WO2003000901A2 - Acides nucleiques codant des proteines kinases - Google Patents

Acides nucleiques codant des proteines kinases Download PDF

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WO2003000901A2
WO2003000901A2 PCT/IB2002/002358 IB0202358W WO03000901A2 WO 2003000901 A2 WO2003000901 A2 WO 2003000901A2 IB 0202358 W IB0202358 W IB 0202358W WO 03000901 A2 WO03000901 A2 WO 03000901A2
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protein kinase
nucleic acid
polypeptide
agent
kinase gene
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PCT/IB2002/002358
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WO2003000901A3 (fr
WO2003000901A8 (fr
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Roger A. Moraga Martinez
Gunnar Thor Sigurdsson
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Decode Genetics Ehf.
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Publication of WO2003000901A8 publication Critical patent/WO2003000901A8/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • Protein kinases are enzymes that catalyze the phosphorylation of target protein substrates.
  • the phosphorylation is usually a transfer reaction of a phosphate group from ATP to the protein substrate.
  • At least 400 enzymes have been identified as protein kinases.
  • the specific structure in the target substrate to which the phosphate is transferred is frequently a tyrosine, serine or threonine residue, and such protein kinase enzymes are commonly referred to as tyrosine kinases or serine/threonine kinases.
  • Another class of protein kinases phosphorylates histidine residues. Most protein kinases recognize a simple amino acid motif associated with a nearby residue.
  • the phosphorylation reactions, and counteracting phosphatase reactions are involved in countless cellular processes that underlie responses to diverse intracellular signals (typically mediated through cellular receptors), regulation of cellular functions, and activation or deactivation of cellular processes.
  • a cascade of protein kinases often participate in intracellular signal transduction and are necessary for the realization of these cellular processes. Because of their ubiquity in these processes, the protein kinases can be found as an integral part of the plasma membrane or as cytoplasmic enzymes or localized in the nucleus, often as components of enzyme complexes. In many instances, these protein kinases are an essential element of enzyme and structural protein complexes that determine where and when a cellular process occurs within a cell.
  • Protein kinases are regulated by a variety of mechanisms. Some protein kinases are constitutively active, while others are activated by the binding of a messenger (c AMP -dependent protein kinase, e.g. , protein kinase A; cGMP-dependent protein kinase; protein kinase C). Some are activated by calcium and
  • calmodulin e.g. , Ca /calmodulin-dependent protein kinase II, phosphorylase kinase, myosin light chain kinase, elongation factor 2 kinase. Protein kinases that are activated by such a messenger tend to regulate most or all of the intracellular responses to the messenger.
  • protein kinases are activated by an extracellular signal, and are also called the receptor protein kinases (e.g., polypeptide hormones receptors, growth factors, insulin, epidermal growth factor, platelet-derived growth factor).
  • the catalytic domain of such protein kinases is also the cytoplasmic domain of a transmembrane receptor, the the extracellular domain binds an extracellular messenger. Binding to the domain activates the protein kinase inside the membrane.
  • protein kinases are in turn regulated by other protein kinases, and multiple such relationships between protein kinases can exist, forming a protein kinase cascade, in which each protein kinase phosphorylates the next.
  • Such kinases include, e.g. , c AMP-dependent protein kinase, AMP-activated protein kinase, MAP kinase kinase kinase, MAP kinase kinase, MAP kinase and S6 kinase.
  • the present invention relates to human protein kinase genes, particularly nucleic acids comprising protein kinase genes, and the amino acids encoded by such nucleic acids. These genes are shown in Tables I and II and the sequence listing. In Tables I and II, each kinase entry lists the name (e.g., "MOOSE03013"), the University of California at Santa Cruz contig designation from which the sequence was analyzed (e.g., "ctgl4435”), and the exon locations (e.g., "1134827 . . 1135076, . .”).
  • Sub-family information on the sequences is shown in Table III. For each sequence, the following information is provided: the University of California at Santa Cruz contig designation from which the sequence was analyzed (e.g.,
  • the isolated nucleic acid molecule comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-80 (odd numbers), as shown in Tables I and II, and the complements thereof.
  • the invention further relates to a nucleic acid molecule which hybridizes under high stringency conditions to a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-80 (odd numbers), as shown in Tables I and II, and the complements thereof.
  • the invention additionally relates to isolated nucleic acid molecules (e.g., cDNA molecules) encoding a protein kinase polypeptide (e.g., encoding a polypeptide selected from the group consisting of SEQ ID NOs: 1-80 (even numbers).
  • isolated nucleic acid molecules e.g., cDNA molecules
  • a protein kinase polypeptide e.g., encoding a polypeptide selected from the group consisting of SEQ ID NOs: 1-80 (even numbers).
  • the invention further provides a method for assaying a sample for the presence of a nucleic acid molecule comprising all or a portion of a protein kinase in a sample, comprising contacting said sample with a second nucleic acid molecule comprising a nucleotide sequence encoding a protein kinase polypeptide (e.g., one of SEQ ED NOs: 1-80 (even numbers, or the complement of one of SEQ ID NOs: 1- 80 (even numbers); a nucleotide sequence encoding one of SEQ JJ) NOs: 1-80 (odd numbers), or a fragment or derivative thereof, under conditions appropriate for selective hybridization.
  • a nucleotide sequence encoding a protein kinase polypeptide e.g., one of SEQ ED NOs: 1-80 (even numbers, or the complement of one of SEQ ID NOs: 1- 80 (even numbers); a nucleotide sequence encoding one of SEQ JJ) NOs
  • the invention additionally provides a method for assaying a sample for the level of expression of a protein kinase polypeptide, or fragment or derivative thereof, comprising detecting (directly or indirectly) the level of expression of the protein kinase polypeptide, fragment or derivative thereof.
  • the invention also relates to a vector comprising an isolated nucleic acid molecule of the invention operatively linked to a regulatory sequence, as well as to a recombinant host cell comprising the vector.
  • the invention also provides a method for preparing a polypeptide encoded by an isolated nucleic acid molecule described herein (a protein kinase polypeptide), comprising culturing a recombinant host cell of the invention under conditions suitable for expression of said nucleic acid molecule.
  • the invention further provides an isolated polypeptide encoded by isolated nucleic acid molecules of the invention (e.g., protein kinase polypeptide), as well as fragments or derivatives thereof.
  • the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-80 (even numbers).
  • the invention also relates to an isolated polypeptide comprising an amino acid sequence which is greater than about 90 percent identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-80 (even numbers), preferably about 95, 96, 97, 98 or 99 percent identical.
  • the invention also relates to an antibody, or an antigen-binding fragment thereof, which selectively binds to a polypeptide of the invention, as well as to a method for assaying the presence of a polypeptide encoded by an isolated nucleic acid molecule of the invention in a sample, comprising contacting said sample with an antibody which specifically binds to the encoded polypeptide.
  • the invention further relates to methods of diagnosing a predisposition to a condition mediated by a protein kinase.
  • the methods of diagnosing such a predisposition in an individual include detecting the presence of a mutation in a protein kinase, as well as detecting alterations in expression of a protein kinase polypeptide, such as the presence of different splicing variants of protein kinase polypeptides.
  • the alterations in expression can be quantitative, qualitative, or both quantitative and qualitative.
  • the invention additionally relates to an assay for identifying agents that alter
  • a cell, cellular fraction, or solution containing a protein kinase polypeptide or a fragment or derivative thereof can be contacted with an agent to be tested, and the level of protein kinase polypeptide expression or activity can be assessed.
  • the activity or expression of more than one protein kinase polypeptide can be assessed concurrently (e.g., the cell, cellular fraction, or solution can contain more than one type of protein kinase polypeptide, such as different splicing variants, and the levels of the different polypeptides or splicing variants can be assessed).
  • the invention relates to assays to identify polypeptides which interact with one or more protein kinase polypeptides.
  • a first vector is used which includes a nucleic acid encoding a DNA binding domain and also a protein kinase polypeptide, splicing variant, or fragment or derivative thereof
  • a second vector is used which includes a nucleic acid encoding a transcription activation domain and also a nucleic acid encoding a polypeptide which potentially may interact with the protein kinase polypeptide, splicing variant, or fragment or derivative thereof (e.g., a protein kinase polypeptide binding agent or receptor).
  • Incubation of yeast containing both the first vector and the second vector under appropriate conditions allows identification of polypeptides which interact with the protein kinase polypeptide or fragment or derivative thereof, and thus can be agents which alter the activity of expression of a protein kinase polypeptide.
  • Agents that enhance or inhibit protein kinase polypeptide expression or activity are also included in the current invention, as are methods of altering (enhancing or inhibiting) protein kinase polypeptide expression or activity by contacting a cell containing protein kinase and/or polypeptide, or by contacting the protein kinase polypeptide, with an agent that enhances or inhibits expression or activity of protein kinase or polypeptide.
  • the invention pertains to pharmaceutical compositions comprising the nucleic acids of the invention, the polypeptides of the invention, and/or the agents that alter activity of protein kinase polypeptide.
  • the invention further pertains to methods of treating conditions mediated by protein kinases, by administering protein kinase therapeutic agents, such as nucleic acids of the invention, polypeptides of the invention, the agents that alter activity of protein kinase polypeptide, or compositions comprising the nucleic acids, polypeptides, and/or the agents that alter activity of protein kinase polypeptide.
  • the present invention relates to nucleic acids comprising protein kinases, and the protein kinase amino acids encoded by those nucleic acids.
  • Protein kinases are involved in regulation of cellular functions, and activation or deactivation of cellular processes, and many of the cellular processes in response to intracellular signals. Cascades of protein kinases often participate in intracellular signal transduction and are necessary for the realization of these cellular processes.
  • Protein kinases are the major modes through which extracellular signal molecules produce intracellular effects. They are also involved in stress responses, and protect the cell against changes in metabolite levels that would otherwise be toxic to the cell. They are also the major way in which mechanisms such as DNA synthesis and mitosis are timed.
  • the receptor tyrosine kinases comprise a large family of transmembrane receptors with diverse biological activities. At least nineteen distinct receptor tyrosine kinase subfamilies have been identified. This family includes receptors that are crucial for the growth and differentiation of a variety of cell types (Yarden and Ullrich, Ann. Rev. Biochem. 57:433-478 (1988); Ullrich and Schlessinger, Cell 61 :243-254 (1990)). The intrinsic function of receptor tyrosine kinases is activated upon ligand binding, which results in phosphorylation of the receptor and multiple cellular substrates, and subsequently in a variety of cellular responses (Ullrich & Schlessinger, Cell 61:203-212 (1990)).
  • receptor tyrosine kinase-mediated signal transduction is initiated by extracellular interaction with a specific growth factor (ligand), typically followed by receptor dimerization, stimulation of the intrinsic protein tyrosine kinase activity and receptor trans-phosphorylation.
  • ligand a specific growth factor
  • Binding sites are thereby created for intracellular signal transduction molecules and lead to the formation of complexes with a spectrum of cytoplasmic signaling molecules that facilitate the appropriate cellular response (e.g., cell division, differentiation, metabolic effects, changes in the extracellular microenvironment).
  • cytoplasmic signaling molecules e.g., cell division, differentiation, metabolic effects, changes in the extracellular microenvironment.
  • receptor tyrosine kinases such as FGFR-1, PDGFR, TIE-2 and c-Met, and growth factors that bind them, have been suggested to play a role in angiogenesis, although some may promote angiogenesis indirectly (Mustonen and Alitalo, J. Cell Biol. 129:895-898 (1995)).
  • FLK-1 fetal liver kinase 1
  • KDR kinase insert domain- containing receptor
  • VEGFR-2 vascular endothelial cell growth factor receptor 2
  • the murine version of FLK-l/VEGFR-2 has also been called NYK (Oelrichs et al, Oncogene 8(1):11-15 (1993)).
  • VEGF and FLK-1 /KDR/VEGFR-2 are a ligand-receptor pair that play an important role in the proliferation of vascular endothelial cells, and formation and sprouting of blood vessels, termed vasculo genesis and angiogenesis, respectively.
  • Protein tyrosine kinases catalyse the phosphorylation of specific tyrosine residues in cellular proteins, and this post-translational modification of these substrate proteins, which can be enzymes themselves, acts as a molecular switch regulating cell proliferation, activation or differentiation (for review, see
  • Angiogenesis-mediated diseases include, but are not limited to, cancers, solid tumors, blood-born tumors (e.g., leukemias), tumor metastasis, benign tumors (e.g., hemangiomas, acoustic neuromas, neurofibromas, trachomas, and pyogenic granulomas), infantile hemangiomas, rheumatoid arthritis, psoriasis, ocular angiogenic diseases (e.g., diabetic retinopathy, retinopathy of prematurity, choroidal neovascularization due to age-related macular degeneration, corneal graft rejection, neovascular glaucoma, retrolental fibroplasia, rubeosis), Osier- Webber Syndrome, myocardial angiogenesis, plaque neovascularization, telangiectasia, hemophiliac joints, angiofibroma, and wound granulation.
  • benign tumors e.
  • G protein-coupled receptor superfamily Some intracellular protein kinases specifically interact with various members of the G protein-coupled receptor superfamily, and are able to desensitize them and thereby weaken the signal or prevent it from being effected.
  • the G protein-coupled receptors are involved in the transmission of signals that originate from low molecular weight ligands such as adrenaline or from peptide ligands such as chemokines and a variety of hormones such as melanocyte stimulating hormone (MSH). So far, six of these kinases have been discovered (GRKl through GRK6). Some of the GRKs are restricted to a small number of tissues (e.g., GRK-1), while GRK2 and 3, known also as ⁇ ARKl and ⁇ ARK2, are ubiquitously expressed. A comprehensive review is provided, for example, by M. Bunemann and M.M. Hosey,
  • G-Protein Coupled Receptor Kinases as Modulators of G-Protein Signalling
  • Methods and substances that modulate (increase or decrease) the function of 7TM receptors could be used to treat a wide variety of diseases and conditions associated with 7TM receptor function.
