EP1088079A2 - Nek-verwandte und bub1-verwandte proteinkinasen - Google Patents

Nek-verwandte und bub1-verwandte proteinkinasen

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Publication number
EP1088079A2
EP1088079A2 EP99930298A EP99930298A EP1088079A2 EP 1088079 A2 EP1088079 A2 EP 1088079A2 EP 99930298 A EP99930298 A EP 99930298A EP 99930298 A EP99930298 A EP 99930298A EP 1088079 A2 EP1088079 A2 EP 1088079A2
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EP
European Patent Office
Prior art keywords
seq
amino acid
kinase
kinase polypeptide
acid sequence
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EP99930298A
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English (en)
French (fr)
Inventor
Gregory Plowman
Ricardo Martinez
Yingfang Zhu
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Sugen LLC
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Sugen LLC
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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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders

Definitions

  • the present invention relates to novel kinase polypeptides, nucleotide sequences encoding the novel kinase polypeptides, as well as various products and methods useful for the diagnosis and treatment of various kinase-related diseases and conditions.
  • Cellular signal transduction is a fundamental mechanism whereby external stimuli that regulate diverse cellular processes are relayed to the interior of cells.
  • One of the key biochemical mechanisms of signal transduction involves the reversible phosphorylation of proteins, which enables regulation of the activity of mature proteins by altering their structure and function.
  • protein kinases in eukaryotes phosphorylate proteins on the hydroxyl substituent of serine, threonine and tyrosine residues. These kinases largely fall into two groups, those specific for phosphorylatmg serines and threonines, and those specific for phosphorylatmg tyrosines. Some kinases, referred to as "dual specificity" kinases, are able to phosphorylate on tyrosine as well as serine/threonine residues. Protein kinases can also be characterized by their location within the cell.
  • kinases are transmembrane receptor-type proteins capable of directly altering their catalytic activity in response to the external environment such as the binding of a ligand. Others are non-receptor-type proteins lacking any transmembrane domain. They can be found in a variety of cellular compartments from the inner surface of the cell membrane to the nucleus.
  • kinases are involved in regulatory cascades wherein their substrates may include other kinases whose activities are regulated by their phosphorylation state. Ultimately the activity of some downstream effector is modulated by phosphorylation resulting from activation of such a pathway.
  • Protein kinases are one of the largest families of eukaryotic proteins with several hundred known members. These proteins share a 250-300 amino acid domain that can be subdivided into 12 distinct subdomains that comprise the common catalytic core structure. These conserved protein motifs have recently been exploited using PCR-based cloning strategies leading to a significant expansion of the known kinases.
  • tyrosine kinases dual-specificity kinases
  • STK's serine/threonine kinases
  • the latter subfamily includes cyclic-nucleotide- dependent kinases, calcium/calmodulin kinases, cyclin-dependent kinases (CDK's), MAP- kinases, serine-threonine kinase receptors, and several other less defined subfamilies
  • Cell cycle checkpoints are regulatory pathways that control the order and timing of cell cycle transitions and ensure that critical events such as DNA replication and chromosome segregation are completed with high fidelity.
  • the mitotic checkpoint ensures proper chromosome segregation by delaying anaphase until all chromosomes are aligned on the spindles. Loss of cell cycle checkpoints in mitosis can result in cell death or uncontrolled cell division.
  • Cancer cells differ from normal cells in many important characteristics including differentiation, increased invasiveness and decreased drug sensitivity.
  • CDK's play a pivotal role in the regulation of mitosis during cell division.
  • the process of cell division occurs in four stages: S phase, the period during which
  • chromosomes duplicate; G2; mitosis; and Gl, or interphase. During mitosis the duplicated chromosomes are evenly segregated, allowing each daughter cell to receive a complete copy of the genome.
  • a key mitotic regulator in all eukaryotic cells is the STK cdc2, a CDK regulated by cyclin B.
  • Novel serine/threonine kinases have been identified and their protein sequence predicted by as part of the instant invention.
  • Mammalian members of the NEK family were identified through the use of a bioinformatics strategy, and the human BUB1 kinase was identified through the use of a targeted PCR cloning strategy.
  • the partial or complete sequences of these kinases are presented here, together with their classification, predicted or deduced protein structure, and an assessment of their pattern of expression.
  • the NEK kinases of the invention function in cell cycle regulation. Consequently, the deregulation of these kinases through overexpression and/or mutational events plays a role in cancer. Similarly, they also play a role in neurodegenerative and/or immune disorders through aberrant modulation of cell division, differentiation, and apoptosis. Thus, modulators of NEK kinases will play a role in the treatment and/or prevention of cancers and neurodegenerative or immune disorders in which a NEK kinase is functioning aberrantly through overexpression or as the result of a mutational event.
  • the human bubl gene was found to be a checkpoint gene in mitosis.
  • the biological and clinical therapeutic relevance of BUB 1 was demonstrated by expression analysis which showed that the bubl transcript was expressed at very low levels in normal human tissues, but was strikingly overexpressed in numerous tumor cell lines.
  • the high expression of the bubl gene in many malignant tumor cells indicates that deregulation of BUB1 may play a role in the development of cancer.
  • Overexpression of the bubl gene in cancer cells also suggests that it may be an attractive target for cancer therapy.
  • BUB1 expression was found to be cell cycle regulated and located in kinetochores, indicating that BUBl is a centromeric protein involved in chromosome segregation during mitosis. In addition to its potential role in tumorigenesis, BUBl may also be the target of human autoimmune diseases such as CREST syndrome typified by the presence of auto-antibodies to centromeric proteins.
  • a first aspect of the invention features an isolated, enriched, or purified nucleic acid molecule encoding a kinase polypeptide selected from the group consisting of a BUBl kinase having the amino acid sequence set forth in SEQ LD NO:2, a NEK4a kinase having the amino acid sequence set forth in SEQ LD NO:8, a NEK4b kinase having the amino acid sequence set forth in SEQ LD NO: 10, a NEK5 kinase having the amino acid sequence set forth in SEQ ID NO:4, and a NEK6 kinase having the amino acid sequence set forth in SEQ ID NO:6.
  • nucleic acid in reference to nucleic acid is meant a polymer of 6 (preferably 21, more preferably 39, most preferably 75) or more nucleotides conjugated to each other, including DNA and RNA that is isolated from a natural source or that is synthesized.
  • longer nucleic acids are preferred, for example those of 300, 600, 900 or more nucleotides and/or those having at least 50%, 60%, 75%, 90%, 95% or 99% identity to the sequence shown in SEQ ID NO:l, SEQ ID NO:3, SEQ ID NO 5, SEQ ID NO:7, or SEQ ID NO:9.
  • the isolated nucleic acid of the present invention is unique in the sense that it is not found in a pure or separated state in nature.
  • isolated indicates that a naturally occurring sequence has been removed from its normal cellular (i.e., chromosomal) environment. Thus, the sequence may be in a cell-free solution or placed in a different cellular environment. The term does not imply that the sequence is the only nucleotide chain present, but that it is essentially free (about 90 - 95% pure at least) of non-nucleotide material naturally associated with it, and thus is distinguished from isolated chromosomes.
  • enriched in reference to nucleic acid is meant that the specific DNA or RNA sequence constitutes a significantly higher fraction (2- to 5-fold) of the total DNA or RNA present in the cells or solution of interest than in normal or diseased cells or in the cells from which the sequence was taken. This could be caused by a person by preferential reduction in the amount of other DNA or RNA present, or by a preferential increase in the amount of the specific DNA or RNA sequence, or by a combination of the two. However, it should be noted that enriched does not imply that there are no other DNA or RNA sequences present, just that the relative amount of the sequence of interest has been significantly increased.
  • the term "significant" is used to indicate that the level of increase is useful to the person making such an increase, and generally means an increase relative to other nucleic acids of about at least 2-fold, more preferably at least 5- to 10-fold or even more.
  • the term also does not imply that there is no DNA or RNA from other sources.
  • the other source DNA may, for example, comprise DNA from a yeast or bacterial genome, or a cloning vector such as pUC19. This term distinguishes from naturally occurring events, such as viral infection, or tumor type growths, in which the level of one mRNA may be naturally increased relative to other species of mRNA. That is, the term is meant to cover only those situations in which a person has intervened to elevate the proportion of the desired nucleic acid.
  • nucleotide sequence be in purified form.
  • purified in reference to nucleic acid does not require absolute purity (such as a homogeneous preparation). Instead, it represents an indication that the sequence is relatively more pure than in the natural environment (compared to the natural level this level should be at least 2- to 5-fold greater, e.g., in terms of mg/mL).
  • Individual clones isolated from a cDNA library may be purified to electrophoretic homogeneity. The claimed DNA molecules obtained from these clones could be obtained directly from total DNA or from total RNA.
  • the cDNA clones are not naturally occurring, but rather are preferably obtained via manipulation of a partially purified naturally occurring substance (messenger RNA).
  • a cDNA library from mRNA involves the creation of a synthetic substance (cDNA) and pure individual cDNA clones can be isolated from the synthetic library by clonal selection of the cells carrying the cDNA library.
  • cDNA synthetic substance
  • the process which includes the construction of a cDNA library from mRNA and isolation of distinct cDNA clones yields an approximately 10 6 -fold purification of the native message.
  • purification of at least one order of magnitude, preferably two or three orders, and more preferably four or five orders of magnitude is expressly contemplated.
  • kinase polypeptide 32 (preferably 40, more preferably 45, most preferably 55) or more contiguous amino acids set forth in the amino acid sequence of SEQ ID NO:2, 33 (preferably 40, more preferably 45, most preferably 55) or more contiguous amino acids set forth in the amino acid sequence of SEQ ID NO:4, or 8 (preferably 15, more preferably 20, most preferably 30) or more contiguous amino acids set forth in the amino acid sequence of SEQ LD NO:6, 33 (preferably 40, more preferably 45, most preferably 55) or more contiguous amino acids set forth in the amino acid sequence of SEQ ID NO:8, 33 (preferably 40, more preferably 45, most preferably 55) or more contiguous amino acids set forth in the amino acid sequence of SEQ ID NO: 10, or functional derivatives thereof as described herein.
  • polypeptides of 100, 200, 300 or more amino acids are preferred.
  • the kinase polypeptide can be encoded by a full-length nucleic acid sequence or any portion of the full-length nucleic acid sequence, so long as a functional activity of the polypeptide is retained. It is well known in the art that due to the degeneracy of the genetic code numerous different nucleic acid sequences can code for the same amino acid sequence. Equally, it is also well known in the art that conservative changes in amino acid can be made to arrive at a protein or polypeptide which retains the functionality of the original. In both cases, all permutations are intended to be covered by this disclosure.
  • amino acid sequence will be substantially similar to the sequence shown in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, or SEQ ID NO: 10, or the corresponding full-length amino acid sequence, or fragments thereof.
  • a sequence that is substantially similar to the sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, or SEQ LD NO: 10 will preferably have at least 90% identity (more preferably at least 95% and most preferably 99-100%) to the sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, or SEQ ID NO:10.
  • identity is meant a property of sequences that measures their similarity or relationship. Identity is measured by dividing the number of identical residues by the total number of residues and gaps and multiplying the product by 100. "Gaps" are spaces in an alignment that are the result of additions or deletions of amino acids. Thus, two copies of exactly the same sequence have 100% identity, but sequences that are less highly conserved, and have deletions, additions, or replacements, may have a lower degree of identity. Those skilled in the art will recognize that several computer programs are available for determining sequence identity using standard parameters, for example Gapped BLAST or PSI-BLAST (Altschul, et al (1997) Nucleic Acids Res.
  • the invention features isolated, enriched, or purified nucleic acid molecules encoding a kinase polypeptide comprising a nucleotide sequence that: (a) encodes a polypeptide having the amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, or SEQ ID NO:10; (b) is the complement of the nucleotide sequence of (a); (c) hybridizes under highly stringent conditions to the nucleotide molecule of (a) and encodes a naturally occurring kinase polypeptide; (d) encodes a kinase polypeptide having the amino acid sequence set forth in SEQ LD NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, or SEQ ID NO:10, except that it lacks one or more, but not all, of the following segments of amino acid residues: 1-785, 786-1028, or 1029-1085 ofSEQ ID NO:2,
  • this domain is selected from the group N-terminal domain, catalytic domain, C-terminal catalytic domain, and C- terminal tail, and for NEK4a kinase, NEK4b kinase, NEK5 kinase or NEK6 kinase this domain is selected from the group N-terminal domain, catalytic domain, C-terminal catalytic domain, and C-terminal domain.
  • the term "complement” refers to two nucleotides that can form multiple favorable interactions with one another. For example, adenine is complementary to thymine as they can form two hydrogen bonds.
  • a nucleotide sequence is the complement of another nucleotide sequence if all of the nucleotides of the first sequence are complementary to all of the nucleotides of the second sequence.
  • domain refers to a region of a polypeptide which contains a particular function.
  • N-terminal or C-terminal domains of signal transduction proteins can serve functions including, but not limited to, binding molecules that localize the signal transduction molecule to different regions of the cell or binding other signaling molecules directly responsible for propagating a particular cellular signal.
  • Some domains can be expressed separately from the rest of the protein and function by themselves, while others must remain part of the intact protein to retain function. The latter are termed functional regions of proteins and also relate to domains.
  • N-terminal domain refers to the extracatalytic region located between the initiator methionine and the catalytic domain of the protein kinase.
  • the N-terminal domain can be identified following a Smith-Waterman alignment of the protein sequence against the non-redundant protein database to define the N-terminal boundary of the catalytic domain.
  • the N-terminal domain may or may not play a regulatory role in kinase function.
  • PAK65 An example of a protein kinase whose N-terminal domain has been shown to play a regulatory role is PAK65, which contains a CRIB motif used for Cdc42 and rac binding (Burbelo, P.D. et al (1995) J. Biol. Chem.
  • the N-terminal domain spans amino acid residues 1-785 of the sequence set forth in SEQ ID NO:2; 1-31 of the sequence set forth in SEQ LD NO:4; 1-22 of the sequence set forth in SEQ ID NO:6; and 1-42 of the sequence set forth in SEQ ID NO:8.
  • catalytic domain refers to a region of the protein kinase that is typically 25-300 amino acids long and is responsible for carrying out the phosphate transfer reaction from a high-energy phosphate donor molecule such as ATP or GTP to itself (autophosphorylation) or to other proteins (exogenous phosphorylation).
  • the catalytic domain of protein kinases is made up of 12 subdomains that contain highly conserved amino acid residues, and are responsible for proper polypeptide folding and for catalysis.
  • the catalytic domain can be identified following a Smith- Waterman alignment of the protein sequence against the non-redundant protein database.
  • the catalytic domain spans amino acid residues 32-283 of the sequence set forth in SEQ LD NO:4; and 23-302 of the sequence set forth in SEQ ID NO:6; and 43-294 of the sequence set forth in SEQ ID NO:8; and 1-259 of the sequence set forth in SEQ LD NO:10.
  • the C-terminal catalytic domain spans amino acid residues 786-1028 of the sequence set forth in SEQ ID NO:2.
  • the term "catalytic activity”, as used herein, defines the rate at which a kinase catalytic domain phosphorylates a substrate. Catalytic activity can be measured, for example, by determining the amount of a substrate converted to a phosphorylated product as a function of time. Catalytic activity can be measured by methods of the invention by holding time constant and determining the concentration of a phosphorylated substrate
  • Phosphorylation of a substrate occurs at the active-site of a protein kinase.
  • the active-site is normally a cavity in which the substrate binds to the protein kinase and is phosphorylated.
  • substrate refers to a molecule phosphorylated by a kinase of the invention.
  • Kinases phosphorylate substrates on serine/threonine or tyrosine amino acids.
  • the molecule may be another protein or a polypeptide.
  • C-terminal domain refers to the region located between the catalytic domain or the last (located closest to the C-terminus) functional domain and the carboxy- terminal amino acid residue of the protein kinase.
  • functional domain is meant any region of the polypeptide that may play a regulatory or catalytic role as predicted from amino acid sequence homology to other proteins or by the presence of amino acid sequences that may give rise to specific structural conformations (e.g. N-terminal domain).
  • the C-terminal domain can be identified by using a Smith- Waterman alignment of the protein sequence against the non-redundant protein database to define the C-terminal boundary of the catalytic domain or of any functional C-terminal extracatalytic domain.
  • the C-terminal domain may or may not play a regulatory role in kinase function.
  • An example of a protein kinase whose C- terminal domain may play a regulatory role is PAK3 which contains a heterotrimeric G t , subunit-binding site near its C-terminus (Leeuw, T. et al (1998) Nature, 391, 191-195).
  • the C-terminal domain also comprises the catalytic domain (above).
  • C-terminal catalytic domain spans amino acid residues 786-1028 of the sequence set forth in SEQ TD NO:2; 284-302 of the sequence set forth in SEQ ID NO:4; 295-313 of the sequence set forth in SEQ ID NO:8; and 260-268 of the sequence set forth in SEQ ID NO: 10.
  • C-terminal tail refers to a C-terminal domain of a protein kinase, that by homology extends or protrudes past the C-terminal amino acid of its closest homolog.
  • C-terminal tails can be identified by using a Smith- Waterman sequence alignment of the protein sequence against the non-redundant protein database, or by means of a multiple sequence alignment of homologous sequences using the DNAStar program Megalign. Depending on its length, a C-terminal tail may or may not play a regulatory role in kinase function. The C-terminal tail spans amino acid residues 1029- 1085 of the sequence set forth in SEQ ID NO:2.
  • signal transduction pathway refers to the molecules that propagate an extracellular signal through the cell membrane to become an intracellular signal. This signal can then stimulate a cellular response.
  • the polypeptide molecules involved in signal transduction processes are typically receptor and non-receptor protein tyrosine kinases, receptor and non-receptor protein phosphatases, SRC homology 2 and 3 domains, phosphotyrosine binding proteins (SRC homology 2 (SH2) and phosphotyrosine binding (PTB and PH) domain containing proteins), proline-rich binding proteins (SH3 domain containing proteins), nucleotide exchange factors, and transcription factors.
  • low or high stringency hybridization conditions may be used depending upon the specificity and selectivity desired. These conditions are well-known to those skilled in the art. Under stringent hybridization conditions only highly complementary nucleic acid sequences hybridize. Preferably, such conditions prevent hybridization of nucleic acids having more than 1 or 2 mismatches out of 20 contiguous nucleotides, more preferably, such conditions prevent hybridization of nucleic acids having more than 1 or 2 mismatches out of 50 contiguous nucleotides, most preferably, such conditions prevent hybridization of nucleic acids having more than 1 or 2 mismatches out of 100 contiguous nucleotides. In some instances, the conditions may prevent hybridization of nucleic acids having more than 5 mismatches in the full-length sequence.
  • stringent hybridization assay conditions hybridization assay conditions at least as stringent as the following: hybridization in 50% formamide, 5X SSC, 50 mM NaH 2 PO 4 , pH 6.8, 0.5% SDS, 0.1 mg/mL sonicated salmon sperm DNA, and 5X Denhardt's solution at 42 °C overnight; washing with 2X SSC, 0.1% SDS at 45 °C; and washing with 0.2X SSC, 0.1% SDS at 45 °C. Under some of the most stringent hybridization assay conditions, the second wash can be done with 0.1X SSC at a temperature up to 70 °C (Berger et al.
  • the invention features isolated, enriched, or purified nucleic acid molecules encoding kinase polypeptides, further comprising a vector or promoter effective to initiate transcription in a host cell.
  • the invention also features recombinant nucleic acid, preferably in a cell or an organism.
  • the recombinant nucleic acid may contain a sequence set forth in SEQ ID NO:l, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, or SEQ ID NO:9, or a functional derivative thereof and a vector or a promoter effective to initiate transcription in a host cell.
  • the recombinant nucleic acid can alternatively contain a transcriptional initiation region functional in a cell, a sequence complementary to an RNA sequence encoding a kinase polypeptide and a transcriptional termination region functional in a cell.
  • vector relates to a single or double-stranded circular nucleic acid molecule that can be transfected into cells and replicated within or independently of a cell genome.
  • a circular double-stranded nucleic acid molecule can be cut and thereby linearized upon treatment with restriction enzymes.
  • restriction enzymes An assortment of nucleic acid vectors, restriction enzymes, and the knowledge of the nucleotide sequences cut by restriction enzymes are readily available to those skilled in the art.
  • a nucleic acid molecule encoding a kinase can be inserted into a vector by cutting the vector with restriction enzymes and ligating the two pieces together.
  • transfecting defines a number of methods to insert a nucleic acid vector or other nucleic acid molecules into a cellular organism. These methods involve a variety of techniques, such as treating the cells with high concentrations of salt, an electric field, detergent, or DMSO to render the outer membrane or wall of the cells permeable to nucleic acid molecules of interest or use of various viral transduction strategies.
  • promoter refers to nucleic acid sequence needed for gene sequence expression. Promoter regions vary from organism to organism, but are well known to persons skilled in the art for different organisms. For example, in prokaryotes, the promoter region contains both the promoter (which directs the initiation of RNA transcription) as well as the DNA sequences which, when transcribed into RNA, will signal synthesis initiation. Such regions will normally include those 5'-non-coding sequences involved with initiation of transcription and translation, such as the TATA box, capping sequence, CAAT sequence, and the like.
  • the isolated nucleic acid comprises, consists essentially of, or consists of a nucleic acid sequence set forth in SEQ ID NO:l, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, or SEQ ID NO:9 which encodes the amino acid sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, a functional derivative thereof, or at least 35, 40, 45, 50, 60, 75, 100, 200, or 300 contiguous amino acids of SEQ ID NO:2, or at least 35, 40, 45, 50, 60, 75, 100, 200, or 300 contiguous amino acids of SEQ ID NO:4, or at least 10, 15, 20, 30, 40, 45, 50, 60, 75, 100, 200, or 300 contiguous amino acids of SEQ ID NO:6, or at least 35, 40, 45, 50, 60, 75, 100, 200, or 300 contiguous amino acids of SEQ ID NO:6, or at least 35, 40, 45, 50, 60, 75
  • a BUBl kinase polypeptide comprises, consists essentially of, or consists of at least 35, 40, 45, 50, 60, 75, 100, 200, or 300 contiguous amino acids of SEQ ID NO:2.
  • a NEK kinase polypeptide comprises, consists essentially of, or consists of at least 35, 40, 45, 50, 60, 75, 100, 200, or 300 contiguous amino acids of SEQ ID NO:4, at least 10, 15, 20, 30, 40, 45, 50, 60, 75, 100, 200, or 300 contiguous amino acids of SEQ ID NO:6, of at least 35, 40, 45, 50, 60, 75, 100, 200, or 300 contiguous amino acids of SEQ ID NO:8, or of at least 35, 40, 45, 50, 60, 75, 100, 200, or 300 contiguous amino acids of SEQ LD NO:10.
