Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Patent
1995-01-20
2000-02-15
Teng, Sally P.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
435 15, 435 691, 435194, 435325, C12Q 148
Patent
active
06025145&
DESCRIPTION:
BRIEF SUMMARY
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to a kinase receptor activation (KIRA) assay. In particular, the invention relates to an assay for measuring autophosphorylation of the kinase domain of a receptor protein tyrosine kinase (rPTK) using a kinase receptor activation, enzyme-linked immunosorbent assay (KIRA ELISA).
2. Description of Related Art
One mechanism for signal transduction in animals involves protein phosphorylation. Protein phosphorylation involves the action of protein kinase, an enzyme that transfers a phosphate group from a phosphate donor onto an acceptor amino acid in a substrate protein. Protein phosphatases provide a means for reversing the signal when the stimulus is removed.
Protein kinases have multiple substrates, and classification of the protein kinases is based on the acceptor amino acid specificity. The two most well characterized protein kinases are the protein kinases with a protein alcohol group as acceptor called protein serine/threonine kinases and the protein kinases with a protein phenolic group as acceptor called protein tyrosine kinases (Hunter, Methods in Enzymology 200: 3-9 [1991]).
The most well known type of signal-transducing protein kinases are growth factor receptor protein tyrosine kinases (rPTKs). rPTKs usually comprise a large, glycosylated, extracellular ligand binding domain (ECD) and an intracellular domain (ICD) which contains a tyrosine kinase catalytic domain. A single hydrophobic transmembrane (TM) domain connects the ECD and ICD. Examples of rPTKs include the insulin receptor, epidermal growth factor receptor (EGF-R), platelet-derived growth factor receptor (PDGF-R), insulin-like growth factor 1 receptor (IGF-1-R), and the HER2 receptor, to name a few. See, for example, Ullrich and Schlessinger Cell 61: 203-212 (1990) and Fantl et al., Annu. Rev. Biochem. 62: 453-481 (1993). rPTKs can phosphorylate exogenous protein substrates and intrinsic tyrosine residues via their catalytic tyrosine kinase domain. The intrinsic tyrosine residues normally reside in the ICD of the rPTK (see FIG. 1 herein). Activation of the intracellular kinase domain of rPTKs appears to be mediated by receptor oligomerization which results from the conformational alteration of the ECD upon ligand binding thereto. See Ullrich and Schlessinger, supra.
Serine-threonine kinases have also been disclosed in the literature. While most of the known protein serine-threonine kinases are cytoplasmic proteins, a family of mammalian transmembrane receptors with serine-threonine kinase domains has recently been found. Members of this receptor family have been described as binding TGF-.beta.'s and activin. For reviews of serine-threonine kinases, see Sale, G., Biochem. Soc. Transactions 20: 664-670 (1992); ten Dijke et al., Prog. in Growth Factor Res. 5: 55-72 (1994); and Mathews, L., Endoc. Rev. 15(3): 310-325 (1994).
Various assays have been developed which measure tyrosine kinase activity. Some of these assays measure the ability of a tyrosine kinase enzyme to phosphorylate a synthetic substrate polypeptide. For example, an assay has been developed which measures growth factor-stimulated tyrosine kinase activity by measuring the ability of the kinase to catalyze the transfer of the .gamma.-phosphate of ATP to a suitable acceptor substrate. See Pike, L., Methods of Enzymology 146: 353-362 (1987) and Hunter, Journal of Biological Chemistry 257(9): 4843-4848 (1982), for example. In this assay, the use of [.gamma.-.sup.32 P]ATP permits the radioactive labeling of the phosphorylated substrate, which is a synthetic tyrosine-containing peptide. Others have described protein kinase assays wherein incorporation of .sup.32 P into a tyrosine kinase receptor, such as the EGF receptor (see Donato et al., Cell Growth Differ. 3: 259-268 [1992]), insulin receptor (see Kasuga et al., Journal of Biological Chemistry 257(17): 9891-9884 [1982] and Kasuga et al. , Methods in Enzymology 109: 609-621 [1985]), and liver growth hormone receptor (see Wang et al., Journal of Biologica
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Godowski Paul J.
Mark Melanie R.
Sadick Michael Daniel
Wong Wai Lee Tan
Genentech Inc.
Kresnak Mark T.
Silva Robin M.
Teng Sally P.
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