  • G protein-coupled receptors for instance, are involved in an enormous range of biological processes, and have been found to regulate such processes as hydrolysis
  • the GPCR superfamily includes the receptors for many important signaling pathways, including, but not limited to, hormone receptors, growth factors, viral receptors, neuroreceptors, etc., such as acetylcholine, adrenocorticotropin (ACTH), adenosine, ⁇ -adrenergic receptors, ⁇ -adrenergic receptors, angiotensin, bombesin, bradykinin, C5a, calcitonin, cAMP, cannabinoid, C-C chemokine, cholecystokinin/gastrin (CCK/gastrin), cytomegalovirus, dopamine, endothelial differentiation gene-1, endothelin, formyl peptide, glutamate (metabotropic), gonadotropin-releasing hormone, growth hormone-releasing hormone, histamine, 5- hydroxytryptamine, interleukin-8, kinin, luteinizing hormone/follicle-stimulating hormone/t
  • These receptors are involved in the treatment of infections and various diseases and conditions, including, but not limited to, bacterial, fungal, protozoan and viral infections, particularly infections caused by HJV-1 or HIV-2; cancers; diabetes; asthma; Parkinson's disease; both acute and congestive heart failure; hypotension; hypertension; urinary retention; osteoporosis; angina pectoris; myocardial infarction; ulcers; asthma; allergies; benign prostatic hypertrophy (benign prostatic hyperplasia); chronic renal failure; renal disease; impaired glucose tolerance; seizure disorder; depression; anxiety; obsessive compulsive disorder; affective neurosis/disorder; depressive neurosis/disorder; anxiety neurosis; dysthymic disorder; behavior disorder; mood disorder; shizophrenia; psychosexual dysfunction; sex disorder; sexual disorder; disturbed biological and circadian rhythms; feeding disorders, such as anorexia, bulimia, cachexia, and obesity; Cushing's syndrome/disease; basophil a
  • HMMs Hidden Markov Models
  • ClustalW Thimpson et al, Nucleic Acids Res. 22:4673-4680 (1994) package to measure the distance between different sequences.
  • DiAlign 2 Merge-Met al. 15(3):211-8(1999).
  • DiAlign works based on segment-to-segment comparisons instead of arbitrary thresholds for gap opening and extension, which makes it ideally suited for building models that represent an entire, full-length sequence, since the alignments built this way have more match states that would be assigned as insertion states when using other alignment algorithms.
  • the models were built using the standard HMMer package. To search for new genes, a genome-wide scan was done on the University of
  • a classification was obtained in which the sequences are grouped by length and similarity. Each one of these groups was then used to build a HMM profile representing this group of sequences.
  • This approach aims to have models that can represent the full length of the encoded proteins for a whole range of proteins, without being too specific for any one of them or being too general, as would be a HMM built for large groups of sequences.
  • This classification was based either on existing expert-supervised classifications, or by retrieval of sequences and classification based on pairwise alignment distances.
  • a number of the genes were linked with markers known to be associated with human diseases genes. These are shown in Table JV.
  • the diseases were linked to the HMM genes in the following manner: (1) the HMM gene models were compared to the consensus of the human genome sequence, located and the results kept in a relational database; (2) all possible markers (Sequence Tagged Sites (STS's)) (public or deCODE genetics) are also located in the same consensus using ePCR or BLAT and results kept in a relational database; and (3) LOD scores for diseases are linked to markers. A span of one LOD drop around the marker was also given. A computer program takes each LOD peak and links it to the consensus through the markerhit in the database. The database is then queried for all HMM genes within the span of one LOD drop or a minimum of 15 Mb in each direction from the marker. The output is the name of the peak marker and its distance to the HMM gene.
  • protein kinase refers to an isolated nucleic acid molecule selected from the group consisting of SEQ ID NOs: 1-80 (odd numbers), and also to a portion or fragment of the isolated nucleic acid molecule (e.g., cDNA or the gene) that encodes protein kinase polypeptide (e.g., a polypeptide selected from the group consisting of SEQ JD NOs: 1-80 (even numbers)).
  • the isolated nucleic acid molecule comprises a nucleic acid molecule selected from the group consisting of SEQ ID NOs: 1-80 (odd numbers) or the complement of such a nucleic acid molecule.
  • the isolated nucleic acid molecules of the present invention can be RNA, for example, mRNA, or DNA, such as cDNA and genomic DNA.
  • DNA molecules can be double-stranded or single-stranded; single stranded RNA or DNA can be either the coding, or sense, strand or the non-coding, or antisense, strand.
  • the nucleic acid molecule can include all or a portion of the coding sequence of the gene and can further comprise additional non-coding sequences such as introns and non-coding 3' and 5' sequences (including regulatory sequences, for example). Additionally, the nucleic acid molecule can be fused to a marker sequence, for example, a sequence that encodes a polypeptide to assist in isolation or purification of the polypeptide. Such sequences include, but are not limited to, those that encode a glutathione-S- transferase (GST) fusion protein and those that encode a hemagglutinin A (HA) polypeptide marker from influenza.
  • GST glutathione-S- transferase
  • HA hemagglutinin A
  • an "isolated" nucleic acid molecule is one that is separated from nucleic acids that normally flank the gene or nucleotide sequence (as in genomic sequences) and/or has been completely or partially purified from other transcribed sequences (e.g., as in an RNA library).
  • an isolated nucleic acid of the invention may be substantially isolated with respect to the complex cellular milieu in which it naturally occurs, or culture medium when produced by recombinant techniques, or chemical precursors or other chemicals when chemically synthesized. In some instances, the isolated material will form part of a composition
  • an isolated nucleic acid molecule comprises at least about 50, 80 or 90% (on a molar basis) of all macromolecular species present.
  • the term "isolated” also can refer to nucleic acid molecules that are separated from the chromosome with which the genomic DNA is naturally associated.
  • the isolated nucleic acid molecule can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of nucleotides which flank the nucleic acid molecule in the genomic DNA of the cell from which the nucleic acid molecule is derived.
  • nucleic acid molecule can be fused to other coding or regulatory sequences and still be considered isolated.
  • recombinant DNA contained in a vector is included in the definition of "isolated” as used herein.
  • isolated nucleic acid molecules include recombinant DNA molecules in heterologous host cells, as well as partially or substantially purified DNA molecules in solution.
  • isolated nucleic acid molecules also encompass in vivo and in vitro RNA transcripts of the DNA molecules of the present invention.
  • An isolated nucleic acid molecule or nucleotide sequence can include a nucleic acid molecule or nucleotide sequence that is synthesized chemically or by recombinant means. Therefore, recombinant DNA contained in a vector is included in the definition of "isolated” as used herein.
  • isolated nucleotide sequences include recombinant DNA molecules in heterologous organisms, as well as partially or substantially purified DNA molecules in solution. In vivo and in vitro RNA transcripts of the DNA molecules of the present invention are also encompassed by "isolated" nucleotide sequences.
  • Such isolated nucleotide sequences are useful in the manufacture of the encoded polypeptide, as probes for isolating homologous sequences (e.g., from other mammalian species), for gene mapping (e.g., by in situ hybridization with chromosomes), or for detecting expression of the gene in tissue (e.g., human tissue), such as by Northern blot analysis.
  • homologous sequences e.g., from other mammalian species
  • gene mapping e.g., by in situ hybridization with chromosomes
  • tissue e.g., human tissue
  • the present invention also pertains to nucleic acid molecules which are not necessarily found in nature but which encode a protein kinase polypeptide (e.g., a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOS: 1-80 (even numbers)), or another splicing variant of a protein kinase polypeptide or polymo ⁇ hic variant thereof.
  • a protein kinase polypeptide e.g., a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOS: 1-80 (even numbers)
  • DNA molecules which comprise a sequence that is different from the naturally-occurring nucleotide sequence but which, due to the degeneracy of the genetic code, encode a protein kinase polypeptide of the present invention are also the subject of this invention.
  • the invention also encompasses nucleotide sequences encoding portions (fragments), or encoding variant polypeptides such as analogues or derivatives of a protein kinase polypeptide.
  • variants can be naturally-occurring, such as in the case of allelic variation or single nucleotide polymorphisms, or non-naturally- occurring, such as those induced by various mutagens and mutagenic processes.
  • Intended variations include, but are not limited to, addition, deletion and substitution of one or more nucleotides that can result in conservative or non-conservative amino acid changes, including additions and deletions.
  • nucleotide (and/or resultant amino acid) changes are silent or conserved; that is, they do not alter the characteristics or activity of a protein kinase polypeptide.
  • nucleotide sequences are fragments that comprise one or more polymo ⁇ hic microsatellite markers.
  • nucleotide sequences are fragments that comprise one or more single nucleotide polymo ⁇ hisms in a protein kinase gene.
  • nucleic acid molecules of the invention can include, for example, labeling, methylation, intemucleotide modifications such as uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoamidates, carbamates), charged linkages (e.g., phosphorothioates, phosphorodithioates), pendent moieties (e.g., polypeptides), intercalators (e.g., acridine, psoralen), chelators, alkylators, and modified linkages (e.g., alpha anomeric nucleic acids).
  • synthetic molecules that mimic nucleic acid molecules in the ability to bind to designated sequences via hydrogen bonding and other chemical interactions. Such molecules include, for example, those in which peptide linkages substitute for phosphate linkages in the backbone of the molecule.
  • the invention also pertains to nucleic acid molecules that hybridize under high stringency hybridization conditions, such as for selective hybridization, to a nucleotide sequence described herein (e.g., nucleic acid molecules which specifically hybridize to a nucleotide sequence encoding polypeptides described herein, and, optionally, have an activity of the polypeptide).
  • the invention includes variants described herein which hybridize under high stringency hybridization conditions (e.g., for selective hybridization) to a nucleotide sequence comprising a nucleotide sequence selected from the group consisting of SEQ JD NOs: 1-80 (odd numbers).
  • the invention includes variants described herein which hybridize under high stringency hybridization conditions
  • nucleic acid molecules can be detected and/or isolated by specific hybridization (e.g., under high stringency conditions).
  • Specific hybridization refers to the ability of a first nucleic acid to hybridize to a second nucleic acid in a manner such that the first nucleic acid does not hybridize to any nucleic acid other than to the second nucleic acid (e.g., when the first nucleic acid has a higher similarity to the second nucleic acid than to any other nucleic acid in a sample wherein the hybridization is to be performed).
  • “Stringency conditions” for hybridization is a term of art which refers to the incubation and wash conditions, e.g., conditions of temperature and buffer concentration, which permit hybridization of a particular nucleic acid to a second nucleic acid; the first nucleic acid may be perfectly (i.e., 100%) complementary to the second, or the first and second may share some degree of complementarity which is less than perfect (e.g., 70%, 75%, 85%, 90%, 95%).
  • certain high stringency conditions can be used which distinguish perfectly complementary nucleic acids from those of less complementarity.
  • “High stringency conditions”, “moderate stringency conditions” and “low stringency conditions” for nucleic acid hybridizations are explained on pages 2.10.1-2.10.16 and pages 6.3.1 -6.3.6 in Current Protocols in Molecular
  • equivalent conditions can be determined by varying one or more of these parameters while maintaining a similar degree of identity or similarity between the two nucleic acid molecules.
  • conditions are used such that sequences at least about 60%, at least about 70%, at least about 80%, at least about 90% or at least about 95% or more identical to each other remain hybridized to one another.
  • washing conditions are described in Krause, M.H. and S.A. Aaronson, Methods in Enzymology 200:546-556 (1991), and in, Ausubel, et al, "Current Protocols in Molecular Biology", John Wiley & Sons, (1998), which describes the determination of washing conditions for moderate or low stringency conditions. Washing is the step in which conditions are usually set so as to determine a minimum level of complementarity of the hybrids. Generally, starting from the lowest temperature at which only homologous hybridization occurs, each °C by which the final wash temperature is reduced (holding SSC concentration constant) allows an increase by 1% in the maximum extent of mismatching among the sequences that hybridize.
  • the washing temperature can be determined empirically for high, moderate or low stringency, depending on the level of mismatch sought.
  • a low stringency wash can comprise washing in a solution containing 0.2X SSC/0.1% SDS for 10 minutes at room temperature;
  • a moderate stringency wash can comprise washing in a prewarmed solution (42°C) solution containing 0.2X SSC/0.1% SDS for 15 minutes at 42°C;
  • a high stringency wash can comprise washing in prewarmed (68°C) solution containing 0. IX SSC/0.1 %SDS for 15 minutes at 68°C.
  • washes can be performed repeatedly or sequentially to obtain a desired result as known in the art.
  • Equivalent conditions can be determined by varying one or more of the parameters given as an example, as known in the art, while maintaining a similar degree of identity or similarity between the target nucleic acid molecule and the primer or probe used.
  • the length of a sequence aligned for comparison pu ⁇ oses is at least 30%, preferably at least 40%, more preferably at least 60%, and even more preferably at least 70%, 80%, 90% or 95% of the length of the reference sequence.
  • the percent identity between two amino acid sequences can be accomplished using the GAP program in the GCG software using either a BLOSUM63 matrix or a PAM250 matrix, and a gap weight of 12, 10, 8, 6, or 4 and a length weight of 2, 3, or 4.
  • the percent identity between two nucleic acid sequences can be accomplished using the GAP program in the GCG software package, using a gap weight of 50 and a length weight of3.
  • the present invention also provides isolated nucleic acid molecules that contain a fragment or portion that hybridizes under highly stringent conditions to a nucleotide sequence comprising a nucleotide sequence selected from the group consisting of SEQ JD NOs: 1-80 (odd numbers), or the complement of such a sequence, and also provides isolated nucleic acid molecules that contain a fragment or portion that hybridizes under highly stringent conditions to a nucleotide sequence encoding an amino acid sequence selected SEQ JD NOs: 1-80 (even numbers), or polymo ⁇ hic variant thereof.
  • the nucleic acid fragments of the invention are at least about 15, preferably at least about 18, 20, 23 or 25 nucleotides, and can be 30, 40, 50, 100, 200 or more nucleotides in length. Longer fragments, for example, 30 or more nucleotides in length, which encode antigenic polypeptides described herein are particularly useful, such as for the generation of antibodies as described below.
  • nucleic acid fragments of the invention are used as probes or primers in assays such as those described herein.
  • Probes or “primers” are oligonucleotides that hybridize in a base-specific manner to a complementary strand of nucleic acid molecules.
  • probes and primers include polypeptide nucleic acids, as described in Nielsen et al, Science 254: 1497-1500 (1991).