  • the nucleic acid may be isolated from a natural source by cDNA cloning or by subtractive hybridization.
  • the natural source may be mammalian, preferably human, blood, semen, or tissue, and the nucleic acid may be synthesized by the triester method or by using an automated DNA synthesizer.
  • mice refers preferably to such organisms as mice, rats, rabbits, guinea pigs, sheep, and goats, more preferably to cats, dogs, monkeys, and apes, and most preferably to humans.
  • the nucleic acid is a conserved or unique region, for example those useful for: the design of hybridization probes to facilitate identification and cloning of additional polypeptides, the design of PCR probes to facilitate cloning of additional polypeptides, obtaining antibodies to polypeptide regions, and designing antisense oligonucleotides.
  • conserved nucleic acid regions regions present on two or more nucleic acids encoding a kinase polypeptide, to which a particular nucleic acid sequence can hybridize under lower stringency conditions. Examples of lower stringency conditions suitable for screening for nucleic acid encoding kinase polypeptides are provided in Abe, et al. (J. Biol. Chem. 19:13361-13368, 1992), hereby incorporated by reference herein in its entirety, including any drawings, figures, or tables. Preferably, conserved regions differ by no more than 5 out of 20 nucleotides, even more preferably 2 out of 20 nucleotides or most preferably 1 out of 20 nucleotides.
  • nucleic acid region is meant a sequence present in a nucleic acid coding for a kinase polypeptide that is not present in a sequence coding for any other naturally occurring polypeptide.
  • Such regions preferably encode 32 (preferably 40, more preferably 45, most preferably 55) or more contiguous amino acids set forth in the full- length amino acid sequence of SEQ ID NO:2, 33 (preferably 40, more preferably 45, most preferably 55) or more contiguous amino acids set forth in the amino acid sequence of SEQ ID NO:4, or 8 (preferably 15, more preferably 20, most preferably 30) or more contiguous amino acids set forth in the amino acid sequence of SEQ ID NO:6, 33
  • a unique nucleic acid region is preferably of mammalian origin.
  • a second aspect of the invention features a nucleic acid probe for the detection of nucleic acid encoding a BUB 1 kinase polypeptide having the amino acid sequence set forth in SEQ ID NO:2 in a sample or a NEK kinase polypeptide in a sample.
  • the nucleic acid probe encodes a kinase polypeptide that is a fragment of the protein encoded by the amino acid sequence set forth in SEQ ID NO:4 or SEQ LD NO:6.
  • the NEK kinase polypeptide is selected from the group consisting of NEK4, NEK5, and NEK6; preferably NEK4 includes NEK4a having the amino acid sequence set forth in SEQ ID NO: 8 and NEK4b having the amino acid sequence set forth in SEQ ID NO: 10; more preferably the kinase polypeptide is selected from the group consisting of NEK5 having the amino acid sequence set forth in SEQ ID NO:4 and NEK6; and most preferably the kinase polypeptide is NEK 6 having the amino acid sequence set forth in SEQ ID NO:6.
  • the nucleic acid probe contains a nucleotide base sequence that will hybridize to the sequence set forth in SEQ ID NO: 1, SEQ ID NO:3 or SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, or a functional derivative thereof.
  • the nucleic acid probe hybridizes to nucleic acid encoding at least 12, 32, 75, 90, 105, 120, 150, 200, 250, 300 or 350 contiguous amino acids of the full-length sequence set forth in SEQ ID NO:2, at least 35, 75, 90, 105, 120, 150, 200, 250, 300 or 350 contiguous amino acids of the full-length sequence set forth in SEQ ID NO:4, at least 12, 32, 75, 90, 105, 120, 150, 200, 250, 300 or 350 contiguous amino acids of the full-length sequence set forth in SEQ ID NO:6, at least 35, 75, 90, 105, 120, 150, 200, 250, 300 or 350 contiguous amino acids of the full-length sequence set forth in SEQ ID NO:8, at least 35, 75, 90, 105, 120, 150, 200, 250, 300 or 350 contiguous amino acids of the full-length sequence set forth in SEQ ID NO: 10, or a functional derivative thereof.
  • Methods for using the probes include detecting the presence or amount of kinase RNA in a sample by contacting the sample with a nucleic acid probe under conditions such that hybridization occurs and detecting the presence or amount of the probe bound to kinase RNA.
  • the nucleic acid duplex formed between the probe and a nucleic acid sequence coding for a kinase polypeptide may be used in the identification of the sequence of the nucleic acid detected (Nelson et al, in Nonisotopic DNA Probe Techniques, Academic Press, San Diego, Kricka, ed., p. 275, 1992, hereby incorporated by reference herein in its entirety, including any drawings, figures, or tables).
  • Kits for performing such methods may be constructed to include a container means having disposed therein a nucleic acid probe.
  • the invention describes a recombinant cell or tissue comprising a nucleic acid molecule encoding a BUB 1 kinase polypeptide having the amino acid sequence set forth in SEQ ID NO:2 or encoding a NEK kinase polypeptide.
  • the NEK kinase polypeptide is selected from the group consisting of NEK4, NEK5, and NEK6; preferably NEK4 includes NEK4a having the amino acid sequence set forth in SEQ ID NO:8, and NEK4b having the amino acid sequence set forth in SEQ ID NO: 10; more preferably the kinase polypeptide is selected from the group consisting of NEK5 having the amino acid sequence set forth in SEQ ID NO:4 and NEK6 having the amino acid sequence set forth in SEQ ID NO:6; and most preferably the kinase polypeptide is
  • the nucleic acid may be under the control of the genomic regulatory elements, or may be under the control of exogenous regulatory elements including an exogenous promoter.
  • exogenous it is meant a promoter that is not normally coupled in vivo transcriptionally to the coding sequence for the kinase polypeptides.
  • the polypeptide is preferably a fragment of the protein encoded by the full-length amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, or SEQ ID NO: 10.
  • fragment is meant an amino acid sequence present in a kinase polypeptide.
  • such a sequence comprises at least 32, 45, 50, 60, 100, 200, or 300 contiguous amino acids of the full-length sequence set forth in SEQ ID NO:2, comprises at least 35, 45, 50, 60, 100, 200, or 300 contiguous amino acids of the sequence set forth in SEQ ID NO:4, comprises at least 10, 15, 20, 30, 45, 50, 60, 100, 200, or 300 contiguous amino acids of the sequence set forth in SEQ ID NO:6, comprises at least 35, 45, 50, 60, 100, 200, or 300 contiguous amino acids of the sequence set forth in SEQ ID NO:8, or comprises at least 35, 45, 50, 60, 100, 200, or 300 contiguous amino acids of the sequence set forth in SEQ ID NO: 10.
  • the invention features an isolated, enriched, or purified BUBl kinase polypeptide having the amino acid sequence set forth in SEQ D NO:2 or an isolated, enriched, or purified NEK kinase polypeptide.
  • the NEK kinase polypeptide is selected from the group consisting of NEK4, NEK5, and NEK6; preferably NEK4 includes NEK4a having the amino acid sequence set forth in SEQ ID NO:8 and NEK4b having the amino acid sequence set forth in SEQ ID NO: 10; more preferably the kinase polypeptide is selected from the group consisting of NEK5 having the amino acid sequence set forth in SEQ ID NO:4 and NEK6 having the amino acid sequence set forth in SEQ ID NO:6; and most preferably the kinase polypeptide is NEK 6.
  • isolated in reference to a polypeptide is meant a polymer of 6 (preferably 12, more preferably 18, most preferably 25, 32, 40, or 50) or more amino acids conjugated to each other, including polypeptides that are isolated from a natural source or that are synthesized. In certain aspects longer polypeptides are preferred, such as those with 100, 200, 300, 400, or more contiguous amino acids of the full-length sequence set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, or SEQ ID NO: 10.
  • isolated polypeptides of the present invention are unique in the sense that they are not found in a pure or separated state in nature.
  • Use of the term "isolated” indicates that a naturally occurring sequence has been removed from its normal cellular environment. Thus, the sequence may be in a cell-free solution or placed in a different cellular environment. The term does not imply that the sequence is the only amino acid chain present, but that it is essentially free (about 90 - 95% pure at least) of non-amino acid-based material naturally associated with it.
  • enriched in reference to a polypeptide is meant that the specific amino acid sequence constitutes a significantly higher fraction (2- to 5-fold) of the total amino acid sequences present in the cells or solution of interest than in normal or diseased cells or in the cells from which the sequence was taken. This could be caused by a person by preferential reduction in the amount of other amino acid sequences present, or by a preferential increase in the amount of the specific amino acid sequence of interest, or by a combination of the two. However, it should be noted that enriched does not imply that there are no other amino acid sequences present, just that the relative amount of the sequence of interest has been significantly increased.
  • the term significant here is used to indicate that the level of increase is useful to the person making such an increase, and generally means an increase relative to other amino acid sequences of about at least 2-fold, more preferably at least 5- to 10-fold or even more.
  • the term also does not imply that there is no amino acid sequence from other sources.
  • the other source of amino acid sequences may, for example, comprise amino acid sequence encoded by a yeast or bacterial genome, or a cloning vector such as pUC19. The term is meant to cover only those situations in which man has intervened to increase the proportion of the desired amino acid sequence.
  • an amino acid sequence be in purified form.
  • purified in reference to a polypeptide does not require absolute purity (such as a homogeneous preparation); instead, it represents an indication that the sequence is relatively purer than in the natural environment. Compared to the natural level this level should be at least 2-to 5-fold greater (e.g., in terms of mg/mL). Purification of at least one order of magnitude, preferably two or three orders, and more preferably four or five orders of magnitude is expressly contemplated. The substance is preferably free of contamination at a functionally significant level, for example 90%, 95%, or 99% pure.
  • the kinase polypeptide is a fragment of the protein encoded by the full-length amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, or SEQ ID NO:10.
  • the kinase polypeptide contains at least 32, 45, 50, 60, 100, 200, or 300 contiguous amino acids of the full-length sequence set forth in SEQ ID NO:2, at least 35, 45, 50, 60, 100, 200, or 300 contiguous amino acids of the sequence set forth in SEQ LD NO:4, at least 10, 15, 25, 35, 45, 50, 60, 100, 200, or 300 contiguous amino acids of the sequence set forth in SEQ ID NO:6, at least 35, 45, 50, 60, 100, 200, or 300 contiguous amino acids of the sequence set forth in SEQ ID NO:8, or at least 35, 45, 50, 60, 100, 200, or 300 contiguous amino acids of the sequence set forth in SEQ ID NO: 10, or a functional derivative thereof.
  • the kinase polypeptide comprises an amino acid sequence having (a) the amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, or SEQ LD NO: 10; (b) the amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6, except that it lacks one or more of the following segments of amino acid residues: 1-785, 786-1028, or 1029-1085 of SEQ ID NO:2, 1-31, 32-283, or 284-302 of SEQ ID NO:4, or 1-22 or 23-302 of SEQ ID NO:6; (c) the amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:4, or SEQ LD NO:6 from amino acid residues 1-785, 786-1028, or 1029-1085 of SEQ ID NO:2, 1-31, 32-283, or 284-302 of SEQ ID NO:4, or 1-22 or 23-302 of SEQ ID NO:6;
  • the polypeptide can be isolated from a natural source by methods well-known in the art.
  • the natural source may be mammalian, preferably human, blood, semen, or tissue, and the polypeptide may be synthesized using an automated polypeptide synthesizer.
  • the invention includes a recombinant kinase polypeptide selected from the group consisting of BUBl, NEK4a, NEK4b, NEK5 and NEK6 kinase polypeptides.
  • recombinant kinase polypeptide is meant a polypeptide produced by recombinant DNA techniques such that it is distinct from a naturally occurring polypeptide either in its location (e.g., present in a different cell or tissue than found in nature), purity or structure. Generally, such a recombinant polypeptide will be present in a cell in an amount different from that normally observed in nature.
  • the invention features an antibody (e.g., a monoclonal or polyclonal antibody) having specific binding affinity to a kinase polypeptide or a kinase polypeptide domain or fragment where the polypeptide is selected from the group consisting of a BUBl kinase polypeptide having the amino acid sequence set forth in SEQ ID NO:2, a NEK4a kinase polypeptide having the amino acid sequence set forth in SEQ ID NO: 8, a NEK4b kinase polypeptide having the amino acid sequence set forth in SEQ ID NO: 10, a NEK5 kinase polypeptide having the amino acid sequence set forth in SEQ ID NO:4, and a NEK6 kinase polypeptide having the amino acid sequence set forth in SEQ ID NO:6.
  • specific binding affinity is meant that the antibody binds to the target kinase polypeptide with greater affinity than it binds to other polypeptides under
  • Antibodies or antibody fragments are polypeptides that contain regions that can bind other polypeptides.
  • the term "specific binding affinity" describes an antibody that binds to a kinase polypeptide with greater affinity than it binds to other polypeptides under specified conditions.
  • Antibodies can be used to identify an endogenous source of BUB 1 or NEK kinase polypeptides, to monitor cell cycle regulation, and for irnmuno- localization of NEK kinase polypeptides to the centrosomes.
  • polyclonal refers to antibodies that are heterogenous populations of antibody molecules derived from the sera of animals immunized with an antigen or an antigenic functional derivative thereof.
  • various host animals may be immunized by injection with the antigen.
  • Various adjuvants may be used to increase the immunological response, depending on the host species.
  • Monoclonal antibodies are substantially homogenous populations of antibodies to a particular antigen. They may be obtained by any technique which provides for the production of antibody molecules by continuous cell lines in culture. Monoclonal antibodies may be obtained by methods known to those skilled in the art (Kohler et al, Nature 256:495-497, 1975, and U.S. Patent No. 4,376,110, both of which are hereby incorporated by reference herein in their entirety including any figures, tables, or drawings).
  • antibody fragment refers to a portion of an antibody, often the hypervariable region and portions of the surrounding heavy and light chains, that displays specific binding affinity for a particular molecule.
  • a hypervariable region is a portion of an antibody that physically binds to the polypeptide target.
  • Antibodies or antibody fragments having specific binding affinity to a kinase polypeptide of the invention may be used in methods for detecting the presence and/or amount of kinase polypeptide in a sample by probing the sample with the antibody under conditions suitable for kinase-antibody immunocomplex formation and detecting the presence and/or amount of the antibody conjugated to the kinase polypeptide.
  • Diagnostic kits for performing such methods may be constructed to include antibodies or antibody fragments specific for the kinase as well as a conjugate of a binding partner of the antibodies or the antibodies themselves.
  • An antibody or antibody fragment with specific binding affinity to a kinase polypeptide of the invention can be isolated, enriched, or purified from a prokaryotic or eukaryotic organism. Routine methods known to those skilled in the art enable production
  • Antibodies having specific binding affinity to a kinase polypeptide of the invention may be used in methods for detecting the presence and/or amount of kinase polypeptide in a sample by contacting the sample with the antibody under conditions such that an immunocomplex forms and detecting the presence and/or amount of the antibody conjugated to the kinase polypeptide.
  • Diagnostic kits for performing such methods may be constructed to include a first container containing the antibody and a second container having a conjugate of a binding partner of the antibody and a label, such as, for example, a radioisotope. The diagnostic kit may also include notification of an FDA approved use and instructions therefor.
  • the invention features a hybridoma which produces an antibody having specific binding affinity to a kinase ' polypeptide or a kinase polypeptide domain, where the polypeptide is selected from the group consisting of a BUB 1 kinase polypeptide having the amino acid sequence set forth in SEQ ID NO:2, a NEK4a kinase polypeptide having the amino acid sequence set forth in SEQ ID NO:8, aNEK4b kinase polypeptide having the amino acid sequence set forth in SEQ ID NO: 10, a NEK5 kinase polypeptide having the amino acid sequence set forth in SEQ ID NO:4, and a NEK6 kinase polypeptide having the amino acid sequence set forth in SEQ ID NO:6.
  • hybrida an immortalized cell line that is capable of secreting an antibody, for example an antibody to a kinase of the invention.
  • the antibody to the kinase comprises a sequence of amino acids that is able to specifically bind a kinase polypeptide of the invention.
  • the invention features a kinase polypeptide binding agent able to bind to a kinase polypeptide selected from the group consisting of a BUBl kinase polypeptide having the amino acid sequence set forth in SEQ LD NO: 2, a NEK4a kinase polypeptide having the amino acid sequence set forth in SEQ ID NO: 8, a NEK4b kinase polypeptide having the amino acid sequence set forth in SEQ ID NO: 10, aNEK5 kinase polypeptide having the amino acid sequence set forth in SEQ LD NO: 4, and a NEK6 kinase polypeptide having the amino acid sequence set forth in SEQ ID NO:6.
  • a BUBl kinase polypeptide having the amino acid sequence set forth in SEQ LD NO: 2 a NEK4a kinase polypeptide having the amino acid sequence set forth in SEQ ID NO: 8
  • the binding agent is preferably a purified antibody that recognizes an epitope present on a kinase polypeptide of the invention.
  • Other binding agents include molecules that bind to ft kinase polypeptides and analogous molecules that bind to a kinase polypeptide. Such binding agents may be identified by using assays that measure kinase binding partner activity, such as those that measure PDGFR activity.
  • the invention also features a method for screening for human cells containing a kinase polypeptide of the invention or an equivalent sequence.
  • the method involves identifying the novel polypeptide in human cells using techniques that are routine and standard in the art, such as those described herein for identifying the kinases of the invention (e.g., cloning, Southern or Northern blot analysis, in situ hybridization, PCR amplification, etc.).
  • the invention features methods for identifying a substance that modulates kinase activity comprising the steps of: (a) contacting a kinase polypeptide selected from the group consisting of a BUBl kinase polypeptide having the amino acid sequence set forth in SEQ ID NO:2, a NEK4a kinase polypeptide having the amino acid sequence set forth in SEQ ID NO:8, a NEK4b kinase polypeptide having the amino acid sequence set forth in SEQ ID NO: 10, a NEK5 kinase polypeptide having the amino acid sequence set forth in SEQ ID NO:4, and a NEK6 kinase polypeptide having the amino acid sequence set forth in SEQ ID NO: 6 with a test substance; (b) measuring the activity of said polypeptide; and (c) determining whether said substance modulates the activity of said polypeptide.
  • a kinase polypeptide selected from the group consisting of a BUBl kina
  • modulates refers to the ability of a compound to alter the function of a kinase of the invention.
  • a modulator preferably activates or inhibits the activity of a kinase of the invention depending on the concentration of the compound exposed to the kinase.
  • the term "activates” refers to increasing the cellular activity of the kinase.
  • the term inhibit refers to decreasing the cellular activity of the kinase.
  • Kinase activity is preferably the interaction with a natural binding partner.
  • modulates also refers to altering the function of kinases of the invention by increasing or decreasing the probability that a complex forms between the kinase and a natural binding partner.
  • a modulator preferably increases the probability that such a complex forms between the kinase and the natural binding partner, more preferably increases or decreases the probability that a complex forms between the kinase and the natural binding partner depending on the concentration of the compound exposed to the kinase, and most preferably decreases the probability that a complex forms between the kinase and the natural binding partner.
  • complex refers to an assembly of at least two molecules bound to one another.
  • Signal transduction complexes often contain at least two protein molecules bound to one another.
  • a protein tyrosine receptor protein kinase, GRB2, SOS, RAF, and RAS assemble to form a signal transduction complex in response to a mitogenic ligand.
  • natural binding partner refers to polypeptides, lipids, small molecules, or nucleic acids that bind to kinases in cells.
  • a change in the interaction between a kinase and a natural binding partner can manifest itself as an increased or decreased probability that the interaction forms, or an increased or decreased concentration of kinase/natural binding partner complex.
  • contacting refers to mixing a solution comprising the test compound with a liquid medium bathing the cells of the methods.
  • the solution comprising the compound may also comprise another component, such as dimethyl sulfoxide (DMSO), which facilitates the uptake of the test compound or compounds into the cells of the methods.
  • DMSO dimethyl sulfoxide
  • the solution comprising the test compound may be added to the medium bathing the cells by utilizing a delivery apparatus, such as a pipette-based device or syringe-based device.
  • the invention features methods for identifying a substance that modulates kinase activity in a cell comprising the steps of: (a) expressing a kinase polypeptide in a cell, wherein said polypeptide is selected from the group consisting of a BUBl kinase polypeptide having the amino acid sequence set forth in SEQ ID NO:2, a NEK4a kinase polypeptide having the amino acid sequence set forth in SEQ ID NO:8, a NEK4b kinase polypeptide having the amino acid sequence set forth in SEQ LD NO: 10, a NEK5 kinase polypeptide having the amino acid sequence set forth in SEQ ID NO:4, and a NEK6 kinase polypeptide having the amino acid sequence set forth in SEQ ID NO:6; (b) adding a test substance to said cell; and (c) monitoring a change in cell phenotype or the interaction between said polypeptide and a natural binding partner.
  • a test substance
  • expressing refers to the production of kinases of the invention from a nucleic acid vector containing kinase genes within a cell.
  • the nucleic acid vector is transfected into cells using well known techniques in the art as described herein.
  • the invention provides methods for treating a disease by administering to a patient in need of such treatment a substance that modulates the activity of a BUBl kinase polypeptide having the amino acid sequence set forth in SEQ ID NO:2.
  • the disease is cancer, more preferably the cancer is selected from the group consisting of leukemia, cervical cancer, lymphoma, colon cancer, lung cancer, melanoma, ovarian cancer, CNS cancer, prostate cancer, kidney cancer, and breast cancer.
  • the disease or disorder is CREST syndrome.
  • CREST syndrome is named for the primary symptoms of Calcinosis cutis, Raynaud's phenomenon, Esophageal involvement, Sclerodactyly, and Telangeictasia. Patients with CREST syndrome suffer from progressive systemic sclerosis, diffuse systemic scleroderma, primary pulmonary hypertension, idiopathic portal hypertension, as well as increased incidence of malignant cancers.
  • Substances useful for treatment of kinase-related disorders or diseases preferably show positive results in one or more in vitro assays for an activity corresponding to treatment of the disease or disorder in question (Examples of such assays are provided in the references in section VIII, below). Examples of substances that can be screened for favorable activity are provided and referenced in section VIII, below.
  • the substances that modulate the activity of the kinases preferably include, but are not limited to, antisense oligonucleotides and inhibitors of protein kinases, as determined by methods and screens referenced in section VIII, below.
  • preventing refers to decreasing the probability that an organism contracts or develops an abnormal condition.
  • treating refers to having a therapeutic effect and at least partially alleviating or abrogating an abnormal condition in the organism.
  • therapeutic effect refers to the inhibition or activation factors causing or contributing to the abnormal condition.
  • a therapeutic effect relieves to some extent one or more of the symptoms of the abnormal condition.
