Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1999-10-13
2001-09-11
Mertz, Prema (Department: 1646)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S007200, C435S015000, C435S069100, C435S194000, C435S325000
Reexamination Certificate
active
06287784
ABSTRACT:
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-&bgr;'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 &ggr;-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 [&ggr;-
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
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 Biological Chemistry
267(24):17390-17396 [1992]), is measured.
The discovery of anti-phosphotyrosine antibodies has provided a non-radioactive, alternative means for measuring phosphorylation of tyrosine residues. For example, White and Backer (
Methods in Enzymology
201:65-67 [1991]) mention polyclonal antibodies which selectively bind to phosphotyrosine and are considered to be useful for studying rPTKs. An anti-phosphotyrosine monoclonal antibody was used in one of the assays referred to in Madden et al. (
Anal Biochem
199:210-215 [1991]), which measured phosphatase activity toward the insulin receptor. Anti-phosphotyrosine antibodies were also used by Cleaveland et al., in their protein tyrosine kinase ELISA assay. See Cleaveland et al.,
Analytical Biochemistry
190:249-253 (1990). The method of Cleaveland et al. utilizes purified high-activity oncogene tyrosine kinases, v-src and v-fps, and measures the ability of these tyrosine kinases to phosphorylate synthetic polymeric substrates which are coated on an ELISA microtiter plate. The phosphotyrosine produced by src-induced phosphorylation of the polymeric substrate is then quantitated by addition of an anti-phosphotyrosine antibody, the presence of which is detected using a second rabbit anti-mouse antibody which is linked to a reporter enzyme, horseradish peroxidase (HRPO). A similar ELISA assay has been developed by Lazaro et al., which is used for detection of a protein tyrosine kinase. See Lazaro et al.,
Analytical Biochemistry
192:257-261 (1991). Like the assay of Cleaveland et al., this assay also measures the ability of a protein tyrosine kinase to phosphorylate a synthetic substrate which is bound to microELISA wells.
A direct way to assess specific activation of rPTKs is by analysis of receptor autophosphorylation. See, e.g., Hunter and Cooper
Ann Rev Biochem
54:897-930 (1985) and Ullrich and Schlessinger,
Cell
61:203-212 (1990). Using this direct approach, Knutson and Suck disclose assays for measuring autophosphorylation of the insulin receptor under in situ or in vitro conditions (
Archives of Biochemistry and Biophysics
285(2):197-204 [1991]). In the in situ assay, monolayer cultures of embryonic mouse 3T3-C2 fibroblasts (having the endogenous insulin receptor) are incubated with insulin in large cell culture dishes. Following incubation, the insulin receptor is extracted from the membranes. To achieve extraction of the insulin receptor, the cell monolayers are scraped into a buffer containing protease inhibitors and the cells are then disrupted in a homogenizer. The cellular homogenate is subsequently subjected to centrifugation for 60 min., and the pellet which forms is extracted into buffer containing detergent. Following a further centrifugation step, the supernatant (containing the insulin receptor) is incubated with an anti-insulin receptor antibody. Then, the receptor-antibody complex is incubated with protein A-agarose and unoccupied protein A sites are blocked with normal rabbit IgG. The agarose beads are then centrifuged, the supernatants aspirated and the pellets are re-suspended in buffer containing the radiolabelled anti-phosphotyrosine antibody. The amount of bound iodinated anti-phosphotyrosine antibody is consequently measured.
Klein and his colleagues discuss an assay for measuring insulin activation of the insulin receptor (Klein et al.,
Diabetes
42:883-890 [1993]). In this assay, aliquots of a heterogeneous population of mononuclear blood cells (including T cells, B cells, macrophages etc) having the insulin receptor are exposed to insulin in centrifuge tubes. The cells are then lysed in detergent using a motordriven homogenizer and the lysates are concentrated two- to fourfold using vacuum centrifugation. Sometimes, the insulin receptor is also partially purified using wheat germ agglutin agarose. The supernatants which form following centrifugation, are then transferred to anti-i
Godowski Paul J.
Mark Melanie R.
Sadick Michael Daniel
Wong Wai Lee Tan
Carpenter David A.
Genentech Inc.
Mertz Prema
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