  • a probe or primer comprises a region of nucleotide sequence that hybridizes to at least about 15, typically about 20-25, and more typically about 40, 50 or 75, consecutive nucleotides of a nucleic acid molecule comprising a contiguous nucleotide sequence selected from the group consisting of SEQ JD NOs:
  • a probe or primer comprises 100 or fewer nucleotides, preferably from 6 to 50 nucleotides, preferably from 12 to 30 nucleotides. In other embodiments, the probe or primer is at least
  • the probe or primer further comprises a label, e.g., radioisotope, fluorescent compound, enzyme, or enzyme co-factor.
  • nucleic acid molecules of the invention such as those described above can be identified and isolated using standard molecular biology techniques and the sequence information provided herein.
  • nucleic acid molecules can be amplified and isolated by the polymerase chain reaction using synthetic oligonucleotide primers designed based on one or more of the sequences selected from the group consisting of SEQ ID NOs: 1-80 (odd numbers), or the complement of such a sequence, or designed based on nucleotides based on sequences encoding one or more of the amino acid sequences provided herein. See generally PCR Technology: Principles and Applications for DNA Amplification (ed. H.A. Erlich,
  • the nucleic acid molecules can be amplified using cDNA, mRNA or genomic DNA as a template, cloned into an appropriate vector and characterized by DNA sequence analysis.
  • LCR ligase chain reaction
  • ssRNA single stranded RNA
  • dsDNA double stranded DNA
  • the amplified DNA can be radiolabelled and used as a probe for screening a cDNA library derived from human cells, mRNA in zap express, ZIPLOX or other suitable vector.
  • Corresponding clones can be isolated, DNA can obtained following in vivo excision, and the cloned insert can be sequenced in either or both orientations by art recognized methods to identify the correct reading frame encoding a polypeptide of the appropriate molecular weight.
  • the direct analysis of the nucleotide sequence of nucleic acid molecules of the present invention can be accomplished using well-known methods that are commercially available. See, for example, Sambrook et al, Molecular Cloning, A Laboratory Manual (2nd Ed., CSHP, New York 1989); Zyskind et al. , Recombinant DNA Laboratory Manual,
  • polypeptide and the DNA encoding the polypeptide can be isolated, sequenced and further characterized.
  • Antisense nucleic acid molecules of the invention can be designed using the nucleotide sequences of one or more of SEQ JD NOs: 1-80 (odd numbers) and/or the complement of one or more of SEQ ID NOs: 1-80 (odd numbers), and/or a portion of one or more of SEQ ID NOs: 1-80 (odd numbers), or the complement of one or more of SEQ ID NOs: 1-80 (odd numbers) and or a sequence encoding the amino acid sequences of one or more of SEQ JD NOs: 1-80 (even numbers), or encoding a portion of one or more of SEQ JD NOs: 1-80 (even numbers), and constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art.
  • an antisense nucleic acid molecule e.g., an antisense oligonucleotide
  • an antisense nucleic acid molecule can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used.
  • the antisense nucleic acid molecule can be produced biologically using an expression vector into which a nucleic acid molecule has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid molecule will be of an antisense orientation to a target nucleic acid of interest).
  • the isolated nucleic acid sequences of the invention can be used as molecular weight markers on Southern gels, and as chromosome markers that are labeled to map related gene positions.
  • the nucleic acid sequences can also be used to compare with endogenous DNA sequences in patients to identify one or more of the disorders described above, and as probes, such as to hybridize and discover related DNA sequences or to subtract out known sequences from a sample.
  • the nucleic acid sequences can further be used to derive primers for genetic finge ⁇ rinting, to raise anti-polypeptide antibodies using DNA immunization techniques, and as an antigen to raise anti-DNA antibodies or elicit immune responses.
  • nucleotide sequences identified herein can be used in numerous ways as polynucleotide reagents. For example, these sequences can be used to: (i) map their respective genes on a chromosome; and, thus, locate gene regions associated with genetic disease; (ii) identify an individual from a minute biological sample (tissue typing); and (iii) aid in forensic identification of a biological sample. Additionally, the nucleotide sequences of the invention can be used to identify and express recombinant polypeptides for analysis, characterization or therapeutic use, or as markers for tissues in which the corresponding polypeptide is expressed, either constitutively, during tissue differentiation, or in diseased states.
  • nucleic acid sequences can additionally be used as reagents in the screening and/or diagnostic assays described herein, and can also be included as components of kits (e.g., reagent kits) for use in the screening and/or diagnostic assays described herein.
  • kits e.g., reagent kits
  • nucleic acid constructs containing a nucleic acid molecule selected from the group consisting of SEQ ID NOs: 1-80 (odd numbers) and the complements thereof (or a portion thereof).
  • nucleic acid constructs containing a nucleic acid molecule encoding an amino acid sequence of SEQ JD NOs: 1-80 (even numbers) or polymo ⁇ hic variant thereof.
  • the constructs comprise a vector (e.g., an expression vector) into which a sequence of the invention has been inserted in a sense or antisense orientation.
  • vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • vector refers to a circular double stranded DNA loop into which additional DNA segments can be ligated.
  • viral vector Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome.
  • Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome.
  • certain vectors, expression vectors are capable of directing the expression of genes to which they are operably linked.
  • expression vectors of utility in recombinant DNA techniques are often in the form of plasmids.
  • the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses) that serve equivalent functions.
  • viral vectors e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses
  • recombinant expression vectors of the invention comprise a nucleic acid molecule of the invention in a form suitable for expression of the nucleic acid molecule in a host cell.
  • the recombinant expression vectors include one or more regulatory sequences, selected on the basis of the host cells to be used for expression, which is operably linked to the nucleic acid sequence to be expressed.
  • operably linked or “operatively linked” is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequence(s) in a manner which allows for expression of the nucleotide sequence (e.g., in an in vitro transcription translation system or in a host cell when the vector is introduced into the host cell).
  • regulatory sequence is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel, "Gene Expression Technology", Methods in Enzymology 185, Academic Press, San Diego, CA (1990).
  • Regulatory sequences include those which direct constitutive expression of a nucleotide sequence in many types of host cell and those which direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed and the level of expression of polypeptide desired.
  • the expression vectors of the invention can be introduced into host cells to thereby produce polypeptides, including fusion polypeptides, encoded by nucleic acid molecules as described herein.
  • the recombinant expression vectors of the invention can be designed for expression of a polypeptide of the invention in prokaryotic or eukaryotic cells, e.g., bacterial cells such as E. coli, insect cells (using baculovirus expression vectors), yeast cells or mammalian cells. Suitable host cells are discussed further in Goeddel, supra.
  • the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.
  • host cell and “recombinant host cell” are used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but also to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.
  • a host cell can be any prokaryotic or eukaryotic cell.
  • a nucleic acid molecule of the invention can be expressed in bacterial cells (e.g., E.
  • Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques.
  • transformation and “transfection” are intended to refer to a variety of art- recognized techniques for introducing a foreign nucleic acid molecule (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, or electroporation. Suitable methods for transforming or transfecting host cells can be found in Sambrook, et al. (supra), and other laboratory manuals.
  • a gene that encodes a selectable marker (e.g., for resistance to antibiotics) is generally introduced into the host cells along with the gene of interest.
  • selectable markers include those that confer resistance to drugs, such as G418, hygromycin and methotrexate.
  • Nucleic acid molecules encoding a selectable marker can be introduced into a host cell on the same vector as the nucleic acid molecule of the invention or can be introduced on a separate vector.
  • Cells stably transfected with the introduced nucleic acid molecule can be identified by drug selection (e.g., cells that have inco ⁇ orated the selectable marker gene will survive, while the other cells die).
  • a host cell of the invention such as a prokaryotic or eukaryotic host cell in culture, can be used to produce (i.e., express) a polypeptide of the invention.
  • the invention further provides methods for producing a polypeptide using the host cells of the invention.
  • the method comprises culturing the host cell of invention (into which a recombinant expression vector encoding a polypeptide of the invention has been introduced) in a suitable medium such that the polypeptide is produced.
  • the method further comprises isolating the polypeptide from the medium or the host cell.
  • a host cell of the invention is a fertilized oocyte or an embryonic stem cell into which a nucleic acid molecule of the invention has been introduced (e.g., an exogenous protein kinase gene, or an exogenous nucleic acid encoding a protein kinase polypeptide).
  • a nucleic acid molecule of the invention e.g., an exogenous protein kinase gene, or an exogenous nucleic acid encoding a protein kinase polypeptide.
  • Such host cells can then be used to create non-human transgenic animals in which exogenous nucleotide sequences have been introduced into the genome or homologous recombinant animals in which endogenous nucleotide sequences have been altered.
  • transgenic animal is a non-human animal, preferably a mammal, more preferably a rodent such as a rat or mouse, in which one or more of the cells of the animal include a transgene.
  • rodent such as a rat or mouse
  • transgenic animals include non-human primates, sheep, dogs, cows, goats, chickens and amphibians.
  • a transgene is exogenous DNA which is integrated into the genome of a cell from which a transgenic animal develops and which remains in the genome of the mature animal, thereby directing the expression of an encoded gene product in one or more cell types or tissues of the transgenic animal.
  • an "homologous recombinant animal” is a non-human animal, preferably a mammal, more preferably a mouse, in which an endogenous gene has been altered by homologous recombination between the endogenous gene and an exogenous DNA molecule introduced into a cell of the animal, e.g., an embryonic cell of the animal, prior to development of the animal.
  • the present invention also pertains to isolated polypeptides encoded by protein kinases ("protein kinase polypeptides”) and fragments and variants thereof, as well as polypeptides encoded by nucleotide sequences described herein (e.g., other splicing variants).
  • protein kinase polypeptides encoded by protein kinases
  • polypeptides encoded by nucleotide sequences described herein e.g., other splicing variants.
  • polypeptide refers to a polymer of amino acids, and not to a specific length; thus, peptides, oligopeptides and proteins are included within the definition of a polypeptide.
  • a polypeptide is said to be “isolated” or “purified” when it is substantially free of cellular material when it is isolated from recombinant and non-recombinant cells, or free of chemical precursors or other chemicals when it is chemically synthesized.
  • a polypeptide can be joined to another polypeptide with which it is not normally associated in a cell (e.g., in a "fusion protein") and still be “isolated” or “purified.”
  • polypeptides of the invention can be purified to homogeneity. It is understood, however, that preparations in which the polypeptide is not purified to homogeneity are useful. The critical feature is that the preparation allows for the desired function of the polypeptide, even in the presence of considerable amounts of other components. Thus, the invention encompasses various degrees of purity.
  • the language "substantially free of cellular material” includes preparations of the polypeptide having less than about 30% (by dry weight) other proteins (i.e., contaminating protein), less than about 20% other proteins, less than about 10% other proteins, or less than about 5% other proteins.
  • a polypeptide When a polypeptide is recombinantly produced, it can also be substantially free of culture medium, i.e., culture medium represents less than about 20%, less than about 10%, or less than about 5% of the volume of the polypeptide preparation.
  • the language “substantially free of chemical precursors or other chemicals” includes preparations of the polypeptide in which it is separated from chemical precursors or other chemicals that are involved in its synthesis. In one embodiment, the language “substantially free of chemical precursors or other chemicals” includes preparations of the polypeptide having less than about 30% (by dry weight) chemical precursors or other chemicals, less than about 20% chemical precursors or other chemicals, less than about 10% chemical precursors or other chemicals, or less than about 5% chemical precursors or other chemicals.
  • a polypeptide of the invention comprises an amino acid sequence encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-80 (odd numbers), or the complement of such a nucleic acid, or portions thereof, e.g., SEQ ED NO: 1-80 (odd numbers), or a portion or polymo ⁇ hic variant thereof.
  • the polypeptides of the invention also encompass fragment and sequence variants. Variants include a substantially homologous polypeptide encoded by the same genetic locus in an organism, i.e., an allelic variant, as well as other splicing variants.
  • Variants also encompass polypeptides derived from other genetic loci in an organism, but having substantial homology to a polypeptide encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-80 (odd numbers), or a complement of such a sequence, or portions thereof, or having substantial homology to a polypeptide encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of nucleotide sequences encoding SEQ JD NOs: 1-80 (odd numbers), or polymo ⁇ hic variants thereof.
  • Variants also include polypeptides substantially homologous or identical to these polypeptides but derived from another organism, i.e., an ortholog. Variants also include polypeptides that are substantially homologous or identical to these polypeptides that are produced by chemical synthesis. Variants also include polypeptides that are substantially homologous or identical to these polypeptides that are produced by recombinant methods.
  • two polypeptides are substantially homologous or identical when the amino acid sequences are at least about 45-55%, typically at least about 70-75%, more typically at least about 80- 85%, and most typically greater than about 90% or 95% or more homologous or identical.
  • a substantially homologous amino acid sequence will be encoded by a nucleic acid molecule hybridizing to one or more of SEQ ID NOs: 1-80 (odd numbers), or portion thereof, under stringent conditions as more particularly described above, or will be encoded by a nucleic acid molecule hybridizing to a nucleic acid sequence encoding one of SEQ ED NOs: 1-80 (odd numbers), a portion thereof or polymo ⁇ hic variant thereof, under stringent conditions as more particularly described thereof.
  • the sequences are aligned for optimal comparison pu ⁇ oses (e.g., gaps can be introduced in the sequence of one polypeptide or nucleic acid molecule for optimal alignment with the other polypeptide or nucleic acid molecule).
  • the amino acid residues or nucleotides at co ⁇ esponding amino acid positions or nucleotide positions are then compared. When a position in one sequence is occupied by the same amino acid residue or nucleotide as the co ⁇ esponding position in the other sequence, then the molecules are homologous at that position.
  • amino acid or nucleic acid "homology" is equivalent to amino acid or nucleic acid "identity”.
  • the percent homology between the two sequences is a function of the number of identical positions shared by the sequences (i.e., percent homology equals the number of identical positions/total number of positions times 100).