  • a therapeutic effect can refer to one or more of the following: (a) an increase in the proliferation, growth, and/or differentiation of cells; (b) inhibition (i.e., slowing or stopping) of cell death; (c) inhibition of degeneration; (d) relieving to some extent one or more of the symptoms associated with the abnormal condition; and (e) enhancing the function of the affected population of cells.
  • Compounds demonstrating efficacy against abnormal conditions can be identified as described herein.
  • abnormal condition refers to a function in the cells or tissues of an organism that deviates from their normal functions in that organism.
  • An abnormal condition can relate to cell proliferation, cell differentiation, or cell survival.
  • Abnormal conditions include leukemia, cervical, lymphoma, colon, lung, melanoma, ovarian, CNS, prostate, kidney, and breast cancer, as well as CREST syndrome.
  • Abnormal cell proliferative conditions include cancers such as fibrotic and mesangial disorders, abnormal angiogenesis and vasculogenesis, wound healing, psoriasis, diabetes mellitus, and inflammation, as well as the cancers of the invention.
  • Abnormal differentiation conditions include, but are not limited to neurodegenerative disorders, slow wound healing rates, and slow tissue grafting healing rates.
  • Abnormal cell survival conditions relate to conditions in which programmed cell death (apoptosis) pathways are activated or abrogated.
  • a number of protein kinases are associated with the apoptosis pathways. Aberrations in the function of any one of the protein kinases could lead to cell immortality or premature cell death.
  • aberration in reference to the function of a kinase in a signal transduction process, refers to a kinase that is over- or under-expressed in an organism, mutated such that its catalytic activity is lower or higher than wild-type protein kinase activity, mutated such that it can no longer interact with a natural binding partner, is no longer modified by another protein kinase or protein phosphatase, or no longer interacts with a natural binding partner.
  • administering relates to a method of incorporating a compound into cells or tissues of an organism.
  • the abnormal condition can be prevented or treated when the cells or tissues of the organism exist within the organism or outside of the organism.
  • Cells existing outside the organism can be maintained or grown in cell culture dishes.
  • many techniques exist in the art to administer compounds including (but not limited to) oral, parenteral, dermal, injection, and aerosol applications.
  • multiple techniques exist in the art to administer the compounds including (but not limited to) cell microinjection techniques, transformation techniques, and carrier techniques.
  • the abnormal condition can also be prevented or treated by administering a compound to a group of cells having an aberration in a signal transduction pathway to an organism.
  • the effect of administering a compound on organism function can then be monitored.
  • the organism is preferably a mouse, rat, rabbit, guinea pig, or goat, more preferably a monkey or ape, and most preferably a human.
  • the eleventh aspect of the invention features methods for detection of nucleic acid encoding a BUBl kinase polypeptide having the amino acid sequence set forth in SEQ LD NO:2 in a sample as a diagnostic tool for a disease or a disorder, wherein the method comprises the steps of: (a) contacting the sample with a nucleic acid probe which hybridizes under hybridization assay conditions to a nucleic acid target region of bubl, the probe comprising the nucleic acid sequence encoding the polypeptide, fragments thereof, and the complements of the sequences and fragments; and (b) detecting the presence or amount of the probe:target region hybrid as an indication of the disease or the disorder.
  • the disease or disorder is cancer. More preferably the cancer is selected from the group consisting of leukemia, cervical cancer, lymphoma, colon cancer, lung cancer, melanoma cancer, ovarian cancer, CNS cancer, prostate cancer, kidney cancer, and breast cancer. Alternatively, the disease or disorder is CREST syndrome.
  • target region is the full-length nucleotide base sequence set forth in SEQ ID NO:l, a functional derivative thereof, or a fragment thereof, to which the nucleic acid probe will specifically hybridize. Specific hybridization indicates that in the presence of other nucleic acids the probe only hybridizes detectably with the kinase of the invention's target region. Putative target regions can be identified by methods well known in the art consisting of alignment and comparison of the most closely related sequences in the database.
  • the nucleic acid probe hybridizes to a nucleic acid target region encoding at least 12, 32, 50, 75, 90, 105, 120, 150, 200, 250, 300 or 350 contiguous amino acids of the full-length sequence set forth in SEQ ID NO:2, or a functional derivative thereof.
  • Hybridization conditions should be such that hybridization occurs only with the kinase genes in the presence of other nucleic acid molecules. Under stringent hybridization conditions only highly complementary nucleic acid sequences hybridize. Preferably, such conditions prevent hybridization of nucleic acids having 1 or 2 mismatches out of 20 contiguous nucleotides. Such conditions are defined supra.
  • the diseases for which detection of kinase genes in a sample could be diagnostic include diseases in which nucleic acids (DNA and/or RNA) encoding the BUBl kinase are amplified in comparison to normal cells.
  • amplification is meant increased numbers of kinase DNA or RNA in a cell compared with normal cells.
  • kinases are typically found as single copy genes.
  • the chromosomal location of the kinase genes may be amplified, resulting in multiple copies of the gene, or amplification.
  • Gene amplification can lead to amplification of kinase RNA, or kinase RNA can be amplified in the absence of kinase DNA amplification.
  • RNA can be the detectable presence of kinase RNA in cells, since in some normal cells there is no basal expression of kinase RNA. In other normal cells, a basal level of expression of kinase exists, therefore in these cases amplification is the detection of at least 1- to 2-fold, and preferably more, kinase RNA, compared to the basal level.
  • the diseases that could be diagnosed by detection of nucleic acid encoding a BUBl kinase polypeptide in a sample preferably include cancers and CREST syndrome.
  • the test samples suitable for nucleic acid probing methods of the present invention include, for example, cells or nucleic acid extracts of cells, or biological fluids.
  • the samples used in the above-described methods will vary based on the assay format, the detection method and the nature of the tissues, cells or extracts to be assayed. Methods for preparing nucleic acid extracts of cells are well known in the art and can be readily adapted in order to obtain a sample which is compatible with the method utilized.
  • the invention provides methods for treating a disease by administering to a patient in need of such treatment a substance that modulates the activity of a NEK kinase polypeptide.
  • a substance that modulates the activity of a NEK kinase polypeptide is selected from the group consisting of NEK4, NEK5, and NEK6.
  • NEK4 includes NEK4a kinase polypeptide having the amino acid sequence set forth in SEQ ID NO:8 and NEK4b kinase polypeptide having the amino acid sequence set forth in SEQ ID NO: 10; more preferably the kinase polypeptide is selected from the group consisting of NEK5 kinase polypeptide having the amino acid sequence set forth in SEQ ID NO:4 and NEK6 kinase polypeptide having the amino acid sequence set forth in SEQ ID NO:6; and most preferably the kinase polypeptide is NEK 6.
  • Substances useful for treatment of kinase-related disorders or diseases preferably show positive results in one or more in vitro assays for an activity corresponding to treatment of the disease or disorder in question (Examples of such assays are provided in the references in section IX, and Examples 22-24, below). Examples of substances that zs can be screened for favorable activity are provided and referenced in section IX, below.
  • the substances that modulate the activity of the kinases preferably include, but are not limited to, antibodies, antisense oligonucleotides and inhibitors of protein kinases, as determined by methods and screens referenced in section IX and Examples 19, and 22-24, below.
  • the substance is an antisense oligonucleotide for the inhibition of the expression of NEK kinase polypeptide and fragments thereof.
  • the kinase polypeptide is selected from the group consisting of NEK4, NEK5, and NEK6.
  • NEK4 includes NEK4a and NEK4b, and the antisense oligonucleotides are synthesized as phosphorothioates.
  • abnormal condition with respect to a NEK kinase refers to a function in the cells or tissues of an organism that deviates from their normal functions in that organism. An abnormal condition can relate to cell proliferation, cell differentiation, or cell survival.
  • Abnormal conditions include: cancer and neurodegenerative and immune disorders.
  • the thirteenth aspect of the invention features methods for detection of nucleic acid encoding a NEK kinase polypeptide in a sample as a diagnostic tool for a disease or a disorder, wherein said method comprises: (a) contacting the sample with a nucleic acid probe which hybridizes under hybridization assay conditions to a nucleic acid target region of the kinase polypeptide, the probe comprising the nucleic acid sequence encoding the kinase polypeptide, fragments thereof, and the complements of the sequences and fragments; and (b) detecting the presence or amount of the probe:target region hybrid as an indication of the disease or the disorder.
  • the kinase polypeptide is selected from the group consisting of NEK4, NEK5, and NEK6.
  • NEK4 includes NEK4a kinase polypeptide having the amino acid sequence set forth in SEQ ID NO: 8 and NEK4b kinase polypeptide having the amino acid sequence set forth in SEQ ID NO: 10; more preferably the kinase polypeptide is selected from the group consisting of NEK5 kinase polypeptide having the amino acid sequence set forth in SEQ LD NO:4 and NEK6 kinase polypeptide having the amino acid sequence set forth in SEQ LD NO:6; and most preferably the kinase polypeptide is NEK 6.
  • the disease or disorder is selected from the group consisting of cancer and neurodegenerative and immune disorders. More preferably the disease or disorder is cancer.
  • the nucleic acid "target region" is the full-length nucleotide base sequence set forth in SEQ ID NO:3, SEQ ID NO:5, SEQ LD NO:7, or SEQ LD NO:9, a functional derivative thereof, or a fragment thereof, to which the nucleic acid probe will specifically hybridize. Specific hybridization indicates that in the presence of other nucleic acids the probe only hybridizes detectably with the kinase of the invention's target region. Putative target regions can be identified by methods well known in the art consisting of alignment and comparison of the most closely related sequences in the database.
  • the nucleic acid probe hybridizes to a nucleic acid target region encoding at least 6, 12, 32, 50, 75, 90, 105, 120, 150, 200, 250, 300 or 350 contiguous amino acids of the sequence set forth in SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, or SEQ ID NO:10, or a functional derivative thereof.
  • Hybridization conditions should be such that hybridization occurs only with the kinase genes, even in the presence of other nucleic acid molecules. Under stringent hybridization conditions only highly complementary nucleic acid sequences hybridize. Preferably, such conditions prevent hybridization of nucleic acids having 1 or 2 mismatches out of 20 contiguous nucleotides. Such conditions are defined supra.
  • the diseases for which detection of kinase genes in a sample could be diagnostic include diseases in which NEK kinase polypeptide nucleic acid (DNA and/or RNA) is amplified in comparison to normal cells.
  • the diseases that could be diagnosed by detection of nucleic acid encoding a NEK kinase polypeptide in a sample preferably include cancer and neurodegenerative and immune disorders.
  • the test samples suitable for nucleic acid probing methods of the present invention include, for example, cells or nucleic acid extracts of cells, or biological fluids. The samples used in the above-described methods will vary based on the assay format, the detection method and the nature of the tissues, cells or extracts to be assayed. Methods for preparing nucleic acid extracts of cells are well known in the art and can be readily adapted in order to obtain a sample that is compatible with the method utilized.
  • the fourteenth aspect of the invention features antisense oligonucleotides for the inhibition of the expression of a BUBl kinase polypeptide having the amino acid sequence set forth in SEQ ID NO:2 or a NEK kinase polypeptide.
  • the kinase polypeptide is selected from the group consisting of BUBl, NEK4, NEK5, and NEK6, and fragments thereof.
  • NEK4 includes NEK4a having the amino acid sequence set forth in SEQ ID NO:8 and NEK4b kinase polypeptide having the amino acid sequence set forth in SEQ ID NO: 10; more preferably the kinase polypeptide is selected from the group consisting of NEK5 kinase polypeptide having the amino acid sequence set forth in SEQ ID NO:4 and NEK6 kinase polypeptide having the amino acid sequence set forth in SEQ ID NO:6; and most preferably the kinase polypeptide is NEK 6.
  • the oligonucleotide is the complement of a sequence encoding a fragment of the protein encoded by the amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, or SEQ ID NO: 10.
  • the antisense oligonucleotides are synthesized as phosphorothioates.
  • Antisense oligonucleotides are preferably designed to specifically inhibit expression of a BUB 1 or NEK kinases. Preferably this is accomplished by identifying sequences unique to kinase polypeptides of the invention (one or more). Preferably, these sequences are not present in other non-BUBl or non-NEK kinase polypeptides present in a sample or in a patient. Methods to identify such sequences are well-known in the art and thus are only briefly described.
  • antisense oligonucleotides are 10 to 100 oligonucleotides, more preferably 15 to 30 nucleotides, and most preferably 18 to 25 nucleotides in length.
  • the antisense oligonucleotides of the invention are preferably used to inhibit BUB 1 protein or NEK protein expression in vivo in normal and tumor cells.
  • Antisense oligonucleotides can be used either singly or in combination.
  • expression of a BUB 1 kinase or a NEK kinase is significantly reduced, and more preferably reduced to below the limit of detection.
  • treatment with the antisense oligonucleotides of the invention inhibits growth and/or induces apoptosis in cells.
  • Antisense oligonucleotides can also be used to inhibit BUBl or NEK protein expression in human tumor cell xenografts in nude mice.
  • antisense oligonucleotides are used as a treatment in various human diseases and disorders in which BUB 1 kinase polypeptides or NEK kinase polypeptides are overexpressed or aberrantly expressed.
  • antisense oligonucleotides and effective combinations can be identified by methods well known in the art. Briefly, cells or tissues overexpressing BUBl or NEK kinase polypeptides can be contacted with antisense oligonucleotides, either singly or in combination, and the expression of bubl or nek RNA, and/or BUBl kinase or NEK kinase polypeptides can be determined by methods described herein. Preferably, treatment with antisense oligonucleotides to BUBl kinases or NEK kinases causes a decrease in the
  • Figures 1A-C show a human bubl nucleic acid sequence (SEQ ID NO:l).
  • Figure 2 shows a human BUBl amino acid sequence (SEQ ID NO:2).
  • Figures 3 A & B show a human BUBl (SEQ ID NO:2), mouse BUBl (SEQ ID NO:22), and S. cerevisiae BUBl (SEQ ID NO:23) multiple sequence alignment.
  • Figures 4A, 4B, 4C, and 4D show the partial or full-length nucleic acid sequences of human NEK4a (SEQ ID NO:7), human NEK4b (SEQ ID NO:9), human NEK5 (SEQ ID NO:3), and murine NEK6 (SEQ LD NO:5) kinase polypeptides.
  • Figure 5 shows the partial or full-length amino acid sequences of human NEK4a (SEQ ID NO: 8), human NEK4b (SEQ ID NO: 10), human NEK5 (SEQ YD NO:4), and murine NEK6 (SEQ ID NO:6) kinase polypeptides.
  • Figure 6 shows a multiple sequence alignment of the amino acid sequences of NEK-subfamily kinases, including: human NEK4b (SEQ ID NO: 10), human NEK5 (SEQ ID NO:4), mouse NEK6 (SEQ ID NO:6), Emericella nidulans NLMA (SEQ ID NO:l 1), human NEK2 (SEQ ID NO: 12), and human GAK (SEQ ID NO: 13) kinase polypeptides.
  • the C-termini of NLMA, NEK2 and GAK are not shown in this figure, since NEK4, NEK5, and NEK6 lack long C-terminal regions.
  • Figure 7 shows a sequence alignment of the amino acid sequences of NEK4a (SEQ ID NO: 10), human NEK5 (SEQ ID NO:4), mouse NEK6 (SEQ ID NO:6), Emericella nidulans NLMA (SEQ ID NO:l 1), human NEK2 (
  • NEK4b SEQ ID NO:10
  • the full length human bubl cDNA (SEQ ID NO:l) is 3408 bp long and consists of a 3255 bp ORF (45-3300) flanked by a 44 bp (1-44) 5' UTR and a 108 bp 3' UTR.
  • 1 ⁇ potential polyadenylation signal begins at position 3404.
  • the sequence flanking the first ATG conforms to the Kozak consensus (Kozak, M., Nucleic Acids Res. 15, 8125-8148 (1987)) for an initiating methionine, and is believed to be the translational start site for the human BUBl.
  • Two human bubl isoforms were originally identified as PCR fragments:
  • SuSTK34a (R51-40-18, 238 bp), and SuSTK34b (R51-40-4, 246 bp).
  • SuSTK34b is identical to SuSTK34a except for the presence of a 15 nucleotide deletion within kinase subdomain VII, just N-terminal to the typically conserved "DFG" motif in the activation loop. This isoform predicts the in frame deletion of the 5 amino acid sequence, "GLALI”.
  • Eight human EST fragments are present in the dbEST database: R94348 spans human bubl cDNA from nucleotide 898-1259; the other seven span nucleotides 2049-3422 of human bubl.
  • the full-length human NEK4a cDNA (SEQ ID NO:7) is 1,449 bp long and consists of a 1,107 bp open reading frame (ORF) flanked by a 121 bp 5' UTR and a 200 bp 3' UTR that is followed by a 21 nucleotide polyadenylated region. No polyadenylation signal (AATAAA) was found preceding the polyadenylated region.
  • the sequence flanking the first ATG conforms to the Kozak consensus (Kozak, M. (1987) Nucleic Acids Res. 15:8125-8148) for an initiating methionine, and is believed to be the translational start site for NEK4a.
  • NEK4a isoform (NEK4b) was also identified whose sequence diverges from NEK4a beginning at position 1005 as a result of the substitution of the terminal 75 amino acids of NEK4a with a unique 19 amino acid C-terminal sequence.
  • the partial human NEK4b cDNA (SEQ ID NO:9) is 1,346 bp long, and consists of a 804 bp ORF flanked by a 499 bp 3' UTR that is followed by a 41 nucleotide polyadenylated region. No polyadenylation signal (AATAAA) was found preceding the polyadenylated region.
  • AATAAA polyadenylation signal
  • the full-length human NEK5 cDNA (SEQ ID NO:3) is 3,854 bp long and consists of a 906 bp ORF flanked by a 31 bp 5' UTR and a 2,897 bp 3' UTR that is followed by a 20 nucleotide polyadenylated region.
  • a polyadenylation signal (AATAAA) is found at positions 3817-3822. The sequence flanking the first ATG conforms to the Kozak consensus for an initiating methionine, and is believed to be the translational start site for NEK5.
  • At least one EST fragment, AA181165, spans the complete NEK5 sequence.
  • E. Mammalian NEK6 The partial murine NEK6 cDNA (SEQ ID NO: 5) is 1 ,310 bp long and consists of a
  • One EST fragment, AA030068, represents the partial NEK6 sequence.
  • the 3408 bp human bubl nucleotide sequence encodes a polypeptide of 1085 amino acids (SEQ ID NO:2). Analysis of the deduced amino acid sequence predicts BUB 1 protein to be an intracellular serine/threonine kinase, lacking both a signal sequence and transmembrane domain.
  • BUBl protein contains a large N terminal region (amino acid 1 to 785) followed by a C-terminal kinase domain (amino acid 786 to 1028) and a small C-terminal tail (amino acid 1029 to 1085) (FIG 3).
  • the 785 amino acid N-terminal domain of human BUBl shares 68% amino acid identity to murine BUBl.
  • a 140 amino acid subdomain in this N-terminal region of human BUBl shares 81%, 31%, and 29% amino acid sequence identity to murine BUBl (AF002823) Saccharomyces cerevisiae BUBl (L32027), and to the N-terminal domain of the non-kinase S. cerevisiae MAD3 protein (P47074), respectively.
  • the 243 amino acid predicted catalytic domain of human BUBl shares 83% and 36% amino acid identity to murine BUBl and S. cerevisiae BUBl, respectively.
  • the 57 amino acid C-terminal tail of human BUBl exhibits 72% and 24% amino acid identity to murine BUBl and S. cerevisiae BUBl, respectively.
  • B. Mammalian NEK4a " Long C-terminal Isoform)
  • the 1,449 bp human NEK4a nucleotide sequence encodes a polypeptide of 369 amino acids (SEQ ID NO:8) with a predicted molecular mass of 41,825 daltons. Analysis of the deduced amino acid sequence predicts NEK4a to be an intracellular serine/threonine kinase, lacking both a signal sequence and a transmembrane domain. NEK4a contains a 42 amino acid N-terminal domain, a 252 amino acid catalytic domain with all the motifs characteristic of a serine/threonine kinase, and a 75 amino acid C-terminal domain.
  • NEK4a is most closely related to human NEK5 (SEQ ID NO:4), a C. elegans kinase F19H6.1 (GB:Z68115), murine NEKl (GB:S45828), human NEK3 (GB:Z29067), human NEK2 (GB:U11050), and Emericella nidulans NLMA (GB:M20249), sharing 82.6%, 78.8%, 41.2%, 40.2%, 44.2%, 42.6%, and 30.4% amino acid identity, respectively.
  • NEK4a also shares 97.2% identity with human HPK1, a partial protein kinase disclosed in the International Publication No. WO98/11234.
  • NEK4a (369 amino acids) and NEK4b (268 amino acids) have identical amino acid (and nucleotide) sequence up to amino acids 304 and 249 in NEK4a and NEK4b, respectively, where these two proteins diverge, most likely as result of alternative splicing (FIG. 10).
  • the 42 amino acid N-terminal domain of human NEK4a does not reveal any significant homologies following a Smith- Waterman search of the non-redundant protein database. Human NEK4a lacks a glycine residue at position 2, and is therefore unlikely to undergo myristylation.
  • NEK4a contains the potential "TPY” motif regulatory phosphorylation site in its activation loop. This "TPY” motif is also present in NEK5.
  • NEK4a and NEK5 also contain the catalytic motif HPN(I/V) located C-terminal to the conserved catalytic lysine (position 102-105 in NEK4a, FIG.9) that is characteristic of the NIMA/NEK-subfamily of STK's.
  • the kinase domain of NEK4a also shares 97.2% identity with human HPKl, a partial protein kinase disclosed in the International Publication No. WO98/11234.
  • the 75 amino acid C-terminal domain of human NEK4a is most closely related to human NEK5 (SEQ ID NO:4), sharing 61.5% amino acid identity.
  • the C-terminus of NEK4a does not display any detectable homology with human HPKl , a partial protein kinase disclosed in the International Publication No. WO98/11234.
  • the 75 amino acid C-terminal region unique to NEK4a contains additional potential phosphorylation sites at 308 (Cdc2) and 344 (casein kinase II).
  • Cdc2 The 75 amino acid C-terminal region unique to NEK4a contains additional potential phosphorylation sites at 308 (Cdc2) and 344 (casein kinase II).
  • Cdc2 The 75 amino acid C-terminal region unique to NEK4a contains additional potential phosphorylation sites at 308 (Cdc2) and 344 (casein kinase II).
  • C. Mammalian NEK4b Short C-terminal Isoform
  • NEK4a (369 amino acids) and NEK4b (268 amino acids) have identical amino acid (and nucleotide) sequence up to amino acids 304 and 249 in NEK4a and NEK4b, respectively, where these two proteins diverge, most likely as result of alternative splicing (FIG. 4). This divergence occurs immediately following the predicted kinase domain, causing these two proteins to have divergent C-terminal tails.
