EP0981642A1 - Kinase inhibitrice de nf-kappa b kappa b, sous-unites de la kinase kappa b et procedes d'utilisation - Google Patents

Kinase inhibitrice de nf-kappa b kappa b, sous-unites de la kinase kappa b et procedes d'utilisation

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Publication number
EP0981642A1
EP0981642A1 EP98908673A EP98908673A EP0981642A1 EP 0981642 A1 EP0981642 A1 EP 0981642A1 EP 98908673 A EP98908673 A EP 98908673A EP 98908673 A EP98908673 A EP 98908673A EP 0981642 A1 EP0981642 A1 EP 0981642A1
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EP
European Patent Office
Prior art keywords
ikk
subunit
seq
protein
activity
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
EP98908673A
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German (de)
English (en)
Other versions
EP0981642A4 (fr
Inventor
Michael Karin
Joseph A. Didonato
David M. Rothwarf
Makio Hayakawa
Ebrahim Zandi
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University of California
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University of California
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Priority claimed from US08/810,131 external-priority patent/US6268194B1/en
Application filed by University of California filed Critical University of California
Publication of EP0981642A1 publication Critical patent/EP0981642A1/fr
Publication of EP0981642A4 publication Critical patent/EP0981642A4/fr
Ceased legal-status Critical Current

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6842—Proteomic analysis of subsets of protein mixtures with reduced complexity, e.g. membrane proteins, phosphoproteins, organelle proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A61P11/06—Antiasthmatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/11—Protein-serine/threonine kinases (2.7.11)
    • C12Y207/1101—IkappaB kinase (2.7.11.10)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/91—Transferases (2.)
    • G01N2333/912—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • G01N2333/91205—Phosphotransferases in general
    • G01N2333/9121—Phosphotransferases in general with an alcohol group as acceptor (2.7.1), e.g. general tyrosine, serine or threonine kinases

Definitions

  • the present invention relates generally to molecular biology and biochemistry and more specifically to a protein kinase, I ⁇ B kinase, which is activated in response to environmental stresses and proinflammatory signals to phosphorylate inhibitors of the NF- ⁇ B transcription factors and to methods of using the protein kinase.
  • the induction of gene expression due to exposure of a cell to a specific stimulus is a tightly controlled process. Depending on the inducing stimulus, it can be critical to survival of the cell that one or more genes be rapidly induced, such that the expressed gene product can mediate its effect.
  • an inflammatory response stimulated due to an injury to or infection of a tissue results in rapid vasodilation in the area of the injury and infiltration of effector cells such as macrophages . Vasodilation occurs within minutes of the response and is due, in part, to the expression of cytokines in the injured region.
  • the rapid induction for example, of an inflammatory response or an immune response, requires that the transcription factors involved in regulating such responses be present in the cell in a form that is amenable to rapid activation. Thus, upon exposure to an inducing stimulus, the response can occur quickly. If, on the other hand, such transcription factors were not already present in a cell in an inactive state, the factors first would have to be synthesized upon exposure to an inducing stimulus, greatly reducing the speed with which a response such as an inflammatory response could occur.
  • a transcription factor that exists in an inactive state in a cell can be activated by a post- translational modification such as phosphorylation on one or more serine, threonine or tyrosine residues.
  • a transcription factor can be inactive due to an association with a regulatory factor, which, upon exposure to an inducing stimulus, is released from the transcription factor, thereby activating the transcription factor.
  • an inactive transcription factor may have to associate with a second protein in order to have transcriptional activity.
  • the inducing stimulus interacts directly with the inactive transcription factor, rendering it active and resulting in the induction of gene expression. More often, however, an inducing stimulus initiates the induced response by interacting with a specific receptor present on the cell membrane or by entering the cell and interacting with an intracellular protein. Furthermore, the signal generally is transmitted along a pathway, for example, from the cell membrane to the nucleus, due to a series of interactions of proteins. Such signal transduction pathways allow for the rapid transmission of an extracellular inducing stimulus such that the appropriate gene expression is rapidly induced.
  • a clearer understanding of the proteins involved in such pathways can allow a description, for example, of the mechanism of action of a drug that is known to interfere with the expression of genes regulated by a particular pathway, but the target of which is not known.
  • the understanding of such pathways can allow the identification of a defect in the pathway that is associated with a disease such as cancer.
  • the altered expression of cell adhesion molecules is associated with the ability of a cancer cell to metastasize.
  • the critical proteins involved in the signal transduction pathway leading to expression of cell adhesion molecules have not been identified.
  • the present invention satisfies this need and provides related advantages as well .
  • the present invention provides isolated nucleic acid molecules encoding full length human serine protein kinases, designated I ⁇ B kinase (IKK) subunits IKK ⁇ and IKK ⁇ .
  • IKK I ⁇ B kinase
  • the disclosed IKK subunits share substantial sequence homology and are activated in response to proinflammatory signals to phosphorylate proteins (I B'S) that inhibit the activity of the NF- ⁇ B transcription factor.
  • the invention provides a nucleic acid molecule having the nucleotide sequence shown as SEQ ID NO: 1, which encodes a cytokine inducible I ⁇ B kinase subunit designated IKK ⁇ , particularly the sequence shown as nucleotides -35 to 92 in SEQ ID NO : 1, and nucleic acid molecules encoding the amino acid sequence shown as SEQ ID NO: 2, as well as nucleotide sequences complementary thereto.
  • the invention provides a nucleic acid molecule having the nucleotide sequence shown as SEQ ID NO: 14, which encodes a second cytokine inducible I ⁇ B kinase subunit, designated IKK ⁇ , and nucleic acid molecules encoding the amino acid sequence shown as SEQ ID NO: 15, as well nucleotide sequences complementary thereto.
  • the invention also provides vectors comprising the nucleic acid molecules of the invention and host cells containing such vectors.
  • the invention provides nucleotide sequences that bind to a nucleic acid molecule of the invention, including to nucleotides -35 to 92 as shown in SEQ ID NO: 1.
  • Such nucleotide sequences of the invention are useful as probes, which can be used to identify the presence of a nucleic acid molecule encoding an IKK subunit in a sample, and as antisense molecules, which can be used to inhibit the expression of a nucleic acid molecule encoding an IKK subunit.
  • the present invention also provides isolated full length human IKK subunits, which can phosphorylate an I ⁇ B protein.
  • the invention provides an IKK ⁇ polypeptide having the amino acid sequence shown as SEQ ID NO: 2, particularly the amino acid sequence comprising amino acids 1 to 31 at the N-terminus of the polypeptide of SEQ ID NO: 2.
  • the invention provides an IKK ⁇ polypeptide having the amino acid sequence shown as SEQ ID NO: 15.
  • the invention also provides peptide portions of an IKK subunit, including, for example, peptide portions comprising one or more contiguous amino acids of the N-terminal amino acids shown as residues 1 to 31 in SEQ ID NO: 2.
  • a peptide portion of an IKK subunit can comprise the kinase domain of the IKK subunit or can comprise a peptide useful for eliciting production of an antibody that specifically binds to an I ⁇ B kinase or to the IKK subunit.
  • the invention also provides anti-IKK antibodies that specifically bind to an IKK complex comprising an IKK subunit, particularly to the IKK subunit, for example, to an epitope comprising at least one of the amino acids shown as residues 1 to 31 of SEQ ID NO: 2, and also provides IKK subunit-binding fragments of such antibodies.
  • the invention provides cell lines producing anti-IKK antibodies or IKK-binding fragments thereof .
  • an IKK complex can have an apparent molecular mass of about 900 kDa or about 300 kDa.
  • An IKK complex is characterized, in part, in that it comprises an IKK ⁇ subunit, an IKK ⁇ subunit, or both and can phosphorylate an I ⁇ B protein.
  • the present invention further provides methods for isolating an IKK complex or an IKK subunit, as well as methods of identifying an agent that can alter the association of an IKK complex or an IKK subunit with a second protein that associates with the IKK in vi tro or in vivo .