  • the invention also encompasses polypeptides having a lower degree of identity but having sufficient similarity so as to perform one or more of the same functions performed by a polypeptide encoded by a nucleic acid molecule of the invention. Similarity is determined by conserved amino acid substitution. Such substitutions are those that substitute a given amino acid in a polypeptide by another amino acid of like characteristics. Conservative substitutions are likely to be phenotypically silent. Typically seen as conservative substitutions are the replacements, one for another, among the aliphatic amino acids Ala, Val, Leu and
  • variant polypeptide can differ in amino acid sequence by one or more substitutions, deletions, insertions, inversions, fusions, and truncations or a combination of any of these. Further, variant polypeptides can be fully functional or can lack function in one or more activities. Fully functional variants typically contain only conservative variation or variation in non-critical residues or in non- critical regions. Functional variants can also contain substitution of similar amino acids that result in no change or an insignificant change in function. Alternatively, such substitutions may positively or negatively affect function to some degree. Non- functional variants typically contain one or more non-conservative amino acid substitutions, deletions, insertions, inversions, or truncation or a substitution, insertion, inversion, or deletion in a critical residue or critical region.
  • Amino acids that are essential for function can be identified by methods known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham et al, Science 244: 1081-1085 (1989)). The latter procedure introduces single alanine mutations at every residue in the molecule. The resulting mutant molecules are then tested for biological activity in vitro, or in vitro proliferative activity. Sites that are critical for polypeptide activity can also be determined by structural analysis such as crystallization, nuclear magnetic resonance or photoaffinity labeling (Smith et al, J. Mol. Biol. 224:899-904 (1992); de Vos et al, Science 255:306-312 (1992)).
  • the invention also includes polypeptide fragments of the polypeptides of the invention. Fragments can be derived from a polypeptide encoded by a nucleic acid molecule comprising one of SEQ ED NOs: 1-80 (odd numbers), or a complement of such a nucleic acid (e.g., SEQ ED NOs: 1-80 (odd numbers), or other variants). However, the invention also encompasses fragments of the variants of the polypeptides described herein. As used herein, a fragment comprises at least 6 contiguous amino acids. Useful fragments include those that retain one or more of the biological activities of the polypeptide as well as fragments that can be used as an immunogen to generate polypeptide-specific antibodies. Biologically active fragments (peptides which are, for example, 6, 9, 12, 15,
  • 16, 20, 30, 35, 36, 37, 38, 39, 40, 50, 100 or more amino acids in length can comprise a domain, segment, or motif that has been identified by analysis of the polypeptide sequence using well-known methods, e.g. , signal peptides, extracellular domains, one or more transmembrane segments or loops, ligand binding regions, zinc finger domains, DNA binding domains, acylation sites, glycosylation sites, or phosphorylation sites.
  • Fragments can be discrete (not fused to other amino acids or polypeptides) or can be within a larger polypeptide. Further, several fragments can be comprised within a single larger polypeptide. In one embodiment a fragment designed for expression in a host can have heterologous pre- and pro-polypeptide regions fused to the amino terminus of the polypeptide fragment and an additional region fused to the carboxyl terminus of the fragment.
  • the invention thus provides chimeric or fusion polypeptides.
  • These comprise a polypeptide of the invention operatively linked to a heterologous protein or polypeptide having an amino acid sequence not substantially homologous to the polypeptide.
  • “Operatively linked” indicates that the polypeptide and the heterologous protein are fused in-frame.
  • the heterologous protein can be fused to the N-terminus or C-terminus of the polypeptide.
  • the fusion polypeptide does not affect function of the polypeptide per se.
  • the fusion polypeptide can be a GST-fusion polypeptide in which the polypeptide sequences are fused to the C-terminus of the GST sequences.
  • fusion polypeptides include, but are not limited to, enzymatic fusion polypeptides, for example beta-galactosidase fusions, yeast two-hybrid GAL fusions, poly-His fusions and Ig fusions.
  • fusion polypeptides particularly poly-His fusions, can facilitate the purification of recombinant polypeptide.
  • expression and or secretion of a polypeptide can be increased using a heterologous signal sequence. Therefore, in another embodiment, the fusion polypeptide contains a heterologous signal sequence at its N-terminus.
  • EP-A-O 464 533 discloses fusion proteins comprising various portions of immunoglobulin constant regions. The Fc is useful in therapy and diagnosis and thus results, for example, in improved pharmacokinetic properties (EP-A 0232 262).
  • this invention also encompasses soluble fusion polypeptides containing a polypeptide of the invention and various portions of the constant regions of heavy or light chains of immunoglobulins of various subclasses (IgG, IgM, IgA, IgE).
  • a chimeric or fusion polypeptide can be produced by standard recombinant DNA techniques. For example, DNA fragments coding for the different polypeptide sequences are ligated together in- frame in accordance with conventional techniques.
  • the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers.
  • PCR amplification of nucleic acid fragments can be carried out using anchor primers which give rise to complementary overhangs between two consecutive nucleic acid fragments which can subsequently be annealed and re-amplified to generate a chimeric nucleic acid sequence (see Ausubel et al, Current Protocols in Molecular Biology, 1992).
  • many expression vectors are commercially available that already encode a fusion moiety (e.g., a GST protein).
  • a nucleic acid molecule encoding a polypeptide of the invention can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the polypeptide.
  • the isolated polypeptide can be purified from cells that naturally express it, purified from cells that have been altered to express it (recombinant), or synthesized using known protein synthesis methods.
  • the polypeptide is produced by recombinant DNA techniques. For example, a nucleic acid molecule encoding the polypeptide is cloned into an expression vector, the expression vector introduced into a host cell and the polypeptide expressed in the host cell. The polypeptide can then be isolated from the cells by an appropriate purification scheme using standard protein purification techniques.
  • polypeptides of the present invention can be used as a molecular weight marker on SDS-PAGE gels or on molecular sieve gel filtration columns using art-recognized methods.
  • the polypeptides of the present invention can be used to raise antibodies or to elicit an immune response.
  • the polypeptides can also be used as a reagent, e.g., a labeled reagent, in assays to quantitatively determine levels of the polypeptide or a molecule to which it binds (e.g., a ligand) in biological fluids.
  • the polypeptides can also be used as markers for cells or tissues in which the co ⁇ esponding polypeptide is preferentially expressed, either constitutively, during tissue differentiation, or in a diseased state.
  • the polypeptides can be used to isolate a co ⁇ esponding binding agent, e.g., ligand, such as, for example, in an interaction trap assay, and to screen for peptide or small molecule antagonists or agonists of the binding interaction.
  • Polyclonal and/or monoclonal antibodies that specifically bind one form of the gene product but not to the other form of the gene product are also provided.
  • Antibodies that bind a portion of either the variant or the reference gene product that contains the polymo ⁇ hic site or sites are also provided.
  • the invention provides antibodies to the polypeptides and polypeptide fragments of the invention, e.g., having an amino acid sequence of one of SEQ ID NOs: 1-80 (even numbers) or a portion thereof, or having an amino acid sequence encoded by a nucleic acid molecule comprising all or a portion of SEQ ED NOs: 1-80 (odd numbers), or a complement or another variant or portion thereof.
  • antibody refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that specifically binds an antigen.
  • a molecule that specifically binds to a polypeptide of the invention is a molecule that binds to that polypeptide or a fragment thereof, but does not substantially bind other molecules in a sample, e.g., a biological sample, which naturally contains the polypeptide.
  • immunologically active portions of immunoglobulin molecules include F(ab) and F(ab')2 fragments which can be generated by treating the antibody with an enzyme such as pepsin.
  • the invention provides polyclonal and monoclonal antibodies that bind to a polypeptide of the invention.
  • a monoclonal antibody composition thus typically displays a single binding affinity for a particular polypeptide of the invention with which it immunoreacts.
  • Polyclonal antibodies can be prepared as described above by immunizing a suitable subject with a desired immunogen, e.g., polypeptide of the invention or fragment thereof.
  • the antibody titer in the immunized subject can be monitored over time by standard techniques, such as with an enzyme linked imrnunosorbent assay (ELISA) using immobilized polypeptide.
  • ELISA enzyme linked imrnunosorbent assay
  • the antibody molecules directed against the polypeptide can be isolated from the mammal (e.g., from the blood) and further purified by well-known tecliniques, such as protein A chromatography to obtain the IgG fraction.
  • antibody-producing cells can be obtained from the subject and used to prepare monoclonal antibodies by standard techniques, such as the hybridoma technique originally described by Kohler and Milstein, Nature 256:495-497 (1975), the human B cell hybridoma technique (Kozbor et al, Immunol. Today 4:72 (1983)), the EBV-hybridoma technique (Cole et al. , Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, 1985, Inc., pp. 77-96) or rrioma technique.
  • standard techniques such as the hybridoma technique originally described by Kohler and Milstein, Nature 256:495-497 (1975), the human B cell hybridoma technique (Kozbor et al, Immunol. Today 4:72 (1983)), the EBV-hybridoma technique (Cole et al. , Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, 1985, Inc., pp. 77-96) or
  • hybridomas The technology for producing hybridomas is well known (see generally Current Protocols in Immunology (1994) Coligan et al. (eds.) John Wiley & Sons, Inc., New York, NY). Briefly, an immortal cell line (typically a myeloma) is fused to lymphocytes (typically splenocytes) from a mammal immunized with an immunogen as described above, and the culture supernatants of the resulting hybridoma cells are screened to identify a hybridoma producing a monoclonal antibody that binds a polypeptide of the invention.
  • lymphocytes typically splenocytes
  • a monoclonal antibody to a polypeptide of the invention can be identified and isolated by screening a recombinant combinatorial immunoglobulin library (e.g. , an antibody phage display library) with the polypeptide to thereby isolate immunoglobulin library members that bind the polypeptide.
  • Kits for generating and screening phage display libraries are commercially available (e.g., the Pharmacia Recombinant
  • Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art.
  • antibodies of the invention e.g., a monoclonal antibody
  • a polypeptide-specific antibody can facilitate the purification of natural polypeptide from cells and of recombinantly produced polypeptide expressed in host cells.
  • an antibody specific for a polypeptide of the invention can be used to detect the polypeptide (e.g., in a cellular lysate, cell supernatant, or tissue sample) in order to evaluate the abundance and pattern of expression of the polypeptide.
  • Antibodies can be used diagnostically to monitor protein levels in tissue as part of a clinical testing procedure, e.g., to, for example, determine the efficacy of a given treatment regimen. Detection of the antibody can be facilitated by coupling the antibody to a detectable substance.
  • detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials.
  • suitable enzymes include horseradish peroxidase, alkaline phosphatase, bata-galactosidase, or acetylcholinesterase;
  • suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin;
  • suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin;
  • an example of a luminescent material includes luminol;
  • bioluminescent materials include luciferase, luciferin,
  • radioactive material examples include I, I, S or
  • the present invention also pertains to a method of diagnosing or aiding in the diagnosis of a disease or condition associated with a protein kinase gene or gene product in an individual.
  • Diagnostic assays can be designed for assessing protein kinase gene expression, or for assessing activity of protein kinase polypeptides of the invention.
  • the assays are used in the context of a biological sample (e.g., blood, serum, cells, tissue) to thereby determine whether an individual is afflicted with a disease or condition associated with a protein kinase, or a defect in a protein kinase.
  • the invention also provides for prognostic (or predictive) assays for determining whether an individual is susceptible to a disease of condition associated with a protein kinase. For example, mutations in the gene can be assayed in a biological sample. Such assays can be used for prognostic or predictive pu ⁇ ose to thereby prophylactically treat an individual prior to the onset of symptoms associated with a susceptibility to a disease or condition associated with a protein kinase.
  • Another aspect of the invention pertains to assays for monitoring the influence of agents (e.g., drugs, compounds or other agents) on the gene expression or activity of polypeptides of the invention, as well as to assays for identifying agents that bind to a polypeptides.
  • nucleic acids, probes, primers, polypeptides and antibodies described herein can be used in methods of diagnosis of a susceptibility to a disease or condition associated with a protein kinase, as well as in kits useful for diagnosis of a susceptibility to a disease or condition associated with a protein kinase.
  • susceptibility to a disease or condition associated with a protein kinase is diagnosed by detecting a polymo ⁇ hism in a protein kinase as described herein.
  • the polymo ⁇ hism can be a mutation in a protein kinase, such as the insertion or deletion of a single nucleotide, or of more than one nucleotide, resulting in a frame shift mutation; the change of at least one nucleotide, resulting in a change in the encoded amino acid; the change of at least one nucleotide, resulting in the generation of a premature stop codon; the deletion of several nucleotides, resulting in a deletion of one or more amino acids encoded by the nucleotides; the insertion of one or several nucleotides, such as by unequal recombination or gene conversion, resulting in an interruption of the coding sequence of the gene; duplication of all or a part of the gene; transposition of all or a part
  • More than one such mutation may be present in a single gene.
  • sequence changes cause a mutation in the polypeptide encoded by a protein kinase gene.
  • the mutation is a frame shift mutation
  • the frame shift can result in a change in the encoded amino acids, and/or can result in the generation of a premature stop codon, causing generation of a truncated polypeptide.
  • a polymo ⁇ hism associated with a susceptibility to a disease or condition associated with a protein kinase can be a synonymous mutation in one or more nucleotides (i.e., a mutation that does not result in a change in the polypeptide encoded by a protein kinase gene).
  • a protein kinase gene that has any of the mutations described above is refe ⁇ ed to herein as a "mutant gene.”
  • hybridization methods such as Southern analysis, Northern analysis, or in situ hybridizations, can be used (see Current Protocols in Molecular Biology, Ausubel, F. et al. , eds., John Wiley & Sons, including all supplements through 1999).
  • a biological sample from a test subject (a "test sample") of genomic DNA, RNA, or cDNA, is obtained from an individual suspected of having, being susceptible to or predisposed for, or ca ⁇ ying a defect for, a susceptibility to a disease or condition associated with a protein kinase (the "test individual").
  • the individual can be an adult, child, or fetus.
  • the test sample can be from any source which contains genomic DNA, such as a blood sample, sample of amniotic fluid, sample of cerebrospinal fluid, or tissue sample from skin, muscle, buccal or conjunctival mucosa, placenta, gastrointestinal tract or other organs.
  • a test sample of DNA from fetal cells or tissue can be obtained by appropriate methods, such as by amniocentesis or chorionic villus sampling.