  • the 19 amino acid C-terminal domain of human NEK4b is most related to human NEK5 (SEQ ID NO:4), sharing 61.5% amino acid identity.
  • the C-terminal domain of NEK4b also shares 89.5% identity with human HPKl, a partial protein kinase disclosed in the International Publication No. WO98/11234.
  • the 3,854 bp human NEK5 nucleotide sequence encodes a polypeptide of 302 amino acids (SEQ ID NO:4) with a predicted molecular mass of 34,550 daltons. Analysis of the deduced amino acid sequence predicts NEK5 to be an intracellular serine/threonine kinase, lacking both a signal sequence and transmembrane domain. NEK5 contains a 31 amino acid N-terminal domain, a 252 amino acid catalytic domain with all the motifs characteristic of a serine/threonine kinase, and a 19 amino acid C-terminal domain.
  • NEK5 is most closely related to human NEK4a (SEQ ID NO:8), a C. elegans kinase F19H6.1 (GB:Z68115), murine NEKl (GB:S45828), human NRK2 (GB:P51957),
  • NEK5 also shares 86.1% identity with human HPKl, a partial protein kinase disclosed in the International Publication No. WO98/11234. The 31 amino acid N-terminal domain of human NEK5 is 34.6% identical to the
  • Human NEK5 lacks a glycine residue at position 2, and is therefore unlikely to undergo myristylation. A Smith- Waterman search of the nonredundant protein database does not reveal any significant homologies that might suggest a potential function for this domain.
  • the 252 amino acid catalytic domain of human NEK5 is most related to human
  • NEK4a (SEQ ID NO:8), a C. elegans kinase F19H6.1 (GB:Z68115), murine NEKl (GB:S45828), human NEK2 (GB:U11050), human NRK2 (GB:P51957), human NEK3 (GB:Z299067), and E. nidulans NLMA (GB:M20249), sharing 88.5%, 79.8%, 39.0%, 42.3%, 40.2%, 44.1%, and 32.2% amino acid identity, respectively.
  • NEK5 contains the potential "TPY” motif regulatory phosphorylation site in its activation loop. This "TPY" motif is also present in NEK4a.
  • NEK4a and NEK5 also share with NLMA, as well as with other NEK-subfamily kinases, the catalytic motif HPN(I/V) located C-terminal to the conserved catalytic lysine (position 102-105 in NEK4a, FIG. 9).
  • the kinase domain of NEK5 shares 88.1% identity with human HPKl, a partial protein kinase disclosed in International Publication No. WO98/11234.
  • the 19 amino acid C-terminal domain of human NEK5 is most related to human NEK4a (SEQ ID NO:8), sharing 61.5% amino acid identity.
  • NEK4 and NEK5 are conserved in NEK4 and NEK5, as well as in their C. elegans orthologue, at the indicated positions in NEK4a (FIG. 9): PKC at 165, casein kinase II at 111, tyrosine kinase at 224, and Cdc2 kinase at 245.
  • PKC at 165
  • casein kinase II at 111
  • tyrosine kinase at 224
  • Cdc2 kinase Cdc2 kinase at 245.
  • the 1,310 bp murine NEK6 partial cDNA nucleotide sequence encodes a polypeptide of 302 amino acids (SEQ ID NO:6), with a predicted molecular mass of 33,590 daltons. Analysis of the deduced amino acid sequence predicts NEK6 to be an intracellular serine/threonine kinase, lacking both a signal sequence and transmembrane domain. NEK6 contains a 22 amino acid N-terminal domain, and a 280 amino acid catalytic domain with all the motifs characteristic of a serine/threonine kinase. The NEK6
  • 1 ⁇ sequence is partial and, therefore, does not contain the C-terminal end of the catalytic domain nor the carboxyl-terminus encoded by the gene.
  • NEK6 is most closely related to a Drosophila heteroneura kinase (GB:AF052296), a C. elegans kinase (GB:Z46242), a Saccharomyces cerevisiae kinase (GB:YILO95W), human GAK (GB:D88435), human NEKl (GB:S45828), human NEK3 (GB:Z299067), and human NRK (GB:L20321), sharing 67.8%, 43.7%, 37.0%, 39.5%, 30.5%, 33.9%, and 28.4% amino acid identity, respectively.
  • the 22 amino acid N-terminal domain of murine NEK6 lacked any significant homologies following a Smith- Waterman search of the non-redundant protein database.
  • the 281 amino acids of the catalytic domain of murine NEK6 are most related to a
  • D. heteroneura kinase (GB:AF052296), a C. elegans kinase (GB:Z46242), a S. cerevisiae kinase (GB:YILO95W), human GAK (GB:D88435), human NEKl (GB:S45828), human NEK3 (GB:Z299067), and human NRK (GB:L20321), sharing 67.8%, 43.7%, 37.0%, 39.5%, 30.5%, 33.9%, and 28.4% amino acid identity, respectively.
  • NEK6 shares with other NEK6-sub family kinases three features that differentiate them from other NEK-subfamily members: (1) an insert between the catalytic glycine-rich loop and the catalytic lysine; (2) a three amino acid insert in the activation loop; and (3) the lack of a "TPY" motif regulatory phosphorylation motif in the activation loop.
  • NEK6 contains the following potential nucleotide sequence phosphorylation sites at the indicated positions: cAMP-dependent kinase [RK](2)-X-[ST] (PROSITE: PS00004) at 221; PKC at 9, 199, 226, 235, and 242; and casein kinase II at 7, 47, 115, 147, 241, and 278.
  • the PKC site at 199 two amino acids C-terminal to the "DFG" catalytic motif
  • NEK6-related kinases murine and Drosophila NEK; A. thaliana, rat, and human GAK; and the Saccharomyces NEK-related kinases such as YILO95W.
  • a significant, yet lower, degree of conservation is seen for the PKC site at position 235.
  • a group of yeast cell cycle checkpoint genes was demonstrated in the G2-M transition and mitosis including BUBl, BUB2, BUB3, MAD1, MAD2, MAD3, and MPS1.
  • Human BUB 1 is a novel gene encoding a serine/threonine kinase apparently functioning as a mitotic checkpoint.
  • Human BUBl is a centromeric protein which interacts with microtubules during the process of mitotic division.
  • Improper functioning of the mitotic spindle at metaphase can arrest cell cycle progression. For example, chromosome segregation at the metaphase-anaphase transition is prevented if one or more chromosomes are not yet congressed at the metaphase plate. Moreover, the initiation of a new cell cycle is prevented if mitosis was not completed in the previous cell cycle because of inhibition of microtubule assembly. Mitotic defects can cause genomic changes (aneuploid or tetraploid) and chromosome aberrations. Studying the biological function of the hBUBl gene will improve the understanding of mitotic checkpoint control and cancer development.
  • Human NEK4 and NEK5 are most closely related to the C. elegans kinase F19H6.1, and to four mammalian NEK-subfamily kinases: murine NEKl, and human NRK2, NEK2 and NEK3. They also demonstrate distant homology to the E. nidulans NIMA kinase.
  • Murine NEKl (NIMA Related Kinase) is a 774 amino acid STK with a predicted molecular weight of 88.4 kDa. NEKl has a 526 amino acid extracatalytic C-terminal domain containing four putative nuclear-localization signals. The percent amino acid sequence identity between NEKl and NIMA over the catalytic and extracatalytic regions is 42% and 14%, respectively.
  • NEKl is expressed predominantly in male and female germ cells, suggesting a role for this kinase in meiosis.
  • NEKl is enriched in postmitotic neurons from peripheral ganglia, indicative of a role for this STK in differentiated cells.
  • NEKl behaves as a dual-specificity kinase, phosphorylatmg serine/threonine and also tyrosine residues in vitro. Dual phosphorylation in vivo remains to be demonstrated (Arama, E. et al. (1998) Oncogene 16:1813-1823)
  • Human NRK2 (NIMA-related kinase 2) is an 841 amino acid STK with a predicted molecular weight of 94.6 kDa.
  • NRK2 contains a 580 amino acid extracatalytic C-terminal domain whose sequence is unrelated to the C-termini found in NIMA and other NEK-subfamily kinases. The percent amino acid sequence identity between NRK2 and NIMA over the catalytic region is 39%.
  • NRK2 is expressed predominantly in liver, skeletal muscle, kidney, and pancreas.
  • NRK2 transcript levels do not vary throughout the cell cycle.
  • the NRK2 gene localizes to chromosome 3p21.1, a region that is frequently altered in renal carcinomas (Levedakou, E. et al. (1994) Oncogene 9:1977-1988).
  • Human NEK2 is a 445 amino acid STK with a predicted molecular weight of 51.7 kDa.
  • the percent amino acid sequence identity between NEK2 and NLMA over the catalytic region and C-terminal extracatalytic regions is 47% and 19%, respectively.
  • NEK2 expression is highest in germ cells where it is found in meiotic pachytene- stage spermatocytes and oocytes. NEK2 levels increase at the G2/M transition of the meiotic cell cycle (Rhee, K and Wolgemuth, D. (1997) Development 124:2167-2177). NEK2 may also have a mitotic role, since the levels and activity of this kinase are maximal during the S and G2 phases of the cell cycle. A role in centrosome separation during mitosis has been postulated for NEK2 (Fry, A. et al. (1998) EMBO J. 17:470-481). The NEK2 gene is localized to human chromosome 14ql2 (Schultz, S.
  • Human NEK3 is a 459 amino acid STK with a predicted molecular weight of 52.3 kDa.
  • the database entry for NEK3 (GB:Z29067) is missing the N-terminal catalytic- region and start methionine of this protein.
  • the percent amino acid sequence identity if- between NEK3 and NIMA over the catalytic region and C-terminal extracatalytic regions is 43% and 19%, respectively (Schultz, S. et al. (1994) Cell Growth and Diff. 5:625-635).
  • NIMA an STK found in the filamentous fungi, Aspergillus and E. nidulans, is a 699 amino acid protein with a predicted molecular weight of 78.9 kDa. Mitosis in A. nidulans requires both Cdc2 and NIMA.
  • NIMA expression and kinase activity are coordinately regulated during the cell cycle, being lowest in Gl and highest through the S and G2 phases.
  • Full NLMA activation requires multiple phosphorylation events, some of which may be carried out by Cdc2. Exit from mitosis into Gl requires the inactivation of both NLMA and Cdc2, possibly through ubiquitin-mediated proteolysis.
  • the C-terminus of NIMA displays consensus Cdc2 phosphorylation sites as well as PEST motifs that may mediate the function of this protein through phosphorylation and proteolysis.
  • NIMA-like pathway in mitosis in higher eukaryotes comes from the observation that expression of a dominant-negative form of NLMA in Hela cells causes an arrest in G2, and that overexpression of NIMA in Aspergillus, as well as in higher eukaryotes, triggers chromatin condensation (Fry, A. and Nigg, E.A. (1995) Current Biology, 5:1122-1125).
  • NEK4 and NEK5 contrast sharply with all other NEK-subfamily kinases in that they lack long extracatalytic C-termini.
  • the function of these extracatalytic regions in NIMA and NEK2 is to regulate their catalytic activity and turnover in a cell cycle- dependent manner.
  • NEK4 and NEK5 are predicted to have longer half-lives than other NEK-subfamily kinases because they lack motifs that target proteolysis such as those found in NIMA and NEK2.
  • the short C-terminal extracatalytic tail found in NEK4a could play a regulatory role in kinase activity and/or substrate recognition.
  • Modulation of NEK4 function may be carried out in part through alternative splicing of the C-terminus generating the NEK4a and NEK4b isoforms.
  • NEK4 and NEK5 may also be modulated through phosphorylation, as these proteins have multiple conserved consensus sites for phosphorylation by various kinases.
  • NEK4 and NEK5 are predicted to be small, compact kinases akin to MAPK's and
  • MAPK's are regulated through phosphorylation of the "TXY" residues in the activation loop by dual-specificity MAPK kinases (MEK's). These phosphorylations reorient the activation loop, resulting in over a 1000-fold enzymatic activation.
  • MEK's dual-specificity MAPK kinases
  • the mechanism of this rearrangement involves the interaction of the 50 amino acid C-terminus of MAPK ( ⁇ -helix LI 6) with the phosphorylated residues in the activation loop.
  • the rearranged activation loop forms a tight pocket (P+l loop) that defines the substrate specificity of MAPK's as proline-directed kinases capable of phosphorylatmg only those substrates whose phosphorylation serine/threonine residues are followed by a proline (Canagarajah BJ. et al. (1997) Cell, 90:859-869).
  • NEK4 and NEK5 Three features of NEK4 and NEK5 suggest that they may behave like MAPK's: (1) their predicted small, compact nature; (2) the presence of the potential dual phosphorylation "TPY" site in the activation loop; and (3) the presence of short C-termini (19 amino acids in NEK4b and NEK5 and 75 amino acids in NEK4a) that could play a role similar to the 50 amino acid C-terminal ⁇ -helix L16 of MAPK in generating an active site.
  • NEK4 and NEK5 may also be subject to activation by MEK's through dual phosphorylation in their activation loop.
  • NEK4 and NEK5, like MAPK's and CDK's may be proline-directed kinases, a frequently observed specificity among kinases that phosphorylate transcription and cell cycle-regulatory molecules.
  • NEK4 and NEK5 define a new subclass of NEK-subfamily kinases that may potentially transduce signals originated at the level of activated membrane receptors to the nucleus where their targets could be transcriptional or cell-cycle modulator proteins.
  • Murine NEK6 is most closely related to the D. heteroneura kinase AF052296, to the S. cerevisiae kinase (GB:YILO95W), and to the mammalian kinases GAK, NEKl, NEK3 and NRK2, with distal homology to E. Nidulans NIMA.
  • Human GAK is a 1,311 amino acid STK with a predicted molecular weight of 143.1 kDa.
  • GAK contains a 998 amino acid extracatalytic C-terminal domain whose sequence is unrelated to the C-termini found in NIMA and in other NEK-subfamily kinases.
  • the C-terminus of GAK contains a tensin/auxilin-like domain.
  • GAK expression and kinase activity oscillate during the cell cycle with maximum levels found in Gl.
  • the GAK gene maps to human chromosome 4pl6 (Kimura, S.H. et al. (1997) Genomics, 44:179-187).
  • Cyclin G is a member of the cyclin family of regulatory subunits of the cyclin-dependent family of STK's. Unlike cyclins A and B, which regulate the G2/M transition, cyclin G regulates the Gl to S phase transition of the cell cycle (Tamura, K. et al. 1993)
  • Cyclin G is a transcriptional target of the p53 tumor suppressor protein. Its overexpression may override a p53-mediated cell cycle checkpoint resulting in a hypersensitivity to the cytotoxic effects of cancer chemotherapy (Smith, M.L. et al. (1997) Exp. Cell Res. 230:61-68). Based on the sequence homology between NEK6 and GAK, NEK6 may also have a long C-terminal region, possibly containing a tensin/auxilin-like domain like GAK.
  • At least three regions unique to the kinase domain of NEK6 are likely to exert a strong influence in its substrate specificity: (1) NEK6, and related kinases like human GAK, the S. cerevisiae kinases YNL020C, YIL095W ,and YBR059C, and the C. elegans Z46242, have in common, relative to other STK's, the presence of a variable-length insert located between the glycine-rich loop (subdomain I) and the conserved nucleotide-binding lysine residue (subdomain II) (FIG.
  • NEK6 and GAK 3 only NEK6 and GAK shown); (2) NEK6 and its related kinases display a six amino acid insert between the "DFG” (subdomain VII) and “TPF” activation loop (subdomain VIII) catalytic motifs; and (3) the activation loop of NEK6 and its related kinases lack the "TPY” motif observed in NEK4 and NEK5 and in its place contain two contiguous uniformly conserved threonine residues.
  • NEK6 defines another subclass of NEK-subfamily kinases that are GAK-like, and consequently may be subject to modulation by regulatory subunits such as cyclins and by domains that may be found within its extracatalytic region.
  • a nucleic acid probe of the present invention may be used to probe an appropriate chromosomal or cDNA library by usual hybridization methods to obtain other nucleic acid molecules of the present invention.
  • a chromosomal DNA or cDNA library may be prepared from appropriate cells according to recognized methods in the art (cf. "Molecular Cloning: A Laboratory Manual", second edition, Cold Spring Harbor Laboratory, Sambrook, Fritsch, & Maniatis, eds., 1989).
  • nucleic acid probes having nucleotide sequences which correspond to N-terminal and C-terminal portions of the amino acid sequence of the polypeptide of interest.
  • the synthesized nucleic acid probes may be used as primers in a polymerase chain reaction (PCR) carried out in accordance with recognized PCR techniques, essentially according to PCR Protocols, "A Guide to Methods and Applications", Academic Press, Michael, et al, eds., 1990, utilizing the appropriate chromosomal or cDNA library to obtain the fragment of the present invention.
  • PCR polymerase chain reaction
  • the hybridization probes of the present invention can be labeled by standard labeling techniques such as with a radiolabel, enzyme label, fluorescent label, biotin-avidin label, chemiluminescence, and the like. After hybridization, the probes may be visualized using known methods.
  • the nucleic acid probes of the present invention include RNA, as well as DNA probes, such probes being generated using techniques known in the art.
  • the nucleic acid probe may be immobilized on a solid support.
  • solid supports include, but are not limited to, plastics such as polycarbonate, complex carbohydrates such as agarose and sepharose, and acrylic resins, such as polyacrylamide and latex beads. Techniques for coupling nucleic acid probes to such solid supports are well known in the art.
  • test samples suitable for nucleic acid probing methods of the present invention include, for example, cells or nucleic acid extracts of cells, or biological fluids.
  • the samples used in the above-described methods will vary based on the assay format, the detection method and the nature of the tissues, cells or extracts to be assayed. Methods for preparing nucleic acid extracts of cells are well known in the art and can be readily adapted in order to obtain a sample which is compatible with the method utilized.
  • One method of detecting the presence of nucleic acids of the invention in a sample comprises (a) contacting said sample with the above-described nucleic acid probe under conditions such that hybridization occurs, and (b) detecting the presence of said probe bound to said nucleic acid molecule.
  • One skilled in the art would select the nucleic acid probe according to techniques known in the art as described above. Samples to be tested include but should not be limited to RNA samples of human tissue.
  • a kit for detecting the presence of nucleic acids of the invention in a sample comprises at least one container means having disposed therein the above-described nucleic acid probe.
  • the kit may further comprise other containers comprising one or more of the following: wash reagents and reagents capable of detecting the presence of bound nucleic acid probe.
  • detection reagents include, but are not limited to radiolabelled probes, enzymatic labeled probes (horseradish peroxidase, alkaline l phosphatase), and affinity labeled probes (biotin, avidin, or steptavidin).
  • the kit further comprises instructions for use.
  • a compartmentalized kit includes any kit in which reagents are contained in separate containers.
  • Such containers include small glass containers, plastic containers or strips of plastic or paper.
  • Such containers allow the efficient transfer of reagents from one compartment to another compartment such that the samples and reagents are not cross-contaminated and the agents or solutions of each container can be added in a quantitative fashion from one compartment to another.
  • Such containers will include a container which will accept the test sample, a container which contains the probe or primers used in the assay, containers which contain wash reagents (such as phosphate buffered saline, Tris-buffers, and the like), and containers which contain the reagents used to detect the hybridized probe, bound antibody, amplified product, or the like.
  • wash reagents such as phosphate buffered saline, Tris-buffers, and the like
  • the present invention also relates to a recombinant DNA molecule comprising, 5* to 3', a promoter effective to initiate transcription in a host cell and the above-described nucleic acid molecules.
  • the present invention relates to a recombinant DNA molecule comprising a vector and an above-described nucleic acid molecule.
  • the present invention also relates to a nucleic acid molecule comprising a transcriptional region functional in a cell, a sequence complementary to an RNA sequence encoding an amino acid sequence corresponding to the above-described polypeptide, and a transcriptional termination region functional in said cell.
  • the above-described molecules may be isolated and/or purified DNA molecules.
  • the present invention also relates to a cell or organism that contains an above- described nucleic acid molecule and thereby is capable of expressing a polypeptide.
  • the polypeptide may be purified from cells which have been altered to express the polypeptide.
  • a cell is said to be "altered to express a desired polypeptide" when the cell, through genetic manipulation, is made to produce a protein which it normally does not produce or which the cell normally produces at lower levels.
  • One skilled in the art can readily adapt procedures for introducing and expressing either genomic, cDNA, or synthetic sequences into either eukaryotic or prokaryotic cells.
  • a nucleic acid molecule such as DNA, is said to be "capable of expressing" a polypeptide if it contains nucleotide sequences which contain transcriptional and translational regulatory information and such sequences are “operably linked” to nucleotide sequences which encode the polypeptide.
  • An operable linkage is a linkage in which the regulatory DNA sequences and the DNA sequence sought to be expressed are connected in such a way as to permit gene sequence expression.
  • the precise nature of the regulatory regions needed for gene sequence expression may vary from organism to organism, but shall in general include a promoter region which, in prokaryotes, contains both the promoter (which directs the initiation of RNA transcription) as well as the DNA sequences which, when transcribed into RNA, will signal synthesis initiation. Such regions will normally include those 5'-non-coding sequences involved with initiation of transcription and translation, such as the TATA box, capping sequence, CAAT sequence, and the like.
  • the non-coding region 3' to the sequence encoding a kinase of the invention may be obtained by the above-described methods.
  • This region may be retained for its transcriptional termination regulatory sequences, such as termination and polyadenylation.
  • the transcriptional termination signals may be provided. Where the transcriptional termination signals are not satisfactorily functional in the expression host cell, then a 3' region functional in the host cell may be substituted.
  • Two DNA sequences are said to be operably linked if the nature of the linkage between the two DNA sequences does not (1) result in the introduction of a frame- shift mutation, (2) interfere with the ability of the promoter region sequence to direct the transcription of a gene sequence encoding a kinase of the invention, or (3) interfere with the ability of the gene sequence of a kinase of the invention to be transcribed by the promoter region sequence.
  • a promoter region would be operably linked to a DNA sequence if the promoter were capable of effecting transcription of that DNA sequence.
  • the present invention encompasses the expression of a gene encoding a kinase of the invention (or a functional derivative thereof) in either prokaryotic or eukaryotic cells.
  • Prokaryotic hosts are, generally, very efficient and convenient for the production of recombinant proteins and are, therefore, one type of preferred expression system for kinases of the invention.
  • Prokaryotes most frequently are represented by various strains of E. coli. However, other microbial strains may also be used, including other bacterial strains.
  • plasmid vectors that contain replication sites and control sequences derived from a species compatible with the host may be used.