  • Such a second protein can be, for example, another IKK subunit; an I ⁇ B protein, which is a substrate for IKK activity and is involved in a signal transduction pathway that results in the regulated expression of a gene; a protein that is upstream of the I ⁇ B kinase in a signal transduction pathway and regulates IKK activity; or a protein that acts as a regulatory subunit of the I ⁇ B kinase or of an IKK subunit and is necessary for full activation of the IKK complex.
  • An agent that alters the association of an IKK subunit with a second protein can be, for example, a peptide, a polypeptide, a peptidomimetic or a small organic molecule. Such agents can be useful for modulating the level of phosphorylation of I ⁇ B in a cell, thereby modulating the activity of NF- ⁇ B in the cell and the expression of a gene regulated by NF-KB.
  • the invention also provides methods of identifying proteins that can interact with an I ⁇ B kinase, including with an IKK subunit, such proteins which can be a downstream effector of the IKK such as a member of the I ⁇ B family of proteins or an upstream activator or a regulatory subunit of an IKK.
  • proteins that interact with an IKK complex or the IKK subunit can be isolated, for example, by coprecipitation with the IKK or by using the IKK subunit as a ligand, and can be involved, for example, in tissue specific regulation of NF- ⁇ B activation and consequent tissue specific gene expression.
  • Figure 1 shows a nucleotide sequence (SEQ ID NO: 1; lower case letter) and deduced amino acid sequence (SEQ ID NO: 2; upper case letters) of full length human IKK ⁇ subunit of an IKK complex. Nucleotide positions are indicated to the right and left of the sequence; the "A” of the ATG encoding the initiator methionine is shown as position 1. Underlined amino acid residues indicate the peptide portions of the protein (“peptide 1" and "peptide 2”) that were sequenced and used to design oligonucleotide probes. The asterisk indicates the sequence encoding the STOP codon.
  • Figure 2 shows a nucleotide sequence (SEQ ID NO: 1
  • Figure 3 shows an alignment of the deduced amino acid sequences of IKK ⁇ (" ⁇ ", SEQ ID NO: 2) and IKK ⁇ (" ⁇ " , SEQ ID NO: 15). Numbers to the right of the sequences indicate the respective amino acid positions. Underlined amino acid residues indicate peptide portions of the IKK ⁇ subunit that were sequenced and used to search an EST database (see Example III) . Vertical bars between amino acid residues indicate identical amino acids; two dots between amino acid residues indicates very similar amino acids (e.g., Glu and Asp; Arg and Lys) and one dot between amino acid residues indicates a lesser degree of similarity. A dot within an amino acid sequence indicates a space introduced to maintain sequence homology.
  • the kinase domains in the N-terminal half of the sequences and helix-loop-helix domains in the C-terminal half of the sequences are bracketed and the leucine residues involved in the leucine zippers are indicated by the filled circles above the IKK ⁇ sequence.
  • the present invention provides isolated nucleic acid molecules encoding polypeptide subunits of human serine protein kinase complex, the I ⁇ B kinase (IKK) , which is activated in response to proinflammatory signals and phosphorylates proteins (IKB'S) that bind to and inhibit the activity of NF- ⁇ B transcription factors.
  • IKK I ⁇ B kinase
  • the invention provides an isolated nucleic acid molecule (SEQ ID NO: 1) encoding a full length human IKK ⁇ subunit having the amino acid sequence shown as SEQ ID NO: 1
  • the invention provides an isolated nucleic acid molecule (SEQ ID NO: 14; Figure 2) encoding a full length human IKK ⁇ subunit having the amino acid sequence shown as SEQ ID NO: 15 ( Figure 3) .
  • isolated when used in reference to a nucleic acid molecule of the invention, means that the nucleic acid molecule is relatively free from contaminating lipids, proteins, nucleic acids or other cellular material normally associated with a nucleic acid molecule in a cell.
  • An isolated nucleic acid molecule of the invention can be obtained, for example, by chemical synthesis of the nucleotide sequence shown as SEQ ID NO : 1 or SEQ ID NO: 14 or by cloning the molecule using methods such as those disclosed in Examples II and III.
  • an isolated nucleic acid molecule comprises at least about 30% of a sample containing the nucleic acid molecule, and generally comprises about 50% or 70% or 90% of a sample, preferably 95% or 98% of the sample.
  • Such an isolated nucleic acid molecule can be identified by comparing, for example, a sample containing the isolated nucleic acid molecule with the material from which the sample originally was obtained.
  • an isolated nucleic acid molecule can be identified, for example, by comparing the relative amount of the nucleic acid molecule in fraction of a cell lysate obtained following gel electrophoresis with the relative amount of the nucleic acid molecule in the cell, itself.
  • IKK ⁇ and IKK ⁇ have been designated IKK subunits because they are components of an approximately 900 kDa complex having I ⁇ B kinase (IKK) activity and because they share substantial nucleotide and amino acid sequence homology.
  • IKK ⁇ and IKK ⁇ are related members of a family of IKK catalytic subunits (see Figure 3) .
  • the 900 kDa I ⁇ B kinase complex can be isolated in a single step, for example, by immunoprecipitation using an antibody specific for an IKK subunit or by using metal ion chelation chromatography methods (see Example IV) .
  • a 300 kDa IKK complex also can be isolated as disclosed herein and has kinase activity for an I ⁇ B substrate (see Example III) .
  • Nucleic acid molecules related to SEQ ID NO: 1 previously have been described (Connelly and Marcu, Cell . Mol. Biol. Res. 41:537-549 (1995), which is incorporated herein by reference) .
  • Connelly and Marcu describe a 3466 base pair (bp) nucleic acid molecule (GenBank Accession #U12473; Locus MMU 12473), which is incorporated herein by reference) , which encodes a full length mouse polypeptide having an apparent molecular mass of 85 kiloDaltons (kDa) and designated CHUK.
  • a 2146 bp nucleic acid molecule (GenBank Accession #U22512; Locus HSU 22512) , which is incorporated herein by reference) , which encodes a portion of the polypeptide shown in SEQ ID NO: 2 also was described. However, the amino acid sequence deduced from #U22512 lacks amino acids 1 to 31 as shown in SEQ ID NO : 2 and, therefore, is not a full length protein.
  • nucleotide differences occur in SEQ ID NO: 1 as compared to the sequence of #U22512, including nucleotide changes that encode different amino acids at positions 543, 604, 679, 680, 684 and 685 of SEQ ID NO: 2; silent nucleotide changes also occur at codons 665 and 678.
  • the polypeptides encoded by the nucleotide sequences of GenBank Accession #U12473 and #U22512 share about 95% identity at the amino acid level and are substantially similar to that shown in SEQ ID NO: 2. No function has been demonstrated for the polypeptides described by Connelly and Marcu, although Regnier et al . (Cell 90:373-383 (1997)) recently have confirmed that human CHUK corresponds to IKK ⁇ , as disclosed herein.
  • a nucleic acid molecule of the invention is exemplified by the nucleotide sequences shown as SEQ ID NO: 1, which encodes a full length human IKK ⁇ (SEQ ID NO: 2; Figure 1), the activity of which is stimulated by a cytokine or other proinflammatory signal, and as SEQ ID NO: 1, which encodes a full length human IKK ⁇ (SEQ ID NO: 2; Figure 1), the activity of which is stimulated by a cytokine or other proinflammatory signal, and as SEQ ID NO: 1, which encodes a full length human IKK ⁇ (SEQ ID NO: 2; Figure 1), the activity of which is stimulated by a cytokine or other proinflammatory signal, and as SEQ ID NO: 1, which encodes a full length human IKK ⁇ (SEQ ID NO: 2; Figure 1), the activity of which is stimulated by a cytokine or other proinflammatory signal, and as SEQ ID NO: 1, which encodes a full length human IKK ⁇ (SEQ ID NO: 2; Figure 1)
  • nucleic acid NO: 14 which encodes a full length IKK ⁇ (SEQ ID NO: 15) . Due to the degeneracy of the genetic code and in view of the disclosed amino acid sequence of a full length human IKK ⁇ (SEQ ID NO: 2) and of the IKK ⁇ (SEQ ID NO: 15), additional nucleic acid molecules of the invention would be well known to those skilled in the art. Such nucleic acid molecules, respectively, have a nucleotide sequence that is different from SEQ ID NO: 1 but, nevertheless, encodes the amino acid sequence shown as SEQ ID NO: 2, or have a nucleotide sequence that is different from SEQ ID NO:
  • nucleic acid molecule comprising a nucleotide sequence encoding the amino acid sequence of a full length human IKK ⁇ as shown in SEQ ID NO: 2 or of IKK ⁇ as shown in SEQ ID NO: 15.
