Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-07-11
2002-11-26
Jones, W. Gary (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S006120, C435S069100, C435S091100, C435S091200, C536S023100, C536S024100, C530S350000
Reexamination Certificate
active
06485910
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to a mammalian cDNA which encodes a mammalian Ras association domain containing protein and to the use of the cDNA and the encoded protein in the diagnosis and treatment of cell proliferative and inflammatory disorders.
BACKGROUND OF THE INVENTION
Phylogenetic relationships among organisms have been demonstrated many times, and studies from a diversity of prokaryotic and eukaryotic organisms suggest a more or less gradual evolution of molecules, biochemical and physiological mechanisms, and metabolic pathways. Despite different evolutionary pressures, the proteins of nematode, fly, rat, and man have common chemical and structural is features and generally perform the same cellular function. Comparisons of the nucleic acid and protein sequences from organisms where structure and/or function are known accelerate the investigation of human sequences and allow the development of model systems for testing diagnostic and therapeutic agents for human conditions, diseases, and disorders.
Signal transduction is the general process by which cells respond to extracellular signals. In typical signal transduction pathways, binding of a signaling molecule such as a hormone, neurotransmitter, or growth factor to a cell membrane receptor is coupled to the action of an intracellular second messenger. G protein-coupled receptors (GPCRs) control intracellular processes through the activation of guanine nucleotide-binding proteins (G proteins). G proteins are heterotrimeric and consist of &agr;, &bgr;, and &ggr; subunits. The &agr; subunit contains a guanine nucleotide binding domain and has GTPase activity. When GTP binds to the &agr; subunit, it dissociates from the &bgr; and &ggr; subunits and interacts with cellular target molecules. Hydrolysis of GTP to GDP serves as a molecular switch controlling the interactions of the &agr; subunit with other proteins. The GDP bound form of the &agr; subunit dissociates from its cellular target and reassociates with the &bgr; and &ggr; subunits. A number of accessory proteins modulate G protein function by controlling their nucleotide state or membrane association. These regulatory molecules include exchange factors (GEFs) which stimulate GDP-GTP exchange, GTPase activating proteins (GAPs) which promote GTP hydrolysis, and guanine nucleotide dissociation inhibitors (GDIs) which inhibit guanine nucleotide dissociation and stabilize the GDP-bound form. G proteins can be classified into at least five subfamilies: Ras, Rho, Ran, Rab, and ADP-ribosylation factor, and they regulate various cell functions including cell growth and differentiation, cytoskeletal organization, and intracellular vesicle transport and secretion.
The Ras subfamily transduces signals from tyrosine kinase receptors, non-tyrosine kinase receptors, and heterotrimeric GPCRs (Fantl et al. (1993) Annu Rev Biochem 62:453-481; Woodrow et al. (1993) J Immunol 150:3853-3861; and Van Corven et al. (1993) Proc Natl Acad Sci 90:1257-1261). Stimulation of cell surface receptors activates Ras which, in turn, activates cytoplasmic kinases that control cell growth and differentiation. The first Ras targets identified were the Raf kinases (Avruch et al. (1994) Trends Biochem Sci 19:279-283). Interaction of Ras and Raf leads to activation of the MAP kinase cascade of serine/threonine kinases, which activate key transcription factors that control gene expression and protein synthesis (Barbacid (1987) Ann Rev Biochem 56:779-827; Treisman (1994) Curr Opin Genet Dev 4:96-101). Mutant Ras proteins, which bind but do not hydrolyze GTP, are constitutively activated, and cause continuous cell proliferation and cancer (Bos (1989) Cancer Res 49:4682-4689; Grunicke and Maly (1993) 4:389-402).