  • the DNA, RNA, or cDNA sample is then examined to determine whether a polymo ⁇ hism in a protein kinase is present, and or to determine which splicing variant(s) encoded by the protein kinase is present.
  • the presence of the polymo ⁇ hism or splicing variant(s) can be indicated by hybridization of the gene in the genomic DNA, RNA, or cDNA to a nucleic acid probe.
  • a “nucleic acid probe”, as used herein, can be a DNA probe or an RNA probe; the nucleic acid probe can contain at least one polymo ⁇ hism in a protein kinase or contains a nucleic acid encoding a particular splicing variant of a protein kinase.
  • the probe can be any of the nucleic acid molecules described above (e.g., the gene, a fragment, a vector comprising the gene, a probe or primer, etc.).
  • the test sample containing a protein kinase is contacted with at least one nucleic acid probe to form the hybridization sample.
  • a prefe ⁇ ed probe for detecting mRNA or genomic DNA is a labeled nucleic acid probe capable of hybridizing to mRNA or genomic DNA sequences described herein.
  • the nucleic acid probe can be, for example, a full-length nucleic acid molecule, or a portion thereof, such as an oligonucleotide of at least 15, 30, 50, 100, 250 or 500 nucleotides in length and sufficient to specifically hybridize under stringent conditions to appropriate mRNA or genomic DNA.
  • the nucleic acid probe can be all or a portion of one of SEQ ID NOs: 1-80 (odd numbers), or the complement thereof, or a portion thereof; or can be a nucleic acid encoding a portion of one of SEQ JD NOs: 1-80 (odd numbers).
  • Other suitable probes for use in the diagnostic assays of the invention are described above (see e.g., probes and primers discussed under the heading, "Nucleic Acids of the Invention").
  • hybridization sample is maintained under conditions that are sufficient to allow specific hybridization of the nucleic acid probe to a protein kinase.
  • Specific hybridization indicates exact hybridization (e.g. , with no mismatches).
  • Specific hybridization can be performed under high stringency conditions or moderate stringency conditions, for example, as described above. In a particularly prefe ⁇ ed embodiment, the hybridization conditions for specific hybridization are high stringency.
  • Specific hybridization if present, is then detected using standard methods. If specific hybridization occurs between the nucleic acid probe and the protein kinase in the test sample, then the protein kinase has the polymo ⁇ hism, or is the splicing variant, that is present in the nucleic acid probe. More than one nucleic acid probe can also be used concu ⁇ ently in this method. Specific hybridization of any one of the nucleic acid probes is indicative of a polymo ⁇ hism in the protein kinase, or of the presence of a particular splicing variant encoding the protein kinase and is therefore diagnostic for a susceptibility to a susceptibility to a disease or condition associated with a protein kinase.
  • RNA is obtained from the individual by appropriate means.
  • Specific hybridization of a nucleic acid probe, as described above, to RNA from the individual is indicative of a polymo ⁇ hism in a protein kinase, or of the presence of a particular splicing variant encoded by a protein kinase, and is therefore diagnostic for a susceptibility to a susceptibility to a disease or condition associated with a protein kinase.
  • nucleic acid probes For representative examples of use of nucleic acid probes, see, for example, U.S. Patents No. 5,288,611 and 4,851,330.
  • PNA peptide nucleic acid
  • a nucleic acid probe can be used instead of a nucleic acid probe in the hybridization methods described above.
  • PNA is a DNA mimic having a peptide-like, inorganic backbone, such as N-(2-aminoethyl)glycine units, with an organic base (A, G, C, T or U) attached to the glycine nitrogen via a methylene carbonyl linker (see, for example, Nielsen, P.E. et al, Bioconjugate Chemistry 5, American Chemical Society, p. 1 (1994).
  • the PNA probe can be designed to specifically hybridize to a gene having a polymo ⁇ hism associated with a susceptibility to a susceptibility to a disease or condition associated with a protein kinase. Hybridization of the PNA probe to a protein kinase is diagnostic for a susceptibility to a susceptibility to a disease or condition associated with a protein kinase.
  • mutation analysis by restriction digestion can be used to detect a mutant gene, or genes containing a polymo ⁇ hism(s), if the mutation or polymo ⁇ hism in the gene results in the creation or elimination of a restriction site.
  • a test sample containing genomic DNA is obtained from the individual. Polymerase chain reaction (PCR) can be used to amplify a protein kinase (and, if necessary, the flanking sequences) in the test sample of genomic
  • DNA from the test individual is RFLP analysis is conducted as described (see Current Protocols in Molecular Biology, supra).
  • the digestion pattern of the relevant DNA fragment indicates the presence or absence of the mutation or polymo ⁇ hism in the protein kinase, and therefore indicates the presence or absence of this susceptibility to a susceptibility to a disease or condition associated with a protein kinase.
  • Sequence analysis can also be used to detect specific polymo ⁇ hisms in a protein kinase.
  • a test sample of DNA or RNA is obtained from the test individual.
  • PCR or other appropriate methods can be used to amplify the gene, and/or its flanking sequences, if desired.
  • the sequence of a protein kinase, or a fragment of the gene, or cDNA, or fragment of the cDNA, or mRNA, or fragment of the mRNA, is determined, using standard methods.
  • the sequence of the gene, gene fragment, cDNA, cDNA fragment, mRNA, or mRNA fragment is compared with the known nucleic acid sequence of the gene, cDNA (e.g., one or more of SEQ ID NOs: 1-80 (odd numbers), or a complement thereof, or a nucleic acid sequence encoding one of SEQ ED NOs: 1-80 (odd numbers) or a fragment thereof) or mRNA, as appropriate.
  • cDNA e.g., one or more of SEQ ID NOs: 1-80 (odd numbers), or a complement thereof, or a nucleic acid sequence encoding one of SEQ ED NOs: 1-80 (odd numbers) or a fragment thereof
  • the presence of a polymo ⁇ hism in the protein kinase indicates that the individual has a susceptibility to a susceptibility to a disease or condition associated with a protein kinase.
  • Allele-specific oligonucleotides can also be used to detect the presence of a polymo ⁇ hism in a protein kinase, through the use of dot-blot hybridization of amplified oligonucleotides with allele-specific oligonucleotide (ASO) probes (see, for example, Saiki, R. et al, Nature 324:163-166 (1986)).
  • ASO allele-specific oligonucleotide
  • an “allele-specific oligonucleotide” (also refe ⁇ ed to herein as an “allele-specific oligonucleotide probe”) is an oligonucleotide of approximately 10-50 base pairs, preferably approximately 15-30 base pairs, that specifically hybridizes to a protein kinase, and that contains a polymo ⁇ hism associated with a susceptibility to a susceptibility to a disease or condition associated with a protein kinase.
  • An allele-specific oligonucleotide probe that is specific for particular polymo ⁇ hisms in a protein kinase can be prepared, using standard methods (see Current Protocols in Molecular Biology, supra).
  • a test sample of DNA is obtained from the individual.
  • PCR can be used to amplify all or a fragment of a protein kinase, and its flanking sequences.
  • the DNA containing the amplified protein kinase (or fragment of the gene) is dot-blotted, using standard methods (see Current Protocols in Molecular Biology, supra), and the blot is contacted with the oligonucleotide probe. The presence of specific hybridization of the probe to the amplified protein kinase is then detected.
  • Specific hybridization of an allele-specific oligonucleotide probe to DNA from the individual is indicative of a polymo ⁇ hism in the protein kinase, and is therefore indicative of a susceptibility to a susceptibility to a disease or condition associated with a protein kinase.
  • a ⁇ ays of oligonucleotide probes that are complementary to target nucleic acid sequence segments from an individual can be used to identify polymo ⁇ hisms in a protein kinase.
  • an oligonucleotide a ⁇ ay can be used.
  • Oligonucleotide a ⁇ ays typically comprise a plurality of different oligonucleotide probes that are coupled to a surface of a substrate in different known locations.
  • These oligonucleotide a ⁇ ays also described as "GenechipsTM," have been generally described in the art, for example, U.S. Pat. No. 5,143,854 and PCT patent publication Nos.
  • WO 90/15070 and 92/10092 can generally be produced using mechanical synthesis methods or light directed synthesis methods which inco ⁇ orate a combination of photolithographic methods and solid phase oligonucleotide synthesis methods. See Fodor et al, Science 25 :161-111 (1991), Pirrung et al, U.S. Pat. No. 5,143,854 (see also PCT Application No. WO 90/15070) and Fodor et al, PCT Publication No. WO 92/10092 and U.S. Pat. No. 5,424,186, the entire teachings of each of which are inco ⁇ orated by reference herein. Techniques for the synthesis of these a ⁇ ays using mechanical synthesis methods are described in, e.g., U.S. Pat. Nos. 5,384,261; the entire teachings of which are inco ⁇ orated by reference herein.
  • a nucleic acid of interest is hybridized with the a ⁇ ay and scanned for polymo ⁇ hisms.
  • Hybridization and scanning are generally carried out by methods described herein and also in, e.g., published PCT Application Nos. WO 92/10092 and WO 95/11995, and U.S. Pat. No. 5,424,186, the entire teachings of which are inco ⁇ orated by reference herein.
  • a target nucleic acid sequence that includes one or more previously identified polymo ⁇ hic markers is amplified by well known amplification techniques, e.g., PCR.
  • Asymmetric PCR techniques may also be used.
  • Amplified target generally inco ⁇ orating a label, is then hybridized with the a ⁇ ay under appropriate conditions.
  • the a ⁇ ay is scanned to determine the position on the array to which the target sequence hybridizes.
  • the hybridization data obtained from the scan is typically in the form of fluorescence intensities as a function of location on the a ⁇ ay.
  • arrays can include multiple detection blocks, and thus be capable of analyzing multiple, specific polymo ⁇ hisms.
  • detection blocks may be grouped within a single a ⁇ ay or in multiple, separate a ⁇ ays so that varying, optimal conditions may be used during the hybridization of the target to the a ⁇ ay. For example, it may often be desirable to provide for the detection of those polymo ⁇ hisms that fall within G-C rich stretches of a genomic sequence, separately from those falling in A-T rich segments. This allows for the separate optimization of hybridization conditions for each situation.
  • nucleic acid analysis can be used to detect polymo ⁇ hisms in a protein kinase or variants encoding by a protein kinase.
  • Representative methods include direct manual sequencing (Church and Gilbert, Proc. Natl. Acad. Sci. USA 81 :1991-1995 (1988); Sanger, F. et al, Proc. Natl. Acad. Sci. USA 74:5463-5467 (1977); Beavis et al. U.S. Pat. No.
  • CMC chemical mismatch cleavage
  • RNase protection assays Myers, R.M. et al, Science 230: 1242 (1985)
  • polypeptides which recognize nucleotide mismatches such as E. coli mutS protein
  • allele-specific PCR for example.
  • diagnosis of a susceptibility to a susceptibility to a disease or condition associated with a protein kinase can also be made by examining expression and/or composition of a protein kinase polypeptide, by a variety of methods, including enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations and immunofluorescence.
  • ELISAs enzyme linked immunosorbent assays
  • a test sample from an individual is assessed for the presence of an alteration in the expression and/or an alteration in composition of the polypeptide encoded by a protein kinase, or for the presence of a particular variant encoded by a protein kinase.
  • An alteration in expression of a polypeptide encoded by a protein kinase can be, for example, an alteration in the quantitative polypeptide expression (i.e., the amount of polypeptide produced); an alteration in the composition of a polypeptide encoded by a protein kinase is an alteration in the qualitative polypeptide expression (e.g., expression of a mutant protein kinase polypeptide or of a different splicing variant).
  • diagnosis of susceptibility to a disease or condition associated with a protein kinase is made by detecting a particular splicing variant encoded by that protein kinase, or a particular pattern of splicing variants.
  • An "alteration" in the polypeptide expression or composition refers to an alteration in expression or composition in a test sample, as compared with the expression or composition of polypeptide by a protein kinase in a control sample.
  • a control sample is a sample that co ⁇ esponds to the test sample (e.g., is from the same type of cells), and is from an individual who is not affected by a susceptibility to a disease or condition associated with a protein kinase.
  • An alteration in the expression or composition of the polypeptide in the test sample, as compared with the control sample, is indicative of a susceptibility to a susceptibility to a disease or condition associated with a protein kinase.
  • the presence of one or more different splicing variants in the test sample, or the presence of significantly different amounts of different splicing variants in the test sample, as compared with the control sample is indicative of a susceptibility to a susceptibility to a disease or condition associated with a protein kinase.
  • Various means of examining expression or composition of the polypeptide encoded by a protein kinase can be used, including speetroscopy, colorimetry, electrophoresis, isoelectric focusing, and immunoassays (e.g., David et al, U.S. Pat. No. 4,376,110) such as immunoblotting (see also Current Protocols in Molecular Biology, particularly Chapter 10).
  • an antibody capable of binding to the polypeptide e-g-, as described above
  • Antibodies can be polyclonal, or more preferably, monoclonal.
  • an intact antibody, or a fragment thereof can be used.
  • labeled with regard to the probe or antibody, is intended to encompass direct labeling of the probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling of the probe or antibody by reactivity with another reagent that is directly labeled.
  • indirect labeling include detection of a primary antibody using a fluorescently labeled secondary antibody and end-labeling of a DNA probe with biotin such that it can be detected with fluorescently labeled streptavidin.
  • Western blotting analysis using an antibody as described above that specifically binds to a polypeptide encoded by a mutant protein kinase, or an antibody that specifically binds to a polypeptide encoded by a non-mutant gene, or an antibody that specifically binds to a particular splicing variant encoded by a protein kinase, can be used to identify the presence in a test sample of a particular splicing variant or of a polypeptide encoded by a polymo ⁇ hic or mutant protein kinase, or the absence in a test sample of a particular splicing variant or of a polypeptide encoded by a non-polymo ⁇ hic or non-mutant gene.
  • the presence of a polypeptide encoded by a polymo ⁇ hic or mutant gene, or the absence of a polypeptide encoded by a non-polymo ⁇ hic or non-mutant gene, is diagnostic for a susceptibility to a susceptibility to a disease or condition associated with a protein kinase, as is the presence (or absence) of particular splicing variants encoded by the protein kinase gene.
