel, supra; Itakura et al. Science 198:1056-1063 (1977)) and Riggs (U.S. Pat. No. 4,366,246 (1982); Marston, Biochem. J. 240:1-12 (1986); Nagai et al., (Nature 309:810-812 (1984); (Germino et al., Proc. Natl. Acad. Sci. USA 81:692-4696 (1984); Scholtissek et al., Gene 62:55-64 (1988); Smith et al., Gene 67:31-40 (1988); Knott et al., Eur. J. Biochem, 174:405-410 (1988); and Dykes et al., Eur. J, Biochem. 174:411-416 (1988), which references are all entirely incorporated herein by reference.
The term "selective cleavage site" refers to an amino acid residue or residues which can be selectively cleaved with either chemicals or enzymes and where cleavage can be achieved in a predictable manner. A selective enzymatic cleavage site is an amino acid or a peptide sequence which is recognized and hydrolyzed by a proteolytic enzyme. Examples of such sites include trypsin or chymotrypsin cleavage sites. In a preferred embodiment of this invention, the selective cleavage site is comprised of the sequence Ile-Glu-Gly-Arg (SEQ ID NO: 15), which is recognized and cleaved by blood coagulation factor Xa. In another embodiment, the selective cleavage site has the sequence Leu-Val-Pro-Arg (SEQ ID NO:16), which is recognized and cleaved by thrombin.
In constructing the tyrosine kinase-like derivative, an oligonucleotide sequence, 5' to the sequence coding for the enzyme recognition site can be included, and may vary in length. For example, in one embodiment, 13 nucleotides are situated between the codon for Ile (the start of the factor Xa recognition site) and the 3' end of the sequence encoding the tyrosine kinase domain.
Thus, in one embodiment of the present invention, the Ile-Glu-Gly-Arg (SEQ ID NO:15) sequence is introduced between the tyrosine kinase domain and the C-terminal domain. In another embodiment, the Leu-Val-Pro-Arg (SEQ ID NO:16) sequence is introduced. The proteins having this cleavage site are expressed in bacteria using standard methods. Thereafter, autophosphorylation of the C-terminal domain, preferably with (.gamma.32P) adenosine triphosphate, is allowed to occur, followed by selective cleavage of the tyrosine-phosphorylated C-terminal domain with the appropriate cleaving agent, e.g., factor Xa.
The present invention also provides a method for mapping a gene, preferably a human gene, which encodes a target protein for a tyrosine kinase (such as a GRB protein as defined herein), to a particular human chromosome. This method combines the new expression cloning method described herein with one of several known techniques for mapping a gene to a particular chromosome. Thus, according to the present invention, a clone, such as a lambda gt11 clone, containing a DNA insert encoding a GRB protein, is identified using the expression cloning methods disclosed herein. The insert may be further subcloned, if desired, using methods well-known in the art, and a probe constructed, either by direct labeling of the nucleic acid of the clone or by producing an oligonucleotide probe corresponding to a unique portion of the clone's sequence (see: Sambrook, J. et al. (Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989); and Ausubel, supra). This labeled probe is then used in a hybridization assay with commercially available blots, such Chromosome Blots from Bios Corporation (New Haven, Connecticut) which contain DNA from a panel of human-hamster somatic cell hybrids (Kouri, R. E. et al., Cytogenet. Cell Genet. 51:1025 (1989)). By comparison of which human chromosomes remain in the human-hamster hybrid cell and the hybridization of the probe specific for the GRB gene of interest, the gene is mapped to a particular human chromosome. In this way, linkage is established to known human genes (or diseases caused by mutations therein) present on this chromosome. Using methods well-known in the art for finer mapping, e.g., using known human deletion mutations, the GRB gene can be mapped more precisely to other human genes.
The tyrosine-phosphorylated tyrosine kinase C-terminal probe polypeptide of the present invention, as well as the GRB proteins of the present invention, and additional yet unknown GRB proteins which are discovered using the methods of this invention, are useful in methods for screening drugs and other agents which are capable of modulating cell growth control that occurs via signal transduction through tyrosine kinases. By attaching a tyrosine-phosphorylated probe polypeptide or a GRB protein, or fragments thereof, to a solid phase carrier matrix, an affinity probe is created which can be used to isolate and purify molecules from complex mixtures which are capable of binding to the affinity probe. Furthermore, such an affinity probe is useful for detecting the presence in a biological fluid of a molecule capable of binding the tyrosine-phosphorylated probe or the GRB protein. Similarly, chemical agents can be tested for their capacity to interact with the probe or GRB.
Methods for coupling proteins and peptides to the solid phase, the solid phase substances useful in these methods, and means for elution, are well known to those of skill in the art.
In the case of growth factor receptors which are receptor tyrosine kinases (including as non-limiting examples EDGFR, PDGFR and FGFR), tyrosine phosphorylation is linked to cell growth and to oncogenic transformation. Disruption of the action of a GRB in the cell may prevent or inhibit growth, and might serve as means to counteract development of a tumor. Furthermore, a mutation in the C-terminal portion of the tyrosine kinase or the GRB, or a disregulation in their mutual interactions, may promote susceptibility to cancer.
The insulin receptor (InsR) is also a receptor tyrosine kinase, and tyrosine phosphorylation in cells bearing InsR is associated with normal physiological function. In contrast to the case of cell growth and cancer, disruption of normal interactions between of the tyrosine-phosphorylated portion of the receptor and the GRB would counteract insulin effects. Subnormal levels or activity of a GRB protein may act to remove a normal counterregulatory mechanisms. It is expected that overexpression or overactivity of a GRB protein could inhibit or totally prevent the action of insulin on cells, leading to diabetes (of an insulin-resistant variety). Thus susceptibility to diabetes may be associated with GRB protein dysregulation.
Therefore methods of the present invention for identifying normal or mutant GRB protein genes, or for detecting the presence or the amount of GRB protein in a cell, can serve as methods for identifying susceptibility to cancer, diabetes, or other diseases associated with alterations in cellular metabolism mediated by tyrosine kinase pathways.
The present invention provides methods for evaluating the presence, and the level of normal or mutant GRB protein in a subject. Altered expression of these proteins, or presence of a mutant GRB protein, in an individual may serve as an important predictor of susceptibility to oncogenic transformation and the development of cancer. Alternatively, altered expression of GRB protein may serve as an important predictor of susceptibility to diabetes.
Oligonucleotide probes encoding various portions of the GRB protein are used to test cells from a subject for the presence DNA or RNA sequences encoding the GRB protein. A preferred probe would be one directed to the nucleic acid sequence encoding at least 4 amino acid residues, and preferably at least 5 amino acid residues of the GRB-1, GRB-2, GRB-3, GRB-4 or GRB-7, protein of the present invention, such as 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50 amino acids. Qualitative or quantitative assays can be performed using such probes. For example, Northern analysis (see Example III, below) is used to measure expression of an GRB protein mRNA in a cell or tissue preparation.
Such methods can be used even with very small amounts of DNA obtained from an individual, following use of selective amplification techniques. Recombinant DNA methodologies capable of amplifying purified nucleic acid fragments have long been recognized. Typically, such methodologies involve the introduction of the nucleic acid fragment into a DNA or RNA vector, the clonal amplification of the vector, and the recovery of the amplified nucleic acid fragment. Examples of such methodologies are provided by Cohen et al. (U.S. Pat. No. 4,237,224), Sambrook et al. (supra). Ausubel et al, supra, etc.
Recently, an in vitro, enzymatic method has been described which is capable of increasing the concentration of such desired nucleic acid molecules. This method has been referred to as the "polymerase chain reaction" or "PCR" (Mullis, K. et al., Cold Spring Harbor Symp. Quant. Biol. 51:263-273 (1986); Erlich H. et al., EP 50,424; EP 84,796, EP 258,017, EP 237,362; Mullis, K., EP 201,184; Mullis K. et al., U.S. Pat. No. 4,683,202; Erlich, H., U.S. Pat. No. 4,582,788; and Saiki, R. et al., U.S. Pat. No. 4,683,194; Mullis, K. B. (Cold Spring Harbor Symp. Quant. Biol. 51:263-273 (1986)); Saiki, R. K., et al, (Bio/Technology 3:1008-1012 (1985)); and Mullis, K. B., et al., (Meth. Enzymol. 55:335-350 (1987), which references are entirely incorporated herein by reference).
In one embodiment, the invention is directed to target proteins of eukaryotic tyrosine kinases, which include, as non-limiting examples, GRB proteins such as GRB-1, GRB-2, GRB-3, GRB-4 or GRB-7 proteins are included. In another embodiment, the invention is directed to recombinant eukaryotic GRB proteins. The invention provides the naturally occurring protein molecule substantially free of other proteins with which it is natively associated. "Substantially free of other proteins or glycoproteins" indicates that the protein has been purified away from at least 90 per cent (on a weight basis), and from even at least 99 per cent if desired, of other proteins and glycoproteins with which it is natively associated, and is therefore substantially free of them. That can be achieved by subjecting the cells, tissue or fluids containing the GRB-1, GRB-2, GRB-3, GRB-4 or GRB-7 protein to standard protein purification techniques such as immunoadsorbent columns bearing monoclonal antibodies reactive against the protein.
The nucleotide sequence of the GRB-1 gene (SEQ ID NO:1), and the amino acid sequence of the GRB-1 protein (SEQ ID NO:5), are shown in FIG. 4A-4I (SEQ ID NO:5). The partial nucleotide sequence of GRB-2 (1-949 of SEQ ID NO:2) and the partial amino acid sequence, are shown in FIG. 16A-16C, and the complete amino acid sequence is shown in FIG. 26A-26C (SEQ ID NO:6), as well as the complete nucleotide sequence.
In a preferred embodiment, GRB-1, GRB-2, GRB-3, GRB-4 or GRB-7or other eukaryotic GRB proteins can be isolated and purified using as an affinity probe, the probe of the present invention which is a tyrosine-phosphorylated C-terminal domain of a tyrosine kinase, or a functional derivative thereof.
Alternatively, the purification can be achieved by a combination of standard methods, such as ammonium sulfate precipitations molecular sieve chromatography, and ion exchange chromatography.
It will be understood that the GRB-1 proteins of the present invention can be biochemically purified from a variety of cell or tissue sources. For preparation of naturally occurring GRB protein, tissues such as mammalian placenta or brain are preferred.
Alternatively, because the gene for GRB1, GRB-2, GRB-3, GRB-4 or GRB-7 can be isolated or synthesized, the polypeptide can be synthesized substantially free of other proteins or glycoproteins of mammalian origin in a prokaryotic organism or in a non-mammalian eukaryotic organism, if desired. As intended by the present invention, a recombinant GRB-1, GRB-2, GRB-3, GRB-4 or GRB-7 molecule produced in mammalian cells, such as transfected COS, NIH-3T3, or CHO cells, for example, is either a naturally occurring protein sequence or a functional derivative thereof. Where a naturally occurring protein or glycoprotein is produced by recombinant means, it is provided substantially free of the other proteins and glycoproteins with which it is natively associated.
Alternatively, methods are well known for the synthesis of polypeptides of desired sequence on solid phase supports and their subsequent separation from the support or carrier. In particular, the tyrosine-phosphorylated C-terminal domain probe of the present invention, or a functional derivative thereof, can be synthesized using a peptide synthesis method wherein phosphotyrosine is provided in place of tyrosine, resulting in direct synthesis of the phosphorylated form of the polypeptide. See, e.g., Staerkaer et al., Tetrahedron Letters 32:5289-5392 (1991); Shoelson et al Tetrahedron Letters 32:6061 (1991), which references are entirely incorporated herein by reference).
The present invention also provides "functional derivatives" of the tyrosine-phosphorylated C-terminal domain polypeptide and or the GRB-1, GRB-2, GRB-3, GRB-4 or GRB-7 proteins.
By "functional derivative" is meant a "fragment," "variant," "analog," or "chemical derivative" of the GRB protein, which terms are defined below. A functional derivative retains at least a portion of the function of the native protein which permits its utility in accordance with the present invention.
A "fragment" of any of the proteins or polypeptides of the present invention refers to any subset of the molecule, that is, a shorter peptide.
A "variant" of the protein refers to a molecule substantially similar to either the entire peptide or a fragment thereof. Variant peptides may be conveniently prepared by direct chemical synthesis of the variant peptide, using methods well known in the art.
The term "substantially corresponding to the amino acid sequence of" in the context of the present refers to a protein containing conservative amino acid substitutions, known in the art and as described herein, that would be expected to maintain the functional biological activity of the referenced sequence, and/or target protein binding characteristics.
Such substitutions can be readily determined without undue experimentation by using known conservative substitutions, as known in the art. Alternatively, known software can be used to provide such conservative substitutions according to the present invention. As a non-limiting example the program "BESTFIT" can be used to provide conservative amino acid substitutions of a define sequence, e.g., defined as having a score of .gtoreq.0.4, 0.6, 0.8 or 1.0 depending on the type of protein used. See e.g., Gribskov and Burgess, Nucl. Acid. Res. 14:6745 (1984), which is entirely incorporated by reference. Variant peptides may be conveniently prepared by direct chemical synthesis of the variant peptide using methods well-known in the art.
Alternatively, amino acid sequence variants of the peptide can be prepared by mutations in the DNA which encodes the synthesized peptide. Such variants include, for example, deletions from, or insertions or substitutions of, residues within the amino acid sequence. Any combination of deletion, insertion, and substitution may also be made to arrive at the final construct, provided that the final construct possesses the desired activity. Mutations that will be made in the DNA encoding the variant peptide must not alter the reading frame and preferably will not create complementary regions that could produce secondary mRNA structure (see European Patent Publication No. EP 75,444).
At the genetic level, these variants ordinarily are prepared by site-directed mutagenesis (as exemplified by Adelman et al., DNA 2:183 (1983)) of nucleotides in the DNA encoding the peptide molecule, thereby producing DNA encoding the variant, and thereafter expressing the DNA in recombinant cell culture (see below). The variants typically exhibit the same qualitative biological activity as the nonvariant peptide.
Amino acid substitutions in the context of the present invention include substitutions wherein at least one amino acid residue in the peptide molecule, and preferably, only one, has been removed and a different residue inserted in its place. For a detailed description of protein chemistry and structure, see Schulz, G. E. et al., Principles of Protein Structure, Springer-Verlag, New York, 1978, and Creighton, T. E., Proteins: Structure and Molecule Properties, W. H. Freeman & Co., San Francisco, 1983, which are hereby incorporated by reference. The types of substitutions which may by made in the protein or peptide molecule of the present invention may be based on analysis of the frequencies of amino acid changes between a homologous protein of different species, such as those presented in Table 1-2 of Schultz et al. (supra) and FIG. 3-9 of Creighton (supra). Base on such an analysis, conservative substitutions are defined herein as exchanges within one of the following five groups:
1. Small aliphatic, nonpolar or slightly polar residues: ala, ser, thr (pro, gly);
2. Polar, negatively charged residues and their amides: asp, asn, glu, gly;
3. Polar, positively charged residues: his, arg, lys;
4. Large aliphatic, nonpolar residues: Met, leu, ile, val (cys); and
5. Large aromatic residues: phe, tyr, trp.
Accordingly, amino acid sequences substantially corresponding to a given sequence can be made without undue experimentation and then routinely screened for tyrosine kinase binding activity using known methods or those disclosed herein, such that one of ordinary skill in the art can determine which substitutions provide tyrosine kinase target proteins according to the present invention. For example, once target protein sequences are determined, such as for GRB-1, GRB-2, GRB-3, GRB-4 or GRB-7, conservative amino acid substitutions can be made to provide target proteins having amino acid sequences which substantially correspond to the determined target protein sequences.