  • suitable plasmid vectors may include pBR322, pUCl 18, pUCl 19 and the like; suitable phage or bacteriophage vectors may include ⁇ gtlO, ⁇ gtl 1 and the like; and suitable virus vectors may include pMAM-neo, pKRC and the like.
  • the selected vector of the present invention has the capacity to replicate in the selected host cell.
  • prokaryotic hosts include bacteria such as E. coli, Bacillus, Streptomyces, Pseudomonas, Salmonella, Serratia, and the like. However, under such conditions, the polypeptide will not be glycosylated.
  • the prokaryotic host must be compatible with the replicon and control sequences in the expression plasmid.
  • a kinase of the invention (or a functional derivative thereof) in a prokaryotic cell, it is necessary to operably link the sequence encoding the kinase of the invention to a functional prokaryotic promoter.
  • promoters may be either constitutive or, more preferably, regulatable (i.e., inducible or derepressible).
  • constitutive promoters include the int promoter of bacteriophage ⁇ , the bla promoter of the ⁇ - lactamase gene sequence of pBR322, and the cat promoter of the chloramphenicol acetyl transferase gene sequence of pPR325, and the like.
  • inducible prokaryotic promoters examples include the major right and left promoters of bacteriophage ⁇ (P L and P R ), the trp, recA, ⁇ acZ, ⁇ acI, and gal promoters of E. coli, the ⁇ -amylase (Ulmanen et al, J. Bacteriol. 162:176-182, 1985) and the ⁇ -28-specific promoters of B.
  • subtilis (Gilman et al, Gene Sequence 32:11-20, 1984), the promoters of the bacteriophages of Bacillus (Gryczan, In: The Molecular Biology of the Bacilli, Academic Press, Inc., NY, 1982), and Streptomyces promoters (Ward et al, Mol. Gen. Genet. 203:468-478, 1986).
  • Prokaryotic promoters are reviewed by Glick (Ind. Microbiot. 1:277-282, 1987), Cenatiempo (Biochimie 68:505-516, 1986), and Gottesman (Ann. Rev. Genet. 18:415-442, 1984).
  • ribosome-binding site upstream of the gene sequence-encoding sequence.
  • Such ribosome-binding sites are disclosed, for example, by Gold et al. (Ann. Rev. Microbiol. 35:365-404, 1981).
  • the selection of control sequences, expression vectors, transformation methods, and the like, are dependent on the type of host cell used to express the gene.
  • progeny may be used interchangeably and all such designations include progeny.
  • transformationants or “transformed cells” include the primary subject cell and cultures derived therefrom, without regard to the number of transfers. It is also understood that all progeny may not be precisely identical in DNA content, due to deliberate or inadvertent mutations. However, as defined, mutant progeny have the same functionality as that of the originally transformed cell.
  • Host cells which may be used in the expression systems of the present invention are not strictly limited, provided that they are suitable for use in the expression of the kinase polypeptide of interest. Suitable hosts may often include eukaryotic cells. Preferred eukaryotic hosts include, for example, yeast, fungi, insect cells, mammalian cells either in vivo, or in tissue culture. Mammalian cells which may be useful as hosts include HeLa cells, cells of fibroblast origin such as VERO or CHO-K1, or cells of lymphoid origin and their derivatives. Preferred mammalian host cells include SP2/0 and J558L, as well as neuroblastoma cell lines such as IMR 332, which may provide better capacities for correct post-translational processing.
  • plant cells are also available as hosts, and control sequences compatible with plant cells are available, such as the cauliflower mosaic virus 35S and 19S, and nopaline synthase promoter and polyadenylation signal sequences.
  • Another preferred host is an insect cell, for example the Drosophila larvae. Using insect cells as hosts, the Drosophila alcohol dehydrogenase promoter can be used (Rubin, Science
  • baculovirus vectors can be engineered to express large amounts of kinases of the invention in insect cells (Jasny, Science 238:1653, 1987; Miller et al, In: Genetic Engineering, Vol. 8, Plenum, Setlow et al, eds., pp. 277-297, 1986).
  • Any of a series of yeast expression systems can be utilized which incorporate promoter and termination elements from the actively expressed sequences coding for glycolytic enzymes that are produced in large quantities when yeast are grown in mediums rich in glucose.
  • Known glycolytic gene sequences can also provide very efficient
  • Yeast provides substantial advantages in that it can also carry out post-translational modifications.
  • Yeast recognizes leader sequences on cloned mammalian genes and secretes peptides bearing leader sequences (i.e., pre- peptides).
  • leader sequences i.e., pre- peptides.
  • Several possible vector systems are available for the expression of kinases of the invention in a mammalian host.
  • transcriptional and translational regulatory sequences may be employed, depending upon the nature of the host.
  • the transcriptional and translational regulatory signals may be derived from viral sources, such as adenovirus, bovine papilloma virus, cytomegalovirus, simian virus, or the like, where the regulatory signals are associated with a particular gene sequence which has a high level of expression.
  • promoters from mammalian expression products such as actin, collagen, myosin, and the like, may be employed.
  • Transcriptional initiation regulatory signals may be selected which allow for repression or activation, so that expression of the gene sequences can be modulated.
  • regulatory signals which are temperature- sensitive so that by varying the temperature, expression can be repressed or initiated, or are subject to chemical (such as metabolite) regulation.
  • eukaryotic regulatory regions Such regions will, in general, include a promoter region sufficient to direct the initiation of RNA synthesis.
  • Preferred eukaryotic promoters include, for example, the promoter of the mouse metallothionein I gene sequence (Hamer et al, J. Mol. Appl. Gen. 1:273-288, 1982); the TK promoter of Herpes virus (McKnight, Cell 31:355-365, 1982); the SV40 early promoter (Benoist et al, Nature (London) 290:304-31, 1981); and the yeast gal4 gene sequence promoter (Johnston et al, Proc. Natl. Acad. Sci. (USA) 79:6971-6975, 1982; Silver et al, Proc. Natl. Acad. Sci. (USA) 81:5951-5955, 1984).
  • eukaryotic mRNA Translation of eukaryotic mRNA is initiated at the codon which encodes the first methionine. For this reason, it is preferable to ensure that the linkage between a eukaryotic promoter and a DNA sequence which encodes a kinase of the invention (or a functional derivative thereof) does not contain any intervening codons which are capable of encoding a methionine (i.e., AUG). The presence of such codons results either in the formation of a fusion protein (if the AUG codon is in the same reading frame as the kinase
  • a nucleic acid molecule encoding a kinase of the invention and an operably linked promoter may be introduced into a recipient prokaryotic or eukaryotic cell either as a nonreplicating DNA or RNA molecule, which may either be a linear molecule or, more preferably, a closed covalent circular molecule. Since such molecules are incapable of autonomous replication, the expression of the gene may occur through the transient expression of the introduced sequence. Alternatively, permanent expression may occur through the integration of the introduced DNA sequence into the host chromosome.
  • a vector may be employed which is capable of integrating the desired gene sequences into the host cell chromosome.
  • Cells which have stably integrated the introduced DNA into their chromosomes can be selected by also introducing one or more markers which allow for selection of host cells which contain the expression vector.
  • the marker may provide for prototrophy to an auxotrophic host, biocide resistance, e.g., antibiotics, or heavy metals, such as copper, or the like.
  • the selectable marker gene sequence can either be directly linked to the DNA gene sequences to be expressed, or introduced into the same cell by co-transfection. Additional elements may also be needed for optimal synthesis of mRNA. These elements may include splice signals, as well as transcription promoters, enhancers, and termination signals. cDNA expression vectors incorporating such elements include those described by Okayama (Mol. Cell. Biol. 3:280- 289, 1983).
  • the introduced nucleic acid molecule can be incorporated into a plasmid or viral vector capable of autonomous replication in the recipient host. Any of a wide variety of vectors may be employed for this purpose. Factors of importance in selecting a particular plasmid or viral vector include: the ease with which recipient cells that contain the vector may be recognized and selected from those recipient cells which do not contain the vector; the number of copies of the vector which are desired in a particular host; and whether it is desirable to be able to "shuttle" the vector between host cells of different species.
  • Preferred prokaryotic vectors include plasmids such as those capable of replication in E. coli (such as, for example, pBR322, ColEl, pSClOl, pACYC 184, ⁇ VX; "Molecular Cloning: A Laboratory Manual", 1989, supra).
  • Bacillus plasmids include pC 194, pC221, pT127, and the like (Gryczan, In: The Molecular Biology of the Bacilli, Academic Press, NY, pp. 307-329, 1982).
  • Suitable Streptomyces plasmids include plJlOl (Kendall et al, J. Bacteriol.
  • Preferred eukaryotic plasmids include, for example, BPV, vaccinia, SV40, 2- micron circle, and the like, or their derivatives.
  • Such plasmids are well known in the art (Botstein et al, Miami Wntr. Symp. 19:265-274, 1982; Broach, In: "The Molecular Biology of the Yeast Saccharomyces: Life Cycle and Inheritance", Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, p. 445-470, 1981; Broach, Cell 28:203-204, 1982; Bollon et al, J. Clin. Hematol. Oncol. 10:39-48, 1980; Maniatis, In: Cell Biology: A Comprehensive Treatise, Vol. 3, Gene Sequence Expression, Academic Press, NY, pp. 563-608, 1980).
  • the DNA construct(s) may be introduced into an appropriate host cell by any of a variety of suitable means, i.e., transformation, transfection, conjugation, protoplast fusion, electroporation, particle gun technology, calcium phosphate- precipitation, direct micro injection, and the like.
  • recipient cells are grown in a selective medium, which selects for the growth of vector- containing cells. Expression of the cloned gene(s) results in the production of a kinase of the invention, or fragments thereof.
  • the present invention relates to an antibody having binding affinity to a kinase of the invention.
  • the polypeptide may have the amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10, or a functional derivative thereof, or at least 9 contiguous amino acids thereof (preferably, at least 20, 30, 35, or 40 contiguous amino acids thereof).
  • the present invention also relates to an antibody having specific binding affinity to a kinase of the invention.
  • an antibody may be isolated by comparing its binding affinity to a kinase of the invention with its binding affinity to other polypeptides.
  • Those which bind selectively to a kinase of the invention would be chosen for use in methods requiring a distinction between a kinase of the invention and other polypeptides.
  • Such methods could include, but should not be limited to, the analysis of altered kinase expression in tissue containing other polypeptides.
  • the BUB 1 or NEK kinases of the present invention can be used in a variety of procedures and methods, such as for the generation of antibodies, for use in identifying pharmaceutical compositions, and for studying DNA protein interaction.
  • the kinases of the present invention can be used to produce antibodies or hybridomas.
  • One skilled in the art will recognize that if an antibody is desired, such a peptide could be generated as described herein and used as an immunogen.
  • the antibodies of the present invention include monoclonal and polyclonal antibodies, as well fragments of these antibodies, and humanized forms. Humanized forms of the antibodies of the present invention may be generated using one of the procedures known in the art such as chimerization or CDR grafting.
  • the present invention also relates to a hybridoma which produces the above- described monoclonal antibody, or binding fragment thereof.
  • a hybridoma is an immortalized cell line which is capable of secreting a specific monoclonal antibody.
  • the polypeptide may be modified or administered in an adjuvant in order to increase the peptide antigenicity.
  • Methods of increasing the antigenicity of a polypeptide are well known in the art. Such procedures include coupling the antigen with a heterologous protein (such as globulin or ⁇ -galactosidase) or through the inclusion of an adjuvant during immunization.
  • a heterologous protein such as globulin or ⁇ -galactosidase
  • spleen cells from the immunized animals are removed, fused with myeloma cells, such as SP2/0-Agl4 myeloma cells, and allowed to become monoclonal antibody producing hybridoma cells.
  • myeloma cells such as SP2/0-Agl4 myeloma cells
  • Any one of a number of methods well known in the art can be used to identify the hybridoma cell which produces an antibody with the desired characteristics. These include screening the hybridomas with an ELISA assay, western blot analysis, or radioimmunoassay (Lutz et al, Exp. Cell Res. 175:109- 124, 1988).
  • Hybridomas secreting the desired antibodies are cloned and the class and subclass are determined using procedures known in the art (Campbell, "Monoclonal
  • antibody-containing antisera is isolated from the immunized animal and is screened for the presence of antibodies with the desired specificity using one of the above-described procedures.
  • the above-described antibodies may be detectably labeled.
  • Antibodies can be detectably labeled through the use of radioisotopes, affinity labels (such as biotin, avidin, and the like), enzymatic labels (such as horseradish peroxidase, alkaline phosphatase, and the like) fluorescent labels (such as FITC or rhodamine, and the like), paramagnetic atoms, and the like. Procedures for accomplishing such labeling are well-known in the art, for example, see Stemberger et al, J.
  • the labeled antibodies of the present invention can be used for in vitro, in vivo, and in situ assays to identify cells or tissues which express a specific peptide.
  • the above-described antibodies may also be immobilized on a solid support.
  • Such solid supports include plastics such as polycarbonate, complex carbohydrates such as agarose and sepharose, acrylic resins such as polyacrylamide and latex beads. Techniques for coupling antibodies to such solid supports are well known in the art (Weir et al, "Handbook of Experimental Immunology” 4th Ed., Blackwell Scientific Publications, Oxford, England, Chapter 10, 1986; Jacoby et al, Meth. Enzym. 34, Academic Press, N.Y., 1974).
  • the immobilized antibodies of the present invention can be used for in vitro, in vivo, and in situ assays as well as in immunochromotography.
  • Anti-peptide peptides can be generated by replacing the basic amino acid residues found in the peptide sequences of the kinases of the invention with acidic residues, while maintaining hydrophobic and uncharged polar groups. For example, lysine, arginine, and/or histidine residues are replaced with aspartic acid or glutamic acid and glutamic acid residues are replaced by lysine, arginine or histidine.
  • the present invention also encompasses a method of detecting a BUBl -related or NEK kinase polypeptide in a sample, comprising: (a) contacting the sample with an above-described antibody, under conditions such that immunocomplexes form, and (b) detecting the presence of said antibody bound to the polypeptide.
  • the methods comprise incubating a test sample with one or more of the antibodies of the present invention and assaying whether the antibody binds to the test sample. Altered levels of a kinase of the invention in a sample as compared to normal levels may indicate disease. Conditions for incubating an antibody with a test sample vary.
  • Incubation conditions depend on the format employed in the assay, the detection methods employed, and the type and nature of the antibody used in the assay.
  • immunological assay formats such as radioimmunoassays, enzyme-linked immunosorbent assays, diffusion-based Ouchterlony, or rocket immunofluorescent assays
  • Examples of such assays can be found in Chard ("An Introduction to Radioimmunoassay and Related Techniques” Elsevier Science Publishers, Amsterdam, The Netherlands, 1986), Bullock et al. (“Techniques in Immunocytochemistry,” Academic Press, Orlando, FL Vol. 1, 1982; Vol. 2, 1983; Vol. 3, 1985), Tijssen (“Practice and Theory of Enzyme Immunoassays: Laboratory Techniques in Biochemistry and Molecular Biology,” Elsevier Science Publishers, Amsterdam, The Netherlands, 1985).
  • the immunological assay test samples of the present invention include cells, protein or membrane extracts of cells, or biological fluids such as blood, serum, plasma, or urine.
  • the test samples used in the above-described method will vary based on the assay format, nature of the detection method and the tissues, cells or extracts used as the sample to be assayed. Methods for preparing protein extracts or membrane extracts of cells are well known in the art and can readily be adapted in order to obtain a sample which is testable with the system utilized.
  • a kit contains all the necessary reagents to carry out the previously described methods of detection.
  • the kit may comprise: (i) a first container means containing an above-described antibody, and (ii) second container means containing a conjugate comprising a binding partner of the antibody and a label.
  • the kit further comprises one or more other containers comprising one or more of the following: wash reagents and reagents capable of detecting the presence of bound antibodies.
  • detection reagents include, but are not limited to, labeled secondary antibodies, or in the alternative, if the primary antibody is labeled, the chromophoric, enzymatic, or antibody binding reagents which are capable of reacting with the labeled antibody.
  • the compartmentalized kit may be as described above for nucleic acid probe kits.
  • the antibodies described in the present invention can readily be incorporated into one of the established kit formats which are well known in the art.
  • the present invention also relates to a method of detecting a compound capable of binding to a BUBl -related or NEK kinase of the invention comprising incubating the compound with a kinase of the invention and detecting the presence of the compound bound to the kinase.
  • the compound may be present within a complex mixture, for example, serum, body fluid, or cell extracts.
  • the present invention also relates to a method of detecting an agonist or antagonist of kinase activity or kinase binding partner activity comprising incubating cells that produce a kinase of the invention in the presence of a compound and detecting changes in the level of kinase activity or kinase binding partner activity.
  • the compounds thus identified would produce a change in activity indicative of the presence of the compound.
  • the compound may be present within a complex mixture, for example, serum, body fluid, or cell extracts. Once the compound is identified it can be isolated using techniques well known in the art.
  • the present invention also encompasses a method of agonizing (stimulating) or antagonizing kinase associated activity in a mammal comprising administering to said mammal an agonist or antagonist to a kinase of the invention in an amount sufficient to effect said agonism or antagonism.
  • a method of treating diseases in a mammal with an agonist or antagonist of BUB 1 -related or NEK kinase activity comprising administering the agonist or antagonist to a mammal in an amount sufficient to agonize or antagonize BUBl -related or NEK kinase associated functions is also encompassed in the present application.
  • indolinone compounds form classes of acid resistant and membrane permeable organic molecules.
  • WO 96/22976 published August 1, 1996 by Ballinari et al. describes hydrosoluble indolinone compounds that harbor tetralin, naphthalene, quinoline, and indole substituents fused to the oxindole ring. These bicyclic substituents are in turn substituted with polar moieties including hydroxylated alkyl, phosphate, and ether moieties.
  • U.S. Patent Application Serial Nos. 08/702,232 filed August 23, 1996, entitled "Indolinone Combinatorial Libraries and Related Products and Methods for the Treatment of Disease" by Tang et al. (Lyon & Lyon Docket No.
  • substances capable of modulating kinase activity include, but are not limited to, tyrphostins, quinazolines, quinoxolines, and quinolines.
  • the quinazolines, tyrphostins, quinolines, and quinoxolines referred to above include well known compounds such as those described in the literature.
  • representative publications describing quinazolines include Barker et al, EPO Publication No. 0 520 722 Al ; Jones et al, U.S. Patent No. 4,447,608; Kabbe et al, U.S. Patent No. 4,757,072; Kaul and Vougioukas, U.S. Patent No.
  • oxindolinones such as those described in U.S. patent application Serial No. 08/702,232 filed August 23, 1996, incorporated herein by reference in its entirety, including any drawings.
  • DNA can be injected into the pronucleus of a fertilized egg
  • Embryos can be infected with viruses, especially retroviruses, modified to carry inorganic-ion receptor nucleotide sequences of the invention.
  • Pluripotent stem cells derived from the inner cell mass of the embryo and stabilized in culture can be manipulated in culture to incorporate nucleotide sequences of the invention.
  • a transgenic animal can be produced from such cells through implantation into a blastocyst that is implanted into a foster mother and allowed to come to term. Animals suitable for transgenic experiments can be obtained from standard commercial sources such as Charles River (Wilmington, MA), Taconic (Germantown, NY), Harlan Sprague Dawley (Indianapolis, IN), etc.
  • transgenic mouse female mice are induced to superovulate. Females are placed with males, and the mated females are sacrificed by CO 2 asphyxiation or cervical dislocation and embryos are recovered from excised oviducts. Surrounding cumulus cells are removed. Pronuclear embryos are then washed and stored until the time of injection. Randomly cycling adult female mice are paired with vasectomized males. Recipient females are mated at the same time as donor females. Embryos then are transferred surgically. The procedure for generating transgenic rats is similar to that of mice (Hammer et al, Cell 63:1099-1112, 1990).
  • a clone containing the sequence(s) of the invention is co-transfected with a gene encoding resistance.
  • the gene encoding neomycin resistance is physically linked to the sequence(s) of the invention.
  • DNA molecules introduced into ES cells can also be integrated into the chromosome through the process of homologous recombination (Capecchi, Science 244:1288-1292, 1989).
  • Methods for positive selection of the recombination event (i.e., neo resistance) and dual positive-negative selection (i.e., neo resistance and gancyclovir resistance) and the subsequent identification of the desired clones by PCR have been described by Capecchi, supra and Joyner et al. (Nature 338:153-156, 1989), the teachings of which are incorporated herein in their entirety including any drawings.
  • the final phase of the procedure is to inject targeted ES cells into blastocysts and to transfer the blastocysts into pseudopregnant females.
  • the resulting chimeric animals are bred and the offspring are analyzed by Southern blotting to identify individuals that carry the transgene.
  • Procedures for the production of non-rodent mammals and other animals have been discussed by others (Houdebine and Chourrout, supra; Pursel et al, Science 244:1281- 1288, 1989; and Simms et al, Bio/Technology 6:179-183, 1988).
  • the invention provides transgenic, nonhuman mammals containing a transgene encoding a kinase of the invention or a gene affecting the expression of the kinase.
  • Such transgenic nonhuman mammals are particularly useful as an in vivo test system for studying the effects of introduction of a kinase, or regulating the expression of a kinase (i.e., through the introduction of additional genes, antisense nucleic acids, or ribozymes).
  • transgenic animal is an animal having cells that contain DNA which has been artificially inserted into a cell, which DNA becomes part of the genome of the animal which develops from that cell.
  • Preferred transgenic animals are primates, mice, rats, cows, pigs, horses, goats, sheep, dogs and cats.
  • the transgenic DNA may encode human STE20-related kinases. Native expression in an animal may be reduced by providing an amount of antisense RNA or DNA effective to reduce expression of the receptor.
  • BUBl -related or NEK kinases or their genetic sequences will also be useful in gene therapy (reviewed in Miller, Nature 357:455-460, 1992). Miller states that advances have resulted in practical approaches to human gene therapy that have demonstrated positive initial results. The basic science of gene therapy is described in Mulligan (Science 260:926-931, 1993).
  • an expression vector containing BUBl -related or NEK kinase coding sequence is inserted into cells, the cells are grown in vitro and then infused in large numbers into patients.
  • a DNA segment containing a promoter of choice (for example a strong promoter) is transferred into cells containing an endogenous gene encoding kinases of the invention in such a manner that the promoter segment enhances expression of the endogenous kinase gene (for example, the promoter segment is transferred to the cell such that it becomes directly linked to the endogenous kinase gene).
  • the gene therapy may involve the use of an adenovirus containing kinase cDNA targeted to a tumor, systemic kinase increase by implantation of engineered cells, injection with kinase-encoding virus, or injection of naked kinase DNA into appropriate tissues.
