Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
1999-03-29
2002-05-21
Ulm, John (Department: 1648)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S252300, C435S320100, C530S350000, C530S388220, C536S023500
Reexamination Certificate
active
06391584
ABSTRACT:
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-&ggr; (PLC-&ggr;) 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 (1989); 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-&ggr;, GAP, and cellular tyrosine kinases such as pp60
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-&ggr; 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 receptors. SH2 domains are conserved sequences of about 100 amino acids found in cytoplasmic non-receptor tyrosine kinases such as pp6Osrc, PLC-&ggr;, 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 (
s
rc
h
omology
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. 10:435441 (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 carboxyterminal (C-terminal) fragment of the EGFR, which contains all the known autophosphorylation sites, binds specifically to the SH2 domains of GAP and PLC-&ggr; (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 lines 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 carboxyterminus (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 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, GRB-7 or GRB-10, because of their lack of similarity at the DNA level.
Another object of the present invention is to provide a method for identifying compounds that disrupt or inhibit the interaction between activated tyrosine kinase molecules and those proteins (e. adaptor proteins) they bind.
The methods of the present invention take advantage of the discover that the C-terminus of the EGFR protein in which the tyrosine residues are phosphorylated can bind substrates as described herein. By
Margolis Benjamin L.
Schlessinger Joseph
Skolnik Edward Y.
New York University Medical Center
Pennie & Edmonds LLP
Ulm John
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