The preferred bacterial host for this invention is E. coli. In other embodiments, other bacterial species can be used. In yet other embodiments, eukaryotic cells may be utilized, such as, for example, yeast, filamentous fungi, or the like. Use of these cell types are well known in the art. Any host may be used to express the protein which is compatible with replicon and control sequences in the expression plasmid. In general, vectors containing replicon and control sequences are derived from species compatible with a host cell are used in connection with the host. The vector ordinarily carries a replicon site, as well as specific genes which are capable of providing phenotypic selection in infected or in transformed cells. The expression of the fusion protein can also be placed under control with other regulatory sequences which may be homologous to the organism in its untransformed state. Preferred promoters can include a T7 promoter. Such preferred promoters express the human gene as a fusion protein such as the T7 capsid protein P10 under control of the T7 promoter. Such expression systems are commercially available, e.g, as the .lambda.EXlox vector from Novagen, Inc. (Madison, Wis.). In such fusion protein expression systems, the recombinant T7 vector containing a human gene, encoding such proteins obtainable by methods of the present invention, such as GRB-1, GRB-2, GRB-3, GRB-4 and GRB-7, as, e.g., a T10 fusion protein. The recombinant T7 vector can then be used to transform a bacteria, such as E. coli, by infection with a phage containing the recombinant T7 vector under lac control, such lacUV5 control. Induction of the infected, successfully transformed bacteria or other suitable host cell, by IPTG generates the T7 polymerase which then initiates transcription of the fusion protein encoded by the phage library. Because such resulting T7 vector infected bacteria provide human gene library plaques that have stronger signals than obtained by the use of bacterial RNA polymerases, such as E. coli RNA polymerase. According to the present invention, the use of a T7 polymerase expression system is particularly suitable for library screening when there as thousands of small plaques per plate. The major advantage of the use of a T7 expression system is the high level of protein expression due to the greater activity of the T7 polymerase versus E. coli RNA polymerase, and because fusion proteins using the smaller phage fusion protein genes such as the T10 gene fragment (26 kd versus the 110 kd B-galactosidase of .lambda.gt11 expression library) yields more stable expression and that its hydrophobic character promotes binding to nitrocellulose. In addition to directional cloning, the use of T7 phages also allow for automatic conversion to a PET plasmid (see, e.g., Palazzalo et al., Gene 88, 25 (1990)) which can be useful for expression of a fusion protein for antibody production.
This invention is also directed to an antibody specific for an epitope of the GRB-1, GRB-2, GRB-3, GRB-4 or GRB-7 protein and the use of such an antibody to detect the presence of, or measure the quantity or concentration of, the GRB protein in a cells a cell or tissue extract, or a biological fluid.
The term "antibody" is meant to include polyclonal antibodies, monoclonal antibodies (mAbs), chimeric antibodies, and anti-idiotypic (anti-Id) antibodies.
Polyclonal antibodies are heterogeneous populations of antibody molecules derived from the sera of animals immunized with an antigen.
Monoclonal antibodies are a substantially homogeneous population of antibodies to specific antigens. MAbs may be obtained by methods known to those skilled in the art. See, for example Kohler and Milstein, Nature 256:495-497 (1975) and U.S. Pat. No. 4,376,110. Such antibodies may be of any immunoglobulin class including IgG, IgM, IgE, IgA, GILD and any subclass thereof. The hybridoma producing the mAbs of this invention may be cultivated in vitro or in vivo. Production of high titers of mAbs in vivo production makes this the presently preferred method of production. Briefly, cells from the individual hybridomas are injected intraperitoneally into pristane-primed BALB/c mice to produce ascites fluid containing high concentrations of the desired mAbs. MAbs of isotype IgM or IgG may be purified from such ascites fluids, or from culture supernatants, using column chromatography methods well known to those of skill in the art.
Chimeric antibodies are molecules different portions of which are derived from different animal species , such as those having variable region derived from a murine mAb and a human immunoglobulin constant region. Chimeric antibodies and methods for their production are known in the art (Cabilly et al., Proc. Natl. Acad. Sci. USA 81:3273-3277 (1984); Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984); Boulianne et al., Nature 312:643-646 (1984); Cabilly et al., European Patent Application 125023 (published Nov. 14, 1984); Neuberger et al., Nature 314:268-270 (1985); Taniguchi et al., European Patent Application 171496 (published Feb. 19, 1985); Morrison et al., European Patent Application 173494 (published Mar. 5, 1986); Neuberger et al., PCT Application WO 86/01533, (published Mar. 13, 1986); Kudo et al., European Patent Application 184187 (published Jun. 11, 1986); Morrison et al., European Patent Application 173494 (published Mar. 5, 1986); Sahagan et al., J. Immunol. 137:1066-1074 (1986); Robinson et al., International Patent Publication #PCT/US86/02269 (published 7 May 1987); Liu et al., Proc. Natl. Acad. Sci. USA 84:3439-3443 (1987); Sun et al., Proc. Natl. Acad. Sci. USA 84:214- 218 (1987); Better et al., Science 240:1041-1043 (1988); and Harlow and Lane ANTIBODIES: A LABORATORY MANUAL Cold Spring Harbor Laboratory (1988)). These references are hereby entirely incorporated by reference.
An anti-idiotypic (anti-Id) antibody is an antibody which recognizes unique determinants generally associated with the antigen-binding site of an antibody. An Id antibody can be prepared by immunizing an animal of the same species and genetic type (e.g., mouse strain) as the source of the mAb with the mAb to which an anti-Id is being prepared. The immunized animal will recognize and respond to the idiotypic determinants of the immunizing antibody by producing an antibody to these idiotypic determinants (the anti-Id antibody).
The anti-Id antibody may also be used as an "immunogen" to induce an immune response in yet another animal, producing a so-called anti-anti-Id antibody. The anti-anti-Id may be epitopically identical to the original mAb which induced the anti-Id. Thus, by using antibodies to the idiotypic determinants of a mAb, it is possible to identify other clones expressing antibodies of identical specificity.
Accordingly, mAbs generated against the GRB protein of the present invention may be used to induce anti-Id antibodies in suitable animals, such as BALB/c mice. Spleen cells from such immunized mice are used to produce anti-Id hybridomas secreting anti-Id mAbs. Further, the anti-Id mAbs can be coupled to a carrier such as keyhole limpet hemocyanin (KLH) and used to immunize additional BALB/c mice. Sera from these mice will contain anti-anti-Id antibodies that have the binding properties of the original mAb specific for a GRB protein epitope.
The anti-Id mAbs thus have their own idiotypic epitopes, or "idiotopes" structurally similar to the epitope being evaluated, such as GRB protein-.alpha..
The term "antibody" is also meant to include both intact molecules as well as fragments thereof, such as, for example, Fab and F(ab').sub.2, which are capable of binding antigen. Fab and F(ab').sub.2 fragments lack the Fc fragment of intact antibody, clear more rapidly from the circulation, and may have less non-specific tissue binding than an intact antibody (Wahl et al., J. Nucl. Med. 24:316-325 (1983)).
It will be appreciated that Fab and F(ab').sub.2 and other fragments of the antibodies useful in the present invention may be used for the detection and quantitation of GRB protein according to the methods disclosed herein for intact antibody molecules. Such fragments are typically produced by proteolytic cleavage, using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab').sub.2 fragments).
An antibody is said to be "capable of binding" a molecule if it is capable of specifically reacting with the molecule to thereby bind the molecule to the antibody. The term "epitope" is meant to refer to that portion of any molecule capable of being bound by an antibody which can also be recognized by that antibody. Epitopes or "antigenic determinants" usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and have specific three dimensional structural characteristics as well as specific charge characteristics.
An "antigen" is a molecule or a portion of a molecule capable of being bound by an antibody which is additionally capable of inducing an animal to produce antibody capable of binding to an epitope of that antigen. An antigen may have one, or more than one epitope. The specific reaction referred to above is meant to indicate that the antigen will react, in a highly selective manner, with its corresponding antibody and not with the multitude of other antibodies which may be evoked by other antigens.
The antibodies, or fragments of antibodies, useful in the present invention may be used to quantitatively or qualitatively detect the presence of cells which express the GRB protein. This can be accomplished by immunofluorescence techniques employing a fluorescently labeled antibody (see below) coupled with light microscopic, flow cytometric, or fluorometric detection.
The antibodies (of fragments thereof) useful in the present invention may be employed histologically, as in immunofluorescence or immunoelectron microscopy, for in situ detection of GRB proteins. In situ detection may be accomplished by removing a histological specimen form a patient, and providing the a labeled antibody of the present invention to such a specimen. The antibody (or fragment) is preferably provided by applying or by overlaying the labeled antibody (or fragment) to a biological sample. Through the use of such a procedure, it is possible to determine not only the presence of the GRB protein but also its distribution on the examined tissue. Using the present invention, those of ordinary skill will readily perceive that any of wide variety of histological methods (such as staining procedures) can be modified in order to achieve such in situ detection.
Such assays for GRB protein typically comprises incubating a biological sample, such as a biological fluid, a tissue extract, freshly harvested cells such as lymphocytes or leukocytes, or cells which have been incubated in tissue culture, in the presence of a detectably labeled antibody capable of identifying GRB protein, and detecting the antibody by any of a number of techniques well-known in the art,
The biological sample may be treated with a solid phase support or carrier such as nitrocellulose, or other solid support or carrier which is capable of immobilizing cells, cell particles or soluble proteins. The support or carrier may then be washed with suitable buffers followed by treatment with the detectably labeled GRB protein-specific antibody. The solid phase support or carrier may then be washed with the buffer a second time to remove unbound antibody. The amount of bound label on said solid support or carrier may then be detected by conventional means.
By "solid phase support", "solid phase carrier", "solid support", "solid carrier", "support" or "carrier" is intended any support or carrier capable of binding antigen or antibodies, Well-known supports or carriers, include glass, polystyrene, polypropylene, polyethylene, dextran, nylon amylases, natural and modified celluloses, polyacrylamides, gabbros, and magnetite. The nature of the carrier can be either soluble to some extent or insoluble for the purposes of the present invention. The support material may have virtually any possible structural configuration so long as the coupled molecule is capable of binding to an antigen or antibody. Thus, the support or carrier configuration may be spherical, as in a bead, or cylindrical, as in the inside surface of a test tube, or the external surface of a rod. Alternatively, the surface may be flat such as a sheet, test strip, etc. Preferred supports or carriers include polystyrene beads. Those skilled in the art will know many other suitable carriers for binding antibody or antigen, or will be able to ascertain the same by use of routine experimentation.
The binding activity of a given lot of anti-GRB-1, anti-GRB-2, anti-GRB-3, Anti-GRB-4 or anti-GRB-7, antibody may be determined according to well known methods. Those skilled in the art will be able to determine operative and optimal assay conditions for each determination by employing routine experimentation.
Other such steps as washing, stirring, shaking, filtering and the like may be added to the assays as is customary or necessary for the particular situation.
One of the ways in which a GRB-specific antibody can be detectably labeled is by linking the same to an enzyme and use in an enzyme immunoassay (EIA). This enzyme, in turns when later exposed to an appropriate substrate, will react with the substrate in such a manner as to produce a chemical moiety which can be detected, for examples by spectrophotometric, fluorometric or by visual means. Enzymes which can be used detectably label the antibody include, but are not limited to, malate dehydrogenase, staphylococcal nuclease, delta-5-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase and acetylcholinesterase. The detection can be accomplished by colorimetric methods which employ a chromoenic substrate for the enzyme. Detection may also be accomplished by visual comparison of the extent of enzymatic reaction of a substrate in comparison with similarly prepared standards.
Detection may be accomplished using any of a variety of other immunoassays. For example, by radioactivity labeling the antibodies or antibody fragments, it is possible to detect R-PTPase through the use of a radioimmunoassay (RIA). A good description of RIA maybe found in Laboratory Techniques and Bio chemistry in Molecular Biology, by Work, T. S. et al., North Holland Publishing Company, N.Y. (1978) with particular reference to the chapter entitled "An Introduction to Radioimmune Assay and Related Techniques" by Chard, T., incorporated by reference herein. The radioactive isotope can be detected by such means as the use of a .gamma. counter or a scintillation counter or by autoradiography.
It is also possible to label the antibody with a fluorescent compound. When the fluorescently labeled antibody is exposed to light of the proper wave length, its presence can be then be detected due to fluorescence. Among the most commonly used fluorescent labelling compounds are fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine.
The antibody can also be detectably labeled using fluorescence emitting metals such as .sup.152 EU, or others of the lanthanide series. These metals can be attached to the antibody using such metal chelating groups as diethylenetriamine pentaacetic acid (EDTA).
The antibody also can be detectably labeled by coupling it to a chemiluminescent compound. The presence of the chemiluminescent-tagged antibody is then determined by detecting the presence of luminescence that arises during the course of a chemical reaction. Examples of particularly useful chemiluminescent labeling compounds are luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester.
Likewise, a bioluminescent compound may be used to label the antibody of the present invention. Bioluminescence is a type of chemiluminescence found in biological systems in which a catalytic protein increases the efficiency of the chemiluminescent reaction. The presence of a bioluminescent protein is determined by detecting the presence of luminescence. Important bioluminescent compounds for purposes of labeling are luciferin, luciferase and aequorin.
The antibody molecules of the present invention may be adapted for utilization in a immunometric assay, also known as a "two-site" or "sandwich" assay. In a typical immunometric assay, a quantity of unlabeled antibody (or fragment of antibody) is bound to a solid support or carrier and a quantity of detectably labeled soluble antibody is added to permit detection and/or quantitation of the ternary complex formed between solid-phase antibody, antigen, and labeled antibody.
Typical, and preferred, immunometric assays include "forward" assays in which the antibody bound to the solid phase is first contacted with the sample being tested to extract the antigen form the sample by formation of a binary solid phase antibody-antigen complex. After a suitable incubation period, the solid support or carrier is washed to remove the residue of the fluid sampler including unreacted antigen, if any, and then contacted with the solution containing an unknown quantity of labeled antibody (which functions as a "reporter molecule"). After a second incubation period to permit the labeled antibody to complex with the antigen bound to the solid support or carrier through the unlabeled antibody, the solid support or carrier is washed a second time to remove the unreacted labeled antibody.
In another type of "sandwich" assay, which my also be useful with the antigens of the present invention, the so-called "simultaneous" and "reverse" assays are used. A "simultaneous" and "reverse" assays are used. A simultaneous assay involves a single incubation step as the antibody bound to the solid support or carrier and labeled antibody are both added to the sample being tested at the same time. After the incubation is completed, the solid support or carrier is washed to remove the residue of fluid sample and uncomplexed labeled antibody. The presence of labeled antibody associated with the solid support or carrier is then determined as it would be in a conventional "forward" sandwich assay.
In the "reverse" assay, stepwise addition first of a solution of labeled antibody to the fluid sample followed by the addition of unlabeled antibody bound to a solid support or carrier after a suitable incubation period is utilized. After a second incubation, the solid phase is washed in conventional fashion to free it of the residue of the sample being tested and the solution of unreacted labeled antibody. The determination of labeled antibody associated with a solid support or carrier is then determined as in the "simultaneous" and "forward" assays.
The following example are presented by way of further explanation of the present invention, and not by way of limitation.
EXAMPLE I
A study was performed to determine the detectability of binding of the C-terminal domain of EGFR to a protein containing the SH2 domain immobilized on nitrocellulose filters. For this purpose, the binding of the C-terminal domain to a bacterially expressed fusion protein was assessed (see FIG. 1).
A. Isolation and Labelling of the Carboxyterminal Domain of the EGFR
The intracellular portion of the EGFR, which includes the tyrosine kinase domain and the carboxy terminal domain, was purified from recombinant baculovirus which expressed cDNA complementary to the intracellular domain of the human EGFR, as described previously (Hsu, C-Y. et al., Cell Growth and Differentiation 1:191-200 (1990)). The recombinant protein (2 .mu.g) was then phosphorylated with (.gamma.-.sup.32 P)ATP (200 .mu.Ci, 6000 Ci/Mmol), at 4.degree. C. in HNTG (20 mM HEPES, pH 7.5, 150mM NaCl, 0.1% Triton X-100, and 10% glycerol) buffer which contained 5mM MnCl.sub.2. In order to remove unincorporated (y-32P) ATP, the phosphorylated kinase was diluted to 1 ml with 20 mM HEPES, pH 7.5, containing 100 .mu.g BSA and then concentrated in a Centricon-10 to a volume of 50 .mu.l. This procedure was repeated 3 times resulting in the removal of >99% of the unincorporated ATP. To separate the C-terminal domain from the kinase domain, the concentrated protein was then digested with cyanogen bromide (CNBr) in 70% formic acid for 14 hours at room temperature (see also Example VI, below). Samples were then washed three times with water, dried and resuspended in binding buffer to a concentration of 2.times.10.sup.6 cpm/ml.