  • Target cell populations may be modified by introducing altered forms of one or more components of the protein complexes in order to modulate the activity of such complexes. For example, by reducing or inhibiting a complex component activity within target cells, an abnormal signal transduction event(s) leading to a condition may be decreased, inhibited, or reversed. Deletion or missense mutants of a component, that retain the ability to interact with other components of the protein complexes but cannot function in signal transduction, may be used to inhibit an abnormal, deleterious signal transduction event.
  • Expression vectors derived from viruses such as retroviruses, vaccinia virus, adenovirus, adeno-associated virus, herpes viruses, several RNA viruses, or bovine papilloma virus may be used for delivery of nucleotide sequences (e.g., cDNA) encoding recombinant kinase of the invention protein into the targeted cell population (e.g., tumor cells).
  • nucleotide sequences e.g., cDNA
  • Methods which are well known to those skilled in the art can be used to construct recombinant viral vectors containing coding sequences (Maniatis et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, N.Y., 1989; Ausubel et al.
  • nucleic acid molecules encoding protein sequences can be used as naked DNA or in a reconstituted system e.g., liposomes or other lipid systems for delivery to target cells (e.g., Feigner et al, Nature 337:387-8, 1989).
  • target cells e.g., Feigner et al, Nature 337:387-8, 1989.
  • gene transfer can be performed by simply injecting minute amounts of DNA into the nucleus of a cell, through a process of microinjection (Capecchi, Cell 22:479-88, 1980). Once recombinant genes are introduced into a cell, they can be recognized by the cell's normal mechanisms for transcription and translation, and a gene product will be expressed. Other methods have also been attempted for introducing DNA into larger numbers of cells. These methods include: transfection, wherein DNA is precipitated with calcium phosphate and taken into cells by pinocytosis (Chen et al, Mol. Cell Biol.
  • adenovirus proteins are capable of destabilizing endosomes and enhancing the uptake of DNA into cells.
  • the admixture of adenovirus to solutions containing DNA complexes, or the binding of DNA to polylysine covalently attached to adenovirus using protein crosslinking agents substantially improves the uptake and expression of the recombinant gene (Curiel et al, Am. J. Respir. Cell. Mol. Biol., 6:247-52, 1992).
  • Gene transfer means the process of introducing a foreign nucleic acid molecule into a cell. Gene transfer is commonly performed to enable the expression of a particular product encoded by the gene.
  • the product may include a protein, polypeptide, anti-sense DNA or RNA, or enzymatically active RNA.
  • Gene transfer can be performed in cultured cells or by direct administration into animals. Generally gene transfer involves the process of nucleic acid contact with a target cell by non-specific or receptor mediated interactions, uptake of nucleic acid into the cell through the membrane or by endocytosis, and release of nucleic acid into the cytoplasm from the plasma membrane or endosome.
  • Gene therapy is a form of gene transfer and is included within the definition of gene transfer as used herein and specifically refers to gene transfer to express a therapeutic product from a cell in vivo or in vitro. Gene transfer can be performed ex vivo on cells which are then transplanted into a patient, or can be performed by direct administration of the nucleic acid or nucleic acid-protein complex into the patient.
  • a vector having nucleic acid sequences encoding a BUBl -related or NEK kinase polypeptide is provided in which the nucleic acid sequence is expressed only in specific tissue.
  • Methods of achieving tissue-specific gene expression are set forth in International Publication No. WO 93/09236, filed November 3, 1992 and published May 13, 1993.
  • nucleic acid sequence contained in the vector may include additions, deletions or modifications to some or all of the sequence of the nucleic acid, as defined above.
  • Gene replacement means supplying a nucleic acid sequence which is capable of being expressed in vivo in an animal and thereby providing or augmenting the function of an endogenous gene which is missing or defective in the animal.
  • Suitable routes of administration may, for example, include oral, rectal, transmucosal, or intestinal administration; parenteral delivery, including intramuscular, subcutaneous, intravenous, intramedullary injections, as well as intrathecal, direct intraventricular, intraperitoneal, intranasal, or intraocular injections.
  • parenteral delivery including intramuscular, subcutaneous, intravenous, intramedullary injections, as well as intrathecal, direct intraventricular, intraperitoneal, intranasal, or intraocular injections.
  • parenteral delivery including intramuscular, subcutaneous, intravenous, intramedullary injections, as well as intrathecal, direct intraventricular, intraperitoneal, intranasal, or intraocular injections.
  • parenteral delivery including intramuscular, subcutaneous, intravenous, intramedullary injections, as well as intrathecal, direct intraventricular, intraperitoneal, intranasal, or intraocular injections.
  • compositions of the present invention may be manufactured in a manner that is itself known, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.
  • compositions for use in accordance with the present invention thus may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.
  • the agents of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiological saline buffer.
  • physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiological saline buffer.
  • penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
  • the compounds can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers well known in the art.
  • Such carriers enable the compounds of the invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated.
  • Suitable carriers include excipients such as, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl- cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP).
  • disintegrating agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.
  • Dragee cores are provided with suitable coatings.
  • concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures.
  • Dyestuffs or pigments may be added to w the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.
  • compositions which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol.
  • the push- fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers.
  • the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols.
  • stabilizers may be added. All formulations for oral administration should be in dosages suitable for such administration.
  • compositions may take the form of tablets or lozenges formulated in conventional manner.
  • the compounds for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebuliser, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofiuoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • a suitable propellant e.g., dichlorodifluoromethane, trichlorofiuoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • the dosage unit may be determined by providing a valve to deliver a metered amount.
  • Capsules and cartridges of e.g. gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.
  • the compounds may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion.
  • Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative.
  • the compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • compositions for parenteral administration include aqueous solutions of the active compounds in water-soluble form. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.
  • the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
  • a suitable vehicle e.g., sterile pyrogen-free water
  • the compounds may also be formulated in rectal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.
  • the compounds may also be formulated as a depot preparation. Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection.
  • the compounds may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
  • a pharmaceutical carrier for the hydrophobic compounds of the invention is a cosolvent system comprising benzyl alcohol, a nonpolar surfactant, a water-miscible organic polymer, and an aqueous phase.
  • the cosolvent system may be the VPD co- solvent system.
  • VPD is a solution of 3% w/v benzyl alcohol, 8% w/v of the nonpolar surfactant polysorbate 80, and 65% w/v polyethylene glycol 300, made up to volume in absolute ethanol.
  • the VPD co-solvent system (VPD:D5W) consists of VPD diluted 1 : 1 with a 5% dextrose in water solution. This co-solvent system dissolves hydrophobic compounds well, and itself produces low toxicity upon systemic administration. Naturally, the proportions of a co-solvent system may be varied considerably without destroying its solubility and toxicity characteristics.
  • co- solvent components may be varied: for example, other low-toxicity nonpolar surfactants may be used instead of polysorbate 80; the fraction size of polyethylene glycol may be varied; other biocompatible polymers may replace polyethylene glycol, e.g. polyvinyl pyrrolidone; and other sugars or polysaccharides may substitute for dextrose.
  • hydrophobic pharmaceutical compounds may be employed.
  • Liposomes and emulsions are well known examples of delivery vehicles or carriers for hydrophobic drugs.
  • Certain organic solvents such as dimethylsulfoxide also may be employed, although usually at the cost of greater toxicity.
  • the compounds may be delivered using a sustained-release system, such as
  • sustained-release materials have been established and are well known by those skilled in the art. Sustained-release capsules may, depending on their chemical nature, release the compounds for a few weeks up to over 100 days. Depending on the chemical nature and the biological stability of the therapeutic reagent, additional strategies for protein stabilization may be employed.
  • compositions also may comprise suitable solid or gel phase carriers or excipients.
  • suitable solid or gel phase carriers or excipients include but are not limited to calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycols.
  • tyrosine or serine/threonine kinase modulating compounds of the invention may be provided as salts with pharmaceutically compatible counterions.
  • Pharmaceutically compatible salts may be formed with many acids, including but not limited to hydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend to be more soluble in aqueous or other protonic solvents that are the corresponding free base forms.
  • compositions suitable for use in the present invention include compositions where the active ingredients are contained in an amount effective to achieve its intended purpose. More specifically, a therapeutically effective amount means an amount of compound effective to prevent, alleviate or ameliorate symptoms of disease or prolong the survival of the subject being treated. Determination of a therapeutically effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein. For any compound used in the methods of the invention, the therapeutically effective dose can be estimated initially from cell culture assays.
  • a dose can be formulated in animal models to achieve a circulating concentration range that includes the IC 50 as determined in cell culture (i.e., the concentration of the test compound which achieves a half-maximal inhibition of the tyrosine or serine/threonine kinase activity).
  • IC 50 as determined in cell culture
  • Toxicity and therapeutic efficacy of the compounds described herein can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD 0 (the dose lethal to 50% of the population) and the ED 50 (the dose therapeutically effective in 50% of the population).
  • the dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio between LD 0 and ED 50 .
  • Compounds which exhibit high therapeutic indices are preferred.
  • the data obtained from these cell culture assays and animal studies can be used in formulating a range of dosage for use in human.
  • the dosage of such compounds lies preferably within a range of circulating concentrations that include the ED 50 with little or no toxicity.
  • the dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.
  • the exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See e.g., Fingl et al, 1975, in "The Pharmacological Basis of Therapeutics", Ch. 1 p.l).
  • Dosage amount and interval may be adjusted individually to provide plasma levels of the active moiety which are sufficient to maintain the kinase modulating effects, or minimal effective concentration (MEC).
  • MEC minimal effective concentration
  • the MEC will vary for each compound but can be estimated from in vitro data; e.g., the concentration necessary to achieve 50-90% inhibition of the kinase using the assays described herein. Dosages necessary to achieve the MEC will depend on individual characteristics and route of admimstration. However, HPLC assays or bioassays can be used to determine plasma concentrations.
  • Dosage intervals can also be determined using MEC value.
  • Compounds should be administered using a regimen which maintains plasma levels above the MEC for 10-90% of the time, preferably between 30-90% and most preferably between 50-90%.
  • the effective local concentration of the drug may not be related to plasma concentration.
  • composition administered will, of course, be dependent on the subject being treated, on the subject's weight, the severity of the affliction, the manner of administration and the judgment of the prescribing physician.
  • compositions may, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the active ingredient.
  • the pack may for example comprise metal or plastic foil, such as a blister pack.
  • the pack or dispenser device may be accompanied by instructions for administration.
  • the pack or dispenser may also be accompanied with a notice associated with the container in form prescribed by a governmental agency regulating the manufacture, use, or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the polynucleotide for human or veterinary administration.
  • Such notice for example, may be the labeling approved by the U.S. Food and Drug Administration for prescription drugs, or the approved product insert.
  • compositions comprising a compound of the invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition.
  • Suitable conditions indicated on the label may include treatment of a tumor, inhibition of angiogenesis, treatment of fibrosis, diabetes, and the like.
  • a functional derivative of a polypeptide or nucleic acid of the invention.
  • “functional derivative” is meant a “chemical derivative,” “fragment,” or “variant,” of the polypeptide or nucleic acid of the invention, which terms are defined below.
  • a functional derivative retains at least a portion of the function of the protein, for example reactivity with an antibody specific for the protein, enzymatic activity or binding activity mediated through noncatalytic domains, which permits its utility in accordance with the present invention. It is well known in the art that due to the degeneracy of the genetic code numerous different nucleic acid sequences can code for the same amino acid sequence.
  • portions or all of the BUBl and NEK genes could be synthesized to give a nucleic acid sequence significantly different from that shown in SEQ ID NO:l, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, or SEQ ID NO:9.
  • the encoded amino acid sequence thereof would, however, be preserved.
  • the nucleic acid sequence may comprise a nucleotide sequence which results from the addition, deletion or substitution of at least one nucleotide to the 5'-end and/or the 3 '-end of the nucleic acid formula shown in SEQ ID NO:l, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, or SEQ ID NO:9, or a derivative thereof.
  • Any nucleotide or polynucleotide may be used in this regard, provided that its addition, deletion or substitution does not alter the amino acid sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, or SEQ ID NO:10 which is encoded by the nucleotide sequence.
  • the present invention is intended to include any nucleic acid sequence resulting from the addition of ATG as an initiation codon at the 5'-end of the inventive nucleic acid sequence or its derivative, or from the addition of TTA, TAG or TGA as a termination codon at the 3'-end of the inventive nucleotide sequence or its derivative.
  • the nucleic acid molecule of the present invention may, as necessary, have restriction endonuclease recognition sites added to its 5'-end and/or 3'-end.
  • nucleic acid sequence affords an opportunity to promote secretion and/or processing of heterologous proteins encoded by foreign nucleic acid sequences fused thereto.
  • All variations of the nucleotide sequence of the BUBl and NEK genes of the invention and fragments thereof permitted by the genetic code are, therefore, included in this invention.
  • the two polypeptides are functionally equivalent, as are the two nucleic acid molecules that give rise to their production, even though the differences between the nucleic acid molecules are not related to the degeneracy of the genetic code.
  • a "chemical derivative" of the complex contains additional chemical moieties not normally a part of the protein.
  • Covalent modifications of the protein or peptides are included within the scope of this invention. Such modifications may be introduced into the molecule by reacting targeted amino acid residues of the peptide with an organic derivatizing agent that is capable of reacting with selected side chains or terminal residues, as described below.
  • Cysteinyl residues most commonly are reacted with alpha-haloacetates (and corresponding amines), such as chloroacetic acid or chloroacetamide, to give carboxymethyl or carboxyamidomethyl derivatives. Cysteinyl residues also are derivatized by reaction with bromotrifluoroacetone, chloroacetyl phosphate, N- alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyl disulfide, p- chloromercuribenzoate, 2-chloromercuri-4-nitrophenol, or chloro-7-nitrobenzo-2-oxa-l,3- diazole.
  • Histidyl residues are derivatized by reaction with diethylprocarbonate at pH 5.5- 7.0 because this agent is relatively specific for the histidyl side chain.
  • Para- bromophenacyl bromide also is useful; the reaction is preferably performed in 0.1 M sodium cacodylate at pH 6.0.
  • Lysinyl and amino terminal residues are reacted with succinic or other carboxylic acid anhydrides. Derivatization with these agents has the effect or reversing the charge of the lysinyl residues.
  • Other suitable reagents for derivatizing primary amine containing residues include imidoesters such as methyl picolinimidate; pyridoxal phosphate; pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid; O-methylisourea; 2,4 pentanedione; and transaminase-catalyzed reaction with glyoxylate.
  • Arginyl residues are modified by reaction with one or several conventional reagents, among them phenylglyoxal, 2,3-butanedione, 1,2-cyclohexanedione, and ninhydrin. Derivatization of arginine residues requires that the reaction be performed in alkaline conditions because of the high pK a of the guanidine functional group. Furthermore, these reagents may react with the groups of lysine as well as the arginine alpha- amino group.
  • Tyrosyl residues are well-known targets of modification for introduction of spectral labels by reaction with aromatic diazonium compounds or tefranitromethane.
  • N-acetylimidizol and tetranitromethane are used to form O-acetyl tyrosyl species and 3-nitro derivatives, respectively.
  • Carboxyl side groups (aspartyl or glutamyl) are selectively modified by reaction with carbodumide (R'-N-C-N-R') such as l-cyclohexyl-3-(2-morpholinyl(4-ethyl) carbodumide or l-ethyl-3-(4-azonia-4,4-dimethylpentyl) carbodumide. Furthermore, aspartyl and glutamyl residues are converted to asparaginyl and glutaminyl residues by reaction with ammonium ions.
  • carbodumide R'-N-C-N-R'
  • Glutaminyl and asparaginyl residues are frequently deamidated to the corresponding glutamyl and aspartyl residues. Alternatively, these residues are deamidated under mildly acidic conditions. Either form of these residues falls within the scope of this invention.
  • Derivatization with bifunctional agents is useful, for example, for cross-linking the component peptides of the protein to each other or to other proteins in a complex to a water-insoluble support matrix or to other macromolecular carriers.
  • Commonly used cross-linking agents include, for example, l,l-bis(diazoacetyl)-2-phenylethane, glutaraldehyde, N-hydroxysuccinimide esters, for example, esters with 4-azidosalicylic acid, homobifunctional imidoesters, including disuccinimidyl esters such as 3,3'- dithiobis(succinimidylpropionate), and bifunctional maleimides such as bis-N-maleimido- 1,8-octane.
  • Derivatizing agents such as methyl-3-[p-azidophenyl) dithiolpropioimidate yield photoactivatable intermediates that are capable of forming crosslinks in the presence of light.
  • reactive water-insoluble matrices such as cyanogen bromide- activated carbohydrates and the reactive substrates described in U.S. Patent Nos. 3,969,287; 3,691,016; 4,195,128; 4,247,642; 4,229,537; and 4,330,440 are employed for protein immobilization.
  • Such derivatized moieties may improve the stability, solubility, absorption, biological half life, and the like.
  • the moieties may alternatively eliminate or attenuate any undesirable side effect of the protein complex and the like.
  • Moieties capable of mediating such effects are disclosed, for example, in Remington's Pharmaceutical Sciences, 18th ed., Mack Publishing Co., Easton, PA (1990).
  • fragment is used to indicate a polypeptide derived from the amino acid sequence of the proteins, of the complexes having a length less than the full-length polypeptide from which it has been derived.
  • a fragment may, for example, be produced by proteolytic cleavage of the full-length protein.
  • the fragment is obtained recombinantly by appropriately modifying the DNA sequence encoding the proteins to delete one or more amino acids at one or more sites of the C-terminus, N- terminus, and/or within the native sequence.
  • Fragments of a protein are useful for screening for substances that act to modulate signal transduction, as described herein. It is understood that such fragments may retain one or more characterizing portions of the native complex. Examples of such retained characteristics include: catalytic activity; substrate specificity; interaction with other molecules in the intact cell; regulatory functions; or binding with an antibody specific for the native complex, or an epitope thereof.
  • variant polypeptide which either lacks one or more amino acids or contains additional or substituted amino acids relative to the native polypeptide.
  • the variant may be derived from a naturally occurring complex component by appropriately modifying the protein DNA coding sequence to add, remove, and/or to modify codons for one or more amino acids at one or more sites of the C-terminus, N-terminus, and/or within the native sequence. It is understood that such variants having added, substituted and/or additional amino acids retain one or more characterizing portions of the native protein, as described above.
  • a functional derivative of a protein with deleted, inserted and/or substituted amino acid residues may be prepared using standard techniques well-known to those of ordinary skill in the art.
  • the modified components of the functional derivatives may be produced using site-directed mutagenesis techniques (as exemplified by Adelman et al. , 1983, DNA 2:183) wherein nucleotides in the DNA coding the sequence are modified such that a modified coding sequence is modified, and thereafter expressing this recombinant DNA in a prokaryotic or eukaryotic host cell, using techniques such as those described above.
  • proteins with amino acid deletions, insertions and/or substitutions may be conveniently prepared by direct chemical synthesis, using methods well-known in the art.
  • the functional derivatives of the proteins typically exhibit the same qualitative biological activity as the native proteins.
  • This RNA was used to generate single-stranded cDNA using the Superscript Preamplification System (GIBCO BRL, Gaithersburg, MD; Gerard, GF et al. (1989), FOCUS 11, 66) under conditions recommended by the manufacturer.
  • GEBCO BRL Superscript Preamplification System
  • a typical reaction used 10 ⁇ g total RNA with 1.5 ⁇ g oligo(dT) ⁇ 2- ⁇ 8 in a reaction volume of 60 ⁇ L.
  • the product was treated with RNaseH and diluted to 100 ⁇ L with H 2 0.
  • 3 ⁇ L of this single stranded cDNA was used in each reaction.
  • oligonucleotide primers were synthesized on an Applied Biosystems 3948 DNA synthesizer using established phosphoramidite chemistry, precipitated with ethanol, and used unpurified for PCR.
  • sequence of the degenerate oligonucleotide primers is as follows:
  • BUB HRD 5'- GAGGTGGGCATHATHCAYGGNA-3' (SEQ YD NO: 14).
  • BUB DVW 5'- GATGAATTRTCNGGYTTNAKRTCNCC-3'(SEQ ID NO:15). These primers were derived from the sense and antisense strands of amino acid motifs
  • N A, C, G, or T
  • R A or G
  • Y C or T
  • H A, C or T not G
  • D A, G or T not C.
  • PCR reactions were performed using degenerate primers applied to the NCI colon HCC 2998 single-stranded cDNA.
  • the primers were added at a final concentration of 25 ⁇ M each to a mixture containing 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl 2 , 200 ⁇ M each deoxynucleoside triphosphate, 0.001% gelatin, 1.5 U AmpliTaq DNA Polymerase (Perkin-Elmer/Cetus), and 3 ⁇ L cDNA.
  • PCR fragments migrating between 220 to 250 bp were isolated from 2% agarose gels using a GeneClean Kit (Bio 101) and T-A cloned into the pCRII vector (Invitrogen Corp. U.S.A.) according to the manufacturer's protocol.
  • Colonies were selected for plasmid DNA mini-preparations, the plasmid DNA was purified using Qiagen columns, and was sequenced using a cycle sequencing dye-terminator kit with AmpliTaq DNA Polymerase, FS (ABI, Foster City, CA). Sequencing reaction products were run on an ABI Prism 377 DNA Sequencer and analyzed using the BLAST alignment algorithm (Altschul, S.F. et al (1990) J.Mol.Biol. 215:403-10).
  • a human Leukemia 5 '-Stretch Plus ⁇ gtl 1 cDNA library (Clontech) was probed with the 238 bp PCR fragment of SuSTK34a (R51-40- 18) corresponding to human BUBl. Probes were 32 P-labeled by random priming and used at 2x10 6 cpm/mL following standard techniques for library screening. Pre-hybridization (3 h) and hybridization (overnight) were conducted at 42 °C in 5X SSC, 5X Denhart's solution, 2.5% dextran sulfate, 50 mM Na 2 PO 4 /NaHPO 4 , pH 7.0, 50% formamide with 100 mg/mL denatured salmon sperm DNA.
  • SuSTK34a R51 -40- 18
  • SuSTK34b R51-40-4
  • SuSTK34a and SuSTK34b are identical except for the deletion of 15 nucleotides within kinase subdomain VII of SuSTK34b, resulting in a predicted 5 amino acid deletion.
  • the full length human bubl clone was isolated as four EcoRI cDNA fragments: J6E0.45#9, J7E1.3#3, J6E1.3#5, and J2E0.9#8.
  • a 3436 bp Notl fragment of a single ⁇ clone was subcloned into a pBluescript(SKII+) vector, giving rise to pSGl 10.
  • the full length human bubl cDNA (SEQ ID N0.1) is 3408 bp long and consists of a 3255 bp ORF (45-3300) flanked by a 44 bp (1-44) 5' UTR and a 108 bp 3' UTR.