  • the level or amount of polypeptide encoded by a protein kinase in a test sample is compared with the level or amount of the polypeptide encoded by the protein kinase in a control sample.
  • a level or amount of the polypeptide in the test sample that is higher or lower than the level or amount of the polypeptide in the control sample, such that the difference is statistically significant is indicative of an alteration in the expression of the polypeptide encoded by the protein kinase, and is diagnostic for a susceptibility to a susceptibility to a disease or condition associated with that protein kinase.
  • composition of the polypeptide encoded by a protein kinase in a test sample is compared with the composition of the polypeptide encoded by the protein kinase in a control sample (e.g., the presence of different splicing variants).
  • a difference in the composition of the polypeptide in the test sample, as compared with the composition of the polypeptide in the control sample is diagnostic for a susceptibility to a susceptibility to a disease or condition associated with that protein kinase.
  • both the level or amount and the composition of the polypeptide can be assessed in the test sample and in the control sample.
  • a difference in the amount or level of the polypeptide in the test sample, compared to the control sample; a difference in composition in the test sample, compared to the control sample; or both a difference in the amount or level, and a difference in the composition, is indicative of a susceptibility to a susceptibility to a disease or condition associated with that protein kinase.
  • Kits useful in the methods of diagnosis comprise components useful in any of the methods described herein, including for example, hybridization probes or primers as decribed herein (e.g. , labeled probes or primers), reagents for detection of labeled molecules, restriction enzymes (e.g., for RFLP analysis), allele-specific oligonucleotides, antibodies which bind to mutant or to non-mutant (native) protein kinase polypeptide, means for amplification of nucleic acids comprising a protein kinase, or means for analyzing the nucleic acid sequence of a protein kinase or for analyzing the amino acid sequence of a protein kinase polypeptide, etc.
  • the invention provides methods (also refe ⁇ ed to herein as "screening assays") for identifying the presence of a nucleotide that hybridizes to a nucleic acid of the invention, as well as for identifying the presence of a polypeptide encoded by a nucleic acid of the invention.
  • the presence (or absence) of a nucleic acid molecule of interest in a sample can be assessed by contacting the sample with a nucleic acid comprising a nucleic acid of the invention (e.g., a nucleic acid having the sequence of one of SEQ ID NOs: 1 -80 (odd numbers), or the complement thereof, or a nucleic acid encoding an amino acid having the sequence of one of SEQ ED NOs: 1-80 (odd numbers), or a fragment or variant of such nucleic acids), under stringent conditions as described above, and then assessing the sample for the presence (or absence) of hybridization.
  • a nucleic acid comprising a nucleic acid of the invention e.g., a nucleic acid having the sequence of one of SEQ ID NOs: 1 -80 (odd numbers), or the complement thereof, or a nucleic acid encoding an amino acid having the sequence of one of SEQ ED NOs: 1-80 (odd numbers), or a fragment
  • high stringency conditions are conditions appropriate for selective hybridization.
  • a sample containing the nucleic acid molecule of interest is contacted with a nucleic acid containing a contiguous nucleotide sequence (e.g., a primer or a probe as described above) that is at least partially complementary to a part of the nucleic acid molecule of interest (e.g., a protein kinase nucleic acid), and the contacted sample is assessed for the presence or absence of hybridization.
  • the nucleic acid containing a contiguous nucleotide sequence is completely complementary to a part of the nucleic acid molecule of interest.
  • all or a portion of the nucleic acid of interest can be subjected to amplification prior to performing the hybridization.
  • the presence (or absence) of a polypeptide of interest, such as a polypeptide of the invention or a fragment or variant thereof, in a sample can be assessed by contacting the sample with an antibody that specifically hybridizes to the polypeptide of interest (e.g., an antibody such as those described above), and then assessing the sample for the presence (or absence) of binding of the antibody to the polypeptide of interest.
  • an antibody that specifically hybridizes to the polypeptide of interest e.g., an antibody such as those described above
  • the invention provides methods for identifying agents (e.g., fusion proteins, polypeptides, peptidomimetics, prodrugs, receptors, binding agents, antibodies, small molecules or other drugs, or ribozymes which alter (e.g., increase or decrease) the activity of the polypeptides described herein, or which otherwise interact with the polypeptides herein.
  • agents e.g., fusion proteins, polypeptides, peptidomimetics, prodrugs, receptors, binding agents, antibodies, small molecules or other drugs, or ribozymes which alter (e.g., increase or decrease) the activity of the polypeptides described herein, or which otherwise interact with the polypeptides herein.
  • such agents can be agents which bind to polypeptides described herein (e.g., protein kinase binding agents); which have a stimulatory or inhibitory effect on, for example, activity of polypeptides of the invention; or which change (e.g., enhance or inhibit) the ability of the polypeptides of the invention to interact with protein kinase binding agents (e.g., receptors or other binding agents); or which alter posttranslational processing of the protein kinase polypeptide (e.g. , agents that alter proteolytic processing to direct the polypeptide from where it is normally synthesized to another location in the cell, such as the cell surface; agents that alter proteolytic processing such that more polypeptide is released from the cell, etc.
  • protein kinase binding agents e.g., receptors or other binding agents
  • alter posttranslational processing of the protein kinase polypeptide e.g. , agents that alter proteolytic processing to direct the polypeptide from where it is normally synthesized
  • the invention provides assays for screening candidate or test agents that bind to or modulate the activity of polypeptides described herein (or biologically active portion(s) thereof), as well as agents identifiable by the assays.
  • Test agents can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries; spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the 'one-bead one-compound' library method; and synthetic library methods using affinity chromatography selection.
  • the biological library approach is limited to polypeptide libraries, while the other four approaches are applicable to polypeptide, non-peptide oligomer or small molecule libraries of compounds (Lam, K.S., Anticancer Drug Des. 12:145 (1997)).
  • a cell, cell lysate, or solution containing or expressing a protein kinase polypeptide e.g., one of SEQ ID NOs: 1-80 (odd numbers), or another splicing variant encoded by a protein kinase), or a fragment or derivative thereof (as described above)
  • a protein kinase polypeptide e.g., one of SEQ ID NOs: 1-80 (odd numbers), or another splicing variant encoded by a protein kinase
  • a fragment or derivative thereof as described above
  • (amount) of protein kinase activity is assessed (e.g., the level (amount) of protein kinase activity is measured, either directly or indirectly), and is compared with the level of activity in a control (i.e., the level of activity of the protein kinase polypeptide or active fragment or derivative thereof in the absence of the agent to be tested). If the level of the activity in the presence of the agent differs, by an amount that is statistically significant, from the level of the activity in the absence of the agent, then the agent is an agent that alters the activity of a protein kinase polypeptide.
  • an increase in the level of protein kinase activity relative to a control indicates that the agent is an agent that enhances (is an agonist of) protein kinase activity.
  • a decrease in the level of protein kinase activity relative to a control indicates that the agent is an agent that inhibits (is an antagonist of) protein kinase activity.
  • the level of activity of a protein kinase polypeptide or derivative or fragment thereof in the presence of the agent to be tested is compared with a control level that has previously been established. A level of the activity in the presence of the agent that differs from the control level by an amount that is statistically significant indicates that the agent alters protein kinase activity.
  • the present invention also relates to an assay for identifying agents which alter the expression of a protein kinase gene (e.g., antisense nucleic acids, fusion proteins, polypeptides, peptidomimetics, prodrugs, receptors, binding agents, antibodies, small molecules or other drugs, or ribozymes) which alter (e.g., increase or decrease) expression (e.g., transcription or translation) of the gene or which otherwise interact with the nucleic acids described herein, as well as agents identifiable by the assays.
  • a solution containing a nucleic acid encoding a protein kinase polypeptide e.g., a protein kinase gene
  • an agent to be tested e.g., antisense nucleic acids, fusion proteins, polypeptides, peptidomimetics, prodrugs, receptors, binding agents, antibodies, small molecules or other drugs, or ribozymes
  • the solution can comprise, for example, cells containing the nucleic acid or cell lysate containing the nucleic acid; alternatively, the solution can be another solution that comprises elements necessary for transcription translation of the nucleic acid. Cells not suspended in solution can also be employed, if desired.
  • the level and/or pattern of protein kinase expression e.g., the level and/or pattern of mRNA or of protein expressed, such as the level and/or pattern of different splicing variants
  • a control i.e., the level and/or pattern of the protein kinase expression in the absence of the agent to be tested.
  • the agent is an agent that alters the expression of a protein kinase.
  • Enhancement of protein kinase expression indicates that the agent is an agonist of protein kinase activity.
  • inhibition of protein kinase expression indicates that the agent is an antagonist of protein kinase activity.
  • the level and/or pattern of protein kinase polypeptide(s) e.g., different splicing variants
  • the level and/or pattern of protein kinase polypeptide(s) is compared with a control level and/or pattern that have previously been established.
  • agents which alter the expression of a protein kinase gene or which otherwise interact with the nucleic acids described herein can be identified using a cell, cell lysate, or solution containing a nucleic acid encoding the promoter region of the protein kinase gene operably linked to a reporter gene.
  • the level of expression of the reporter gene (e.g., the level of mRNA or of protein expressed) is assessed, and is compared with the level of expression in a control (i.e., the level of the expression of the reporter gene in the absence of the agent to be tested). If the level in the presence of the agent differs, by an amount or in a manner that is statistically significant, from the level in the absence of the agent, then the agent is an agent that alters the expression of the protein kinase, as indicated by its ability to alter expression of a gene that is operably linked to the protein kinase gene promoter. Enhancement of the expression of the reporter indicates that the agent is an agonist of protein kinase activity.
  • inhibition of the expression of the reporter indicates that the agent is an antagonist of protein kinase activity.
  • the level of expression of the reporter in the presence of the agent to be tested is compared with a control level that has previously been established. A level in the presence of the agent that differs from the control level by an amount or in a manner that is statistically significant indicates that the agent alters f expression.
  • Agents which alter the amounts of different splicing variants encoded by a protein kinase e.g., an agent which enhances activity of a first splicing variant, and which inhibits activity of a second splicing variant
  • agents which are agonists of activity of a first splicing variant and antagonists of activity of a second splicing variant can easily be identified using these methods described above.
  • assays can be used to assess the impact of a test agent on the activity of a polypeptide in relation to a protein kinase binding agent.
  • a cell that expresses a compound that interacts with a protein kinase (herein refe ⁇ ed to as a "protein kinase binding agent", which can be a polypeptide or other molecule that interacts with a protein kinase, such as a receptor) is contacted with a protein kinase in the presence of a test agent, and the ability of the test agent to alter the interaction between the protein kinase and the protein kinase binding agent is determined.
  • a cell lysate or a solution containing the protein kinase binding agent can be used.
  • An agent which binds to the protein kinase or the protein kinase binding agent can alter the interaction by interfering with, or enhancing the ability of the protein kinase to bind to, associate with, or otherwise interact with the protein kinase binding agent. Determining the ability of the test agent to bind to a protein kinase or a protein kinase binding agent can be accomplished, for example, by coupling the test agent with a radioisotope or enzymatic label such that binding of the test agent to the polypeptide can be determined by detecting the labeled with I, S, C or H, either directly or indirectly, and the radioisotope detected by direct counting of radioemmission or by scintillation counting.
  • test agents can be enzymatically labeled with, for example, horseradish peroxidase, alkaline phosphatase, or luciferase, and the enzymatic label detected by determination of conversion of an appropriate substrate to product. It is also within the scope of this invention to determine the ability of a test agent to interact with the polypeptide without the labeling of any of the interactants.
  • a microphysiometer can be used to detect the interaction of a test agent with a protein kinase or a protein kinase binding agent without the labeling of either the test agent, protein kinase, or the protein kinase binding agent. McConnell, H.M. et al, Science 257: 1906-1912 (1992).
  • a "microphysiometer” e.g., CytosensorTM
  • LAPS light-addressable potentiometric sensor
  • Changes in this acidification rate can be used as an indicator of the interaction between ligand and polypeptide.
  • these receptors can be used to screen for compounds that are agonists for use in treating a susceptibility to a disease or condition associated with a protein kinase or antagonists for studying a susceptibility to a disease or condition associated with a protein kinase.
  • Drugs could be designed to regulate protein kinase activation that in turn can be used to regulate signaling pathways and transcription events of genes downstream.
  • assays can be used to identify polypeptides that interact with one or more protein kinase polypeptides, as described herein.
  • a yeast two-hybrid system such as that described by Fields and Song (Fields, S. and Song, O., Nature 340:245-246 (1989)) can be used to identify polypeptides that interact with one or more protein kinase polypeptides.
  • vectors are constructed based on the flexibility of a transcription factor that has two functional domains (a DNA binding domain and a transcription activation domain).
  • transcriptional activation can be achieved, and transcription of specific markers (e.g., nutritional markers such as His and Ade, or color markers such as lacZ) can be used to identify the presence of interaction and transcriptional activation.
  • specific markers e.g., nutritional markers such as His and Ade, or color markers such as lacZ
  • a first vector which includes a nucleic acid encoding a DNA binding domain and also a protein kinase polypeptide, splicing variant, or fragment or derivative thereof
  • a second vector which includes a nucleic acid encoding a transcription activation domain and also a nucleic acid encoding a polypeptide which potentially may interact with the protein kinase polypeptide, splicing variant, or fragment or derivative thereof (e.g., a protein kinase polypeptide binding agent or receptor).
  • yeast containing the first vector and the second vector under appropriate conditions (e.g.
  • mating conditions such as used in the MatchmakerTM system from Clontech (Palo Alto, California, USA) allows identification of colonies that express the markers of interest. These colonies can be examined to identify the polypeptide(s) that interact with the protein kinase polypeptide or fragment or derivative thereof. Such polypeptides may be useful as agents that alter the activity of expression of a protein kinase polypeptide, as described above.
  • binding of a test agent to the polypeptide, or interaction of the polypeptide with a binding agent in the presence and absence of a test agent can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtiter plates, test tubes, and micro-centrifuge tubes.
  • a fusion protein e.g., a glutathione-S-transferase fusion protein
  • a fusion protein e.g., a glutathione-S-transferase fusion protein
  • modulators of expression of nucleic acid molecules of the invention are identified in a method wherein a cell, cell lysate, or solution containing a nucleic acid encoding a protein kinase is contacted with a test agent and the expression of appropriate mRNA or polypeptide (e.g. , splicing variant(s)) in the cell, cell lysate, or solution, is determined.