B. Binding of the C-terminal Domain of the EGFR to Bacterially Expressed TrpE/GAP-SH2 Fusion Protein Immobilized on Nitrocellulose
TrpE and TrpE/GAP-SH2 were obtained from the laboratory of Dr. Tony Pawson and/or prepared as previously described (Moran, M. F. et al., Proc. Natl. Acad. Sci. USA 87:8622-8626 (1990)). Filter binding studies were performed according to published methods (Schneider, W. J. et al., Proc. Natl. Acad. Sci. 76:5577-5581 (1979); Daniel, T. O. et al., J. Biol. Chem. 258:4606-4611 (1983)) with minor modifications. Various concentrations of either bacterially expressed TrpE fusion protein or bacterial protein alone were spotted onto nitrocellulose filters. After blocking the filters for 1 hour at 4.degree. C. in PBS containing 5% Carnation dry milk, .sub.32 P-labelled C-terminal domain of the EGFR was added and incubation was continued overnight at 4.degree. C. After 24 hours, the nitrocellulose filters were washed 3 times at room temperature with PBS containing 0.2% Triton X-100, The filters were dried and exposed to Kodak XAR-5 film at -80.degree. C.
C. Results
The above method permitted detection of specific binding of the EGFR C-terminal domain to less than 5 ng of a bacterially expressed GAP-SH2 fusion protein. The binding was specific, since it required tyrosine phosphorylation of the probe and did not occur when irrelevant proteins were applied to nitrocellulose filters.
The demonstration that the EGFR C-terminal domain could bind specifically to an SH2-containing protein immobilized on nitrocellulose filters encouraged the present inventors to apply this approach to the screening of lambda gt11 expression libraries with the goal of identifying novel EGFR binding proteins.
EXAMPLE II
Screening of Expression Libraries and Isolation of a cDNA Clone Encoding a Novel SH2-Containing Protein
The tyrosine phosphorylated C-terminal tail of the EGFR was used as a probe to screen expression libraries from several different human tissues as described above. The approach to screening is outlined in FIG. 2. Numerous positive clones have been identified so far using this approach, of which two have been analyzed in detail.
A. Screening of cDNA Library
A lambda gt11, library, constructed from mRNA isolated from human brain stem, was obtained from M. Jaye. To screen the library, lambda gt11 phage were plated at a density sufficient to produce 4.times.10.sup.4 plaques per 150 mm agar plate. A total of six plates were initially screened. After incubation of the plates for 4 hours at 42.degree. C., the plates were overlaid with nitrocellulose filters which had been impregnated with isopropyl-B-D-thiogalactopyranoside (IPTG), as previously described (MacGregor, P. F. et al., Oncogene 5:451-458 (1990)). Incubation was continued overnight at 37.degree. C. The filters were then removed, washed with tBST (10 mM Tris-HCl, pH8, 150 mM NaCl, and 0.05% triton X-100) at room temperature, and then blocked in HBB (20 mM HEPES, pH 7.5, 5 mM Mg/Cl, 1 mM KCl) buffer containing 5% carnation dry milk for 1 hour at 4.degree. C., as described (MacGregor et al., supra). Following blocking, labelled tyrosine phosphorylated carboxy-terminus (C-terminus) probe was added at a concentration of 1.6.times.10-4 .mu.g/ml, and incubation was continued overnight. The filters were then washed 3 times at room temperature in PBS containing 0.2% Triton X-100. Filters were dried and exposed to Kodak XAR-5 film at -80.degree. C.
Agar plugs, corresponding to the positive clones, were collect from the plates and placed in 1 ml of SM media. After allowing the phages to diffuse from the agar, the phages were replated and rescreened as described above. Those phages that demonstrated enrichment on subsequent screening were isolated and sequence. Lambda gt11 phage DNA was isolated by the plate lysate method according to Maniatis et al., and subcloned into EcoRI-digested M13 MP19 (Maniatis et al., 1982). Single stranded DNA was isolated and sequenced by the dideoxy chain termination method using the Sequenase DNA sequencing kit (United States Biochemical).
In one experiment, 240,000 pfu from a human brainstem lambda gt11 library were screened. A single plaque, clone ki4 (FIG. 3A) was isolated. On subsequent screening this clone demonstrated enrichment, and on tertiary screening all plaques bound the probe (FIG. 3B). Clone ki4 contained an insert of about 900 nucleotides, which, upon induction of the Lac promoter with IPTG, produced a fusion protein which could bind the EGFR. The size of the fusion protein predicted that the cDNA insert coded for a protein of about 300 amino acids, which was the size expected if the cDNA contained a single large open reading frame. To analyze clone ki4 in more detail, DNA was isolated and the EcoRI fragment, corresponding to the human cDNA insert, was subcloned into M13 and sequenced. Translation of the sequence from this insert demonstrated a single large open reading frame which, upon analysis using the Genbank database, was found to contain a single stretch of about 100 amino acids with sequence homology to SH2 domains of other known proteins (FIGS. 4A-4I and 5, top). However, in other regions, no sequence homology was noted. Thus, using this screening approach, a new SH2-containing protein which could bind to the EGFR was identified,.
B. Isolated of Full Length cDNA
The initial clone isolated encoded for an SH2 domain, but did not contain the 3' or 5' ends of the gene. To isolated the full length cDNA, the library was rescreened using DNA isolated from the initial positive phage. DNA, from recombinant M13 bacteriophage which expressed the positive clone, was amplified using a thermal cycler, Taq1 polymerase and oligonucleotides complementary to the EcoR1 flanking regions of the M13 sequence in information, a second amplified DNA product, corresponding to the most 5' 250 nucleotides of the initial isolated phage, was also generated by using oligonucleotides complementary to sequences at both ends of this region. (.sup.32 P)-labelled DNA probes were then prepared by nick translation of the amplified products.
To rescreen the cDNA library, the library was replated as described above. After incubation of the plates for 8 hours at 37.degree. C., the plates were cooled for 1 hour at 4.degree. C. following which the phage DNA was transferred to nitrocellulose filters. The filters were denatured in a solution of 0.2 N NaOH and 1.5M NaCl and then baked in vacuo for 2 hours at 80.degree. C. (Sambrook, J. et al., (Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989)). After prehybridization of the filters for 1 hour at 42.degree. C., 32P-labelled DNA probe was added and hybridization was continued overnight at 42.degree. C. in a solution containing 5X Denhardt's, 50% formamide, 5X SSC, 0.1% SDS, 200 mM Tris-HCl, pH 7.6 and 100 .mu.g/ml salmon sperm DNA. The filters were then washed in a solution containing 0.1.times. SSC and 0.1% SDS, dried and exposed to Kodak XAR-5 film at -70.degree. C. Positive clones were then isolated and sequenced as described above.
Since the insert from clone ki4 lacked the 3' and 5' ends of the gene, the library was rescreened using two DNA probes which were generated by amplifying DNA from clone ki4. This approach enabled the identification of five additional clones. Three of the clones extended 3' from the initial clone ki4, two of which, clones, ki2.2 and ki2.4, contained a polyadenylation signal and a long 3' untranslated region (>1000 nucleotides). In addition, these clones encoded a protein which contained a second SH2 domain (FIGS. 4A-4I and 5, top).
The other two clones, ki3.0 and ki5.3, extended 5' from clone ki4. Both clones contained long open reading frames and an AUG codon which met the translation initiation criteria as defined by Kozak (Kozak, M. J. Cell. Biol. 108:229-241 (1989)). However, only clone ki3.0, when translated into protein and compared with known sequences in Genbank, was found to contain a domain of 50 amino acids which was homologous to SH3 domains present in other known proteins. The predicted molecular weight of the full length protein encoded by the overlapping clones, ki2.2 and ki3.0, was about 84 kDa. This new protein was termed GRB-1.
EXAMPLE III
GRB-1 Protein Contains SH2 and SH3 domains
Analysis of the GRB-1 protein sequence by comparison to sequences in the Genbank database revealed the presence of two stretches of about 100 amino acids, starting at amino acids 333 and 624, with sequence homology to SH2 domains of other proteins known to interact with the EGFR (FIG. 5, top). While GRB-1 displayed striking homology to other SH2 domains at the protein level, it revealed no significant homology at the DNA level. GRB-1 also contained a segment of about 50 amino acids, located in the N-terminal region, which had sequence homology to SH3 domains (FIG. 4A-4I and 5 bottom).
A comparison of the structural organization of GRB-1 with several other SH2/SH3 containing proteins is shown in FIG. 6. It is apparent from this scheme that the localization of the SH2 and SH3 domains vary from protein to protein. Despite this there are certain similarities and differences among these SH2 containing proteins. GRB-1 is similar to some other substrates which have been found to interact with the EGFR, such as PLC-.gamma. and GAP, in that GRB-1 contains two SH2 domains and a single SH3 domain. However, unlike these substrates, GRB-1 contains no homology to any known catalytic domain, and in this regard resembles the protein encoded by the arian sarcoma virus, v-crk.
Out side of these regions there was no sequence homology with other protein sequences present in Genbank. In particular, GRB-1 lacked a consensus ATP-binding domain, and did nod display sequence homology with any serine/threonine kinase or tyrosine kinase.
The SH2 domain is thought to provide a common motif by which enzymatically distinct signalling molecules can be coupled to activated receptors with tyrosine kinase activity (Moran, M. F. et al., Proc. Natl. Acad. Sci. USA 87:8622-8626 (1990); Anderson, D. et al., Science 250:979-982 (1990)).
The presence of SH2 domains in GRB-1 (FIG. 4A-4I) and in GRB-2 further reinforces the importance of this domain in mediating the interaction of these proteins with the C-terminal tail of the EGFR. Moreover, since many proteins capable of interacting with cytoplasmic or receptor tyrosine kinases remain to be identified, this suggests that additional members of this protein family remain to be discovered.
In addition to containing two SH2 domains, GRB1 also contains an SH3 domain. The SH3 domain is a non-catalytic domain of about 50 amino acid residues which is shared among many SH2-containing proteins. Since SH3 domains are also found in cytoskeletal proteins, such as spectrin and fodrin, the function of this domain could be to localize these proteins to the membrane or submembrane cytoskeleton where they would interact with other molecules.
Comparison of the deduced amino acid sequence of GRB-1 with the protein product encoded by the avian oncogene v-crk may shed light on GRB-1 function. The gene v-crk encodes a protein which is composed primarily of a viral gag protein fused to an SH2 and SH3 domain (Mayer, B. J. et al., Nature 332:272-275 (1988)). Both GRB-1 and the p47.sup.gag-crk protein have no homology with any known catalytic domains. However, chicken embryo fibroblasts transformed with p47.sup.gag-crk display elevated levels of phosphotyrosine-containing proteins (Mayer, B. J. et al., supra; Proc. Natl. Acad. Sci. USA 87:2638-2642 (1990); Matsuda, M. et al., Science 248:1537-1539 (1990)).
Since the v-crk product has been shown to bind several phosphotyrosine-containing proteins in v-Crk transformed cells, it may be that the function of c-crk is to act as a bridge between kinases and substrates. In this regard, it is intriguing that GRB-1 like GAP and PLC-.gamma., contains two SH2 domains, the combination of which may be ideally suited for linking other proteins to activated tyrosine kinase molecules.
EXAMPLE IV
Northern Analysis of GRB-1 Expression
A. Methods
Total cellular RNA was prepared from monkey tissue by the guanidinium isothiocyanate/cesium chloride method described by Sambrook, J. et al., (supra). Poly (A)+RNA was prepared by oligo(dT) cellulose chromatography. For Northern analysis, RNA was size fractionated by electrophoresis in a 1.2% agarose/2.2M formaldehyde gel, transferred onto a nylon membrane by capillary action and baked at 80.degree. C. for 2 hours. Following prehybridization, the blot was hybridized with a (.sup.32 P)-nick-translated DNA probe which was prepared as descried above. Hybridization was carried out overnight at 42.degree. C. in the presence of 50% formamide, 5.times. SSC, 0.1% SDS, and 5X Denhardt's. The membrane was then washed in 0.1.times. SSC, 0.1% SDS at 42.degree. C., and exposed to Kodak XAR film at -70.degree. C. for 12 hours using an intensifying screen.
B. Results
To test for the expression of mRNA corresponding to the newly isolated cDNA, Northern blot analysis of different monkey tissue mRNA, probed with DNA corresponding to the insert from clone ki4, demonstrated the presence of two major bands of 4.4 kb and 7.0 kb in most tissues examined (FIG. 7). Expression was highest in the brain, with heart, spleen, liver and thymus displaying decreasing levels of expression. The 4.4 kb message corresponds to the expected size of the transcript which would encode the isolated clones. In contrast to the 4.4 and 7.0 kb transcripts observed in most tissues, the skin contained two slightly smaller sized mRNAs of 3.6 and 6.6 kb.
The 3.6, 6.6 and 7.0 kb transcripts may represent alternatively spliced forms of mRNA, or may encode for distinct but related mRNA species.
EXAMPLE V
Production of anti-GRB-1 Antibodies and Analysis of GRB-1 Fusion Protein
A. Methods
Polyclonal antibodies were produced by immunizing rabbits with the .beta.-galactosidase fusion protein expressed by the initial isolated phage clone, ki4. E. coli CAG 456 bacteria (obtained from Dr. Michael Snyder, Yale University) were infected with recombinant phage ki4 at a multiplicity-of-infection of 10 and .beta.-galactosidase fusion protein was recovered from the protein pellet after 1.5 hours. Protein extracts were prepared, separated on a 6% SDS-gel, and the band corresponding to the fusion protein excised from gel and used for immunization.
Human glioblastoma cell line U1242, rat bladder carcinoma cell line NBT II, and NIH3T3 cells were grown to confluence in DMEM medium supplemented with 10% fetal bovine serum. Cells were labelled with (.sup.35 S)-methionine (50 .mu.Ci/ml) in 0.5% fetal bovine serum and lysed after 12 hours as previously described (Margolis, B. et al., Cell 57:1101-1107 (1989)). After immunoprecipitation with 10 .mu.l of antibody coupled to protein A-Sepharose, the beads were washed three times with a solution containing 20 mM HEPES, pH 7.5, 300mM NaCl, 10% glycerol, 1% Triton X-100, 0.1% SDS, and 1% sodium deoxycholate. After boiling in sample buffer proteins were separated on a 8% SDS-gel.
B. Results
Polyclonal antibodies were raised against the .beta.-galactosidase fusion protein expressed by the initial isolated phage. Immunoprecipitation experiments, using biosynthetically labelled cells, demonstrated that these antibodies recognized an 85 kDa protein in three different cell lines (FIG. 8, lanes designated "I"). Recognition of the 85 kDa protein by this antiserum was specific since preimmune serum did not recognize this protein (lanes designated "p"). These results provided support for the predicted molecular weight based on the amino acid sequence of cloned GRB-1.
C. Discussion
The finding that the gene for GRB-1 encodes for a protein with an expected molecular weight of 85 kDa, together with the demonstration that antibodies to GRB-1 immunoprecipitated an 85 kDa protein from three different cell lines, suggest that GRB-1 may represent a particular protein which had previously been shown to associate with activated growth factor receptors, namely p85. While the exact function of p85 was unknown, it was presumed to be phosphatidylinositol (PI3)-kinase, since PI3-kinase activity copurified with an 85 kDa protein found in PDGF-stimulated as well as middle T-antigen (MTAg)-transformed cells (Kaplan, D. R. Cell 50:1021-1029 (1987); Whitman, M. et al., Nature 315.:239-242 (1985); Coughlind S. R. et al., Science 243:1191-1194 (1989)). The absence of an ATP binding site argues that GRB-1 is most likely not a phospholipid kinase. GRB-1 exhibits 97% sequence identity with murine and bovine p85. Hence, GRB-1 is the human counterpart of p85. Recombinant p85 is able to bind to the activated PDGFR or EGFR, but does not itself contain intrinsic PI3 kinase activity. p85, however, is found associated with a 110 kDa tyrosine phosphorylated protein which may be the catalytic subunit of the PI3 Kinase. While the exact relationship between PI3 kinase and p85 is not known, overexpression of p85 modulates the interaction between PI3 kinase and the PDGFR. p85 could function as a regulatory subunit or as a bridge between activated receptors and the PI3 kinase.
EXAMPLE VI
The Tyrosine Phosphorylated Carboxy-terminus of the EGF Receptor is a Binding Site for GAP and PLC-.gamma.