  • reference to "a nucleic acid molecule encoding an IKK subunit" indicates 1) the polynucleotide sequence of one strand of a double stranded DNA molecule comprising the nucleotide sequence that codes for the IKK subunit and can be transcribed into an RNA that encodes the IKK subunit, or 2) an RNA molecule, which can be translated into an IKK subunit.
  • a double stranded DNA molecule also comprises a second polynucleotide strand that is complementary to the coding strand and that the disclosure of a polynucleotide sequence comprising a coding sequence necessarily discloses the complementary polynucleotide sequence.
  • the invention provides polynucleotide sequences, including, for example, polydeoxyribonucleotide or polyribonucleotide sequences that are complementary to the nucleotide sequence shown as SEQ ID NO: 1 or as SEQ ID NO: 14, or to a nucleic acid molecule encoding an IKK catalytic subunit having the amino acid sequence shown as SEQ ID NO : 2 or as SEQ ID NO: 15, respectively.
  • polynucleotide is used in its broadest sense to mean two or more nucleotides or nucleotide analogs linked by a covalent bond.
  • oligonucleotide also is used herein to mean two or more nucleotides or nucleotide analogs linked by a covalent bond, although those in the art will recognize that oligonucleotides generally are less than about fifty nucleotides in length and, therefore, are a subset within the broader meaning of the term "polynucleotide.”
  • nucleotides comprising a polynucleotide are naturally occurring deoxyribonucleotides, such as adenine, cytosine, guanine or thymine linked to 2 ' -deoxyribose, or ribonucleotides such as adenine, cytosine, guanine or uracil linked to ribose .
  • a polynucleotide also can comprise nucleotide analogs, including non-naturally occurring synthetic nucleotides or modified naturally occurring nucleotides.
  • nucleotide analogs are well known in the art and commercially available, as are polynucleotides containing such nucleotide analogs (Lin et al., Nucl. Acids Res. 22:5220-5234 (1994); Jellinek et al., Biochemistry 34:11363-11372 (1995); Pagratis et al . , Nature Biotechnol . 15:68-73 (1997)).
  • the covalent bond linking the nucleotides of a polynucleotide generally is a phosphodiester bond.
  • the covalent bond also can be any of numerous other bonds, including a thiodiester bond, a phosphorothioate bond, a peptide-like bond or any other bond known to those in the art as useful for linking nucleotides to produce synthetic polynucleotides (see, for example, Tarn et al . , Nucl . Acids Res. 22:977-986 (1994); Ecker and Crooke, BioTechnolo ⁇ y 13:351360 (1995)).
  • bonds including a thiodiester bond, a phosphorothioate bond, a peptide-like bond or any other bond known to those in the art as useful for linking nucleotides to produce synthetic polynucleotides (see, for example, Tarn et al . , Nucl . Acids Res. 22:977-986 (1994); Ecker and Crooke, BioTechnolo ⁇ y 13:351360 (1995)).
  • nucleotide of the invention where it is desired to synthesize a polynucleotide of the invention, the artisan will know that the selection of particular nucleotides or nucleotide analogs and the covalent bond used to link the nucleotides will depend, in part, on the purpose for which the polynucleotide is prepared. For example, where a polynucleotide will be exposed to an environment containing substantial nuclease activity, the artisan will select nucleotide analogs or covalent bonds that are relatively resistant to the nucleases.
  • a polynucleotide comprising naturally occurring nucleotides and phosphodiester bonds can be chemically synthesized or can be produced using recombinant DNA methods, using an appropriate polynucleotide as a template.
  • a polynucleotide comprising nucleotide analogs or covalent bonds other than phosphodiester bonds generally will be chemically synthesized, although an enzyme such as T7 polymerase can incorporate certain types of nucleotide analogs and, therefore, can be used to produce such a polynucleotide recombinantly from an appropriate template (Jellinek et al . , supra, 1995).
  • the invention also provides nucleotide sequences that can specifically hybridize to a nucleic acid molecule of the invention. Such hybridizing nucleotide sequences are useful, for example, as probes, which can hybridize to a nucleic acid molecule encoding an IKK catalytic subunit and allow the identification of the nucleic acid molecule in a sample.
  • a nucleotide sequence of the invention is characterized, in part, in that it is at least nine nucleotides in length, such sequences being particularly useful as primers for the polymerase chain reaction (PCR) , and can be at least fourteen nucleotides in length or, if desired, at least seventeen nucleotides in length, such nucleotide sequences being particularly useful as hybridization probes, although such sequences also can be used for PCR.
  • PCR polymerase chain reaction
  • a nucleotide sequence of the invention can comprise at least six nucleotides 5' to nucleotide position 92 as shown in SEQ ID NO: 1 ( Figure 1), preferably at least nine nucleotides 5' to position 92, or more as desired, where SEQ ID NO : 1 is shown in the conventional manner from the 5 '-terminus ( Figure 1; upper left) to the 3' -terminus.
  • Such nucleotide sequences of the invention are particularly useful in methods of diagnosing a pathology, for example, a human disease, characterized by aberrant IKK activity.
  • such nucleotide sequences can comprise a kit, which can be made commercially available and can provide a standardized diagnostic assay.
  • a nucleic acid molecule encoding an IKK ⁇ such as the nucleotide sequence shown in SEQ ID NO : 1 diverges from the sequence encoding the mouse homolog (GenBank Accession #U12473) in the region encoding amino acid 30.
  • a nucleotide sequence comprising nucleotides 88 to 90 as shown in SEQ ID NO: 1, which encodes amino acid 30 of human IKK ⁇ can be particularly useful, for example, for identifying the presence of a nucleic acid molecule encoding a human IKK ⁇ in a sample.
  • nucleotide sequences that can hybridize with a nucleic acid molecule encoding a human IKK ⁇ or a human IKK ⁇ or both by designing the sequence to contain conserved or non-conserved nucleotide sequences, as desired. For example, selection of a nucleotide sequence that is highly conserved among SEQ ID NO: 1 and SEQ ID NO: 14 can allow the identification of related members of the IKK subunit family of proteins.
  • nucleotide sequence that is present for example, in SEQ ID NO: 14, but that is not present in SEQ ID NO : 1 or that shares only minimal homology can allow identification of the expression of SEQ ID NO: 14 in a cell, irrespective of whether SEQ ID NO: 1 also is expressed in the cell. It should be recognized, however, that a nucleotide sequence of the invention readily is identifiable in comparison to GenBank Accession #U12473 or #U22512 in that a nucleotide sequence of the invention is not the nucleotide sequence of GenBank Accession #U12473 or #U22512.
  • a nucleotide sequence of the invention can comprise a portion of a coding sequence of a nucleic acid molecule encoding an IKK subunit or of a sequence complementary thereto, depending on the purpose for which the nucleotide sequence is to be used.
  • a mixture of a coding sequence and its complementary sequence can be prepared and, if desired, can be allowed to anneal to produce double stranded molecules .
  • the invention also provides antisense nucleic acid molecules, which are complementary to a nucleic acid molecule encoding an IKK subunit and can bind to and inhibit the expression of the nucleic acid molecule.