  • appropriate mRNA or polypeptide e.g. , splicing variant(s)
  • the level of expression of appropriate mRNA or polypeptide(s) in the presence of the test agent is compared to the level of expression of mRNA or polypeptide(s) in the absence of the test agent.
  • the test agent can then be identified as a modulator of expression based on this comparison.
  • the test agent when expression of mRNA or polypeptide is greater (statistically significantly greater) in the presence of the test agent than in its absence, the test agent is identified as a stimulator or enhancer of the mRNA or polypeptide expression.
  • the test agent when expression of the mRNA or polypeptide is less (statistically significantly less) in the presence of the test agent than in its absence, the test agent is identified as an inhibitor of the mRNA or polypeptide expression.
  • the level of mRNA or polypeptide expression in the cells can be determined by methods described herein for detecting mRNA or polypeptide.
  • This invention further pertains to novel agents identified by the above- described screening assays. Accordingly, it is within the scope of this invention to further use an agent identified as described herein in an appropriate animal model.
  • an agent identified as described herein e.g., a test agent that is a modulating agent, an antisense nucleic acid molecule, a specific antibody, or a polypeptide-binding agent
  • an agent identified as described herein can be used in an animal model to determine the efficacy, toxicity, or side effects of treatment with such an agent.
  • an agent identified as described herein can be used in an animal model to determine the mechanism of action of such an agent.
  • this invention pertains to uses of novel agents identified by the above-described screening assays for treatments as described herein.
  • an agent identified as described herein can be used to alter activity of a polypeptide encoded by a protein kinase, or to alter expression of a protein kinase, by contacting the polypeptide or the gene (or contacting a cell comprising the polypeptide or the gene) with the agent identified as described herein.
  • the present invention also pertains to pharmaceutical compositions comprising nucleic acids described herein, particularly nucleotides encoding the polypeptides described herein; comprising polypeptides described herein (e.g., one or more of SEQ ID NOs: 1-80 (even numbers)); and/or comprising other splicing variants encoded by a protein kinase; and/or an agent that alters (e.g., enhances or inhibits) protein kinase gene expression or protein kinase polypeptide activity as described herein.
  • nucleic acids described herein particularly nucleotides encoding the polypeptides described herein; comprising polypeptides described herein (e.g., one or more of SEQ ID NOs: 1-80 (even numbers)); and/or comprising other splicing variants encoded by a protein kinase; and/or an agent that alters (e.g., enhances or inhibits) protein kinase gene expression or protein kina
  • a polypeptide, protein e.g., a protein kinase receptor
  • an agent that alters protein kinase gene expression or a protein kinase binding agent or binding partner, fragment, fusion protein or prodrug thereof, or a nucleotide or nucleic acid construct (vector) comprising a nucleotide of the present invention, or an agent that alters protein kinase polypeptide activity
  • a physiologically acceptable carrier or excipient can be formulated with a physiologically acceptable carrier or excipient to prepare a pharmaceutical composition.
  • the carrier and composition can be sterile. The formulation should suit the mode of administration.
  • Suitable pharmaceutically acceptable carriers include but are not limited to water, salt solutions (e.g., NaCl), saline, buffered saline, alcohols, glycerol, ethanol, gum arabic, vegetable oils, benzyl alcohols, polyethylene glycols, gelatin, carbohydrates such as lactose, amylose or starch, dextrose, magnesium stearate, talc, silicic acid, viscous paraffin, perfume oil, fatty acid esters, hydroxymethylcellulose, polyvinyl pyrolidone, etc., as well as combinations thereof.
  • the pharmaceutical preparations can, if desired, be mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, flavoring and/or aromatic substances and the like which do not deleteriously react with the active agents.
  • auxiliary agents e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, flavoring and/or aromatic substances and the like which do not deleteriously react with the active agents.
  • the composition can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.
  • the composition can be a liquid solution, suspension, emulsion, tablet, pill, capsule, sustained release formulation, or powder.
  • the composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides.
  • Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, polyvinyl pyrollidone, sodium saccharine, cellulose, magnesium carbonate, etc.
  • compositions of introduction of these compositions include, but are not limited to, intradermal, intramuscular, intraperitoneal, intraocular, intravenous, subcutaneous, topical, oral and intranasal.
  • Other suitable methods of introduction can also include gene therapy (as described below), rechargeable or biodegradable devices, particle acceleration devises ("gene guns") and slow release polymeric devices.
  • the pharmaceutical compositions of this invention can also be administered as part of a combinatorial therapy with other agents.
  • compositions for intravenous administration typically are solutions in sterile isotonic aqueous buffer.
  • the composition may also include a solubilizing agent and a local anesthetic to ease pain at the site of the injection.
  • the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampule or sachette indicating the quantity of active agent.
  • the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water, saline or dextrose/water.
  • composition is administered by injection
  • an ampule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.
  • nonsprayable forms, viscous to semi-solid or solid forms comprising a carrier compatible with topical application and having a dynamic viscosity preferably greater than water, can be employed.
  • Suitable formulations include but are not limited to solutions, suspensions, emulsions, creams, ointments, powders, enemas, lotions, sols, liniments, salves, aerosols, etc., which are, if desired, sterilized or mixed with auxiliary agents, e.g., preservatives, stabilizers, wetting agents, buffers or salts for influencing osmotic pressure, etc.
  • auxiliary agents e.g., preservatives, stabilizers, wetting agents, buffers or salts for influencing osmotic pressure, etc.
  • the agent may be inco ⁇ orated into a cosmetic formulation.
  • sprayable aerosol preparations wherein the active ingredient, preferably in combination with a solid or liquid inert carrier material, is packaged in a squeeze bottle or in admixture with a pressurized volatile, normally gaseous propellant, e.g., pressurized air.
  • a pressurized volatile, normally gaseous propellant e.g., pressurized air.
  • Agents described herein can be formulated as neutral or salt forms.
  • Pharmaceutically acceptable salts include those formed with free amino groups such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with free carboxyl groups such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.
  • the agents are administered in a therapeutically effective amount.
  • the amount of agents which will be therapeutically effective in the treatment of a particular disorder or condition will depend on the nature of the disorder or condition, and can be determined by standard clinical techniques.
  • in vitro or in vivo assays may optionally be employed to help identify optimal dosage ranges.
  • the precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the symptoms of a susceptibility to a disease or condition associated with a protein kinase, and should be decided according to the judgment of a practitioner and each patient's circumstances. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.
  • the invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention.
  • Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use of sale for human administration.
  • the pack or kit can be labeled with information regarding mode of administration, sequence of drug administration (e.g., separately, sequentially or concu ⁇ ently), or the like.
  • the pack or kit may also include means for reminding the patient to take the therapy.
  • the pack or kit can be a single unit dosage of the combination therapy or it can be a plurality of unit dosages.
  • the agents can be separated, mixed together in any combination, present in a single vial or tablet.
  • Agents assembled in a blister pack or other dispensing means is prefe ⁇ ed.
  • unit dosage is intended to mean a dosage that is dependent on the individual pharmacodynamics of each agent and administered in FDA approved dosages in standard time courses.
  • the present invention also pertains to methods of treatment (prophylactic and/or therapeutic) for a susceptibility to a disease or condition associated with a protein kinase, using a protein kinase therapeutic agent.
  • a "protein kinase therapeutic agent” is an agent that alters (e.g., enhances or inhibits) protein kinase polypeptide activity and/or protein kinase gene expression, as described herein (e.g., a protein kinase agonist or antagonist).
  • Protein kinase therapeutic agents can alter protein kinase polypeptide activity or gene expression by a variety of means, such as, for example, by providing additional protein kinase polypeptide or by upregulating the transcription or translation of the protein kinase gene; by altering posttranslational processing of the protein kinase polypeptide; by altering transcription of protein kinase splicing variants; or by interfering with protein kinase polypeptide activity (e.g., by binding to a protein kinase polypeptide), or by downregulating the transcription or translation of a protein kinase gene.
  • means such as, for example, by providing additional protein kinase polypeptide or by upregulating the transcription or translation of the protein kinase gene; by altering posttranslational processing of the protein kinase polypeptide; by altering transcription of protein kinase splicing variants; or by interfering with protein kinase polypeptide activity (
  • Representative protein kinase therapeutic agents include the following: nucleic acids or fragments or derivatives thereof described herein, particularly nucleotides encoding the polypeptides described herein and vectors comprising such nucleic acids (e.g., a gene, cDNA, and/or mRNA, such as a nucleic acid encoding a protein kinase polypeptide or active fragment or derivative thereof, or an oligonucleotide; for example, one of SEQ ED NOs: 1-80 (odd numbers), or a complement thereof, or a nucleic acid encoding one of SEQ JD NOs: 1-80 (odd numbers), or fragments or derivatives thereof); polypeptides described herein (e.g., one or more of SEQ ED NOs: 1-80 (even numbers), and/or other splicing variants encoded by a protein kinase, or fragments or derivatives thereof); other polypeptides (e.g., protein kinase
  • More than one protein kinase therapeutic agent can be used concu ⁇ ently, if desired.
  • a protein kinase therapeutic agent that is a nucleic acid is used in the treatment of a susceptibility to a disease or condition associated with a protein kinase.
  • treatment refers not only to ameliorating symptoms associated with the disease, but also preventing or delaying the onset of the disease, and also lessening the severity or frequency of symptoms of the disease.
  • the therapy is designed to alter (e.g. , inhibit or enhance), replace or supplement activity of a protein kinase polypeptide in an individual.
  • a protein kinase therapeutic agent can be administered in order to upregulate or increase the expression or availability of the protein kinase gene or of specific splicing variants of protein kinase, or, conversely, to downregulate or decrease the expression or availability of the protein kinase gene or specific splicing variants of the protein kinase.
  • Upregulation or increasing expression or availability of a native protein kinase gene or of a particular splicing variant could interfere with or compensate for the expression or activity of a defective gene or another splicing variant; downregulation or decreasing expression or availability of a native protein kinase gene or of a particular splicing variant could minimize the expression or activity of a defective gene or the particular splicing variant and thereby minimize the impact of the defective gene or the particular splicing variant.
  • the protein kinase therapeutic agent(s) are administered in a therapeutically effective amount (i.e., an amount that is sufficient to treat the disease, such as by ameliorating symptoms associated with the disease, preventing or delaying the onset of the disease, and/or also lessening the severity or frequency of symptoms of the disease).
  • a therapeutically effective amount i.e., an amount that is sufficient to treat the disease, such as by ameliorating symptoms associated with the disease, preventing or delaying the onset of the disease, and/or also lessening the severity or frequency of symptoms of the disease.
  • the amount which will be therapeutically effective in the treatment of a particular individual's disorder or condition will depend on the symptoms and severity of the disease, and can be determined by standard clinical techniques.
  • in vitro or in vivo assays may optionally be employed to help identify optimal dosage ranges.
  • a nucleic acid of the invention e.g., a nucleic acid of the invention
  • a nucleic acid encoding a protein kinase polypeptide such as one of SEQ ID NOs: 1-80 (even numbers), or a complement thereof; or another nucleic acid that encodes a protein kinase polypeptide or a splicing variant, derivative or fragment thereof, such as a nucleic acid encoding one of SEQ ED NOs: 1-80 (odd numbers)
  • a nucleic acid encoding a protein kinase polypeptide such as one of SEQ ID NOs: 1-80 (even numbers), or a complement thereof; or another nucleic acid that encodes a protein kinase polypeptide or a splicing variant, derivative or fragment thereof, such as a nucleic acid encoding one of SEQ ED NOs: 1-80 (odd numbers)
  • a protein kinase or a cDNA encoding a protein kinase polypeptide can be introduced into cells (either in vitro or in vivo) such that the cells produce native protein kinase polypeptide. If necessary, cells that have been transformed with the gene or cDNA or a vector comprising the gene or cDNA can be introduced (or re-introduced) into an individual affected with the disease.
  • cells which, in nature, lack native protein kinase expression and activity, or have mutant protein kinase expression and activity, or have expression of a disease- associated protein kinase splicing variant, can be engineered to express the protein kinase polypeptide or an active fragment of the protein kinase polypeptide (or a different variant of the protein kinase polypeptide).
  • nucleic acid encoding a protein kinase polypeptide, or an active fragment or derivative thereof can be introduced into an expression vector, such as a viral vector, and the vector can be introduced into appropriate cells in an animal.
  • Other gene transfer systems including viral and nonviral transfer systems, can be used.
  • nonviral gene transfer methods such as calcium phosphate coprecipitation, mechanical techniques (e.g., microinjection); membrane fusion- mediated transfer via liposomes; or direct DNA uptake, can also be used.
  • a nucleic acid of the invention in another embodiment, can be used in "antisense" therapy, in which a nucleic acid (e.g., an oligonucleotide) which specifically hybridizes to the mRNA and or genomic DNA of a protein kinase is administered or generated in situ.
  • a nucleic acid e.g., an oligonucleotide
  • the antisense nucleic acid that specifically hybridizes to the mRNA and/or DNA inhibits expression of the protein kinase polypeptide, e.g., by inhibiting translation and/or transcription. Binding of the antisense nucleic acid can be by conventional base pair complementarity, or, for example, in the case of binding to DNA duplexes, through specific interaction in the major groove of the double helix.
  • An antisense construct of the present invention can be delivered, for example, as an expression plasmid as described above.
  • the plasmid When the plasmid is transcribed in the cell, it produces RNA that is complementary to a portion of the mRNA and/or DNA which encodes the protein kinase polypeptide.
  • the antisense construct can be an oligonucleotide probe that is generated ex vivo and introduced into cells; it then inhibits expression by hybridizing with the mRNA and or genomic DNA of the protein kinase.
  • the oligonucleotide probes are modified oligonucleotides which are resistant to endogenous nucleases, e.g., exonucleases and/or endonucleases, thereby rendering them stable in vivo.
  • Exemplary nucleic acid molecules for use as antisense oligonucleotides are phosphoramidate, phosphothioate and methylphosphonate analogs of DNA (see also U.S. Pat. Nos. 5,176,996; 5,264,564; and 5,256,775). Additionally, general approaches to constructing oligomers useful in antisense therapy are also described, for example, by Van der Krol et al.