The studies described below confirm that binding of PLC-.gamma. and a fusion protein containing the SH2 and SH3 domains of GAP (trpE/GAP SH2) are specifically controlled by autophosphorylation of the EGFR. The results show that phosphorylation of PLC-.gamma. actually reduces its association with the EGFR. Evidence is presented demonstrating that both PLC-.gamma. and the trpE/GAP SH2 fusion protein bind specifically to the tyrosine phosphorylated C-terminus of the EGFR. In sum, these results indicate that the SH2/SH3 domains interact directly with phosphotyrosine containing regions of the EGF receptor.
A. Materials and Methods
1. Cell lines, mutant receptors and fusion proteins
The cell lines CD126 (Margolis, B. L. et al., J. Biol. Chem. 264:10667-10671 (1989a), HER14, K721 (Honegger, A. M. et al., Cell 51:199-209 (1987); Honegger, A. M. et al., Mol. Cell. Biol. 2:4567-4571 (1987)) were used as sources for wild-type EGF receptor, kinase-negative (kin.sup.31) EGF receptor and C-terminal (C-terminal) truncated EGF receptor, respectively. The intracellular domain of the EGF receptor (EGFR-C) was purified from a baculovirus expression system (Hsu, C. J. et al., Cell Growth Differ 1:191-200 (1990)) (FIG. 9A). 3TP1, a cell line which overexpresses transfected PLC-.gamma. cDNA but has no EGF receptor was used as a source of PLC-65 (Margolis, B. et al., Science 248:607-610 (1990b)).
The preparation of trpE fusion proteins containing the GAP SH2 domain (GAP residues 171-448, FIG. 9B) has been described by Moran, M. F. et al., Proc. Natl. Acad. Sci. USA 87:8622-8626 (1990). Bacterial lysates containing trpE/GAP SH2 fusion proteins were prepared by resuspending 1 g of bacteria in 3 ml of 50 mM Tris pH 7.5, 0.5 mM EDTA, 0.1 mM PMSF. After incubation at 4.degree. C. in 1 mg/ml lysozyme and, 0.2% NP-40, cells were sonicated 5 times for 5 seconds, and the lysate was clarified by centrifugation for 30 min at 10,000 g. Bacterial lysates were diluted 1:100 in the 1% Triton lysis buffer with proteinase and phosphatase inhibitors as described above and were precleared with protein A-Sepharose.
2. Antibodies, immunoprecipitation and immunoblotting
The following anti-EGFR antibodies (FIG. 9A) were used: (a) mAb108, a monoclonal antibody directed against domain III of the extracellular domain (Lax, I. et al., EMBO J. 8:421-427 (1989)); (b) antipeptide antibody RK2 specific for residues 984-996, (c) antipeptide antibody C specific for residues 1176-1186; and (d) antipeptide antibody F, specific for residues 656-676. For immunoprecipitating the trpE fusion proteins, a mouse monoclonal antibody against trpE (Oncogene Science) bound to agarose linked anti-mouse IgG (Sigma) was utilized. For immunoblotting, a polyclonal rabbit antibody against trpE was used (Moran, M. F. et al., Proc. Natl. Acad. Sci. USA 8:8622-8626 (1990)). PLC-.gamma. was immunoblotted and immunoprecipitated with a polyclonal rabbit anti-peptide antibody described previously (Margolis, B. et al., Cell 57:1101-1107 (1989b)).
The techniques used are described in several references from the present inventors' laboratory (Margolis, B. L. et al., J. Biol. Chem. 264:10667-10671 (1989); Cell 57:1101-1107 (1989)). Unstimulated cells were grown to confluence in Dulbecco's Modified Eagle Medium with 10% calf serum and starved overnight in 1% fetal calf serum prior to lysis in a 1% Triton X-100 lysis buffer containing proteinase and phosphatase inhibitors. EGF receptors were immunoprecipitated utilizing antibodies bound to protein A-Sepharose. After washing the receptor material with HNTG (20 mM Hepes, pH 7.5, 150 mM NaCl 0.1% Triton X-100 and 10% glycerol), autophosphorylation was induced by the addition of 5 mM MnCl.sub.2 and 30 .mu.M ATP. Controls were incubated with Mn.sup.2+ only. After further washes with HNTG, lysate containing either PLC-.gamma. (from 3TP1 cells) or the bacterial fusion proteins was added. After allowing binding to proceed for 90 min, three further washes with HNTG were performed and samples were run on an SDS gel and immunoblotted.
3. Cyanogen bromide (CNBr) Cleavage
EGFR-C was phosphorylated at 4.degree. C. with MnCl.sub.2 and ATP sometimes in the presence of (.gamma.-.sup.32 P)ATP (NEN/Dupont, 6000 Ci/mmol). The receptor preparation was then resuspended in 20 mM HEPES, pH 7.5, with 100 .mu.g BSA and concentrated in a Centricon 10 (Amicon) to 50 .mu.l. Then 240 .mu.l 88% formic acid was added with two grains of CNBr and the samples were stored under nitrogen in the dark for 14 h at room temperature. Samples were dried and washed three times with water in a Speed-Vac (Savant) and then resuspended in 1% Triton lysis buffer.
B. RESULTS
A comparison was performed of the binding of PLC-.gamma. to wild-type and mutant EGFRs (FIG. 9A). First, wild-type and mutant receptors from transfected NIH-3T3 cells were immunoprecipitated and some of the receptor immunoprecipitates were allowed to undergo in vitro autophosphorylation with ATP and Mn.sup.2+ et al., Mol. Cell. Biol. 10:435-441 (1990a)). Then, lysates from NIH-3T3 cells which overexpress PLC-.gamma. (Margolis, B. et al, Science 248:607-610 (1990b)) were added and binding allowed to proceed for 90 min. at 4.degree. C. After washing the immunoprecipitates with HNTG, the amount of PLC-.gamma. bound was assessed by immunoblotting. As illustrated in FIG. 10, PLC-.gamma. bound only to the tyrosine phosphorylated wild-type receptor but not to the non-phosphorylated receptor.
To assess the importance of autophosphorylation, two studies with mutant receptors were then undertaken. First to be examined was the binding of PLC-.gamma. to a truncated EGF receptor missing 126 amino acids from the C-terminus (CD126, FIG. 9A) and devoid of four major autophosphorylation sites (Downward, J. et al., Nature 311:483-485 (1984)). This truncated receptor was autophosphorylated, probably at tyrosine 992 (Walton, G. M. et al., J. Biol. Chem. 265:1750-1754 (1990)). However, despite this level of tyrosine autophosphorylation, the binding of PLC-.gamma. was markedly reduced compared to the full length receptor. Reduced association was also observed with CD63, a deletion mutant EGF receptor lacking 63 C-terminal residues containing two autophosphorylation sites. These results suggested a role for the receptor C-terminus in either binding or modulating the binding of PLC-.gamma. to the EGF receptor.
FIG. 10A-10B also demonstrates that PLC-.gamma. cannot bind to the kin.sup.- mutant receptor. To explore the importance of autophosphorylation in this effect, the kin-receptor was cross-phosphorylated with the CD126 receptor (Honegger, A. M. et al., Proc. Natl. Acad. Sci. USA 86:925-929 (1989)). This resulted in normalization of PLC-.gamma. binding to wild-type levels. This suggested that phosphorylation of the kin.sup.- receptor was sufficient to normalize binding to PLC-.gamma..
To confirm that the kin-receptor alone could bind PLC-.gamma. after phosphorylation, this receptor was cross-phosphorylated with a soluble, baculovirus-expressed EGFR cytoplasmic domain (EGFR-C) that does not bind to the mAb 108 (FIG. 9A).
Although cross-phosphorylation was not as strong as with the CD126 mutant, tyrosine phosphorylation of the K721A mutant and binding of PLC-.gamma. were clearly detected. This finding confirms that tyrosine phosphorylation of the EGFR promotes binding of PLC-.gamma..
The role of PLC-.gamma. tyrosine phosphorylation in the interaction between wild-type EGFR and PLC-.gamma. was examined. Tyrosine phosphorylated PLC-.gamma. could be dissociated from the EGFR more readily than non-phosphorylated PLC-.gamma. (FIG. 11), suggesting a lower affinity of tyrosine phosphorylated PLC-.gamma. for the EGFR.
These findings were extended to examination of the binding of a fusion protein containing trpE/GAP SH2 domain (FIG. 9B) to the baculovirus expressed EGFR-C, As with the full length EGFR and PLC-.gamma., the trpE/GAP SH2 fusion protein domain bound only to the tyrosine phosphorylated EGFR-C (FIG. 12A). The trpE protein alone did not bind to EGFR-C. Similarly, phosphorylated EGFR-C bound only to trpE/GAP SH2; however, non-specific binding of non-phosphorylated EGFR-C was high (FIG. 12B). These results demonstrated that the binding site of the EGFR is situated in its intracellular domain.
In general, the trpE/GAP SH2 fusion protein bound with a higher stoichiometry to full length EGFR than did PLC-.gamma.. However, the fusion protein was not tyrosine phosphorylated by the EGFR. The trpE/GAP SH2 protein much better to the phosphorylated full length receptor compared to the CD126 deletion mutant (FIG. 13A). As shown in FIG. 13B, cross-phosphorylation of the kin.sup.- full length EGF receptor by the EGFR-C allowed it to bind the trpE/GAP SH2 protein.
In control groups, the EGFR-C was shown not to enhance the binding to the CD126 receptor probably because this receptor was already maximally tyrosine phosphorylated (FIG. 13A). Also, no binding was observed when EGFR-C was tested in the presence of mAb 108 immunoprecipitate from cells containing no EGF receptor (FIG. 13B). This indicates that the effects of EGFR-C could not be attributed to non-specific binding of tyrosine phosphorylated EGFR-C to sepharose. These studies confirm the importance of autophosphorylation in mediating binding and show that for EGF receptor binding, the GAP SH2 domain behaves similarly to intact PLC-.gamma..
The poor binding to the CD126 deletion mutant suggested that at least part of the binding site for the molecule was in the C-terminus. Yet an effect, possibly allosteric, of this deletion on the overall conformation of the receptor could not be excluded. Therefore, the binding of PLC-.gamma. and trpE/GAP SH2 to a C-terminal fragment of the EGFR was examined. In the EGFR, the most C-terminal methionine residue is found at position 983; CNBr cleavage therefore generates a 203 amino acid fragment which contains all the known autophosphorylation sites. This protein fragment is recognized by an antibody specific for the EGFR C-terminus, anti-C (FIG. 9A).
When this C-terminal fragment was specifically immunoprecipitated and tyrosine phosphorylated, it bound PLC-.gamma. and the trpE/GAP SH2 fusion protein (FIG. 14). CNBr cleavage was complete; no full-length EGFR-C could be detected after proteolysis that could account for the binding. Again, no binding was seen to the non-phosphorylated C-terminal CNBr fragment. CNBr cleavage of EGFR-C also generated a 97 amino acid N-terminal peptide identified by antibody F (FIG. 9A, EGFR residues 645-742). This fragment, immunoprecipitated by antibody F, did not bind trpE/GAP SH2. Additionally, EGFR-C was autophosphorylated with .gamma.-.sup.32 P)ATP and a labeled CNBr C-terminal fragment was generated. As shown in FIG. 15, this fragment bound to the trpE/GAP SH2 fusion protein but not to trpE. In total, these findings demonstrate that direct binding to the tyrosine phosphorylated C-terminus contributes at least in part to the specific binding of SH2 and SH3 domain proteins to the EGFR.
C. Discussion
When taken together, the above findings and several additional lines of evidence argue strongly that the phosphotyrosine residues are part of the actual binding site of the EGFR for SH2 domains. First, P47.sup.gag-crk was found to bind to nearly all phosphotyrosine-containing proteins in v-crk transformed cells (Matsuda, M. et al., Science 248:1537-1539 (1990)). Second, mutations of two autophosphorylation sites on the PDGF receptor greatly decreased the binding of GAP (Kazlauskas, A. et al., Science 247:1578-1581 (1990)). Finally, the results presented above demonstrate specific binding to the C-terminus of the EGFR only when phosphotyrosine is present. Thus, it is concluded that the phosphotyrosine residues either comprise a part of the binding site or locally alter the conformation of this region, allowing binding. It is unlikely that phosphotyrosine alone constitutes the binding site. For example, phosphotyrosine alone cannot interfere with the binding of P47.sup.gag-crk to phosphotyrosine-containing proteins (Matsuda et al., supra). Additionally, PLC-.gamma. does not bind to activated all molecules that contain phosphotyrosine residues, such as the CSF-1 receptor (Downing, J. R. et al., EMBO J. 8:345-3350 (1989)). Similarly, the binding of PLC-.gamma. to PDGFR does not appear to be identical to GAP binding; different SH2 and SH3 domain-containing proteins may have different binding specificities (Kazlauskas et al., supra).
EXAMPLE VII
Cloning Isolation & Characterization of a Target Protein for Receptor Tyrosine Kinase
METHODS: The intracellular domain of the EGFR, which includes the tyrosine kinase and carboxy terminal domain, was purified from a recombinant baculovirus expression system as described (Margolis Mol. Cell. Biol. 10:435-441 (1990) and EMBO J. 9:4375-4390 (1990); Skolnik et al. Cell 65:83-90 (1991). The recombinant protein was phosphorylated with (.sup.32 P) .gamma.-ATP, washed, and cyanogen bromide digested to yield a 204 residue carboxyterminal tail containing all five phosphorylated tyrosine residues (Margolis Mol. Cell Biol. 10:435-441 (1990a) and EMBO J. 9:4375-4390 (1990b). The (.sup.32 P)-carboxyterminal tail was then used as probe to screen a .lambda.gt11 human brainstem expression library, as previously described (Skolnik et al. Cell 65:83-90 (1991)).
An oligo (dT) .gamma.gt11, constructed from mRNA isolated from human brain stem, was obtained from M. Jaye (Rhone Poulenic-Rorer Pharmaceuticals) and is readily available from commercial sources. Screening of the library was performed as previously described (Skolnik et al. Cell 65:83-90 (1991)). cDNA inserts isolated from positive recombinant phage that bound the EGFR were subcloned into M13 and sequenced by the dideoxy chain termination method, using the Sequenase 2.0 kit (U.S.B). Since the initial clone isolated by expression/cloning did not contain the 5' ends of the gene, the library was rescreened, using the clone 2-4 insert as a DNA probe.
Total cellular RNA was prepared with the Stratagene RNA isolation kit. For Northern analysis, RNA was size fractionated on a 1.2% agarose-2.2M formaldehyde gel, transferred by capillary action to a Nytran membrane (Schleicher and Schuell), and prehybridized and hybridized at 65.degree. in 0.5M sodium phosphate pH 7.2, 7% SDS, 1 mM EDTA, 100 ug/ml salmon sperm DNA. The membrane was then washed 1.times. at room temp and then 2.times. at 65.degree. C. in 40 mM sodium phosphate pH 7.2, 1% SDS, 1 mM EDTA.
HER14 are NIH 3T3 cells (clone 2.2) which express approximately 400,000 wild type human EGF receptors per cell (Honeggar et al. Cell 51:199-209 (1987)). HER14 cells were maintained in Dulbecco's modified Eagles medium (DMEM) containing 10% calf serum (CS). Prior to stimulation, cells were cultured for 18 hours in DMEM/1% CS. Cells were then stimulated with either EGF (275 ng/ml) or PDGF-BB (50 ng/ml) Intergen, Purchase, N.Y.) for 2 minutes in DMEM containing 1 mg/ml BSA and 20 mM HEPES pH 7.5, following which the cells were immediately washed and lysed. Lysate protein content was normalized as described (Bradford, 1976). Cell lysis, immunoprecipitation, and immunoblotting were performed as previously described (Margolis et al. Cell 57:1101-1107 (1989)). 293 cells were transfected using a modification of the calcium phosphate precipitation method (Chen and (Okayama Mol. Cell. Biol. 7:2745-272 (1987).