  • expression of an antisense molecule complementary to the nucleotide sequence shown in SEQ ID NO: 1 inhibited the cytokine inducible expression of an NF-KB dependent reporter gene in a cell (Example II.B.).
  • an antisense molecule of the invention can be useful for decreasing IKK activity in a cell, thereby reducing or inhibiting the level of NF- ⁇ B mediated gene expression.
  • Antisense nucleic acid molecules specific for IKK ⁇ or for IKK ⁇ or for both can be designed based on the criteria discussed above for the selection of hybridizing nucleotide sequences.
  • An antisense nucleic acid molecule of the invention can comprise a sequence complementary to the entire coding sequence of an IKK catalytic subunit such as a sequence complementary to SEQ ID NO : 1 or SEQ ID NO : 14, provided the antisense sequence is not complementary in its entirety to the sequences of GenBank Accession #U12473 or #U22512.
  • nucleotide sequence complementary to a portion of a nucleic acid molecule encoding an IKK subunit can be useful as an antisense molecule, particularly a nucleotide sequence complementary to nucleotides -35 to 92 of SEQ ID NO : 1 or, for example, a nucleotide sequence comprising at least 9 nucleotides on each side of the ATG encoding the initiator methionine (complementary to positions -9 to 12 of SEQ ID NO: 1) or, if desired, at least 17 nucleotides on each side of the ATG codon (complementary to positions -17 to 20 of SEQ ID NO: 1) , or to the corresponding sequences of SEQ ID NO: 14.
  • Antisense methods involve introducing the nucleic acid molecule, which is complementary to and can hybridize to the target nucleic acid molecule, into a cell.
  • An antisense nucleic acid molecule can be a chemically synthesized polynucleotide, which can be introduced into the target cells by methods of transfection, or can be expressed from a plasmid or viral vector, which can be introduced into the cell and stably or transiently expressed using well known methods (see, for example, Sambrook et al . , Molecular Cloning: A laboratory manual (Cold Spring Harbor Laboratory Press 1989); Ausubel et al . , Current Protocols in Molecular Biology (Green Publ . , NY 1989), each of which is incorporated herein by reference) .
  • an antisense (or other hybridizing) nucleotide sequence to specifically hybridize to the target nucleic acid sequence depends, for example, on the degree of complementarity shared between the sequences, the GC content of the hybridizing molecules, and the length of the antisense nucleic acid sequence, which can be at least ten nucleotides in length, generally at least thirty nucleotides in length or at least fifty nucleotides in length, and can be up to the full length of a nucleotide sequence of SEQ ID NO : 1 or SEQ ID NO: 14 or a nucleotide sequence encoding an IKK subunit as shown in SEQ ID NO: 2 or in SEQ ID NO: 15 (see Sambrook et al., supra , 1989) .
  • the invention also provides vectors comprising a nucleic acid molecule of the invention and host cells, which are appropriate for maintaining such vectors .
  • Vectors which can be cloning vectors or expression vectors, are well known in the art and commercially available.
  • An expression vector comprising a nucleic acid molecule of the invention, which can encode an IKK- ⁇ or can be an antisense molecule, can be used to express the nucleic acid molecule in a cell.
  • an expression vector contains the expression elements necessary to achieve, for example, sustained transcription of the nucleic acid molecule, although such elements also can be inherent to the nucleic acid molecule cloned into the vector.
  • an expression vector contains or encodes a promoter sequence, which can provide constitutive or, if desired, inducible expression of a cloned nucleic acid sequence, a poly-A recognition sequence, and a ribosome recognition site, and can contain other regulatory elements such as an enhancer, which can be tissue specific.
  • the vector also contains elements required for replication in a procaryotic or eukaryotic host system or both, as desired.
  • Such vectors which include plasmid vectors and viral vectors such as bacteriophage, baculovirus, retrovirus, lentivirus, adenovirus, vaccinia virus, semliki forest virus and adeno-associated virus vectors, are well known and can be purchased from a commercial source (Promega, Madison WI ; Stratagene, La Jolla CA; GIBCO/BRL, Gaithersburg MD) or can be constructed by one skilled in the art (see, for example, Meth. Enzymol.. Vol. 185, D.V. Goeddel, ed. (Academic Press, Inc., 1990); Jolly, Cane. Gene Ther. 1:51-64 (1994); Flotte, J. Bioenerg. Biomemb. 25:37-42 (1993); Kirshenbaum et al . , J. Clin. Invest 92:381-387 (1993), which is incorporated herein by reference) .
  • viral vectors such as bacterioph
  • a nucleic acid molecule including a vector, can be introduced into a cell by any of a variety of methods known in the art (Sambrook et al . , supra, 1989, and in Ausubel et al . , Current Protocols in Molecular Biology. John Wiley and Sons, Baltimore, MD (1994) , which is incorporated herein by reference) . Such methods include, for example, transfection, lipofection, microinjection, electroporation and infection with recombinant vectors or the use of liposomes.
  • a nucleic acid molecule by infection with a viral vector is particularly advantageous in that it can efficiently introduce the nucleic acid molecule into a cell ex vivo or in vivo .
  • viruses are very specialized and typically infect and propagate in specific cell types.
  • their natural specificity can be used to target the nucleic acid molecule contained in the vector to specific cell types.
  • a vector based on HIV-1 can be used to target an antisense IKK subunit molecule to HIV-1 infected cells, thereby reducing the phosphorylation of IKB, which can decrease the high level of constitutive
  • Viral or non-viral vectors also can be modified with specific receptors or ligands to alter target specificity through receptor mediated events.
  • a nucleic acid molecule also can be introduced into a cell using methods that do not require the initial introduction of the nucleic acid molecule into a vector.
  • a nucleic acid molecule encoding an IKK catalytic subunit can be introduced into a cell using a cationic liposomes, which also can be modified with specific receptors or ligands as described above (Morishita et al . , J. Clin. Invest.. 91:2580-2585 (1993), which is incorporated herein by reference; see, also, Nabel et al . , supra , 1993)).
  • a nucleic acid molecule can be introduced into a cell using, for example, adenovirus-polylysine DNA complexes (see, for example, Michael et al . , J. Biol . Chem.. 268:6866-6869 (1993) , which is incorporated herein by reference) .
  • Other methods of introducing a nucleic acid molecule into a cell such that the encoded IKK subunit or antisense nucleic acid molecule can be expressed are well known (see, for example, Goeddel, supra , 1990) .
  • Selectable marker genes encoding, for example, a polypeptide conferring neomycin resistance (Neo R ) also are readily available and, when linked to a nucleic acid molecule of the invention or incorporated into a vector containing the nucleic acid molecule, allows for the selection of cells that have incorporated the nucleic acid molecule.
  • Other selectable markers such as that conferring hygromycin, puromycin or ZEOCIN (Invitrogen) resistance are known to those in the art of gene transfer can be used to identify cells containing the nucleic acid molecule, including the selectable marker gene.
  • a "suicide” gene also can be incorporated into a vector so as to allow for selective inducible killing of a cell containing the gene.
  • a gene such as the herpes simplex virus thymidine kinase gene (TK) can be used as a suicide gene to provide for inducible destruction of such cells.
  • TK herpes simplex virus thymidine kinase gene
  • the cells can be exposed to a drug such as acyclovir or gancyclovir, which can be administered to an individual .
  • TILs tumor infiltrating lymphocytes
  • Neo R Neomycin resistance
  • retroviral vectors have been altered to prevent viral replication by the deletion of viral gag, pol and env genes .
  • Such a method can also be used ex vivo to transduce cells taken from a subject (see Anderson et al . , U.S. Patent No. 5,399,346, issued March 21, 1995, which is incorporated herein by reference) .
  • retroviral vector supernatants used to infect cells will be screened for replication competent virus by standard assays such as PCR and reverse transcriptase assays.