  • a potential polyadenylation signal begins at position 3404.
  • the sequence flanking the ATG conforms to the Kozak consensus for an initiating methionine, and is believed to be the translational start site for the human BUB 1.
  • bubl was analyzed by Northern blots prepared by running 20 ⁇ g of total RNA from 22 human adult tissues (thymus, lung, duodenum, colon, testis, brain, cerebellum, cortex, salivary gland, liver, pancreas, kidney, spleen, stomach, uterus, prostate, skeletal muscle, placenta, mammary gland, bladder, lymph node, adipose tissue), 2 human fetal normal tissues (fetal liver, fetal brain ), and 60 human tumor cell lines (HOP-92, EKVX, NCI-H23, NCI-H226, NCI-H322M, NCI-H460, NCI-H522, A549, HOP-62, OVCAR-3, OVCAR-4, OVCAR-5, OVCAR-8, IGROVl, SK-OV-3, SNB-19, SNB-75, U251, SF-268, SF-295, SF-539, CCRF-CEM, K-562,
  • RNA samples were run on denaturing formaldehyde 1% agarose gels and transferred onto nitrocellulose membranes (BioRad, CA).
  • Filters were hybridized with randomly primed [ ⁇ 32 P]dCTP-labeled probes synthesized from the EcoBI/Pstl 809 bp of human bubl fragment. Hybridization was performed at 42 °C overnight in 6X SSC, 0.1% SDS, IX Denhardt's solution, 100 ⁇ g/mL denatured herring sperm DNA with 1-2 x 10 6 cpm/mL of 32 P-labeled DNA probes. The filters were washed in 0.1X SSC/0.1% SDS, 65 °C, and exposed on a Molecular Dynamics phosphorimager.
  • STK plasmid array blots were prepared by loading 0.5 ⁇ g of denatured plasmid encoding numerous STKs, including bubl (pSGl 10), onto a nylon membrane.
  • the [ ⁇ PJdCTP labeled single stranded DNA probes were synthesized from RNA isolated
  • a single human bubl mRNA transcript of approximately 4.2 kb was identified by Northern analysis, and was found to be expressed in nine human normal tissues (thymus, testis, heart, placenta, lung, lymph node, thymus, bone marrow, fetal liver). The remainder of the 19 human normal tissues (duodenum, colon, brain, cerebellum, cortex, salivary gland, liver, pancreas, kidney, spleen, stomach, uterus, prostate, skeletal muscle, fetal brain, mammary gland, bladder, adipose tissue, peripheral blood leukocyte) were negative. Of the human tumor cell lines, 60 of 64 were positive for expression of human bubl.
  • Human bubl expression was analyzed by hybridization of [ ⁇ 32 P]dCTP-labeled single stranded DNA from human immune cells to a nylon membrane containing plasmid DNA encoding bubl and other STKs. This DNA array-based expression analysis was performed on 14 human immune tissues and cell lines: thymus, dendrocytes, mast cells, monocytes, B cells (primary, Jurkat, RPMI8226, SR), T cells (CD8/CD4+, TH1, TH2, CEM, MOLT4), and K562 (megakaryocytes).
  • bubl was restricted to 4 of these immune sources, RPMI (B cells), MOLT4 (T cells), CEM (T cells), and primary monocytes, and was absent from the other 10 samples (data not shown). This expression profile indicates that human bubl is expressed in several hematopoietic cell types.
  • Expression Plasmid Construction Expression constructs were generated for human bubl including: a) pcDNA3.1 expression plasmids containing: the full length bubl nucleotide base sequence with and without an HA tag; the nucleotide base sequence encoding the N-terminal domain (amino acid 1 to 332) of BUBl with and without an HA tag; the nucleotide base sequence encoding the central-domain (amino acid 333 to 757) of BUBl with and without an HA tag; the nucleotide base sequence encoding the C-terminal kinase domain (amino acid 775 to 1085) of BUBl with and without an HA tag, and the nucleotide base sequence of full length bubl with a Lys to Arg mutation at amino acid 821 of BUBl with and without an HA tag; b) pGEX 4T-3 fusion plasmids containing: the full length nucleotide base sequence of bubl; the nucle
  • EXAMPLE 4 Recombinant Expression of BUBl Serine/threonine Protein Kinase Materials and Methods Transient Expression of BUBl in Mammalian Cells
  • Recombinant protein was detected using various BUBl antisera, or anti-HA specific antisera, by Western analysis.
  • BUBl-antisera was diluted 1 : 1 ,000-fold, and anti-HA monoclonal antibody (Babco, Berkeley, California) was diluted 1 : 10,000-fold for the primary antibody reactions of Western blotting.
  • Goat anti-rabbit HRP BioRad, Richmond, California
  • goat anti-mouse HRP BioRad, Richmond, California
  • ECL reagent (Amersham Life Science,) was used for detection. Results Cell lysates (30 ⁇ g each) were applied to 7.5% SDS PAGE followed by Western blotting.
  • the BUBl protein has a predicted molecular mass of 120 kDa. Recombinant human BUBl migrates as a single polypeptide band of 150 kDa. The higher than predicted molecular mass is likely due to post-translational modification, possibly serine/threonine phosphorylation. The truncated forms migrated as predicted: the N-terminal domain at 45 kDa (with HA tag); the central-domain at 56 kDa.
  • 505A QKYNQRRKHEQWVNE (SEQ ID NO:18; aa257-271; N-terminal); 506A: DDKDEWQSLDQNED (SEQ ID NO: 19; aa485-498; Central domain); 507A: KNIQKFVLKVQK (SEQ ID NO:20; aa813-824; catalytic domain); and 508A: RQKLKKVFQQHYTNK (SEQ ID NO:21; aal051-1065; catalytic domain).
  • Additional immunoreagents were generated by immunizing rabbits with the bacterially expressed GST-fusion proteins containing the N-terminal or C-terminal domain ofBUBl.
  • the link consists of an aldehyde support arm that forms a covalent
  • the purified anti-BUBl IgG was eluted with 1 M propionic acid, neutralized with 3 M Tris, pH 7.4 while eluting, and buffered with 100 mM TEA buffered with 1 M Tris pH 6.8.
  • the purified antibody was dialyzed against phosphate buffered saline pH 7.4, and was stored in 20% glycerol and 0.03% NaN 3 in PBS.
  • the affinity purified anti-BUB 1 antibody was tested by Western analysis using the GST-BUB 1 fusion protein, or the mammalian cell lysates from transiently transfected and expressed BUBl protein.
  • the purified anti-506A was very antigenic, and could be diluted 1 :20,000-fold for testing against the recombinant BUBl protein.
  • Anti-507A and anti-508A can be purified similarly.
  • HeLa cells were synchronized by adding 0.5 M thymidine and 250 ⁇ g/mL aphidicolin. Cells were harvested at 0, 2, 4, 6, 8, 9, 10, 11, and 12 hours after thymidine and aphidicolin were removed. Other experiments were performed by blocking HeLa cells at different cell cycle stages by serum starvation, with thymidine (G1/S/G2), or with nocodozole (M). Total RNA was isolated from the synchronized cells and cell cycle blocked cells. Total RNA (10 ⁇ g) was loaded on the nylon membranes. The RNA blots were hybridized with a bubl EcoRUPstl 809 bp probe. Results
  • RNA derived from HeLa cells either in an exponential growth state or a ⁇ ested in various stages of the cell cycle were probed for expression of bubl.
  • a detectable bubl signal was observed in the exponentially growing cells, whereas no signal was present in samples from cells grown under serum starvation or blocked in the Gj/S/G 2 stages by thymidine/aphidicolin.
  • high levels of bubl expression were observed in cells arrested in mitosis by nocodazole.
  • the bubl RNA was expressed at very low levels immediately following release from synchrony, and peaked at 8 to 10 hours (period of mitotic division) after drug release. The expression levels were decreased at 11 to 12 hours (period of telophase or exit from mitosis) after drug release.
  • bubl is mitotically regulated and is highly expressed in mitosis.
  • EXAMPLE 8 Protein Localization of BUBl Serine/Threonine Protein Kinase Materials and Methods The intracellular location of BUBl protein was determined by immunoflourescence with BUBl-specific antibody. HeLa cells and H1299 cells were grown to confluence. Cells were trypsinized and then plated on 1 cm poly-lysine pre-treated glass cover slips in 60 mm plates and incubated at 37 °C for 1 day. Cells were washed with 5 mL ice-cold PBS after removing medium and then were fixed with -20 °C- cold 100% methanol. Methanol fixed cells can be used for a week or so.
  • BUBl protein exclusively localized to kinetochores during mitosis.
  • BUBl antibody stained all chromosome pairs during prophase and early metaphase in mitosis, demonstrating that BUBl is a centromeric protein.
  • the pcDNA3.1.BUBl.HA expression plasmid was transfected into human 293T cells.
  • the crude protein lysates were extracted from the transfected cells and used for BUBl kinase activity assays.
  • Four different assays were performed: (1) BUBl in vitro kinase autophosphorylation, (2) BUBl phosphorylation of exogenous substrates, (3) BUBl in vivo kinase activity, and (4) BUBl phosphoamino acid analysis.
  • BUBl in vitro Kinase Autophosphorylation Assay Materials and Methods
  • the pcDNA3.1 BUBl expression plasmids (10 ⁇ g DNA/90 mm plate) containing HA-tagged wild-type (BUBl. HA), N-terminally truncated (BUB1.N.HA), or the kinase dead form of BUB 1 (BUB 1.KR.HA), were introduced into human 293T cells with lipofectamine (Gibco BRL, Gaithersburg, MD). After 48 hours, the cells were harvested in 0.4 mL of kinase lysis buffer (50 mM HEPES, pH 7.5, 100 mM KCl, 25 mM NaF, 1 mM sodium orthovanadate, 0.5 mM nonidet P-40 (Sigma, St.
  • kinase lysis buffer 50 mM HEPES, pH 7.5, 100 mM KCl, 25 mM NaF, 1 mM sodium orthovanadate, 0.5 mM nonidet P-40 (Sigma, St.
  • Protein extracts were immunoprecipitated using anti-HA antibody (Babco, Berkeley, CA) at a final dilution of 1:750 and protein G beads (Boehringer Mannheim, Indianapolis, IN), then washed with kinase lysis buffer three times followed by two washes with kinase buffer (20 mM HEPES, pH 7.5, 150 mM KCl, 10 mM MgCl 2 ).
  • kinase reaction mix (IX kinase buffer, 1 mM DTT, 0.1 mM ATP, 10 ⁇ Ci 32 P-r-ATP) was added to the washed beads and the mixture was incubated at 37 °C for 40 minutes with constant agitation. The reaction was stopped by adding 8 ⁇ L 5X PAGE buffer and boiled at 100 °C for 5 minutes. Results
  • the in vitro kinase assay was also used to test whether the N-terminal, truncated form of BUBl could also be a substrate for the BUBl kinase, since a radioactively labeled BUB1.N protein (50 kDa) was detected in the in vivo kinase assay (herein), suggesting that BUB1.N protein is a substrate for an unidentified kinase or for the BUBl kinase in vivo. Autoradiography of the assay using immunoprecipitated BUB1.N as the substrate for immunoprecipitated BUBl, showed that the BUB1.N protein could be detected (results not shown). Therefore, it appears that BUB 1.N protein is a weak substrate for BUBl kinase.
  • HA- tagged full length, truncated, or the kinase dead form of BUBl were introduced into human 293T cells with lipofectamine (Gibco BRL). After 48 hours, the cell medium was changed to phosphate-free medium (Gibco BRL) containing 10% dialyzed fetal calf serum (Gibco BRL). Twenty hours after the medium change, inorganic P-phosphate at 2 mCi/100 mm plate was added to the medium and the cells were incubated for 3 hours. Twelve ⁇ L of pervanadate solution (50 mM sodium orthovanadate, 15% hydrogen peroxide) was added, and the cells were incubated for an additional 10 minutes.
  • pervanadate solution 50 mM sodium orthovanadate, 15% hydrogen peroxide
  • the cell culture dishes were placed on ice, and were rinsed with 4 °C phosphate-buffered saline.
  • the crude protein lysates were extracted with 450 ⁇ L of kinase lysis buffer (50 mM HEPES, pH 7.5, 100 mM KCl, 25 mM NaF, 1 mM sodium orthovanadate, 0.5 mM nonidet P-40 (Sigma), 1 mM DTT, 170 ⁇ g/mL PMSF, 25 ⁇ g/mL leupeptin, 20 ⁇ g/mL trypsin inhibitor, 25 ⁇ g/mL aprotinin), and were immunoprecipitated using anti-HA antibody.
  • kinase lysis buffer 50 mM HEPES, pH 7.5, 100 mM KCl, 25 mM NaF, 1 mM sodium orthovanadate, 0.5 mM nonidet P-40 (Sigma), 1 mM DTT, 170 ⁇ g/m
  • the assay was performed by adding inorganic P to 293T cells transiently transfected with BUB 1 or BUB 1.N. Crude protein was extracted from the radio-labeled cells. Cell lysates were immunoprecipitated by anti-HA antibody and then applied to a 10% polyacrylamide SDS gel. A phosphorylated band was observed at the position with molecular mass of 150 kDa corresponding to BUB 1. No signal was detected in the vector only control. A weak signal was observed from the sample with BUB1.N transfection. This was not seen in the in vitro kinase assay.
  • BUBl Kinase Phosphoamino Acid Analysis Material and Methods Hunter's protocol was employed for BUBl kinase phosphoamino acid analysis.
  • the radio-labeled BUBl band was excised from the dried polyacrylamide gel.
  • the gel slice was rehydrated in 0.5 mL of 50 mM ammonium bicarbonate, pH 7.3 to 7.6, for 5 minutes, and then was homogenized into a fine suspension.
  • the volume of suspension was adjusted to 1 mL with 50 mM ammonium bicarbonate with a final concentration of 5% beta mercaptoethanol (v/v) and 0.1% SDS (w/v).
  • the mixture was incubated at 100 °C for 5 minutes followed by 37 °C for 12 hours with agitation.
  • the parti culate fraction was collected by centrifugation and re-extracted with 50 mM ammonium bicarbonate, pH 7.3 to 7.6, 5% beta mercaptoethanol (v/v), 0.1% SDS (w/v), by incubation at 100 °C for 5 minutes followed by 37 °C for 60 minutes. Following centrifugation, the liquid extract was combined with the prior extract to give a total volume of 1.2 mL. The extract was centrifuged again to remove particulate material, and the supernatant was isolated and chilled to 4 °C.
  • a total of 20 ⁇ g of bovine serum albumin was added to the extract and the proteins were precipitated by addition of 250 ⁇ L of 100% trichloroacetic acid and incubated at 4 °C for 60 minutes.
  • the precipitated proteins were isolated by centrifugation, and the resulting pellets were washed with 500 ⁇ L of 4 °C 100% acetone.
  • the protein pellets were air dried, and then were dissolved in 100 ⁇ L of 5.7 M HCl. The solution was heated at 100 °C for 60 minutes in a screw-capped tube, and then was dried under vacuum.
  • the residual pellet was dissolved in formic acid (2% v/v) and glacial acetic acid (7.3% v/v) containing 67 ⁇ g/mL phosphoserine, 67 ⁇ g/mL phosphothreonine, 67 ⁇ g/mL phosphotyrosine, pH 1.9.
  • a sample containing a total of 600 cpm was spotted onto a cellulose TLC plate (VWR scientific, San Francisco, CA). Two dimension chromatography was performed. The first dimension gel was run in a buffer of formic acid (2% v/v) and glacial acetic acid (7.3% v/v), pH 1.9, for 25 minutes at 1.0 kV.
  • the plate was air-dried, then subjected to the second dimension gel electrophoresis in a buffer of glacial acetic acid (5% v/v), pyridine (0.5% v/v), 0.5 mM EDTA, pH 3.5 for 16 minutes at 1.3 kV.
  • the plate was air-dried again, then sprayed with 0.25% ninhydrin in acetone and developed for 15 minutes at 65 °C. The position and identity of the labeled amino acids were determined by autoradiography.
  • BUBl kinase is a serine/threonine protein kinase.
  • EXAMPLE 10 Development of High Through-Put Screening Protocol for BUBl Protein Kinase Inhibitors Since BUB 1 RNA is overexpressed in many cancer cells, presumably BUB 1 kinase activity is upregulated in many cancer cells, causing a loss of the mitotic check point. Inhibition of BUBl kinase activity might be a way to inhibit uncontrolled cell division and therefore to block tumor cell growth. Overexpression of recombinant BUBl protein can be employed for in vitro kinase inhibitor screening.
  • Recombinant baculoviruses have become widely used as vectors to express heterologous genes in cultured insect cells.
  • Heterologous genes placed under the control of the strong polyhedrin promotor of the Autograhpa California nuclear polyhidrosis virus (AcNPV) are often abundantly expressed during the late stages of infection.
  • the recombinant proteins are processed, modified, and targeted to their appropriate cellular locations, where thy are functionally similar to their authentic counterparts.
  • the baculovirus expression system was purchased from Gibco BRL, Gaithersburg, MD.
  • BUB 1.HA wild type and BUB 1.KR.HA (kinase dead) were subcloned into pFastBacHtb (with the histidine tag at the N-terminal) and pFastBacG2T (the glutathione transferase gene replaced the histidine tag).
  • the recombinant plasmids were constructed in the regular E. coli host strain, XL2-blue.
  • the bacmids were then propagated in E. coli DHlOBac cells.
  • the recombinant bacmid DNA was isolated and transfected into insect cells. Recombinant baculovirus particles were then used to infect insect cells to overexpress recombinant proteins.
  • Protein extracts from overexpressed recombinant BUBl protein insect cells were used for BUBl in vitro kinase activity assays.
  • the protocol for the kinase assay was the same as described in Example 9. Results
  • Protein extracts from BUBl and BUB1.KR fused to HIS tags were isolated from insect cells, and the in vitro kinase activity assay was performed.
  • the radioactively labeled protein was immunoprecipitated by anti-HA antibody and then applied onto a 10% polyacrylamide SDS gel.
  • the 150 kDa BUBl protein was observed only in the sample containing recombinant wild-type BUB 1.
  • the recombinant BUB 1 can be autophosphorylated and can phosphorylate the exogenous substrate histone HI.
  • BUBl overexpressed in the baculovirus system provides a powerful tool for a high throughput screen.
  • Small chemical molecules are one example of compounds to be screened for their use as therapeutic drugs.
  • the invention is meant to also cover the final fonriulation formed by the combination of these excipients.
  • the invention includes formulations in which one to all of the added excipients undergo a reaction during formulation and are no longer present in the final formulation, or are present in modified forms.
  • EXAMPLE 11 Antibody characterization: Materials and Methods A rabbit polyclonal antibody was raised against a specific peptide sequence within the BUBl coding region.
  • the peptide (506A) consists of the following amino acid sequence corresponding to residues 385 to 498 of the BUBl amino acid sequence: DDKDEWQSLDQNED (SEQ ID NO: 19).
  • the IgG fraction was purified by affinity chromatography using protein A agarose beads by standard methodologies. The IgG fraction was dialyzed against PBS and adjusted to 1 mg/ml BSA and frozen in aliquots at -20 °C.
  • the antibodies detect a protein of approximately 150 kDa protein both by immunoprecipitation and immunobloting experiments. The signal was competed by including the immunizing peptide with the antibody. The BUBl antibody immunoprecipitated a kinase activity that could transphosphorylate histone HI. BUBl was also autophosphorylated in kinase assays of BUBl immunocomplexes.
  • EXAMPLE 12 Cell cycle regulation. Materials and Methods
  • HeLa cells were synchronized by a double thymidine/aphidicolin block at the Gl/S transition. The cells were released from the block and time points were taken as the cells proceed through the cell division cycle. Results
  • the amount of BUBl protein as well as the associated kinase activity is cell cycle regulated peaking in mitotis.
  • the activity appears to be maximal during metaphase corresponding to maximal activation of p34 cdc2 (CDK1). Consistent with this observation the activity is also induced by nocodozole treatment which arrest the cells in G2 and mitosis, but not by serum starvation or treatment with thymidine which induce a Gl arrest.
  • An antisense oligonucleotide was identified that specifically down regulates BUBl protein levels.
  • the sequence of this oligo is: GTGCCAAGCCAGCAGATAAAT (SEQ ID NO:24). This corresponds to nucleotides 2889 to 2909 of the hBUBl cDNA sequence. Results
  • This oligonucleotide specifically down regulates BUBl protein levels as determined by immunobloting with BUB 1 specific antisera. The level of ⁇ -tubulin remains unchanged.
  • Other mitotic proteins such as Auroral, Aurora2, cyclin A, cyclin B, and polo-like kinase 1 (PLK1) were up-regulated in cells treated with the BUBl antisense oligonucleotide as compared to control cells.
  • the chromosome appeared to randomly disperse as if the chromosomes were no longer attached to the mitotic spindle. This is consistent with BUBl protein being involved in micro tubule binding to the kinetocore of the chromosome.
  • the cell number of the BUB 1 antisense-treated cells decreased significantly two days post-treatment implying that loss of BUBl was lethal.
  • a TUNEL assay was performed on the cells treated with the BUBl antisense oligonucleotide. The TUNEL assay measures the amount of DNA fragmentation of a given cell population which is a hallmark of apoptotic cell death.
  • mice NIH3T3 cells were transfected with activated H-ras (valinel2) alone or in combination with wild-type BUBl (BUBl WT) or a N-terminal deletion mutant corresponding to amino acids 1-332 (BUB IN).
  • EXAMPLE 15 Isolation of cDNAs Encoding Mammalian NEK-Related Protein Kinases
  • the EST reports were downloaded from ncbi fwww.ncbi .nlm.nih. ov) .
  • the program 'report2est' was used to extract the following information: 1) EST names, 2) GenBank Accession numbers, 3) GenBank gi numbers, 4) Clone Id numbers, 5) the nucleotide sequences of the ESTs, 6) the organism, 7) the library name, 8) the name of the lab, and 9) the institution.
  • the output of 'report2est' is a file in FASTA format with all of the information listed above in the first line of each entry except the sequence, which is listed in the second line of each entry.
  • the resulting file was fomiatted for BLAST using 'pressdb' (available as part of the ncbi tool kit).
  • the program 'Makegene' was used to build a gene or part of a gene from EST's. Input to this program is a query sequence and the organism/species for which a gene is to be built.
  • An initial search of the formatted EST database described above was performed using BLAST (blastn). Any results that contained warnings, such as polyA tails or other repeat elements, were eliminated from future queries.
  • the program 'blast_parse_reports' was used to extract the FASTA header line from the search results and the output was then filtered to extract only FASTA header lines for the desired species.