Several polyclonal antibodies were generated against GRB2. A synthetic peptide derived from the N-terminal SH3 domain (residues 36-50) and the full length GRB2-GST (glutathione-S-transferase) fusion protein were used to produce rabbit polyclonal antisera called Ab 86 and Ab 55, respectively. Both of these antisera are effective at recognizing denatured GRB2 in immunoblots. A third polyclonal rabbit antisera called Ab50 was generated against the GRB2-GST fusion protein containing the C-terminal SH3 domain of GRB2 (residues 167-221), and is capable of immunoprecipitating GRB2 from solubilized cells. Monoclonal antiphosphotyrosine antibodies (1G2) covalently coupled to agarose were purchased from Oncogene Science (Manhasset, N.Y.). Anti-P-Tyr immunoblots were performed with a rabbit polyclonal antibody. Anti-EGF receptor immunoprecipitates were performed with monoclonal antibody mAb m108 (Bellot et al. J. Cell Biol. 110:491-502 (1990).
Anti-EGF receptor immunoblots were performed with anti-C terminus peptide (residues 1176-1186) antisera (Margolis et al. Cell 57:1101-1107 (1989)).
Using the cDNA of GRB2 as a template, DNA fragments corresponding to the various GRB2 domains were synthesized using PCR and oligonucleotides which contained appropriate restriction sites and bordered the domains of interest. The amplified DNA was isolated, digested with BamHI and EcoRI and cloned into pGEX3X (Pharmacia), which was then used to transform E. coli HB 101 to ampicillin resistance. Large scale cultures were then grown, induced with IPTG, and the glutathione S-transferase (GST) fusion proteins purified on glutathione agarose beads as previously described (Smith and Johnson Gene 67:31-40 (1988)).
The following fusion proteins were prepared: GST-GRB2 full length (FL) (amino acids {AA} 2-217); GST-SH2 (AA 50-161); GST-N-terminal SH3 (AA 2-59); GST-C-terminal SH3 (AA 156-217); GST-N-terminal SH3-SH2 (AA-161); GST-SH22-C-terminal SH3 (AA 50-217).
To assay the binding of native growth factor receptors to GST-fusion proteins 500 ul of HER14 cell lysate was incubated for 90 min at 4.degree. C. with approximately 5 ug of fusion protein coupled to glutathione agarose beads. The beads were then washed three times with HNTG, and after boiling in sample buffer, the proteins were separated on 8% SDS-PAGE. Bound proteins were transferred to nitrocellulose and blotted with antibodies as described (Margolis et al. Mol. Cell. Biol. 10:435-411 (1990a), Margolis et al. EMBO J. 9:4375-4380 (1990B); Margolis Cell Growth and Differentiation 3:73-80 (1992); and Margolis et al. Nature 356:71-74 (1992).
Labeling cells with (.sup.32 P)-orthophosphate were carried out as previously described (Li et al. Mol. Biol. Cell 2:641-649, 1991). Briefly, confluent HER14 cells starved for 16 hrs in 1% FCS/DMEM were incubated for two hours in P.sub.i -free media, and labeled for two hours in P.sub.1 -free media, 1% dialyzed FBS, 1mCi/ml orthophosphate (carrier free, 314.5-337.5 TBq/mmole, purchased form NEN, Wilmington, Del.), at 37.degree. C. Where appropriate, cells were incubated with vanadate (200 uM) at 37.degree. C. for the last 20 minutes of cell labeling. Cells were then stimulated for two minutes with EGF (250 ng/ml) or PDGF (50 ng/ml), rapidly washed 2 times with ice cold phosphate-buffered saline (PBS), and solubilized immediately in lysis buffer (10 mM Tris-Cl pH 7.6, 50 mM NaCl, 30 mM sodium pyrophosphate, 50 mM sodium fluoride, 100 uM sodium orthovanadate, 5 uM ZnCl.sub.2, 1 mM PMSF and 0.5% Triton-X-100 ). After nuclei were removed by centrifugation, the lysates where precleared for 1 hour with 50 ul Sepharose G25, and then incubated overnight with anti-GRB2 antiserum (Ab50) at 4.degree. C. The immune complexes were then precipitated with protein A-Sepharose for 45 min at 4.degree. C., washed 8-15 times with RIPA buffer (20 mM Tris-Cl pH 7.6, 300 mM NaCl, 2 mM EDTA, 1% Triton-X-100, 1% sodium deoxycholate and 0.1% SDS), heated in Laemmli sample buffer containing 0.1M B-mercaptoethanol and 1% SDS at 95.degree. C. for 5 mind resolved by SDS PAGE (8-15% gradient), and visualized by autoradiography of dried gels. To isolate tyrosine phosphorylated proteins, the cell lysates were incubated with anti-PY antibody (Oncogene Science) beads for 2 hours at 4.degree. C. The anti-PY beads were washed 5 times with lysis buffer, followed by elution with phenylphosphate (2 mM) in the presence of ovalbumin.
RESULTS: Isolation of a cDNA clone encoding a protein with novel SH2 and SH3 domains.
The carboxyterminal tail of the EGFR was used as a probe to screen a human brain stem .lambda.gt11 protein expression library as previously described (Skolnik et al. Cell 6:4396-4408, 1991). One of the clones isolated utilizing this technique, clone 2-4, contained an insert of 1100 nucleotides found to contain a reading frame encoding novel SH2 and SH3 domains. The insert from clone 2-4 contained a 3' stop codon followed by a polyadenylation signal, but did not contain the 5' start site. To isolate the 5' end of the gene, the library was rescreened using DNA probes generated by amplifying DNA from clone 2-4. This approach enabled identification of clone 10-53, which was found to encode the full length protein. Clone 10-53, while overlapping with clone 2-4 at the 3' end contained a 5' ATG codon meeting Kozak translation initiation criteria (Kozak J. Cell. Biol. 108:229-241 (1989)), giving a 660 bp open reading frame from the initiating methionine (Ficket et al. Nucleic Acids Research 10:5303-5318 (1982)) (FIG. 26A-26C). Analysis of the protein sequence of clone 10-53 using Genbank revealed that the full length protein contained a single SH2 domain flanked by two SH3 domains, and that these three domains comprise the bulk of the protein (FIG. 26B-26F). The SH2 and SH3 domains of GRB2 are compared to those in other proteins in FIG. 26D and 26E. The full length protein encoded by clone 10-53 was named GRB2 (for the second growth factor receptor binding protein identified by the CORT method), and encoded a protein with a predicted molecular weight of about 24.5 kDa. The sequence also contains two potential protein kinase C phosphorylation sites (aa 22 and 102), two potential casein kinase 2 phosphorylation consensus sequences (aa 16 and 131) (Woodget et al. Eur. J. Biochem. 161:177-184 1986; Kishimoto et al. J. Biol. Chem. 260:12492-12499 1985; Marin et al. Eur. J. Biochem. 160:239-244 1986; Kuenzel et al. J. Biol, Chem. 262:9136-9140 1987) and two RGD motifs.
Northern Analysis and Protein Expression
To determine tissue distribution of GRB2, Northern hybridization analysis of various mouse tissue RNAs was performed, using as a probe the insert from clone 10-53. This analysis demonstrated GRB2 expression in every tissue examined, with the highest expression in the brain, spleen, lung, and intestine (FIG. 27A). GRB2 transcripts were visible in the thymus upon longer exposure. We have thus far been unable to identify a tissue or cell line which does not express GRB2, further demonstrating the ubiquitous nature of GRB2 expression. GRB2 hybridized to two transcripts of 1.5 and 3.8 kb. The 1.5 kb transcript corresponds to the expected size of clone 10-53.
Several polyclonal rabbit antisera against GRB2 were generated (see methods section) and used to analyze the GRB2 protein by immunoblotting or immunoprecipitation experiments. FIG. 27B shows that a protein of 25 kDa is recognized by the immune, but not by the preimmune antiserum utilizing either immunoprecipitation analysis of (.sup.35 S) methionine labelled cells or an immunoblotting approach. The various antisera recognized a 25 kDa protein in every cell line and tissue examined, consistent with the distribution of the GRB2 transcript found in Northern analysis.
GRB2 associates with growth factor receptors in living cells. Receptor substrates which contain SH2 domains are endowed with the ability to physically associate with certain activated growth factor receptors. Since the goal of the CORT cloning technique is to identify target proteins for particular growth factor receptors, we assessed whether GRB2 associates with the EGF receptor. HER 14 cells were treated with or without EGF, lysed, and subjected to immunoprecipitation analysis, according to published procedures (Margolis et al. 1990b, 1991b).
Immunoblotting of anti-EGFR immunoprecipitates with antibodies to GRB-2 demonstrated association of the 25 kDa (GRB-2 protein with activated EGFR (FIG. 28, lane 6). As shown for PLC.gamma. the association between EGFR and GRB2 was strictly dependent upon ligand activation and tyrosine autophosphorylation (FIG. 28, lanes 5 and 6) (Anderson et al. Science 250:979-982 (1990); Margolis et al. Cell 57:1101-1107 1989, Mol. Cell. Biol. 10:435-441 1990a, EMBO J. 9:4375-4380 1990b; Wahl et al. Natl. Acad. Sci. USA 8:1568-1572 1989, Meisenhelder Cell 57:1109-1122 1989). Thus, GRB2 associates only with the activated tyrosine phosphorylated EGFR. GRB2 was also demonstrated to have an association with EGFR by immunoprecipitation of GRB2 followed by immunoblotting with anti EGF-receptor antibodies (data not shown). Similar results were obtained with PDGF receptor; activated PDGF receptor associated with GRB2 in HER14 cell sin growth factor dependent manner.
However, no association between GRB2 and the FGF receptor was detected when similar experiments, using anti GRB2 for immunoprecipitation and anti FGF receptor antibodies for immunoblotting, were performed with cell lines expressing FGF-receptor (Mohammadi et al. Mol, Cell. Biol. 11:5068-5078 1991).
Interaction of GRB2 with growth factor receptors is mediated via the SH2 domain. It has been shown that SH2 domains mediate the interaction of signalling molecules, such as PLC.sub.7 or GAP, with tyrosine phosphorylated growth factor receptors (Koch et al. Science 252:668-674 (1991); Heldin et al. Trends in Biol. Sci. 16:450-452 (1991); Margolis et al. Cell Growth and Differentiation 3:73-80 (1992), Margolis et al. Nature 3556:71-74 1992). In order to determine whether the interaction between GRB2 and growth factor receptors is mediated via the SH.sub. 2 domain of GRB2, we constructed bacterial expression vectors which were designed to express GRB2 as well as the various domains of GRB2 as GST-fusion protein (FIG. 4A-4I). These fusion proteins were purified by affinity chromatography on glutathione agarose beads (Smith et al. Gene 67:31-40 1988), and subsequently incubated with lysates from EGF- or PDGF-treated HER 14 cells. The ability of the fusion proteins to bind the activated EGF or PDGF receptors was assessed by immunoblotting the washed complexes with either antiphosphotyrosine or anti-receptor antibodies.
Both the full length GRB2 fusion protein and a fusion protein containing only the SH2 domain of GRB2 were each capable of binding tyrosine phosphorylated proteins which comigrated with the activated EGF or PDGF receptors (FIG. 30, lanes 4, 6, 12 and 14). In contrast, neither receptor bound GST alone (FIG. 30, lane 2) nor a GST-fusion protein containing either the amino or carboxy terminal SH3 domains could bind to activated receptors. Binding was ligand dependent, since immunoblotting with anti-EGFR antibodies revealed association of the EGFR with the fusion proteins only when incubated with lysates from growth factor stimulated cells FIG. 30, lanes 7 through 10). Thus, in agreement with data about other SH2 domain containing proteins, the association between GRB2 and growth factor receptors is mediated by the SH2 domain (Koch et al. Science 252:668-674 1991); Heldin et al. Trends in Biol. Sci. 16:450-452 (1991); Margollis et al. Cell Growth and Differentiation 3:73-80 (1992) and Nature 356:71-74 (1992).
It is noteworthy that the full length GRB2 fusion protein bound several other tyrosine phosphorylated proteins in EGF- and PDGF-stimulated cell lysates (FIG. 30, lanes 3, 4, 11 and 12). While these bound proteins failed to interact with the SH2-GST fusion protein (FIG. 30, lane 6) or either SH3 domain of GRB2 expressed independently, they did interact with a fusion protein containing both the N-terminal SH3 and SH2 domains. The ability of SH3 domain of GRB2 to enhance the binding activity of the SH2 domain suggests that the N-terminal SH3 domain is important for binding to various cellular proteins and that binding to these proteins may require the concerted action of both SH2 and SH3 domains. GRB2 binds to activated growth factor receptors without being phosphorylated in living cells.
After demonstrating that GRB2 was able to bind to activated EGF and PDGF receptors, we were next interested in determining if GRB2 was a substrate for receptor tyrosine kinases. We examined the capacity of EGF to stimulate phosphorylation of GRB2 in HER14 labelled with (.sup.32 P)-orthophosphate. These cells were treated with EGF, lysed and immunoprecipitated with antibodies to GRB2. While anti-GRB2 antibodies immunoprecipitated GRB2 from (.sup.35 S) methionine labeled cell lysates (FIG. 31, lanes 6 and 8), phosphorylated GRB2 was not detected in the anti-GRB2 immunoprecipitates from orthophosphate labelled cells. Despite marked overexposure of this gel, no detectable band corresponding to GRB2 was evident in the orthophosphate labelled immunoprecipitates. In similar experiments, stimulation of HER14 cells with PDGF also did not result in detectable phosphorylation of GRB2. The failure of detect phosphorylated GRB2 was not due to poor stimulation of the cells by EGF, since anti-P-Tyr immunoprecipitation of the (.sup.32 P.sub.i)-labeled lysates demonstrated a marked increase in tyrosine phosphorylation of numerous cellular substrates following EGF stimulation. Similarly anti-phosphotyrosine immunoblotting of GRB2 immunoprecipitated from EGF- or PDGF-stimulated HER14 cell lysates, did not reveal tyrosine phosphorylated GRB2 (data not shown).
To determine if the failure to detect tyrosine phosphorylated GRB2 was due to the rapid dephosphorylation by a protein tyrosine phosphatase, a potent tyrosine phosphatase inhibitor, vanadate, was tested for its effects upon GRB2 phosphorylation. (.sup.32 P)-orthophosphate-labelled cells were incubated with or without vanadate at 37.degree. C. for 20 min prior to the addition of EGF, and GRB2 phosphorylation was assessed as described above. Vanadate treatment of EGF stimulated cells similarly did not result in detectable GRB2 phosphorylation.
The inability to demonstrate GRB2 phosphorylation was further corroborated in a double immunoprecipitation experiment. (.sup.32 P)-labeled HER 14 lysates were immunoprecipitated with anti-PTyr antibodies bound to beads, eluted and the eluates subjected to a second immunoprecipitation with anti-GRB2 antibodies. While clear stimulation of tyrosine phosphorylation was demonstrated in these lysates no significant phosphorylation of the antiP-Tyr-associated GRB2 fraction was detected. Thus, our data demonstrates that while GRB2 associates with the EGF and PDGF-receptors it is not a good substrates for either receptors, and that GRB2 is not phosphorylated by a tyrosine or serine/threonine kinase acting later in the signaling pathway induced by ligand binding. This data suggests that growth factor regulation of GRB2 is not mediated through GRB2 phosphorylation.
GRB2 tyrosine phosphorylation was detected in 293 cells transiently overexpressing PDGFR and GRB2 as determined by anti-PTyr and anti-GRB2 blotting (data not shown). A shift in the mobility of GRB2 was detected on anti-GRB2 (Ab86) blots, in the presence of activated PDGF receptor and the lower mobility form was shown to be tyrosine phosphorylated by anti-PTyr blotting. Similar experiments have confirmed that the immunoprecipitating antibody (Ab50) will recognize tyrosine phosphorylated GRB2. This data suggest that it is possible to tyrosine phosphorylate GRB2 under conditions of overexpression of both receptor and GRB2 protein.
Interestingly, a phosphoprotein of approximately 55 kDa was found to con-immunoprecipitate with GRB2 using immune, but not preimmune sera, in lysates from EGF or PDGF stimulated HER14 cells (FIG. 31, lanes 3, 4 and 7, 8). The association of the 55 kDa protein with GRB2 immunoprecipitates was dependent upon growth factor stimulation, since this interaction was not observed in GRB2 immunoprecipitates from unstimulated cell lysates. The identity of this protein is unknown. GRB2 represents the human homologue of the C. elegans gene product sem-5.