  • a cell requires the precise regulation of expression of nearly all genes. Such gene regulation is accomplished by activation or repression of transcription by various transcription factors, which interact directly with regulatory sequences on nuclear DNA. The ability of transcription factors to bind DNA or activate or repress transcription is regulated in response to external stimuli. In the case of the transcription factor NF- ⁇ B, critical factors involved in the signaling pathway mediating its activation have not been identified (Verma, et al . , Genes Devel. 9:2723-2735 (1995); Baeuerle and Baltimore, Cell 87:13-20 (1996) ) .
  • NF-KB is a member of the Rel family of transcription factors, which are present in most if not all animal cells (Thanos and Maniatis, Cell 80:629-532 (1995)).
  • Rel proteins which include, for example, RelA (p65) , c-Rel, p50, p52 and the Drosophila dorsal and Dif gene products, are characterized by region of about 300 amino acids sharing approximately 35% to 61% homology ("Rel homology domain").
  • the Rel homology domain includes DNA binding and dimerization domains and a nuclear localization signal .
  • Rel proteins are grouped into one of two classes, depending on whether the protein also contains a transcriptional activation domain
  • Rel proteins can from homodimers or heterodimers, which can be transcriptionally activating depending on the presence of a transactivation domain.
  • the most common Rel/NF- ⁇ B dimer which is designated "NF- ⁇ B, " is a p50/p65 heterodimer that can activate transcription of genes containing the appropriate KB binding sites.
  • p50/p65 NF- ⁇ B is present in most cell types and is considered the prototype of the Rel/NF- ⁇ B family of transcription factors.
  • Different dimers vary in their binding to different KB elements, kinetics of nuclear translocation and levels of expression in a tissue (Siebenlist et al . , supra, 1994) .
  • the term "Rel/NF- ⁇ B" is used to refer generally to the
  • NF- ⁇ B Rel family of transcription factors
  • NF-KB originally was identified by its ability to bind a specific DNA sequence present in the immunoglobulin K light chain gene enhancer, the "KB element” (Sen and Baltimore, Cell 46:705-709 (1986)).
  • the KB element has been identified in numerous cellular and viral promotors, including promotors present in human immunodeficiency virus-1 (HIV-1) ; immunoglobulin superfamily genes such as the MHC class 1 (H-2 ⁇ ) gene; cytokine genes such as the tumor necrosis factor ⁇ (TNF ⁇ ) , interleukin-l ⁇ (IL-l ⁇ ), IL-2, IL-6 and the granulocyte-macrophage colony stimulating factor (GM-CSF) gene; chemokine genes such as RANTES and IL-8; and cell adhesion protein genes such as E-selectin.
  • H-2 ⁇ human immunodeficiency virus-1
  • cytokine genes such as the tumor necrosis factor ⁇ (TNF ⁇ ) , interleukin-l ⁇ (IL-l ⁇ ), IL-2, IL-6 and the granulocyte-macrophage colony stimulating factor (GM-CSF) gene
  • chemokine genes such as RANTES and IL-8
  • Rel/NF- ⁇ B is maintained in the cytoplasm in an inactive form complexed with an IKB protein.
  • Rel/NF- ⁇ B transcriptional activity is induced by numerous pathogenic events or stresses, including cytokines, chemokines, viruses and viral products, double stranded RNA, bacteria and bacterial products such as lipopolysaccharide (LPS) and toxic shock syndrome toxin-1, mitogens such as phorbol esters, physical and oxidative stresses, and chemical agents such as okadaic acid and cycloheximide (Thanos and Maniatis, supra, 1995; Siebenlist et al . , supra, 1994).
  • genes encoding agents such as TNF ⁇ , IL-1, IL-6, interferon- ⁇ and various chemokines, which induce NF- ⁇ B activity are, themselves, induced by NF- ⁇ B, resulting in amplification of their signal by a positive, self-regulatory loop (Siebenlist et al . , supra, 1994).
  • Phorbol esters, which activate T cells also activate NF- ⁇ B and immunosuppressants such as cyclosporin A inhibit activation of T cells through T cell receptor mediated signals (Baldwin, Ann. Rev. Immunol. 14:649-681 (1996) , which is incorporated herein by reference) .
  • NF- ⁇ B Regulation of specific genes by NF- ⁇ B can require interaction of NF- ⁇ B with one or more other DNA binding proteins.
  • expression of E-selectin requires an interaction of NF- ⁇ B, the bZIP protein ATF-2 and HMG-I(Y), and expression of the IL-2 receptor ⁇ gene requires an interaction of NF- ⁇ B, HMG-I (Y) and the ets-like protein, ELF-1 (Baldwin, supra, 1996) .
  • NF-KB likely act through various converging signal transduction pathways, including pathways involving activation of protein kinase C, Raf kinase and tyrosine kinases .
  • the ability of antioxidants to inhibit NF- ⁇ B activation by various inducing agents suggests that reactive oxygen species are a converging point of such pathways (Siebenlist et al . , supra, 1994).
  • a Rel/NF- ⁇ B dimer Upon activation by an appropriate inducing agent, a Rel/NF- ⁇ B dimer is translocated into the nucleus, where it can activate gene transcription.
  • the subcellular localization of a Rel/NF- ⁇ B is controlled by specific inhibitory proteins ("inhibitors of Rel/NF- ⁇ B" or "IKB'S”) , which noncovalently bind the Rel/NF- ⁇ B and mask its nuclear localization signal (NLS) , thereby preventing nuclear uptake.
  • IKB'S including, for example, I ⁇ B ⁇ , I ⁇ B ⁇ , Bel-3 and the Drosophila cactus gene product, have been identified (Baeuerle and Baltimore, supra, 1996) .
  • Rel precursor proteins such as pl05 and plOO, which are precursors of p50 and p52, respectively, function as IKB'S (Siebenlist et al . , supra, 1994) .
  • I ⁇ B ⁇ and I ⁇ B ⁇ are expressed in most cell types and generally bind p65- and c-Rel-containing Rel/NF- ⁇ B dimers.
  • Other IKB'S appear to be expressed in a tissue specific manner (Thompson et al . , Cell 80:573-582 (1995) ) .
  • IKB proteins are characterized by the presence of 5 to 8 ankyrin repeat domains, each about 30 amino acids, and a C-terminal PEST domain.
  • I ⁇ B ⁇ contains a 70 amino acid N-terminal domain, a 205 amino acid internal domain containing the ankyrin repeats, and a 42 amino acid C-terminal domain containing the PEST domain (Baldwin, supra, 1996) .
  • IKB proteins interact through their ankyrin repeats with the Rel homology domain of Rel/NF- ⁇ B dimers, binding of particular IKB proteins with particular Rel/NF- ⁇ B proteins appears to be relatively specific.
  • I ⁇ B ⁇ and I ⁇ B ⁇ associate primarily with RelA- and c-Rel- containing Rel/NF- ⁇ B dimers, thereby blocking their nuclear localization signal.
  • the binding of an IKB to NF-KB also interferes with the ability of NF- ⁇ B to bind DNA.
  • I ⁇ B ⁇ is phosphorylated following exposure of cells to tumor necrosis factor (TNF) , IL-1, bacterial lipopolysaccharide (LPS) or phorbol esters
  • TNF tumor necrosis factor
  • IL-1 IL-1
  • LPS bacterial lipopolysaccharide
  • phorbol esters I ⁇ B ⁇ is phosphorylated in certain cell types only in response to LPS or IL-1 (Baldwin, supra, 1996) .
  • I ⁇ B ⁇ is phosphorylated in response to the same signals that induce I ⁇ B ⁇ , although with slower kinetics than I ⁇ B ⁇ (DiDonato et al . , Mol. Cell. Biol. 16:1295-1304 (1996), which is incorporated herein by reference) .
  • Rel/NF- ⁇ B Upon exposure to an appropriate stimulus, the IKB portion of the complex is rapidly degraded and the Rel/NF- ⁇ B portion becomes free to translocate to the cell nucleus.