  • the loop consists of the following steps: 1) when possible, the names of both ends of the EST's are extracted from the database by searching using the 'Clone Id' field or the part of the 'EST name' field before the .r or .s postscript, 2) any EST's that have been used as queries in previous loops are removed from the current query by the program 'subtract', 3) the resulting list of EST's is used to extract the sequences from the database by the program batch_parse_fasta, 4) BLAST is run against the database using each sequence, 5) the output files from BLAST containing warnings are removed, 6) the results are filtered by species, and 7) the loop is reentered if there were new EST's found in the last pass through the loop.
  • the EST's chosen by 'Makegene' were used as input for the program 'mpd2_cluster' (Hide, W., Burke, J, and Davison, D.U. of Houston, unpublished) which clusters overlapping sequences.
  • the programs 'contig' (Kerlavage, T., TIGR, unpublished), 'gde2mult', and 'gde2sing' [Smith, S.W., et al, CABIOS 10:671-675 (1994)], were used to make an alignment and consensus sequence of the overlapping EST's.
  • Plasmid DNA was purified using Qiagen columns, and was sequenced using cycle- sequencing energy transfer BIG DYE-terminator chemistry with Taq, FS DNA polymerase (ABI, Foster City, CA). Sequencing reactions were run on an ABI Prism 377 DNA sequencer, and were analyzed using the BLAST alignment algorithm (Altschul, S.F. et al. (1990) J. Mol. Biol. 215:403-410). Primer-walking sequencing oligonucleotides were synthesized on an Applied Biosystems 3948 DNA synthesizer using established phosphoramidate chemistry (ABI, Foster City, CA).
  • the human nek4a cDNA sequence is represented by the EST fragment AA214523, identified through a Smith-Waterman search of the EST database. The complete sequence of this clone was determined, and was used to generate the predicted full-length NEK4a amino acid sequence (SEQ ID. NO: 8)
  • the full-length human nek4a cDNA (SEQ ID NO:7) is 1,449 bp long, and consists of a 1,107 bp open reading frame (ORF) flanked by a 121 bp 5' UTR and a 200 bp 3' UTR
  • the human nek4b cDNA sequence is represented by the EST fragment AA147800, identified through a Smith-Waterman search of the EST database. The complete sequence of this clone was determined, and was used to generate the predicted full-length NEK4b amino acid sequence (SEQ ID. NO: 10).
  • the partial human nek4b cDNA (SEQ ID NO:9) is 1,346 bp long, and consists of a 804 bp ORF flanked by 499 bp 3' UTR that is followed by a 41 nucleotide polyadenylated region. No polyadenylation signal (AATAAA) was found preceding the polyadenylated region. Since the coding region is open throughout the 5' extent of this sequence, this appears to be a partial cDNA lacking the N-terminal start methionine.
  • AATAAA polyadenylation signal
  • the full-length human nek5 cDNA (SEQ ID NO:3) is 3,854 bp long.
  • NEK5 cDNA contains a 906 bp ORF flanked by a 31 bp 5' UTR (1-31) and a 2,894 bp 3' UTR (941-3,834) that is followed by a 20 nucleotide polyadenylated region.
  • a polyadenylation signal (AATAAA) is found at positions 3817-3822. The sequence flanking the first ATG conforms to the Kozak consensus for an initiating methionine, and is believed to be the translational start site for NEK5.
  • AA181165 contains a 3,854 bp insert and an ORF of 906 bp (302 amino acids).
  • cDNA Cloning and Characterization of NEK6 The EST fragment, AA030068, which represents the partial human NEK6 gene was identified using a Smith- Waterman search of the EST database. The complete sequence of the clone was determined, and was used to generate the partial human NEK6 sequence.
  • the partial murine nek ⁇ cDNA (SEQ ID NO:5) is 1,310 bp long. The murine
  • NEK6 cDNA contains a 907 bp ORF preceded by a 403 bp 5' UTR (1-403).
  • the sequence flanking the first ATG weakly conforms to the Kozak consensus for an initiating methionine
  • it is believed to be the translational start site for NEK6 for four reasons: (1) there is an adenine at the -3 position prior to the start codon, although no pyrimidine at the +4 position; (2) there are multiple in- frame and out-of-frame stop codons 5' of the putative start site; (3) there are no other possible start codons in any frame 5' of the putative strat codon; and (4) the sequence preceding the start site is 57.57% Gs and Cs, moderately characteristic of a 5'UTR region (highly GC- rich).
  • EST fragment AA030068
  • AA030068 contains a 1 ,310 bp insert and an ORF of 907 bp (302 amino acids).
  • EXAMPLE 16 Expression Analysis Of Mammalian Nek-Related Protein Kinases Materials And Methods Dna Array-Based Expression Analysis Plasmid DNA array blots were prepared by loading 0.5 ⁇ g denatured plasmid for each NEK-related kinase on a nylon membrane.
  • the [ ⁇ 32 P]dCTP-labeled single-stranded DNA probes were synthesized from total RNA isolated from several human and rodent immune sources, including primary thymus (B6Thy), mast (MC/9), and T cells (CD4+), as well as from the following cell lines: T (MOLT-4, CEM/Cl, Jurkat), B (2PK3, WEHI- 231, DAUDI, RAMOS, RAJI), TH2 (D10.G4.1, pL104, pL3), THl (pGLlO), CTL (L3), and basophil (RBL-2H3).
  • Hybridization was performed at 42 °C for 16 hours in 6X SSC, 0.1% SDS, IX Denhardt's solution, 100 ⁇ g/mL denatured herring sperm DNA with 10 6 cpm/mL of [ ⁇ 32 P]dCTP labeled single-stranded probe.
  • the filters were washed in 0.1 X SSC/0.1% SDS, 65 °C, and exposed for quantitative analysis on a Molecular Dynamics phosphorimager.
  • NEK4 EST GB:H59937, NEK5 EST GB:AA102912, and NEK6 EST GB:AA030253 The NEK4 probe used in this experiment should detect the NEK4a and NEK4b isoforms with equal efficiency).
  • NEK4 was broadly expressed in the entire immune cell panel with highest and lowest expression observed in mast cells and thymus, respectively.
  • NEK5 was broadly expressed in the entire immune cell panel with highest and lowest expression observed in THl cells and thymus, respectively.
  • NEK6 was broadly expressed in the entire immune cell panel with highest and lowest expression observed in THl and basophil cells, respectively.
  • NEK-subfamily kinases may be candidate targets for various immune disorders. Their broad expression pattern also suggests that they may mediate functions vital to the basic biology of most proliferating cells of the immune system.
  • EXAMPLE 17 nek Gene Expression Plasmid: Construction and Recombinant NEK Expression
  • Expression constructs can be generated for NEK kinases including, for example: a) pcDNA3.1 expression plasmids containing: full-length nek nucleotide base sequences with and without an HA tag; the nucleotide base sequence encoding the N-terminal domains (amino acid 1 to 42, 1-31, or 1-22) of NEK4a, Nek5, or NEK6, respectively, with and without an HA tag; the nucleotide base sequence encoding the catalytic domains (amino acid 43-294, 1-259,32-283, or 23-302) of NEK4a, NEK4b, NEK5, or NEK6, respectively, with and an without HA tag, and the C-terminal domains (amino acids 295- 313, 260-268, or 284-302) of NEK4a, NEK4b, or NEK5, respectively, with and without an HA tag; b) pGEX 4T-3
  • EXAMPLE 18 Recombinant Expression of NEK Protein Kinases Materials and Methods Transient Expression of NEK in Mammalian Cells
  • the pcDNA expression plasmids (10 ⁇ g DNA/100 mm plate) containing sequences encoding the full length or truncated form of a NEK kinase, are introduced into human 293 cells with lipofectamine (Gibco BRL). After 72 hours, the cells are harvested in 0.5 mL solubilization buffer (20 mM HEPES pH 7.4, 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1 mM EGTA, 2 mM phenylmethylsulfonyl fluoride, 1 ⁇ g/mL aprotinin).
  • NEK peptide is covalently coupled to 2 mL Immunolink (Pierce AminoLink Plus).
  • the link consists of an aldehyde support arm that forms a covalent bond to primary amines via a Schiff base intermediate.
  • the NEK peptide (ligand) is immobilized to AminoLink Plus coupling gel. The remaining active sites are blocked by adding the reducing agents. The column is then washed prior to use.
  • the purified anti-NEK IgG is eluted with 1 M propionic acid, is neutralized with 3 M Tris, pH 7.4 while eluting, and is buffered with 100 mM TEA buffered with 1 M Tris pH 6.8.
  • the purified antibody is dialyzed against phosphate buffered saline pH 7.4, and is stored in 20% glycerol and 0.03% NaN 3 in PBS.
  • Anti-NEK antisera is tested by Western analysis using GST-NEK fusion proteins.
  • An affinity purified anti-NEK antibody is tested by Western analysis using a GST- NEK fusion protein, or mammalian cell lysates from transiently transfected and expressed NEK protein.
  • HNTG buffer (20 mM HEPES, pH 7.4, 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1 mM EGTA, 2 mM phenylmethylsulfonylfluoride, 1 ⁇ g/mL aprotinin). The protein concentration is determined by BioRad assay (BioRad, Richmond, California). Protein from each sample (30 ⁇ g each) is tested by Western analysis with the purified anti-NEK antibody.
  • EXAMPLE 21 Cell Cycle Regulation of NEK Materials and Methods
  • Fr HeLa cells are synchronized by adding 0.5 M thymidine and 250 ⁇ g/mL aphidicolin. Cells are harvested at 0, 2, 4, 6, 8, 9, 10, 11, and 12 hours after thymidine and aphidicolin are removed. Other experiments are performed by blocking HeLa cells at different cell cycle stages by serum starvation, with thymidine (G1/S/G2), or with nocodozole (M). Total RNA is isolated from the synchronized cells and cell cycle blocked cells. Total RNA (10 ⁇ g) is loaded on the nylon membranes. The RNA blots are hybridized with a nek probe.
  • Northern blots containing RNA derived from HeLa cells either in an exponential growth state or arrested in various stages of the cell cycle are probed for expression of nek.
  • NEK protein The intracellular location of NEK protein is determined by immunoflourescence with NEK-specific antibody.
  • HeLa cells and H1299 cells are grown to confluence. Cells are trypsinized and are then plated on 1 cm poly-lysine pre-treated glass cover slips in 60 mm plates and are incubated at 37 °C for 1 day. Cells are washed with 5 mL ice-cold PBS after removing medium and then are fixed with -20 °C-cold 100% methanol. Methanol fixed cells can be used for a week or so.
  • Cells are blocked with 30 ⁇ L immunoflourescence blocking buffer (3% BSA, 0.01% Triton X100 in PBS) for 30 min and are washed with 0.01% Triton X100 in PBS.
  • the purified anti-NEK antibody is diluted approx. 1: 100-fold in the blocking buffer.
  • Rhodomine or fluorescein (FITC)-conjugated AffinPure F(ab)2 fragments of goat anti- rabbit IgG are diluted l:400-fold and are used as secondary antibody.
  • Dapi (62.5 ⁇ g/mL) is used for staining nuclei.
  • Immunofluorescence experiments to determine the intracellular location of endogenous NEK are performed using the affinity purified anti-NEK antibody in both HeLa and HI 299 cells, for example. Immunofluorescence experiments are also performed in pcDNA3.1.NEK transiently transfected 293T cells.
  • the pcDNA3.1.NEK.HA expression plasmids are transfected into human 293T cells.
  • the crude protein lysates are extracted from the transfected cells and are used for NEK kinase activity assays. Four different assays are performed: (1) NEK in vitro kinase autophosphorylation, (2) NEK phosphorylation of exogenous substrates, (3) NEK in vivo kinase activity, and (4) NEK phosphoamino acid analysis.
  • the pcDNA3.1 NEK expression plasmids (10 ⁇ g DNA/90 mm plate), containing HA-tagged wild-type (NEK.HA) or N-terminally truncated (NEK.N.HA), are introduced into human 293T cells with lipofectamine (Gibco BRL, Gaithersburg, MD). After 48 hours, the cells are harvested in 0.4 mL of kinase lysis buffer (50 mM HEPES, pH 7.5, 100 mM KCl, 25 mM NaF, 1 mM sodium orthovanadate, 0.5 mM nonidet P-40 (Sigma, St.
  • kinase lysis buffer 50 mM HEPES, pH 7.5, 100 mM KCl, 25 mM NaF, 1 mM sodium orthovanadate, 0.5 mM nonidet P-40 (Sigma, St.
  • Protein extracts are immunoprecipitated using anti-HA antibody (Babco, Berkeley, CA) at a final dilution of 1 :750 and protein G beads (Boehringer Mannheim, Indianapolis, IN), then are washed with kinase lysis buffer three times, followed by two washes with kinase buffer (20 mM HEPES, pH 7.5, 150 mM KCl, 10 mM MgCl 2 ).
  • kinase reaction mix (IX kinase buffer, 1 mM DTT, 0.1 mM ATP, 10 ⁇ Ci P-r-ATP) is added to the washed beads, and the mixture is incubated at 37 °C for 40 minutes with constant agitation. The reaction is stopped by adding 8 ⁇ L 5X PAGE buffer, and is boiled at 100 °C for 5 minutes.
  • Kinase assays using NEK.HA and NEK.N.HA (dominant/negative) in 293T transient cell lysates are performed. The radio-labeled proteins are immunoprecipitated by anti-HA antibody, and are applied to a 10% polyacrylamide SDS gel.
  • the pcDNA3.1.NEK expression plasmids (10 ⁇ g DNA 100 mM plate), containing HA-tagged full length or truncated NEK, are introduced into human 293T cells with lipofectamine (Gibco BRL). After 48 hours, the cell medium is changed to phosphate-free
  • the crude protein lysates are extracted with 450 ⁇ L of kinase lysis buffer (50 mM HEPES, pH 7.5, 100 mM KCl, 25 mM NaF, 1 mM sodium orthovanadate, 0.5 mM nonidet P-40 (Sigma), 1 mM DTT, 170 ⁇ g/mL PMSF, 25 ⁇ g/mL leupeptin, 20 ⁇ g/mL trypsin inhibitor, 25 ⁇ g/mL aprotinin), and are immunoprecipitated using anti-HA antibody.
  • kinase lysis buffer 50 mM HEPES, pH 7.5, 100 mM KCl, 25 mM NaF, 1 mM sodium orthovanadate, 0.5 mM nonidet P-40 (Sigma), 1 mM DTT, 170 ⁇ g/mL PMSF, 25 ⁇ g/mL leupeptin, 20 ⁇ g/mL trypsin inhibitor, 25 ⁇ g
  • the assay is performed by adding inorganic 32 P to 293T cells transiently transfected with NEK or NEK.N. Crude protein is extracted from the radio-labeled cells. Cell lysates are immunoprecipitated by anti-HA antibody, and are then applied to a 10% polyacrylamide SDS gel.
  • NEK Kinase Phosphoamino Acid Analysis is performed by adding inorganic 32 P to 293T cells transiently transfected with NEK or NEK.N. Crude protein is extracted from the radio-labeled cells. Cell lysates are immunoprecipitated by anti-HA antibody, and are then applied to a 10% polyacrylamide SDS gel.
  • the particulate fraction is collected by centrifugation and re-extracted with 50 mM ammonium bicarbonate, pH 7.3 to 7.6, 5% beta mercaptoethanol (v/v), 0.1% SDS (w/v), by incubation at 100 °C for 5 minutes followed by 37 °C for 60 minutes. Following centrifugation, the liquid extract is combined with the prior extract to give a total volume of 1.2 mL. The extract is centrifuged again to remove particulate material, and the supernatant is isolated and chilled to 4 °C.
  • a total of 20 ⁇ g of bovine serum albumin is added to the extract and the proteins are precipitated by addition of 250 ⁇ L of 100% trichloroacetic acid and incubated at 4 °C for 60 minutes.
  • the precipitated proteins are isolated by centrifugation, and the resulting pellets are washed with 500 ⁇ L of 4 °C 100% acetone.
  • the protein pellets are air dried, and then are dissolved in 100 ⁇ L of 5.7 M HCl. The solution is heated at 100 °C for 60 minutes in a screw-capped tube, and then is dried under vacuum.
  • the residual pellet is dissolved in formic acid (2% v/v) and glacial acetic acid (7.3% v/v) containing 67 ⁇ g/mL phosphoserine, 67 ⁇ g/mL phosphothreonine, 67 ⁇ g/mL phosphotyrosine, pH 1.9.
  • a sample containing a total of approx. 600 cpm is spotted onto a cellulose TLC plate (VWR scientific, San Francisco, CA). Two dimension chromatography is performed. The first dimension gel is run in a buffer of formic acid (2% v/v) and glacial acetic acid (7.3% v/v), pH 1.9, for 25 minutes at 1.0 kV.
  • the plate is air-dried, then subjected to the second dimension gel electrophoresis in a buffer of glacial acetic acid (5% v/v), pyridine (0.5% v/v), 0.5 mM EDTA, pH 3.5 for 16 minutes at 1.3 kV.
  • the plate is air-dried again, then sprayed with 0.25% ninhydrin in acetone and is developed for 15 minutes at 65 °C.
  • the position and identity of the labeled amino acids are determined by autoradiography.
  • the Hunter's protocol is performed to determine the phosphorylation sites (amino acid) for the NEK protein kinase.
  • NEK is involved in cell-cycle regulation, deregulation of these kinases through overexpression and/or mutational events plays a role in cancer, for example.
  • NEK kinase activity will inhibit uncontrolled cell division and thereby block tumor cell growth.
  • Overexpression of recombinant NEK protein can be employed for in vitro kinase inhibitor screening.
  • Recombinant baculoviruses have become widely used as vectors to express heterologous genes in cultured insect cells. Heterologous genes placed under the control of the strong polyhedrin promotor of the Autograhpa California nuclear polyhidrosis virus (AcNPV) are often abundantly expressed during the late stages of infection. In most cases, the recombinant proteins are processed, modified, and targeted to their appropriate cellular locations, where they are functionally similar to their authentic counterparts. Expression of Recombinant NEK in a Baculovirus System
  • the baculovirus expression system is purchased from Gibco BRL, Gaithersburg, MD.
  • NEK.HA wild type and NEK.KR.HA (a kinase dead version) are subcloned into pFastBacHtb (with the histidine tag at the N-terminal) and pFastBacG2T (the glutathione transferase gene replaces the histidine tag).
  • the recombinant plasmids are constructed in the regular E. coli host strain, XL2-blue.
  • the bacmids are then propagated in E. coli DHlOBac cells.
  • the recombinant bacmid DNA is isolated and transfected into insect cells. Recombinant baculovirus particles are then used to infect insect cells to overexpress recombinant proteins.
  • Kinase dead constructs can be made by methods well-known in the art, including point mutations, insertion and deletion of nucleic acids such that kinase activity is lost.
  • insect cells 72 hours after baculovirus infection, insect cells are harvested and crude protein lysates are extracted. Expression of recombinant NEK proteins is determined by Western analysis using anti-HA antibody.
  • Protein extracts from overexpressed recombinant NEK protein insect cells were used for NEK in vitro kinase activity assays.
  • the protocol for the kinase assay was the same as described in Example 9.
  • Protein extracts from NEK and NEK.KR fused to HIS tags are isolated from insect cells, and the in vitro kinase activity assay is performed.
  • the radioactively labeled protein is immunoprecipitated by anti-HA antibody and then is applied onto a 10% polyacrylamide SDS gel.
  • NEK overexpressed in the baculovirus system provides a powerful tool for a high throughput screen.
  • Small chemical molecules are one example of substances to be screened for their use as therapeutic drugs.
  • Antisense oligonucleotides are useful for inhibition of NEK expression in normal and tumor cells in order to profile the potential effects of small molecule NEK inhibitors. They can also be used to inhibit NEK expression in human tumor cell xenografts in nude mice to determine the antitumor effects of NEK inhibitors. Another use is as a "drug" to inhibit NEK expression in various human tumors that are characterized by overexpression, or aberrant expression, of a nek gene. Material and Methods: Antisense oligonucleotides to be tested for their inhibition of nek expression can be generated by methods well-known in the art. Typically, the oligonucleotides are synthesized as phosphorothioates, and are tested in the following manner.
  • Human H1299 cells are seeded at -40-50% confluency in a 6 well plate (Falcon).
  • lipofectin (Gibco) and oligo(s) are mixed with OptiMEM (Gibco) such that the final concentration of lipofectin is 100 ⁇ g/mL and the final concentration of each oligo is 1 ⁇ M in a volume of 200 ⁇ L.
  • the lipofectin/oligo/OptiMEM mixture is incubated at room temperature (20-25 °C) for 15 minutes, 800 ⁇ L of OptiMEM is added to the lipofectin/oligo/OptiMEM mixture, and is mixed gently.
  • the growth medium is removed from the HI 299 cells, which are at -80% confluency. Cells are washed once with
  • the OptiMEM is aspirated, the lipofectin oligo/OptiMEM mixture is added, and cultures are incubated at 37 °C for 4 hours.
  • the lipofectin/oligo/OptiMEM mixture is removed and is replaced with normal growth medium containing the antisense oligo(s) at a concentration of 200 nM.
  • the plates are returned to the 37 °C incubator for 16-20 hours after which time the growth medium is removed from the cells, and they are washed once with OptiMEM.
  • the OptiMEM is again aspirated, and the lipofectin/oligo/OptiMEM mixture is added (prepared as described above), and the plates again are incubated at 37 °C for 4 hours.
  • the lipofectin oligo/OptiMEM mixture is removed again, and is replaced with normal growth medium containing the antisense oligo(s) at a concentration of 200 nM.
  • the plates are returned to the 37 °C incubator for 16-20 hours.
  • the cells are harvested (-40 hours after initial treatment), and are analyzed for nek mRNA by Northern blot or NEK protein expression by immunoblot.
  • the invention is meant to also cover the final formulation formed by the combination of these excipients.
  • the invention includes formulations in which one to all of the added excipients undergo a reaction during formulation and are no longer present in the final formulation, or are present in modified forms.

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US6183962B1 (en) * 1998-09-09 2001-02-06 Millennium Pharmaceuticals, Inc. Protein kinase molecules and uses therefor
US20020151005A1 (en) * 2000-06-15 2002-10-17 Meyers Rachel A. 53070, a novel human protein kinase family member and uses thereof
DE10011530A1 (de) * 2000-03-13 2001-09-27 Robert Elez Hochwirksame Antisense-Oligodesoxynucleotide gegen Polio-like Kinasel
AU2001273350A1 (en) * 2000-07-11 2002-01-21 Deltagen, Inc. Transgenic mice containing targeted kinase gene disruptions
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US7033790B2 (en) 2001-04-03 2006-04-25 Curagen Corporation Proteins and nucleic acids encoding same
US20040097441A1 (en) * 2002-11-16 2004-05-20 Isis Pharmaceuticals Inc. Modulation of NIMA-related kinase 6 expression
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