As mentioned earlier, GRB2 is composed of one SH2 domain flanked by two SH3 domains in the order of SH3, SH2, SH3. A C. elegans gene encoding for a protein with similar size and domain order has been cloned in the laboratory of R. Horvitz (Clark et al., 1992). This gene, called sem-5, plays a crucial role in C. elegans development as mutations in sem-5 impair both vulval development and sex myoblast migration. FIG. 32 shows a comparison of the amino acid sequences of GRB2 and sem-5. The N-SH3 domains are 58% (63%) and the C-terminal SH3 domains are 58% identical (60%), respectively. The overall sequence identity (similarity) is 58% (63%). Considering the evolutionary distance between human and nematode, these two genes are very similar suggesting the sem-5 represents the C. elegans homologue of GRB2.
DISCUSSION
A novel EGF receptor binding protein of the present invention was cloned by the CORT expression cloning method of the present invention, designated as GRB2. This 25 kDa protein contains on SH2 domain and two SH3 domains. GRB2 is widely expressed, as determined by Northern analysis in ten different murine tissues. It is also expressed in every human, monkey and murine cell line tested as revealed by Northern blotting, immunoprecipitation and immunoblotting experiments. Also shown is that GRB2 associates with EGF and PDGF receptors in a ligand-dependent manner, both in vitro and in living cells. Like other SH2-domain containing proteins, the association between GRB2 and growth factor receptors is mediated by the SH2 domain, can be dependent upon receptor tyrosine autophosphorylation, and involves a direct interaction between GRB2 and the tyrosine phosphorylated receptors.
Despite the fact that GRB2 forms stable complexes with tyrosine phosphorylated, on tyrosine, serine, or threonine residues at physiologic levels of expression to any significant extent. The fact that pretreatment of cells with vanadate did not increase GRB2 phosphorylation indicates that GRB2 is not rapidly dephosphorylated by tyrosine phosphatases.
The extent of sequence homology between GRB2 and sem-5 is striking considering the evolutionary distance between nematode and man the 58% sequence identity (63% similarity) and the conserved overall architecture of these tow proteins suggest that sem-5 and C. elegans homologue of GRB2 or a closely related member of the same gene family. The similarity between GRB2 and sem-5 is higher than the similarity between let-23 and EGFR; approximately 44% and 28.7% sequence similarities in the catalytic kinase and ligand binding domain, respectively (Aroian et al. Nature 348:693-699 1990).
By detailed genetic studies the laboratories of Horvitz and Sternberg have identified gene crucial for C. elegans vulval development and sex myoblast migration (Horvitz and Sternberg Nature 351:535-341 1991; Aroian et al. Nature 348693-699 1990, Clark et al. Nature In press 1992). It was shown that mutation sin let-23 (EGFR like), sem-5 (GRB2) or let-60 (ras like) lead to defects in vulval development, while sem-5 also functions in sex myoblast migration. It was therefore proposed that the products of these genes lie along the same signal transduction pathway crucial for normal vulval development. Hence, on the basis of genetic studies of C. elegans (Horvitz and Sternberg Nature 351:535-541; Aroian et al. Nature 348:693-699; Clark Nature in press 1992), previous studies on growth factor receptors (Ullrich and Schlessinger Cell 61:203-211 (1990)) and the results presented in this report it is possible to propose a model for the information flow and interaction among these proteins in C. elegans and mammalian cells (FIG. 33). Because of the similarity of sem-5 with GRB2 and let-23 with the EGFR it is likely that sem-5 with GRB2 and let-23 with the EGFR it is likely that sem-5 will bind tyrosine phosphorylated let-23 via its SH2 domain according to the scheme presented in FIG. 8. Since mutations in let-60 cause a similar phenotype as mutations in either let-23 and Sem-5, and since activated ras can rescue let-23 and sem-5 mutations, it is reasonable to assume the let-60/ras functions downstream from EGFR and GRB2 and that GRB2 is somehow involved in regulation of ras activity. In this regard, the 55 kDa phosphoprotein which binds to GRB2 in response to growth factor stimulation is expected to be a downstream signaling molecule regulated upon GRB2 binding to activated growth factor receptors.
EXAMPLE VIII
Utilization of an Alternative Phage Library Expression System For Detecting Proteins of the Present Invention
A T7 phage library expression systems used an alternative to the phage .lambda.gt11 system described in Example II above, was used to express tyrosine kinase target proteins, as presented in the above Examples, with modifications as described below. A T7 polymerase system (Palazzalo et al., Gene 88, 25 (1990); .lambda.EXlox vector, Novagen, Inc.), based on the PET expression systems of Studier and coworkers (Studier et al Meth. Enzymol. 185:60 (1990)) fusing cDNA clones to a fragment of the T7 capsid protein T10 under the control of the T7 promoter. These phages were then used to infect E. coli harboring the T7 polymerase under lacUV5 control. Induction with IPTG generated the T7 polymerase which then initiated transcription of the fusion protein encoded by the phage library. The SH2 domain fragment of PLC-.gamma.1 was incorporated into this phage and analyzed the binding of the phosphorylated EGFR, as described in the above Examples. The DNA fragment containing the human PLC-.gamma.1 (Burgess et al., Mol. Cell. Biol. 10, 4770 (1990)) was amplified by PCT with primers that incorporated EcoR1 sites such that the PLC-.gamma.1 fragment would be in the correct reading frame for .lambda.gt11. The amplified DNA was cut with EcoR1 and ligated into EcoR1 digested .lambda.gt11 DNA (Promega). After packaging (Gigapack, Stragene), the phages were plated and screened with PLC-.gamma.1 antibody using known techniques (Huynh, T. V. et al. In: DNA CLONING, ed. Glover, IRL Press, Oxford, 1:49-78 (1985)). This phage was then tested for binding to a cyanogen bromide generated fragment from .sup.32 P-ATP labelled EGFR as described in the above Examples. An identical approach was taken to clone the two SH2 domains into .lambda.gt11 or .lambda.EXlox vectors.
As can be seen in FIG. 25A-C, uniform binding of the EGFR was seen in the that appeared stronger than was seen with the .lambda.gt11 system (compare FIG. 25A and 25B). We also cloned in a longer fragment which ran from 532-1290 of PLC.gamma.1 and this was also easily seen in the T7 system (FIG. 25C). The T7 plaques although mostly smaller than the Agt11 plaques gave stronger signals. This makes this system particularly suitable for library screening when there as thousands of small plaques per plate. The major advantage of this system is the high level of protein expression due to the greater activity of the T7 polymerase versus E. coli RNA polymerase. It may also be that the fusion proteins using the smaller T10 gene fragment (26 kd versus the 110 kd B-galactosidase of .lambda.gt11) yields more stable expression and that its hydrophobic character promotes binding to nitrocellulose. In addition to directional cloning, the .lambda.EXlox phages also allow for automatic conversion to a PET plasmid (Palazzalo et al., Gene 88, 25 (1990)) which can be useful for expression of a fusion protein for antibody production. Accordingly, screening an T7 expression library is expected to give superior results than for .lambda.gt11 for such a cloning strategy of the present invention.
Of 1.6 million clones of a directional oligo dT primed mouse T7 (.lambda.EXlox) library screened, nine positive clones were obtained. The library from a 16 day mouse embryo was obtained from Novagen. The library was plated at 40,000 phages per plate in E. coli pLysS according to known methods. After growth for 8 hours, plates were covered with nitrocellulose impregnated with 1mM IPTG. Plates were grown overnight and the filters probed as described in the above Examples. Positive clones were selected and reprobed until plaques were purified. Phages were then converted to plasmids utilizing the bacterial strain Bm25.5 per manufacturer's instruction. These plasmids were used to transform bacterial strain DH5.varies. and the resultant plasmids subjected to double stranded sequencing using known techniques (Sequenase Version 2, U.S. Biochemical). Six of nine clones encoded proteins that were similar or identical to other known genes which contained SH2 domains TABLE I--see attached. Figures). The comparison of two of these protein sequences of the present invention, GRB-3 and GRB-4, to their known counterparts is displayed in FIG. 17 and 18. Partial sequence of three clones revealed that they were closely related to the avian oncogene v-crk. GRB-3 has a high degree of identity with v-crk beginning with the methionine at residue 32 and this methionine has been found to be the start site of arian c-crk. In the sequence carboxy-terminus to this methionine, there is 77% amino acid homology (FIG. 17) and 80% DNA similarity between v-crk and GRB-3. GRB-4, was similar to nck (FIG. 18), a human protein composed of three SH3 domains and one SH 2 domain. Our clone contained one SH3 domain and one SH2 domain and was 74% identical at the protein level and 66% similar at the DNA level in the open reading frame. We also cloned two SH2 domain proteins with intrinsic enzymatic activity.
TABLE I______________________________________SH2 DOMAIN CLONESPROTEIN ISOLATED DESCRIPTION______________________________________GRB-3 #19, #76, #80 crk-likeGRB-4 #64 nck-likeGRB-5 #63B fynGRB-6 #88 PLC-.gamma.1GRB-7 #64A, #66, #88 novel protein______________________________________
A remaining clone encoded a new protein with a unique SH2 domain as GRB-7. To obtain a full length DNA clone, the T7 (.lambda.EXlox) library was plated in an E. coli strain without T7 polymerase gene and routine DNA hybridization performed with a 700 base pair EcoR1 fragment from the GRB-7 clone using standard published techniques (Ausubel et al eds., Current Protocols in Molecular Biology, Wiley Interscience, New York, (1987, 1992)). Several overlapping clones were identified which were used for DNA sequencing to obtain the full length GRB-7 protein sequence shown in FIG. 19. A schematic representation of GRB-7 is displayed in FIG. 20 depicting the regions of similarity to known proteins as discussed below. The protein is 535 amino acids in length and has one SH2 domain at its extreme carboxy-terminus. In FIG. 21, the SH2 domain of GRB-7 is compared to other SH2 domains including mouse fyn, human PLC-.gamma.1 and the crk and nck-like proteins we cloned in this project. One interesting aspect is that GRB-7 has an isoleucine at amino acid 448, whereas other SH2 domains have a leucine at this position. To look for other protein motifs in GRB-7, a sequence of 433 amino acids from GRB-7 which excluded the SH2 domain was used to scan the Swissprot and GenEmbl databases, as described herein. Amino acids 242 to 339 of GRB-7, showed similarity to a sequence from the central region of ras GAP. Over this region of 91 amino acids from ras GAP, GRB-7 has 26% identity and 42% similarity allowing for conservative substitutions (FIG. 22). This region of ras GAP lies between the SH2/SH3 domains and he GTPase activating carboxyterminal region and has not been assigned a specific function. The amino-terminal sequence of GRB-7 was found to be proline rich and thus has similarity to many other proline rich proteins. GRB-7 does have an extended region of limited similarity to the catalytic domain of protein phosphatase 2B including this proline rich region (FIG. 23) but no significant similarity was found to other serine/threonine phosphatase such as protein phosphatase 1 or 2A.
A northern blot of GRB-7 in mouse tissues is presented in FIG. 24. Oligo dt selected mRNA was probed with the same EcoR1 fragment used to isolate full length GRB-7. See Ausubel et al eds., Current Protocols in Molecular Biology, Wiley Interscience, New York, (1987, 1992) and Sap et al Proc. Natl. Acad. Sci. USA 87:6112 (1990). The mRNA was extracted from six week old mice tissues by known methods, e.g., as described by Sap et al Proc. Natl. Acad. Sci. USA 87:6112 (1990). Approximately 3 .mu.g was run on a 1.2% agarose formaldehyde gel and blotted to nytran (Schleicher and Scheull). The blot was probed with a DNA fragment that encodes amino acids 297 to 515 and labelled with .sup.32 P-dCTP using a random priming labeling kit (U.S. Biochemical). Blots were probed in 0.5M sodium phosphates pH 7.2, 7% sodium dodecyl sulfate and 1 mM EDTA at 65.degree. C. overnight. Blots were washed in 40 mM sodium phosphate, pH 7.2, 1% SDS and 1 mM EDTA at 65.degree. C. After exposure of the GRB-7 blot for 4 days, blots were stripped and reprobed with actin (exposure 36 hours). The highest signal was detected in liver and kidney, but was also detected in ovary and testes. On longer exposure, a weak signal was detected in lung.
All references cited herein, including journal articles or abstracts, published or corresponding U.S. or foreign patent applications, issued U.S. or foreign patents, or any other references, are entirely incorporated by reference herein, including all data, tables, figures, and text presented in the cited references. Additionally, the contents of the references cited within the references cited herein are also entirely incorporated by reference.
Reference to known method steps, conventional methods steps, known methods or conventional methods is not in any way an admission that any aspect, description or embodiment of the present invention is disclosed, taught or suggested in the relevant art.
The foregoing description of the specific embodiments will so fully reveal the general nature of the invention that others can, by applying knowledge within the skill of the art (including the contents of the references cited herein), readily modify and/or adapt for various applications such specific embodiments, without undue experimentation, without departing from the generic concept of the present invention. Therefore, such adaptations and modifications are intended to be comprehended within the meaning and range of equivalents of the disclosed embodiments, based on the teaching and guidance presented herein. It is to be understood that the phraseology or terminology herein is for the purpose of description and not of limitation, such that the terminology or phraseology of the present specification is to be interpreted by the skilled artisan in light of the teachings and guidance presented herein.