  • activation of a Rel/NF- ⁇ B does not require de novo protein synthesis and, therefore, occurs extremely rapidly. Consequently, activation of gene expression due to a Rel/NF- ⁇ B can be exceptionally rapid and provides an effective means to respond to an external stimulus.
  • Such a rapid response of Rel/NF- ⁇ B transcription factors is particularly important since these factors are involved in the regulation of genes involved in the immune, inflammatory and acute phase responses, including responses to viral and bacterial infections and to various stresses.
  • I ⁇ B ⁇ Upon exposure of a cell to an appropriate inducing agent, I ⁇ B ⁇ , for example, is phosphorylated at serine residue 32 (Ser-32) and Ser-36 (Haskill et al . , Cell 65:1281-1289 (1991)). Phosphorylation of I ⁇ B ⁇ triggers its rapid ubiquitination, which results in proteasome-mediated degradation of the inhibitor and translocation of active NF- ⁇ B to the nucleus (Brown et al., Science 267:1485-1488 (1995); Scherer et al . , Proc . Natl. Acad. Sci .. USA.
  • Rel/NF- ⁇ B activation can be transient or persistent, depending on the inducing agent and the I ⁇ B that is phosphorylated.
  • the inducing agent For example, exposure of a cell to particular cytokines induces I ⁇ B ⁇ phosphorylation and degradation, resulting in NF- ⁇ B activation, which induces the expression of various genes, including the gene encoding I ⁇ B ⁇ .
  • the newly expressed I ⁇ B ⁇ then binds to NF-KB in the nucleus, resulting in its export to the cytoplasm and inactivation and, therefore, a transient NF-KB mediated response.
  • bacterial LPS induces I ⁇ B ⁇ phosphorylation, resulting in NF- ⁇ B activation.
  • the I ⁇ B ⁇ gene is not induced by NF- ⁇ B and, as a result, activation of NF- ⁇ B is more persistent (Thompson et al . , supra, 1995).
  • a constitutively active multisubunit kinase of approximately 700 kDa phosphorylates I ⁇ B ⁇ at Ser-32 and Ser-36 and, in some cases, requires polyubiquitination for activity (Chen et al . , Cell 84:853-862 (1996); Lee et al., Cell 88:213-222 (1997)).
  • the mitogen-activated protein kinase/ERK kinase kinase-1 (MEKK1) phosphorylates several proteins that copurify with this complex and have molecular weights of approximately 105 kDa, 64 kDa and
  • MEKK1 Overexpression of MEKK1 also induces the site- specific phosphorylation of I ⁇ B ⁇ in vivo and can directly activate I ⁇ B ⁇ in vi tro by an ubiquitin-independent mechanism. However, MEKK1 did not phosphorylate I ⁇ B ⁇ at Ser-32 and Ser-36 in the in vi tro experiments, indicating that it is not an I ⁇ B ⁇ kinase, but may act upstream of I ⁇ B ⁇ kinase in a signal transduction pathway (Lee et al . , supra , 1997) .
  • an ubiquitin independent 700 kDa complex phosphorylates I ⁇ B ⁇ Ser-32 and Ser-36, but not a mutant containing threonines substituted for these serines (Baeuerle and Baltimore, supra, 1996) .
  • the specific polypeptides responsible for the IKB kinase activity of these complexes have not been described.
  • a double stranded RNA-dependent protein kinase (PKR) that phosphorylates I ⁇ B ⁇ in vi tro has been described (Kumar et al . , Proc. Natl. Acad. Sci ..
  • a putative serine-threonine protein kinase has been identified in mouse cells by probing for nucleic acid molecules that encode proteins containing a consensus helix-loop-helix domain, which is involved in protein-protein interactions (Connelly and Marcu, supra, 1995) .
  • This putative kinase which is ubiquitously expressed in various established cell lines, but differentially expressed in normal mouse tissues, was named CHUK (conserved helix-loop-helix ubiquitous kinase; GenBank Accession #U12473) .
  • the present invention provides an isolated IKB kinase (IKK) , including isolated full length IKK catalytic subunits.
  • IKK isolated IKB kinase
  • the invention provides an isolated 300 kDa or 900 kDa complex, which comprises an IKK ⁇ or an IKK ⁇ subunit and has IKB kinase activity
  • the invention provides is an isolated human IKK ⁇ catalytic subunit (SEQ ID NO: 2; Example II), which contains a previously undescribed N-terminal amino acid sequence and essentially the C-terminal region of human CHUK (Connelly and Marcu, supra, 1995) and phosphorylates I ⁇ B ⁇ on Ser-32 and Ser-36 and I ⁇ B ⁇ on Ser-19 and Ser-23 (DiDonato et al . , supra, 1996; see, also, Regnier et al . , supra, 1997) .
  • the invention also provides an isolated IKK ⁇ catalytic subunit (SEQ ID NO: 15; Example III), which shares greater than 50% amino acid sequence identity with IKK ⁇ , including conserved homology in the kinase domain, helix-loop-helix domain and leucine zipper domain.
  • isolated when used in reference to an IKB kinase complex or to an IKK catalytic subunit of the invention, means that the complex or the subunit is relatively free from contaminating lipids, proteins, nucleic acids or other cellular material normally associated with an IKK in a cell.
  • An isolated 900 kDa IKB kinase complex or 300 kDa complex can be isolated, for example, by immunoprecipitation using an antibody that binds to an IKK catalytic subunit (see Examples III and IV) .
  • an isolated IKK subunit can be obtained, for example, by expression of a recombinant nucleic acid molecule such as SEQ ID NO: 1 or SEQ ID NO: 14, or can be isolated from a cell by a method comprising affinity chromatography using ATP or IKB as ligands (Example I) or using an anti-IKK subunit antibody.
  • An isolated IKK complex or IKK subunit comprises at least 30% of the material in a sample, generally about 50% or 70% or 90% of a sample, and preferably about 95% or 98% of a sample, as described above with respect to nucleic acids .
  • a polypeptide having the amino acid sequence of the partial human CHUK polypeptide does not have IKB kinase activity when expressed in a cell, indicating that some or all of amino acid residues 1 to 31 are essential for kinase activity.
  • a full length IKK catalytic subunit of the invention is exemplified by human IKK ⁇ , which has an apparent molecular mass of about 85 kDa and phosphorylates I ⁇ B ⁇ on Ser-32 and Ser-36.
  • An IKK catalytic subunit of the invention also is exemplified by IKK ⁇ , which is an 87 kDa polypeptide that shares substantial amino acid sequence homology with IKK ⁇ ( Figure 3) .
  • the term "full length,” when used in reference to an IKK subunit of the invention means a polypeptide having an amino acid sequence of an IKK subunit expressed normally in a cell.
  • Such a normally expressed IKK polypeptide begins with a methionine residue at its N-terminus (Met-1; Figure 3), the Met-1 being encoded by the initiator ATG (AUG) codon, and ends as a result of the termination of translation due to the presence of a STOP codon.
  • a full length human IKK catalytic subunit can be a native IKK polypeptide, which is isolated from a cell, or can be produced using recombinant DNA methods such as by expressing the nucleic acid molecule shown as SEQ ID NO : 1 or SEQ ID NO: 14.
  • the apparent molecular mass of an isolated IKK subunit can be measured using routine methods such as polyacrylamide gel electrophoresis performed in the presence of sodium dodecyl sulfate (SDS-PAGE) or column- chromatography performed under reducing and denaturing conditions.
  • SDS-PAGE sodium dodecyl sulfate
  • column- chromatography performed under reducing and denaturing conditions.
  • the ability of an IKK subunit to phosphorylate I ⁇ B ⁇ on Ser-32 and Ser-36 can be identified using the methods disclosed herein.
  • the apparent molecular mass of a previously unknown protein as determined, for example, by SDS-PAGE is an estimate based on the relative migration of the unknown protein as compared to the migration of several other proteins having known molecular masses.