  • oligodeoxyribonucleotides derived from the translation initiation site are preferred.
  • oligonucleotides mRNA, cDNA or DNA
  • the antisense oligonucleotides bind to protein kinase mRNA transcripts and prevent translation. Absolute complementarity, although prefe ⁇ ed, is not required.
  • a sequence "complementary" to a portion of an RNA indicates that a sequence has sufficient complementarity to be able to hybridize with the RNA, forming a stable duplex; in the case of double-stranded antisense nucleic acids, a single strand of the duplex DNA may thus be tested, or triplex formation may be assayed.
  • the ability to hybridize will depend on both the degree of complementarity and the length of the antisense nucleic acid, as described in detail above. Generally, the longer the hybridizing nucleic acid, the more base mismatches with an RNA it may contain and still form a stable duplex (or triplex, as the case may be).
  • One skilled in the art can ascertain a tolerable degree of mismatch by use of standard procedures.
  • the oligonucleotides used in antisense therapy can be DNA, RNA, or chimeric mixtures or derivatives or modified versions thereof, single-stranded or double-stranded.
  • the oligonucleotides can be modified at the base moiety, sugar moiety, or phosphate backbone, for example, to improve stability of the molecule, hybridization, etc.
  • the oligonucleotides can include other appended groups such as peptides (e.g. for targeting host cell receptors in vivo), or agents facilitating transport across the cell membrane (see, e.g., Letsinger et al, Proc. Nat Acad. Sci. USA 86:6553-6556 (1989); Lemaitre et al, Proc. Natl. Acad. Sci. USA 84:648-652
  • the oligonucleotide may be conjugated to another molecule (e.g., a peptide, hybridization triggered cross-linking agent, transport agent, hybridization-triggered cleavage agent).
  • another molecule e.g., a peptide, hybridization triggered cross-linking agent, transport agent, hybridization-triggered cleavage agent.
  • the antisense molecules are delivered to cells which express protein kinase in vivo.
  • a number of methods can be used for delivering antisense DNA or RNA to cells; e.g., antisense molecules can be injected directly into the tissue site, or modified antisense molecules, designed to target the desired cells (e.g., antisense linked to peptides or antibodies that specifically bind receptors or antigens expressed on the target cell surface) can be administered systemically.
  • a recombinant DNA construct is utilized in which the antisense oligonucleotide is placed under the control of a strong promoter (e.g. , pol
  • RNAs that will form complementary base pairs with the endogenous protein kinase transcripts and thereby prevent translation of the protein kinase mRNA.
  • a vector can be introduced in vivo such that it is taken up by a cell and directs the transcription of an antisense RNA.
  • Such a vector can remain episomal or become chromosomally integrated, as long as it can be transcribed to produce the desired antisense RNA.
  • Such vectors can be constructed by recombinant DNA technology methods standard in the art and described above.
  • a plasmid, cosmid, YAC or viral vector can be used to prepare the recombinant DNA construct that can be introduced directly into the tissue site.
  • viral vectors can be used which selectively infect the desired tissue, in which case administration may be accomplished by another route (e.g., systemically).
  • Endogenous protein kinase expression can also be reduced by inactivating or "knocking out” protein kinase or its promoter using targeted homologous recombination (e.g. , see Smithies et al. , Nature 317:230-234 (1985); Thomas &
  • a mutant, non-functional protein kinase flanked by DNA homologous to the endogenous protein kinase (either the coding regions or regulatory regions of protein kinase) can be used, with or without a selectable marker and or a negative selectable marker, to transfect cells that express the protein kinase in vivo. Insertion of the DNA construct, via targeted homologous recombination, results in inactivation of the protein kinase.
  • the recombinant DNA constructs can be directly administered or targeted to the required site in vivo using appropriate vectors, as described above.
  • expression of non-mutant protein kinases can be increased using a similar method: targeted homologous recombination can be used to insert a DNA construct comprising a non- mutant, functional protein kinase, e.g., a gene having one of SEQ ID NOs: 1-80 (odd numbers), or the complement thereof, or a portion thereof, in place of a mutant protein kinase in the cell, as described above.
  • targeted homologous recombination can be used to insert a DNA construct comprising a nucleic acid that encodes a protein kinase polypeptide variant that differs from that present in the cell.
  • endogenous protein kinase expression can be reduced by targeting deoxyribonucleotide sequences complementary to the regulatory region of a protein kinase (i.e., the protein kinase promoter and/or enhancers) to form triple helical structures that prevent transcription of the protein kinase in target cells in the body.
  • a protein kinase i.e., the protein kinase promoter and/or enhancers
  • the antisense constructs described herein by antagonizing the normal biological activity of one of the protein kinase proteins, can be used in the manipulation of tissue, e.g., tissue differentiation, both in vivo and for ex vivo tissue cultures.
  • tissue e.g., tissue differentiation
  • the anti-sense techniques e.g., microinjection of antisense molecules, or transfection with plasmids whose transcripts are anti- sense with regard to a protein kinase mRNA or gene sequence
  • Such techniques can be utilized in cell culture, but can also be used in the creation of transgenic animals.
  • other protein kinase therapeutic agents as described herein can also be used in the treatment or prevention of a susceptibility to a disease or condition associated with a protein kinase.
  • the therapeutic agents can be delivered in a composition, as described above, or by themselves. They can be administered systemically, or can be targeted to a particular tissue.
  • the therapeutic agents can be produced by a variety of means, including chemical synthesis; recombinant production; in vivo production (e.g., a transgenic animal, such as U.S. Pat. No. 4,873,316 to Meade et al), for example, and can be isolated using standard means such as those described herein.
  • a combination of any of the above methods of treatment e.g., administration of non-mutant protein kinase polypeptide in conjunction with antisense therapy targeting mutant protein kinase mRNA; administration of a first splicing variant encoded by a protein kinase in conjunction with antisense therapy targeting a second splicing encoded by a protein kinase
  • administration of non-mutant protein kinase polypeptide in conjunction with antisense therapy targeting mutant protein kinase mRNA can also be used.
  • MOOSE03177 ctgl6439 524311..524473, 539059..539119, 569811..569849, 571874..571890, 576704..576824, 576955..577078, 578035..578097,
  • MOOSE03288 ctgl7685 34737..34809, 70525..70657, 76156..76460, 79186..79511, 84816.-85274
  • MOOSE03301 ctgl5131 20101431..20101501, 20107338..20107405, 20116288..20116347, 20168182..20168236, 20169109..20169174,
  • LAEAHRRERTGES (SEQ ID NO: 18) agccttaccttactttctcaagaagagcagctagtactctaccgattctccagaggggctttttggaaatatctaga aaggagagagaaaatagaaaaaaatttttaagtgcaaagttttgaaagaaaatatcaccagaaactagaac gtgacttctgaaacatccaaacatcgtgcgcctccatgacagtatttctgaagaagggtttcacta cctcgtgtttgaccttgttaccggcggggagctgtttgaagacattgtggccagagagtactacagtgtggccagccagccactgtatacatcagattc
  • MOOSE03312 ctgl35386574090..6574201, 6574516..6574611, 6592507..6592547, 6603133..6603342, 6605378..6605458, 6606064..6606160, 6613288..6613365, 6613889..6614022, 6620936..6621082, 6621308..6621427, 6629205..6629262, 6632097..6632182
  • MOOSE03332 ctgl59072781758..2781942, 2783326-2783392, 2824027..2824141, 2827236-2827388, 2828296-2828408, 2829233..2829307, 2832037..2832178, 2834312-2834421, 2834490-2834592, 2838446-2838457, 2859043..2859095
  • LNHPNIVKLFEVIETEKT LYLVMEYASAGEVFDYLVSHGRMKEKEARAKFR QEVSAVHYCHQKNIVHRDLKAENLLLDAEANEKIADFGFSNEFTLGSKLDTF CGSPPYAAPELFQGKKYDGPEVDIWSLGVILYTLVSGSLPFDGHNLKELRER VLRGKYRVPFYMSTDCESILRRFLVLNPAKRCTLEQIMKDKWINIGYEGEEL KPYTEPEEDFGDTKJUEVMVGMGYTREEIKESLTSQKYNEVTATYLLLGRKT
  • MOOSE03372 ctg21fm5 107539..107634, 114972-115106, 117564..118777,
  • VALRCLELQPQDRPPSRELLKHPVFRTTW (SEQ ID NO: 46) atggttggcaaactttccagaaggatctacttgagttctgcaagaatggttactacagtaccccatgtgttttcaaaa ctgttagaaatgctgagtgtttccagttccactcacttcaccaggatgcgtcgcgtttgatggctattgcagatgaggtgga aattgccgaagccatccagttgggcgtagaagacactttggatggtcaacaggacagcttcttgcaggcatctgttcccaa caactatctggaaaccacagagaacagttcccctgagtgcacaatccattttagagaaaactggaaaaggattatgtgcta caaaat
  • NLPPLLILVGTFPVECVHVKSAVEIVLSEPPHQVCFCVFFSFEMESCSVPQLEL QKSPVFCVDHVGSSRSELFLFGHLSFLSFLS (SEQ ID NO: 48) atggaaaagtaccacgtgttggagatgattggagaaggctcttttgggagggtgtacaagggtcgaagaaaata cagtgctcaggtcgtggccctgaagttcatcccaaaattggggcgctcagagaaggagctgaggaatttgcaacgaga gattgaaataatgcggggtctgcggcatcccaacattgtgcatatgcttgacagctttgaaactgataaagaggtggtggt ggtgacagactatgctgagggagagctctttcagatcctagaagat
  • MOOSE03455 ctgl5214 6841087-6841115, 6863772-6863842, 6868407-6868463, 6885556-6885770, 6890751..6890831, 6891392-6891423,
  • RGHPCLVPVFKGNASSFCPFKNKDEVRWVR (SEQ ID NO: 52) tacaggaatgtttctcgggctcatgaaaggttgcacagctgcttagttggcctgggagtgtttctgcggggggtg gcctacagggctaagagggagagagatggggaatggctactcagtggagcagtcaaaaaacacatggcattgattaag aacatggctgataaatatctgaaatccttccagcaccaaatgattccttttctttacagagaatggcaggctgtttattgtaatg gaatattgtgatggaggatctcatgaaaaggatcaatagacaacggggtgtgtatttagtgaagatcagatccctcggtttgtgtg
  • MOOSE03605 ctgl67343123770-3123875, 3124050-3124245, 3124825..3124996, 3125298..3125447, 3125740..3125943, 3126172-3126229, 3127039-3127251, 3128405-3128549, 3131441-3131534, 3132511-3132583, 3136796..3136971, 3140646-3140759, 3143510..3143640, 3146484..3146556, 3147052..3147114, 3153441..3153478, 3156029..3156051, 3157718-3157767, 3159767-3159858, 3163220-3163354, 3163842..3164061, 3165485-3165583
  • MOOSE03639 ctgl365734913..34950, 82022-82239, 82529-82663, 82800..82929, 83247..83406, 83533-83643, 84139-84368, 84550-84614, 85310-85415, 85597-85759, 85842-85978, 86211-86431, 86507..86548, 86754-86949, 87087-87254, 87654-87892, 90056..90182, 90381-90595, 92425-92569, 92781-92923, 93023-93326, 94887-95438, 99458-99542
  • MAVPSLWPWGACLPVEFLSLGFGLDTVEVCPSLDERS EVAELRQLENC SVVEGHLQILLMFTATGEDFRGLSFPRLTQVTDYLLLFRVYGLESLRDLFPN LAVERGTRLFLGYALVEFEMPHLRDVALPALGAVLRGAVRVEKNQELCHLS TEDWGLLQPAPGANHEVGNKLGEECADVCPGVLGAAGEPCAKTTFSGHTDY RCWTSSHCQRVCPCPHGMACTARGECCHTECLGGCSQPEDPRACVACRHL
  • EPDSSPTPRDCSPQNGGGRGCREPRSHHC (SEQ ID NO: 60) atggcagtgcctagtctgtggccctggggagcatgcctgcctgtgatcttcctcttgggatttggcctggata cagtagaggtgtgccccagcctggatattcgctcagaggtggcagagcttcgtcagctggagaactgcagcgtggtgg agggccacctgcagatcctgctcatgttcacagccaccggggaggacttccgcggcctcagcttccctcaccca ggtcaccgactacctgctgctcttccgtgtacggactggagagcctgcgcgacctcttcccaacctagcagtcatcccgcaccgactacctgctgct
  • MOOSE03609 ctgl2577 1588402..1588456, 1588545-1588612, 1588711-1588767, 1590533-1590619, 1593252-1593280, 1593323-1593359,
  • AEDLASQMLKGFPRDRVSAQEALVHDYFSALPSQLYQLP (SEQ ID NO: 70) gcctcatcttacttgaacttggagaagctgggtgaaggctcttatgcgacagtttacaaggggattagcAGAat aaatggacaactagtggctttaaaagtcatcagcatgaatgcagaggaaggagtcccatttacagctatccgagaaGC Ttctctcctgaagggtttgaaacatgccaatattgtgctcctgcatgacataatccacaccaaagagacactgacattcgtttt ttgaatacatggtgagtTACctggcccagtatatgtctcagcatccaggagggcttcatcctcataatgtcagactt
  • MOOSE03302 ctgl5540 6561837..6562051, 6568471..6568519, 6570944-6570985, 6581301..6581378, 6581838-6581921, 6583077-6583199,
  • MOOSE03459 ctgl8037 2081197-2081459, 2082567-2082646, 2095897..2095952, 2097096..2097225, 2102594..2102816, 2104449-2104553,
  • MOOSE03376 CaMK II - KENl/SNFl/Niml family MOOSE03459 OPK VI - weel/mikl family MOOSE03283 CMGC Other - CMGK kinases
  • NIDDM Non-insulin dep. Diabetes
  • MI Myocardial Infarction

Abstract

L'invention concerne des acides nucléiques codant des protéines kinases, ainsi que des procédés d'utilisation de ces acides nucléiques.
PCT/IB2002/002358 2001-06-26 2002-06-24 Acides nucleiques codant des proteines kinases WO2003000901A2 (fr)

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