__________________________________________________________________________SEQUENCE LISTING(1) GENERAL INFORMATION:(iii) NUMBER OF SEQUENCES: 16(2) INFORMATION FOR SEQ ID NO:1:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 3372 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:TACAACC AGGCTCAACTGTTGCATGGTAGCAGATTTGCAAACATGAGTGCTGAGGGGTAC60CAGTACAGAGCGCTGTATGATTATAAAAAGGAAAGAGAAGAAGATATTGACTTGCACTTG120GGTGACATATTGACTGTGAATAAAGGGTCCTTAGTAGCTCTTGGATTCAGTG ATGGACAG180GAAGCCAGGCCTCGAAGAAATGGCTGGTTAAATGGCTATAATGAAACCACAGGGGAAAAG240GGGGACTTTCCGGGAACTTACGTAGAATATATTGGAAGGAAAAAAATCTCGCCTCCCACA300CCAAAGCCCCGGCCACCTCGGCCTCTTCCT GTTGCACCAGGTTCTTCGAAAACTGAAGCA360GATGTTGAACAACAAGCTTTGACTCTCCCGGATCTTGCAGAGCAGTTTGCCCCTCCTGAC420ATTGCCCCGCCTCTTCTTATCAAGCTCGTGGAAGCCATTGAAAAGAAAGGTCTGGAATGT480TCAACTC TATACAGAACACAGAGCTCCAGCAACCTGGCAGAATTACGACAGCTTCTTGAT540TGTGATACACCCTCCGTGGACTTGGAAATGATCGATGTGCACGTTTTGGCTGACGCTTTC600AAACGCTATCTCCTGGACTTACCAAATCCTGTCATTCCAGCAGCCGTTTACA GTGAAATG660ATTTCTTTAGCTCCAGAAGTACAAAGCTCCGAAGAATATATTCAGCTATTGAAGAAGCTT720ATTAGGTCGCCTAGCATACCTCATCAGTATTGGCTTACGCTTCAGTATTTGTTAAAACAT780TTCTTCAAGCTCTCTCAAACGTCCAGCAAA AATCTGTTGAATGCAAGAGTACTCTCTGAA840ATTTTCAGCCCTATGCTTTTCAGATTCTCAGCAGCCAGCTCTGATAATACTGAAAACCTC900ATAAAAGTTATAGAAATTTTAATCTCAACTGAATGGAATGAACGACAGCCTGCACCAGCA960CTGCCTC CTAAACCACCAAAACCTACTACTGTAGCCAACAACGGTATGAATAACAATATG1020TCCTTACAAAATGCTGAATGGTACTGGGGAGATATCTCGAGGGAAGAAGTGAATGAAAAA1080CTTCGAGATACAGCAGACGGGACCTTTTTGGTACGAGATGCGTCTACTAAAA TGCATGGT1140GATTATACTCTTACACTAAGGAAAGGGGGAAATAACAAATTAATCAAAATATTTCATCGA1200GATGGGAAATATGGCTTCTCTGACCCATTAACCTTCAGTTCTGTGGTTGAATTAATAAAC1260CACTACCGGAATGAATCTCTAGCTCAGTAT AATCCCAAATTGGATGTGAAATTACTTTAT1320CCAGTATCCAAATACCAACAGGATCAAGTTGTCAAAGAAGATAATATTGAAGCTGTAGGG1380AAAAAATTACATGAATATAACACTCAGTTTCAAGAAAAAAGTCGAGAATATGATAGATTA1440TATGAAG AATATACCCGCACATCCCAGGAAATCCAAATGAAAAGGACAGCTATTGAAGCA1500TTTAATGAAACCATAAAAATATTTGAAGAACAGTGCCAGACCCAAGAGCGGTACAGCAAA1560GAATACATAGAAAAGTTTAAACGTGAAGGCAATGAGAAAGAAATACAAAGGA TTATGCAT1620AATTATGATAAGTTGAAGTCTCGAATCAGTGAAATTATTGACAGTAGAAGAAGATTGGAA1680GAAGACTTGAAGAAGCAGGCAGCTGAGTATCGAGAAATTGACAAACGTATGAACAGCATT1740AAACCAGACCTTATCCAGCTGAGAAAGACG AGAGACCAATACTTGATGTGGTTGACTCAA1800AAAGGTGTTCGGCAAAAGAAGTTGAACGAGTGGTTGGGCAATGAAAACACTGAAGACCAA1860TATTCACTGGTGGAAGATGATGAAGATTTGCCCCATCATGATGAGAAGACATGGAATGTT1920GGAAGCA GCAACCGAAACAAAGCTGAAAACCTGTTGCGAGGGAAGCGAGATGGCACTTTT1980CTTGTCCGGGAGAGCAGTAAACAGGGCTGCTATGCCTGCTCTGTAGTGGTGGACGGCGAA2040GTAAAGCATTGTGTCATAAACAAAACAGCAACTGGCTATGGCTTTGCCGAGC CCTATAAC2100TTGTACAGCTCTCTGAAAGAACTGGTGCTACATTACCAACACACCTCCCTTGTGCAGCAC2160ACCGACTCCCTCAATGTCACACTAGCCTACCCAGTATATGCACAGCAGAGGCGATGAAGC2220GCTTACTCTTTGATCCTTCTCCTGAAGTTC AGCCACCCTGAGGCCTCTGGAAAGCAAAGG2280GCTCCTCTCCAGTCTGATCTGTGAATTGAGCTGCAGAAACGAAGCCATCTTTCTTTGGAT2340GGGACTAGAGCTTTCTTTGACAAAAAAGAAGTAGGGGAAGACATGCAGCCTAAGGCTGTA2400TGATGAC CACACGTTCCTAAGCTGGAGTGCTTATCCCTTCTTTTTCTTTTTTTCTTTGGT2460TTAATTTAAAGCCACAACCACATACAACACAAAGAGAAAAAGAAATGCAAAAATCTCTGC2520GTGCAGGGACAAAGAGGCCTTTAACCATGGTGCTTGTTAATGCTTTCTGAAG CTTTACCA2580GCTGAAAGTTGGGACTCTGGAGAGCGGAGGAGAGAGAGGCAGAAGAACCCTGGCCTGAGA2640AGGTTTGGTCCAGCCTGGTTTAGCCTGGATGTTGCTGTGCACGGTGGACCCAGACACATC2700GCACTGTGGATTATTTCATTTTGTAACAAA TGAACGATATGTAGCAGAAAGGCACGTCCA2760CTCACAAGGGACGCTTTGGGAGAATGTCAGTTCATGTATGTTCAGAAGAAATTCTGTCAT2820AGAAAGTGCCAGAAAGTGTTTAACTTGTCAAAAAACAAAAACCCAGCAACAGAAAAATGG2880AGTTTGG AAAACAGGACTTAAAATGACATTCAGTATATAAAATATGTACATAATATTGGA2940TGACTAACTATCAAATAGATGGATTTGTATCAATACCAAATAGCTTCTGTTTTGTTTTGC3000TGAAGGCTAAATTCACAGCGCTATGCAATTCTTAATTTTCATTAAGTTGTTA TTTCAGTT3060TTAAATGTACCTTCAGAATAAGCTTCCCCACCCCAGTTTTTGTTGCTTGAAAATATTGTT3120GTCCCGGATTTTTGTTAATATTCATTTTTGTTATCCTTTTTTAAAAATAAATGTACAGGA3180TGCCAGTAAAAAAAAAAATGGCTTCAGAAT TAAAACTATGAAATATTTTACAGTTTTTCT3240TGTACAGAGTACTTGCTGTTAGCCCAAGGTTAAAAAGTTCATAACAGATTTTTTTTGGAC3300TGTTTTGTTGGGCAGTGCCTGATAAGCTTCAAAGCTGCTTTATTCAATAAAAAAAAAACC3360CGAATTC ACTGG3372(2) INFORMATION FOR SEQ ID NO:2:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 1072 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:GCCAG TGAATTCGGGGGCTCAGCCCTCCTCCCTCCCTTCCCCCTGCTTCAGGCTGCTGAG60CACTGAGCAGCGCTCAGAATGGAAGCCATCGCCAAATATGACTTCAAAGCTACTGCAGAC120GACGAGCTGAGCTTCAAAAGGGGGGACATCCTCAAGGTTTTGAACGAAGA ATGTGATCAG180AACTGGTACAAGGCAGAGCTTAATGGAAAAGACGGCTTCATTCCCAAGAACTACATAGAA240ATGAAACCACATCCGTGGTTTTTTGGCAAAATCCCCAGAGCCAAGGCAGAAGAAATGCTT300AGCAAACAGCGGCACGATGGGGCCTTTC TTATCCGAGAGAGTGAGAGCGCTCCTGGGGAC360TTCTCCCTCTCTGTCAAGTTTGGAAACGATGTGCAGCACTTCAAGGTGCTCCGAGATGGA420GCCGGGAAGTACTTCCTCTGGGTGGTGAAGTTCAATTCTTTGAATGAGCTGGTGGATTAT480CACAG ATCTACATCTGTCTCCAGAAACCAGCAGATATTCCTGCGGGACATAGAACAGGTG540CCACAGCAGCCGACATACGTCCAGGCCCTCTTTGACTTTGATCCCCAGGAGGATGGAGAG600CTGGGCTTCCGCCGGGGAGATTTTATCCATGTCATGGATAACTCAGACCC CAACTGGTGG660AAAGGAGCTTGCCACGGGCAGACCGGCATGTTTCCCCGCAATTATGTCACCCCCGTGAAC720CGGAACGTCTAAGAGTCAAGAAGCAATTATTTAAAGAAAGTGAAAAATGTAAAACACATA780CAAAAGAATTAAACCCACAAGCTGCCTC TGACAGCAGCCTGTGAGGGAGTGCAGAACACC840TGGCCGGGTCACCCTGTGACCCTCTCACTTTGGTTGGAACTTTAGGGGGTGGGAGGGGGC900GTTGGATTTAAAAATGCCAAAACTTACCTATAAATTAAGAAGAGTTTTTATTACAAATTT960TCACT GCTGCTCCTCTTTCCCCTCCTTTGTCTTTTTTTTCATCCTTTTTTCTCTTCTGTC1020CATCAGTGCATGACGTTTAAGGCCACGTATAGTCCTAGCTGACGCCAATAAT1072(2) INFORMATION FOR SEQ ID NO:3:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 770 base pairs(B ) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:AGCCTGACACCGGAGCCGGTCCGCTGGGCGCGGGCGCCAGGGCTGGAGGGGCGCGCGTGC60CGGCGGCGGCCCAGCGTGAAAGCGCGGAGGCGGCCATGGCGGGCAACTT CGACTCGGAGG120AGCGGAGTAGCTGGTACTGGGGCCGCCTGAGCCGGCAGGAGGCGGTGGCGCTATTGCAGG180GCCAGCGCGACGGGGTGTTCCTGGTGCGGGACTCGAGCACCAGCCCCGGGGACTATGTGC240TTAGCGTCTCCGAAAACTCGCGCGTC TCCCACTACATCATCAACAGCAGCGGCCCGCGCC300CTCCAGTGCCTCCGTCGCCCGCTCAGCCTCCGCCGGGAGTGAGTCCCTCCAGGCTCCGAA360TAGGAGATCAAGAATTTGATTCATTGCCTGCTTTACTGGAATTCTACAAAATACACTATT420TGG ACACTACAACATTGATAGAACCAGTGGCCAGATCAAGGCAGGGTAGTGGAGTGATTC480TCAGGCAGGAGGAGGCAGAGTATGTGCGGGCCCTCTTTGACTTTAATGGGAATGATGAAG540AAGATCTTCCCTTTAAGAAAGGAGACATCCTGAGAATCCGGGATAAGCC TGAAGAGCAGT600GGTGGAATGCAGAGGACAGCGAAGGAAAGAGGGGGATGATTCCTGTCCCTTACGTGGAGA660AGTATAGACCTGCCTCCGCCTCAGTATCGGCTCTGATTGGAGGTAACCAGGAGGGTTCCC720ACCCACAGCCACTGGGTGGCCGGAGC CTGGGCCCTATGCCAACCCAGCGT770(2) INFORMATION FOR SEQ ID NO:4:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 642 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:GTGATTGAGAAGCCGGAGAATGAC CCTGAATGGTGGAAATGCAAAAATGCCCGAGGCCAA60GTGGGCCTGGTCCCCAAAAACTACGTGGTTGTTCTCAGTGATGGGCCTGCTCTGCACCCC120GCTCACACCCCCCAGATCAGCTACACCGGGCCTTCAGCCAGCGGGCGCTTTGCTGGTCGG180G AGTGGTACTATGGCAACGTGACACGGCACCAGGCCGAGTGTGCGCTCAATGAGCGGGGC240GTCGAGGGCGACTTCCTCATTAGGGACAGCGAGTCCTCGCCCAGTGACTTCTCCGTGTCT300CTCAAAGCGTCAGGGAGAAACAAGCACTTCAAGGTGCAGCTGGTGGA CAGCGTCTACTGC360ATTGGGCAGCGGCGGTTCCACAGCATGGACGAGCTTGTGGAGCACTACAAGAAGGCCCCC420ATCTTCACCAGCGAGCACGGGGAGAAGCTCTACCTTGTCCGAGCCCTACAGTGAAAGCAG480CCATTGGCCCCCTCATGCCCTGCC CACTGTGGGCCTCGCTGCCACCTCTGCCTCCCAGAG540CCCAGCACTTCTGGCCACCTCCACCCATGTGGCTTGGATCACCTCTGTGGCCCAGTCTGT600CCTTTCTTTTTCAGCCCTGTTGGTCAACCACGGCTACCTAGG642 (2) INFORMATION FOR SEQ ID NO:5:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 724 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: protein(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:MetSerAlaGluGlyTyrGlnTyrArgAlaLeuTyrAspTyrLysLys 151015GluArgGluGluAspIleAspLeuHisLeuGlyAspIleLeuThrVal202530As nLysGlySerLeuValAlaLeuGlyPheSerAspGlyGlnGluAla354045ArgProArgArgAsnGlyTrpLeuAsnGlyTyrAsnGluThrThrGly 505560GluLysGlyAspPheProGlyThrTyrValGluTyrIleGlyArgLys65707580LysIleSe rProProThrProLysProArgProProArgProLeuPro859095ValAlaProGlySerSerLysThrGluAlaAspValGluGlnGlnAla 100105110LeuThrLeuProAspLeuAlaGluGlnPheAlaProProAspIleAla115120125ProProLeu LeuIleLysLeuValGluAlaIleGluLysLysGlyLeu130135140GluCysSerThrLeuTyrArgThrGlnSerSerSerAsnLeuAlaGlu145 150155160LeuArgGlnLeuLeuAspCysAspThrProSerValAspLeuGluMet165170175IleAspV alHisValLeuAlaAspAlaPheLysArgTyrLeuLeuAsp180185190LeuProAsnProValIleProAlaAlaValTyrSerGluMetIleSer 195200205LeuAlaProGluValGlnSerSerGluGluTyrIleGlnLeuLeuLys210215220LysLeuIleArgSerPr oSerIleProHisGlnTyrTrpLeuThrLeu225230235240GlnTyrLeuLeuLysHisPhePheLysLeuSerGlnThrSerSerLys 245250255AsnLeuLeuAsnAlaArgValLeuSerGluIlePheSerProMetLeu260265270PheArgPhe SerAlaAlaSerSerAspAsnThrGluAsnLeuIleLys275280285ValIleGluIleLeuIleSerThrGluTrpAsnGluArgGlnProAla290 295300ProAlaLeuProProLysProProLysProThrThrValAlaAsnAsn305310315320GlyMetAsnAsn AsnMetSerLeuGlnAsnAlaGluTrpTyrTrpGly325330335AspIleSerArgGluGluValAsnGluLysLeuArgAspThrAlaAsp 340345350GlyThrPheLeuValArgAspAlaSerThrLysMetHisGlyAspTyr355360365ThrLeuThrLeuA rgLysGlyGlyAsnAsnLysLeuIleLysIlePhe370375380HisArgAspGlyLysTyrGlyPheSerAspProLeuThrPheSerSer38539 0395400ValValGluLeuIleAsnHisTyrArgAsnGluSerLeuAlaGlnTyr405410415AsnProLysLe uAspValLysLeuLeuTyrProValSerLysTyrGln420425430GlnAspGlnValValLysGluAspAsnIleGluAlaValGlyLysLys435 440445LeuHisGluTyrAsnThrGlnPheGlnGluLysSerArgGluTyrAsp450455460ArgLeuTyrGluGluTyrThr ArgThrSerGlnGluIleGlnMetLys465470475480ArgThrAlaIleGluAlaPheAsnGluThrIleLysIlePheGluGlu 485490495GlnCysGlnThrGlnGluArgTyrSerLysGluTyrIleGluLysPhe500505510LysArgGluGly AsnGluLysGluIleGlnArgIleMetHisAsnTyr515520525AspLysLeuLysSerArgIleSerGluIleIleAspSerArgArgArg530 535540LeuGluGluAspLeuLysLysGlnAlaAlaGluTyrArgGluIleAsp545550555560LysArgMetAsnSerI leLysProAspLeuIleGlnLeuArgLysThr565570575ArgAspGlnTyrLeuMetTrpLeuThrGlnLysGlyValArgGlnLys58 0585590LysLeuAsnGluTrpLeuGlyAsnGluAsnThrGluAspGlnTyrSer595600605LeuValGluAspAspGl uAspLeuProHisHisAspGluLysThrTrp610615620AsnValGlySerSerAsnArgAsnLysAlaGluAsnLeuLeuArgGly625630 635640LysArgAspGlyThrPheLeuValArgGluSerSerLysGlnGlyCys645650655TyrAlaCysSerVal ValValAspGlyGluValLysHisCysValIle660665670AsnLysThrAlaThrGlyTyrGlyPheAlaGluProTyrAsnLeuTyr675 680685SerSerLeuLysGluLeuValLeuHisTyrGlnHisThrSerLeuVal690695700GlnHisThrAspSerLeuAsnVal ThrLeuAlaTyrProValTyrAla705710715720GlnGlnArgArg(2) INFORMATION FOR SEQ ID NO:6:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 801 amino acids(B) TYPE: amino acid(C ) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: protein(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:MetGluAlaIleAlaLysTyrAspPheLysAlaThrAlaAspAspGlu151015LeuSe rPheLysArgGlyAspIleLeuLysValLeuAsnGluGluCys202530AspGlnAsnTrpTyrLysAlaGluLeuAsnGlyLysAspGlyPheIle 354045ProLysAsnTyrIleGluMetLysProHisProTrpPhePheGlyLys505560IleProArgAlaLysAl aGluGluMetLeuSerLysGlnArgHisAsp65707580GlyAlaPheLeuIleArgGluSerGluSerAlaProGlyAspPheSer 859095LeuSerValLysPheGlyAsnAspValGlnHisPheLysValLeuArg100105110AspGlyAlaG lyLysTyrPheLeuTrpValValLysPheAsnSerLeu115120125AsnGluLeuValAspTyrHisArgSerThrSerValSerArgAsnGln130 