  • one investigator reasonably can estimate, for example, that an unknown protein has an apparent molecular mass of 82 kDa, whereas a second investigator, looking at the same unknown protein under substantially similar conditions, reasonably can estimate that the protein has an apparent molecular mass of 87 kDa.
  • IKB kinase having an apparent molecular mass of "about 85 kDa” indicates that the kinase migrates by SDS-PAGE in an 8% gel under reducing conditions in the range of 80 kDa to 90 kDa, preferably in the range of 82 kDa to 87 kDa.
  • IKK catalytic subunit of the invention is exemplified by the isolated full length polypeptide comprising the amino acid sequence shown as SEQ ID NO: 2 or SEQ ID NO: 15.
  • the invention provides peptide portions of an IKK subunit polypeptide, wherein such peptide portions contain at least three contiguous amino acids as shown in SEQ ID NO: 2 or SEQ ID NO: 15, and generally contain at least six contiguous amino acids or, if desired, at least nine contiguous amino acids, as provided herein.
  • the invention provides peptide portions of IKK ⁇ , containing, for example, at least three contiguous amino acids of SEQ ID NO: 2, including amino acid residue 30, preferably at least four contiguous amino acids, including amino acid residue 30, and more preferably at least six contiguous amino acids, including amino acid residue 30.
  • the invention also provides a peptide portion of IKK ⁇ comprising at least three contiguous amino acids, generally six contiguous amino acids, and preferably ten contiguous amino acids of SEQ ID NO: 15. It is recognized, however, that a peptide of the invention does not consist of a polypeptide disclosed as GenBank Accession #U12473 or #U22512.
  • a peptide portion of an IKK subunit generally is a tripeptide or larger, preferably a hexapeptide or larger, and more preferably a decapeptide or larger, up to a contiguous amino acid sequence having a maximum length that lacks one or more N-terminal or C-terminal amino acids of the full length polypeptide (SEQ ID NO: 2 or SEQ ID NO: 15) .
  • a peptide portion of IKK ⁇ having the amino acid sequence shown as SEQ ID NO: 2 can be from three amino acids long to 744 amino acids long, which is one residue less than the full length polypeptide, except as provided above.
  • a peptide portion of an IKK subunit polypeptide of the invention can be produced by any of several methods well known in the art.
  • a peptide portion of an IKK subunit can be produced by enzymatic cleavage of an IKK subunit protein, which has been isolated from a cell, using a proteolytic enzyme such as trypsin, chymotrypsin, Lys-C or the like, or combinations of such enzymes .
  • proteolytic enzyme such as trypsin, chymotrypsin, Lys-C or the like, or combinations of such enzymes .
  • proteolytic cleavage products can be isolated using methods as disclosed in Example I, to obtain peptide portions of IKK ⁇ and IKK ⁇ , for example.
  • a peptide portion of an IKK subunit also- can be produced using methods of solution or solid phase peptide synthesis or can be expressed from a nucleic acid molecule such as a portion of the coding region of the nucleic acid sequence shown as SEQ ID NO: 1 or SEQ ID NO: 14, or can be purchased from a commercial source.
  • a peptide portion of an IKK subunit can comprise the kinase domain of the IKK subunit and, therefore, can have the ability to phosphorylate an IKB protein.
  • a peptide portion of SEQ ID NO: 2 comprising amino acids 15 to 301 has the characteristics of a serine-threonine protein kinase domain (Hanks and Quinn, Meth. Enzymol . 200:38-62 (1991), which is incorporated herein by reference) .
  • Such a peptide portion of an IKK subunit can be examined for kinase activity by determining that it can phosphorylate I ⁇ B ⁇ at Ser-32 and Ser-36 or I ⁇ B ⁇ at Ser-19 and Ser-23, using methods as disclosed herein.
  • a peptide portion of an IKK subunit can comprise an immunogenic amino acid sequence of the polypeptide and, therefore, can be useful for eliciting production of an antibody that can specifically bind the IKK subunit or to an IKK complex comprising the subunit, particularly to an epitope comprising amino acid residue 30 as shown in SEQ ID NO: 2 or to an epitope of SEQ ID NO: 15, provided said epitope is not present in a CHUK protein.
  • the invention also provides anti-IKK antibodies, which specifically bind to an epitope of an IKK complex, particularly an IKK catalytic subunit, and to IKK subunit binding fragments of such antibodies.
  • the invention provides cell lines producing anti-IKK antibodies or IKK-binding fragments of such antibodies.
  • the term "antibody” is used in its broadest sense to include polyclonal and monoclonal antibodies, as well as antigen binding fragments of such antibodies.
  • anti-IKK antibody of the invention the term "antigen” means an IKK catalytic subunit protein, polypeptide or peptide portion thereof, or an IKK complex comprising an IKK catalytic subunit protein, polypeptide or peptide portion thereof.
  • an anti-IKK antibody can bind to and, for example, immunoprecipitate an IKK complex, the antibody specifically binds an epitope comprising at least a portion of an IKK catalytic subunit.
  • An antibody of the invention also can be used to immunoprecipitate an IKK subunit, free of the IKK complex.
  • an anti-IKK antibody or antigen binding fragment of such an antibody, is characterized by having specific binding activity for an epitope of an IKK subunit of at least about 1 x 10 5 M "1 , generally, at least about 1 x 10 6 M "1 .
  • Fab, F(ab') 2 , Fd and Fv fragments of an anti-IKK antibody which retain specific binding activity for an IKK subunit, are included within the definition of an antibody.
  • an anti-IKK antibody can react with an epitope comprising the N-terminus of IKK ⁇ or with an epitope of IKK ⁇ , but not to a polypeptide having an amino acid sequence shown as residues 32 to 745 of SEQ ID NO : 2.
  • antibody as used herein includes naturally occurring antibodies as well as non-naturally occurring antibodies, including, for example, single chain antibodies, chimeric, bifunctional and humanized antibodies, as well as antigen-binding fragments thereof.
  • non-naturally occurring antibodies can be constructed using solid phase peptide synthesis, can be produced recombinantly or can be obtained, for example, by screening combinatorial libraries consisting of variable heavy chains and variable light chains as described by Huse et al . , Science 246:1275-1281 (1989), which is incorporated herein by reference.
  • These and other methods of making, for example, chimeric, humanized, CDR-grafted, single chain, and bifunctional antibodies are well known to those skilled in the art (Winter and Harris, Immunol .
  • An anti-IKK antibody of the invention can be raised using an isolated IKK subunit or a peptide portion thereof and can bind to a free, uncomplexed form of IKK subunit or can bind to IKK subunit when it is associated with a 300 kDa or 900 kDa IKK complex.
  • an anti-IKK antibody of the invention can be raised against an isolated 300 kDa or 900 kDa I ⁇ B kinase complex, which can be obtained as disclosed herein.
  • an antibody of the invention is referred to generally herein as an "anti-I ⁇ B kinase antibody” or an “anti-IKK antibody.”
  • anti-I ⁇ B kinase antibody an antibody of the invention
  • anti-IKK antibody an antibody of the invention
  • the various antibodies of the invention will have unique antigenic specificities, for example, for a free or complexed IKK subunit, or both, or for a 300 kDa or 900 kDa IKB kinase complex, or both.
  • Anti-IKK antibodies can be raised using as an immunogen an isolated full length IKK catalytic subunit, which can be prepared from natural sources or produced recombinantly, or a peptide portion of an IKK subunit as defined herein, including synthetic peptides as described above.
  • a non-immunogenic peptide portion of an IKK catalytic subunit can be made immunogenic by coupling the hapten to a carrier molecule such bovine serum albumin (BSA) or keyhole limpet hemocyanin (KLH) , or by expressing the peptide portion as a fusion protein.