Ras regulates other signaling pathways by direct interaction with different cellular targets (Katz and McCormick (1997) Curr Opin Genet Dev 7:75-79). One such target is Ral GDS, a guanine nucleotide dissociation stimulator for the Ras-like GTPase, Ral (Albright et al. (1993) EMBO J 12:339-347). Ral GDS couples the Ras and Ral signaling pathways. Epidermal growth factor (EGF) stimulates the association of Ral GDS with Ras in mammalian cells, which activates the GEF activity of Ral GDS (Kikuchi and Williams (1996) J Biol Chem 271:588-594; Urano et al. (1996) EMBO J 15:810-816). Ra1 activation by Ral GDS leads to activation of Src, a tyrosine kinase that phosphorylates other molecules including transcription factors and components of the actin cytoskeleton (Goi et al. (2000) EMBO J 19:623-630). Ral interacts with a number of signaling molecules including Ra1-binding protein, a GAP for the Rho-like GTPases; Cdc42 and Rac, which regulate cytoskeletal rearrangement; and phospholipase D1, which is involved in vesicular trafficking (Feig et al. (1996) Trends Biochem Sci 21:438-441; Voss et al. (1999) J Biol Chem 274:34691-34698).
Norel was identified from a yeast two-hybrid screen as a protein that interacts with Ras and Ras-related protein, Rap1b (Vavvas et al. (1998) J Biol Chem 273:5439-5442). It is a highly basic protein (pI=9.4) of 413 amino acids that contains a cysteine-histidine-rich region predicted to be a diacylglycerol/phorbol ester binding site, a proline-rich region at its N-terminus that may be an SH3 binding domain, and a Ras/Rap binding domain located at its C-terminus. Nore1 binds Ras in vitro in a GTP dependent manner. Experiments in vivo show that the association of Nore1 with Ras is dependent on EGF and 12-O-tetradecanoylphorbol-13-acetate activation in COS-7 cells and on EGF in KB cells.
Ras and other G proteins play roles in regulating the immune inflammatory response. Granulocytes, which include basophils, eosinophils, and neutrophils, play critical roles in inflammation. Eosinophils release toxic granule proteins, which kill microorganisms, and secrete prostaglandins, leukotrienes and cytokines, which amplify the inflammatory response. They sustain inflammation in allergic reactions and their malfunction can cause asthma and other allergic diseases. Interleukin-5 is a cytokine that regulates the growth, activation, and survival of eosinophils. The signal transduction mechanism of IL-5 in eosinophils involves the Ras-MAP kinase and Jak-Stat pathways (Pazdrak et al. (1995) J Exp Med 181:1827-1834; Adachi and Alam (1998) Am J Physiol 275:C623-633). Raf-1 kinase activation by Ras is implicated in eosinophil degranulation.
Neutrophils migrate to inflammatory sites where they eliminate pathogens by phagocytosis and release toxic products from their granules that kill microorganisms. G proteins, including Ras, Ral, Rac 1 and Rap1 regulate neutrophil function (M'Rabet et al. (1999) J Biol Chem 274:21847-21852). Rac1 may be involved in the respiratory burst of neutrophils. Ras and Rap1 are activated in response to the chemotactic agent, formyl methionine leucine phenylalanine (fMLP); the lipid mediator, platelet activating factor (PAF); and the cytokine, granulocyte-macrophage colony-stimulating factor (GM-CSF). Both Ras and Rap1 appear to play roles in neutrophil activation. Ral is activated by fMLP and PAF but not by GM-CSF and may be involved in chemotaxis, phagocytosis, or degranulation. Impairment of neutrophil function is associated with various inflammatory and autoimmune diseases.
The discovery of a mammalian cDNA encoding a new Ras target or effector satisfies a need in the art by providing compositions which are useful in the diagnosis and treatment of cell proliferative and inflammatory disorders.
SUMMARY OF THE INVENTION
The invention is based on the discovery of a mammalian cDNA which encodes a Ras association domain containing protein (RADCP) which is useful in the diagnosis and treatment of cell proliferative and inflammatory disorders, particularly thymus hyperplasia, allergies, asthma, and hypereosoinophilia.
The invention provides an isolated mammalian cDNA or a fragment thereof encoding a mammalian protein or a portion thereof selected from the group consisting of an amino acid sequence of SEQ ID NO:1, a variant having 80% identity to the ami
Klinger Tod M.
Krasnow Randi E.
Walker Michael G.
Incyte Genomics Inc.
Incyte Genomics, Inc.
Jones W. Gary
Taylor Janell E.
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