135140GlnIlePheLeuArgAspIleGluGlnValProGlnGlnProThrTyr145150155160ValGlnAlaLeuPh eAspPheAspProGlnGluAspGlyGluLeuGly165170175PheArgArgGlyAspPheIleHisValMetAspAsnSerAspProAsn 180185190TrpTrpLysGlyAlaCysHisGlyGlnThrGlyMetPheProArgAsn195200205TyrValThrProVal AsnArgAsnValCysAlaAlaAlaAlaGlyAla210215220AlaThrThrAlaAlaAlaCysCysCysAlaCysAlaAlaGlyCysThr225230 235240GlyCysCysThrCysThrGlyAlaCysAlaGlyCysAlaGlyCysCys245250255ThrGlyThrGly AlaGlyGlyGlyAlaGlyThrGlyCysAlaGlyAla260265270AlaCysAlaCysCysGlyThrThrThrThrCysThrThrAlaAlaThr275 280285ThrThrGlyGlyGlyThrGlyThrThrCysGlyAlaCysGlyGlyAla290295300GlyAlaCysThrGlyThrCysG lyThrCysGlyGlyAlaCysAlaCys305310315320ThrCysCysCysThrCysAlaCysGlyThrCysThrThrGlyThrGly32 5330335GlyThrGlyGlyCysCysGlyGlyGlyThrCysAlaCysCysCysThr340345350GlyThrGlyAlaCy sCysCysThrCysThrCysAlaCysThrThrThr355360365GlyGlyThrThrGlyGlyAlaAlaCysThrThrThrAlaGlyGlyGly370 375380GlyGlyThrGlyGlyGlyAlaGlyGlyGlyGlyGlyCysAlaCysCys385390395400GlyGlyCysCysCysAla GlyThrGlyGlyGlyAlaCysAlaCysThr405410415GlyGlyGlyAlaGlyAlaGlyThrGlyAlaAlaAlaCysCysAlaAla420 425430CysCysThrThrGlyAlaAlaAlaThrCysCysCysCysCysAlaCys435440445CysCysThrCysCysCys CysCysGlyGlyThrThrGlyGlyAlaThr450455460ThrThrAlaAlaAlaAlaAlaThrGlyCysCysAlaAlaAlaAlaCys465470 475480ThrThrAlaCysCysThrAlaThrAlaAlaAlaThrThrAlaAlaGly485490495AlaAlaGlyAlaGlyT hrThrThrThrThrAlaThrThrAlaCysAla500505510AlaAlaThrThrThrCysAlaAlaCysCysThrAlaAlaAlaThrThr515 520525ThrThrThrAlaCysGlyGlyThrThrThrThrGlyAlaAlaThrGly530535540GlyAlaThrAlaThrThrThrAlaAl aThrThrCysThrThrCysThr545550555560CysAlaAlaAlaAlaAlaThrAlaAlaThrGlyThrThrThrAlaAla565 570575AlaThrCysAlaCysThrGlyCysThrGlyCysThrCysCysThrCys580585590ThrThrThrCysCysCys CysThrCysCysThrThrThrGlyThrCys595600605ThrThrThrThrThrThrThrThrCysAlaThrCysCysThrThrThr610 615620ThrThrThrCysThrCysThrThrCysThrGlyThrCysAlaGlyThr625630635640GlyAlaCysGlyAlaCysGly AlaGlyGlyAlaGlyAlaAlaAlaGly645650655GlyGlyGlyAlaGlyGlyAlaAlaAlaCysAlaGlyAlaAlaAlaAla660 665670AlaAlaAlaAlaGlyThrAlaGlyGlyAlaAlaAlaAlaAlaAlaGly675680685AlaGlyAlaAlaGlyAlaCysA laGlyCysAlaThrCysAlaGlyThr690695700GlyCysAlaThrGlyAlaCysGlyThrThrThrAlaAlaGlyGlyCys705710 715720CysAlaCysGlyThrAlaThrAlaGlyThrCysCysThrAlaGlyCys725730735ThrGlyAlaCysGlyCysCy sAlaAlaThrAlaAlaThrGlyThrAla740745750GlyThrCysAlaCysGlyThrAlaCysThrGlyCysAlaAlaAlaThr755 760765ThrCysCysGlyGlyThrGlyCysAlaThrAlaThrCysAlaGlyGly770775780AlaThrCysGlyAlaCysThrGlyCysGly GlyThrThrAlaThrThr785790795800Ala(2) INFORMATION FOR SEQ ID NO:7:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 2345 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:CTCTCTCTCTCTCTCTCTCTCCCTCTCTCCTAGCACCTGCTGCTCAGTAGGAAGGGCAAG60AGCAATTCGAGGCCGGTGCATTGTGAGGAGTCTCCACCCCTCCTCCTGCGCTTCCTTCTC120CAGGGAGCCTCTCA GGCCGCCCTCACCTGCCCGAGATAATTTTAGTTTCCCTGGGCCTGG180AATCTGGATACGCAGGGCCTCGCTCTATATTCTCCCGCCTCAACATTCCAAAGGCGGGAT240AGCCTTTCTACCATCTGTAGAGAAGAGAGAAAGGATTCGAAATCAAATCCAAGTGTCTGG 300GATCTCTAGACAGAGCCAGACTTTGGGCCGGGTGTCCGGCTCCTTCTGTTGGAGGTGCTC360CAGGTGCCATGGAACTGGATCTGAGCCCGACTCATCTCAGCAGCTCCCCAGAAGATGTGT420GCCCAACTCCTGCTACCCCTCCTGAGACTCCTCCGCC CCCTGATAACCCTCCGCCAGGGG480ATGTGAAGCGGTCGCAGCCTTTGCCCATCCCCAGCAGCAGGAAACTTCGAGAAGAGGAGT540TTCAGGCAACCTCTCTGCCCTCCATCCCCAACCCCTTCCCTGAGCTCTGCAGCCCACCTT600CACAGAAACCCATT CTTGGTGGTTCCTCCGGTGCAAGGGGGTTGCTTCCTCGAGACTCCA660GCCGCCTCTGTGTGGTGAAGGTGTACAGTGAGGATGGGGCCTGCCGGTCTGTGGAGGTGG720CAGCGGGCGCCACAGCTCGTCACGTGTGTGAGATGCTGGTACAACGAGCTCACGCCCTGA 780GCGACGAGAGCTGGGGACTAGTGGAATCCCACCCCTACCTGGCACTGGAGCGGGGTCTGG840AGGACCATGAATTTGTGGTGGAAGTGCAGGAGGCCTGGCCTGTGGGTGGAGATAGCCGCT900TCATCTTCCGTAAAAACTTCGCCAAGTATGAACTATT CAAGAGCCCCCCACACACCCTGT960TTCCAGAAAAGATGGTCTCGAGCTGTCTGGATGCACAAACAGGCATATCCCATGAAGACC1020TCATCCAGAACTTCCTGAACGCTGGCAGCTTCCCTGAGATCCAGGGCTTCCTGCAGCTGC1080GGGGATCAGGCCGG GGGTCAGGTCGAAAGCTTTGGAAACGTTTCTTCTGCTTTCTGCGTC1140GATCTGGCCTCTACTACTCTACCAAGGGTACCTCCAAGGACCCCAGACACCTACAGTATG1200TGGCAGATGTGAATGAGTCCAATGTCTATGTGGTGACCCAGGGCCGCAAGCTGTATGGGA 1260TGCCCACTGACTTCGGCTTCTGTGTCAAGCCCAACAAGCTTCGAAACGGCCACAAGGGGC1320TCCACATCTTCTGCAGTGAGGATGAGCAGAGTCGGACCTGCTGGCTGGCTGCCTTCCGGC1380TCTTCAAGTACGGGGTACAGCTATATAAGAATTATCA GCAGGCCCAGTCTCGTCACCTGC1440GCCTATCCTATTTGGGGTCTCCACCCTTGAGGAGCGTCTCAGACAATACCCTAGTGGCTA1500TGGACTTCTCTGGCCATGCGGGGCGTGTCATTGATAACCCCCGGGAAGCTCTGAGTGCCG1560CCATGGAGGAGGCC CAGGCCTGGAGGAAGAAGACAAACCACCGTCTGAGCCTGCCCACCA1620CATGCTCTGGCTCGAGCCTCAGCGCAGCCATTCATCGCACCCAGCCCTGGTTTCATGGAC1680GCATCTCTCGGGAGGAGAGCCAGCGGCTAATTGGACAGCAGGGCCTGGTGGATGGTGTGT 1740TCCTGGTCCGGGAGAGCCAGAGGAACCCACAGGGCTTTGTCCTGTCCTTGTGCCATCTGC1800AGAAAGTCAAGCATTATCTCATTTTGCCAAGTGAAGATGAAGGTTGCCTTTACTTCAGCA1860TGGATGAGGGCCAGACCCGTTTCACAGACCTGCTGCA GCTGGTAGAATTCCACCAGCTGA1920ACCGAGGCATCCTGCCCTGCCTGCTGCGCCACTGCTGTGCCCGTGTGGCCCTCTGAGGCC1980GCACAAGCTACTGCAGCCATGGGTTTGCCTACCACCCTTCTGTCCTGTGGACTCGGTGCA2040GGTGGGTGGGGTGG TAAACAGTGGAAGAGCTCCCCCCCCCAATTTTATCCCATTTTTTTT2100AACCTCTCTCAACCAGTGAAACATCCCCTAACCCTGTCCATCCCTGACTCCTGTCCCCAA2160GGGAGGCATTGTGGTCCTGTCCCCTTGGTAGAGCTCCTGAGGTACTGTTCCAGTGAGGGG 2220CATTATGAGAGGAGCGGGGCAGCCCAGGAGGTCTCATACCCCACCCATAATCTGTACAGA2280CTGAGAGGCCAGTTGATCTGCTCTGTTTTATACCAGTAACAATAAAGATTATTTTTTGAT2340ACAAA 2345(2) INFORMATION FOR SEQ ID NO:8:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 256 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: protein(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:ProAspThrGlyAlaGlyProLeuGl yAlaGlyAlaArgAlaGlyGly151015AlaArgValProAlaAlaAlaGlnArgGluSerAlaGluAlaAlaMet20 2530AlaGlyAsnPheAspSerGluGluArgSerSerTrpTyrTrpGlyArg354045LeuSerArgGlnGluAlaValAlaLeuLe uGlnGlyGlnArgAspGly505560ValPheLeuValArgAspSerSerThrSerProGlyAspTyrValLeu657075 80SerValSerGluAsnSerArgValSerHisTyrIleIleAsnSerSer859095GlyProArgProProValProProSerPr oAlaGlnProProProGly100105110ValSerProSerArgLeuArgIleGlyAspGlnGluPheAspSerLeu115120 125ProAlaLeuLeuGluPheTyrLysIleHisTyrLeuAspThrThrThr130135140LeuIleGluProValAlaArgSerArgGlnGlySerGly ValIleLeu145150155160ArgGlnGluGluAlaGluTyrValArgAlaLeuPheAspPheAsnGly165170 175AsnAspGluGluAspLeuProPheLysLysGlyAspIleLeuArgIle180185190ArgAspLysProGluGluGlnTrpTrpAsn AlaGluAspSerGluGly195200205LysArgGlyMetIleProValProTyrValGluLysTyrArgProAla210215 220SerAlaSerValSerAlaLeuIleGlyGlyAsnGlnGluGlySerHis225230235240ProGlnProLeuGlyGlyArgSerLeuGlyProM etProThrGlnArg245250255(2) INFORMATION FOR SEQ ID NO:9:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 157 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:ValIleGluLysProGluAsnAspProGluTrpTrpLysCysLysAsn151015AlaArgGlyGlnValGlyLeuValProLysAsnTy rValValValLeu202530SerAspGlyProAlaLeuHisProAlaHisThrProGlnIleSerTyr3540 45ThrGlyProSerAlaSerGlyArgPheAlaGlyArgGluTrpTyrTyr505560GlyAsnValThrArgHisGlnAlaGluCysAlaLeuAsnGluArgGl y65707580ValGluGlyAspPheLeuIleArgAspSerGluSerSerProSerAsp8590 95PheSerValSerLeuLysAlaSerGlyArgAsnLysHisPheLysVal100105110GlnLeuValAspSerValTyrCysIleGlyGlnArgArgP heHisSer115120125MetAspGluLeuValGluHisTyrLysLysAlaProIlePheThrSer130135140 GluHisGlyGluLysLeuTyrLeuValArgAlaLeuGln145150155(2) INFORMATION FOR SEQ ID NO:10:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 535 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:MetGluLeuAspLeuSerProThrHisLeuSerSerSerProGluAsp151015ValCysProThrProAlaThr ProProGluThrProProProProAsp202530AsnProProProGlyAspValLysArgSerGlnProLeuProIlePro35 4045SerSerArgLysLeuArgGluGluGluPheGlnAlaThrSerLeuPro505560SerIleProAsnProPheProGluLeuCysSer ProProSerGlnLys65707580ProIleLeuGlyGlySerSerGlyAlaArgGlyLeuLeuProArgAsp85 9095SerSerArgLeuCysValValLysValTyrSerGluAspGlyAlaCys100105110ArgSerValGluValAlaAlaGlyAla ThrAlaArgHisValCysGlu115120125MetLeuValGlnArgAlaHisAlaLeuSerAspGluSerTrpGlyLeu130135 140ValGluSerHisProTyrLeuAlaLeuGluArgGlyLeuGluAspHis145150155160GluPheValValGluValGlnGluAlaTrp ProValGlyGlyAspSer165170175ArgPheIlePheArgLysAsnPheAlaLysTyrGluLeuPheLysSer1801 85190ProProHisThrLeuPheProGluLysMetValSerSerCysLeuAsp195200205AlaGlnThrGlyIleSerHisGluAspLeuI leGlnAsnPheLeuAsn210215220AlaGlySerPheProGluIleGlnGlyPheLeuGlnLeuArgGlySer225230235 240GlyArgGlySerGlyArgLysLeuTrpLysArgPhePheCysPheLeu245250255ArgArgSerGlyLeuTyrTyrSerThrLy sGlyThrSerLysAspPro260265270ArgHisLeuGlnTyrValAlaAspValAsnGluSerAsnValTyrVal275280 285ValThrGlnGlyArgLysLeuTyrGlyMetProThrAspPheGlyPhe290295300CysValLysProAsnLysLeuArgAsnGlyHisLysGly LeuHisIle305310315320PheCysSerGluAspGluGlnSerArgThrCysTrpLeuAlaAlaPhe325330 335ArgLeuPheLysTyrGlyValGlnLeuTyrLysAsnTyrGlnGlnAla340345350GlnSerArgHisLeuArgLeuSerTyrLeu GlySerProProLeuArg355360365SerValSerAspAsnThrLeuValAlaMetAspPheSerGlyHisAla370375 380GlyArgValIleAspAsnProArgGluAlaLeuSerAlaAlaMetGlu385390395400GluAlaGlnAlaTrpArgLysLysThrAsnHisA rgLeuSerLeuPro405410415ThrThrCysSerGlySerSerLeuSerAlaAlaIleHisArgThrGln420425 430ProTrpPheHisGlyArgIleSerArgGluGluSerGlnArgLeuIle435440445GlyGlnGlnGlyLeuValAspGlyValPheLeuVa lArgGluSerGln450455460ArgAsnProGlnGlyPheValLeuSerLeuCysHisLeuGlnLysVal465470475 480LysHisTyrLeuIleLeuProSerGluAspGluGlyCysLeuTyrPhe485490495SerMetAspGluGlyGlnThrArgPheThrAsp LeuLeuGlnLeuVal500505510GluPheHisGlnLeuAsnArgGlyIleLeuProCysLeuLeuArgHis515520 525CysCysAlaArgValAlaLeu530535(2) INFORMATION FOR SEQ ID NO:11:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 11 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: protein(ii ) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:GluGluGluGluGluTyrMetProMetXaaXaa1510(2) INFORMATION FOR SEQ ID NO:12:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 11 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single (D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:GluGluGluGluGluTyrValProMetXaaXaa1510(2) INFORMATION FOR SEQ ID NO:13:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 11 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:AspAspAspAspAspTyrMetProMetXaaXaa1510(2) INFORMATION FOR SEQ ID NO:14:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 11 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:AspAspAspAspAspTyrValProMetXaaXaa1510(2) INFORMATION FOR SEQ ID NO:15: (i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 4 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:IleGluGluArg(2) INFORMATION FOR SEQ ID NO:16:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 4 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:LeuValProArg1

Number	Date	Country
9008160	Jul 1990	WOX
9010234	Sep 1990	WOX

Expression-cloning method for identifying target proteins for eukaryotic tyrosine kinases and novel target proteins

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Non-Patent Literature Citations (27)

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Entry
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