  • BSA bovine serum albumin
  • KLH keyhole limpet hemocyanin
  • antibodies of the invention include antibodies that bind with the free, but not the complexed, form of an IKK subunit or, alternatively, with the complexed, but not free, form of an IKK subunit.
  • Antibodies of the invention also include antibodies that bind with the 300 kDa I ⁇ B kinase complex or the 900 kDa IKB kinase complex or both.
  • an antibody specific for the 300 kDa or 900 kDa IKB kinase complex need not recognize an IKK subunit epitope in order to be encompassed within the claimed invention, since, prior to the present disclosure, the 300 kDa and 900 kDa IKK complexes were not known (see DiDonato et al . , Nature 388:548-554 (1997) ) .
  • Antibodies of the invention that bind to an activated IKK but not to an inactive IKK, and, conversely, those that bind to an inactive form of the kinase but not to the activated form also are particularly useful.
  • an IKK can be activated by phosphorylation of an IKK subunit and, therefore, an antibody that recognizes the phosphorylated form of the IKK, but that does not bind to the unphosphorylated form can be obtained.
  • IKK can be activated by release of a regulatory subunit and, therefore, an antibody that recognizes a form of the IKK complex that is not bound to the regulatory subunit can be obtained.
  • Such antibodies are useful for identifying the presence of active IKK in a cell.
  • An anti-IKK antibody is useful, for example, for determining the presence or level of an IKK or of an IKK subunit in a tissue sample, which can be a lysate or a histological section.
  • the identification of the presence or level of an IKK or an IKK subunit in the sample can be made using well known immunoassay and immunohistochemical methods (Harlow and Lane, supra, 1988) .
  • An anti-IKK antibody also can be used to substantially purify an IKB kinase or an IKK subunit from a sample.
  • an anti-IKK antibody can be used in a screening assay to identify agents that alter the activity of an IKB kinase.
  • kits incorporating an anti-IKK antibody which can be specific for the active or inactive form of IKB kinase or can bind to an IKK complex or to an IKK subunit, regardless of the activity state, can be particularly useful.
  • a kit can contain, in addition to an anti-IKK antibody, a reaction cocktail that provides the proper conditions for performing the assay, control samples that contain known amounts of an IKK or IKK subunit and, if desired, a second antibody specific for the anti-IKK antibody.
  • Such an assay also should include a simple method for detecting the presence or amount of an IKK or an IKK subunit in a sample that is bound to the anti-IKK antibody.
  • a protein such as anti-IKK antibody, as well as an IKK subunit or a peptide portion thereof, can be labeled so as to be detectable using methods well known in the art (Hermanson, "Bioconjugate Techniques” (Academic Press 1996) , which is incorporated herein by reference; Harlow and Lane, 1988; chap. 9).
  • a protein can be labeled with various detectable moieties including a radiolabel, an enzyme, biotin or a fluorochrome.
  • Reagents for labeling a protein such as an anti-IKK antibody can be included in a kit containing the protein or can be purchased separately from a commercial source .
  • a labeled antibody can be identified by detecting the particular moiety.
  • a labeled second antibody can be used to identify specific binding of an unlabeled anti-IKK antibody.
  • a second antibody generally will be specific for the particular class of the first antibody. For example, if an anti-I ⁇ B kinase antibody is of the IgG class, a second antibody will be an anti-IgG antibody. Such second antibodies are readily available from commercial sources.
  • the second antibody can be labeled using a detectable moiety as described above.
  • a sample is labeled using a second antibody
  • the sample is first contacted with a first antibody, which is an anti-IKK antibody
  • the sample is contacted with the labeled second antibody, which specifically binds to the anti-IKK antibody and results in a labeled sample.
  • a first antibody which is an anti-IKK antibody
  • the labeled second antibody which specifically binds to the anti-IKK antibody and results in a labeled sample.
  • spleen cells from a mouse immunized with an IKK complex or an IKK subunit or peptide portion thereof can be fused to an appropriate myeloma cell line such as SP/02 myeloma cells to produce hybridoma cells.
  • Cloned hybridoma cell lines can be screened using a labeled IKK subunit to identify clones that secrete anti-IKK monoclonal antibodies.
  • Hybridomas expressing anti-IKK monoclonal antibodies having a desirable specificity and affinity can be isolated and utilized as a continuous source of the antibodies, which are useful, for example, for preparing standardized kits as described above.
  • a recombinant phage that expresses, for example, a single chain anti-IKK also provides a monoclonal antibody that can used for preparing standardized kits.
  • a monoclonal anti-IKK antibody can be used to prepare anti-idiotypic antibodies, which present an epitope that mimics the epitope recognized by the monoclonal antibody used to prepare the anti-idiotypic antibodies.
  • the anti-idiotypic antibody can act as a competitor of IKB and, therefore, can be useful for reducing the level of phosphorylation of IKB and, consequently, the activity of NF- ⁇ B.
  • the present invention further provides methods of identifying an agent that can alter the association of an IKK catalytic subunit with a second protein, which can be an upstream activator, a downstream effector such as IKB, an interacting regulatory protein of the IKK subunit, or an interacting subunit associated with the 300 kDa or 900 kDa IKB kinase complex.
  • associate or “association, " when used in reference to an IKK subunit and a second protein means that the IKK subunit and the second protein have a binding affinity for each other such that they form a bound complex in vivo or in vi tro, including in a cell in culture or in a reaction comprising substantially purified reagents.
  • the term "bind” or “interact” is used interchangeably with the term “associate . "
  • the affinity of binding of an IKK subunit and a second protein such as an IKB or another IKK subunit or other subunit present in an IKK complex is characterized in that it is sufficiently specific such that a bound complex can form in vivo in a cell or can form in vi tro under appropriate conditions as disclosed herein.
  • the formation or dissociation of a bound complex can be identified, for example, using the two hybrid assay or demonstrating coimmunoprecipitation of the second protein with the IKK subunit, as disclosed herein, or using other well known methods such as equilibrium dialysis.
  • Methods for distinguishing the specific association of an IKK subunit and a second protein from nonspecific binding to the IKK subunit are known in the art and, generally, include performing the appropriate control experiments to demonstrate the absence of nonspecific protein binding.
  • second protein refers to a protein that specifically associates with an IKK subunit ("first protein") .
  • IKB proteins including I ⁇ B ⁇ and I ⁇ B ⁇ , which are substrates for IKB kinase activity and are downstream of the IKB kinase in a signal transduction pathway that results in the regulated expression of a gene.
  • second proteins are exemplified by the proteins that, together with the IKK subunits, form a 300 kDa or 900 kDa IKB kinase complex, which coimmunoprecipitates using an anti-IKK antibody (see Example IV) .
  • IKK subunits such as IKK ⁇ and IKK ⁇ interact with each other to form homodimers or heterodimers
  • a second protein also can be a second IKK subunit, which can be the same as or different from the "first" protein.
  • Agents that alter the association of an IKK catalytic subunit and a second protein such as IKB protein or an IKK regulatory subunit can be extremely valuable, for example, for limiting excessive cytokine expression as occurs in an acute phase response by preventing the activation of NF- ⁇ B, thereby preventing NF-KB mediated induction of cytokine gene expression.
  • the IKK subunit can be any protein involved in IKB kinase activity, including, for example, mouse CHUK (Connelly and Marcu, supra, 1995; GenBank Accession #12473) , which, prior to the present disclosure, was not known to have the ability to associate with I ⁇ B or to have IKB kinase activity.
  • a second protein can be a protein that is upstream of IKB kinase in a signal transduction pathway and associates with the IKK complex, particularly with an IKK catalytic subunit of the IKK complex.
  • a second protein which can be an upstream activator of the IKB kinase, can be identified using routine methods for identifying protein-protein interactions as disclosed herein.
  • Such second proteins can be, for example, MEKKl or PKR or CKII, each of which has been reported to be involved in a pathway leading to phosphorylation of IKB and activation of NF- ⁇ B, but neither of which has the characteristics expected of the common I ⁇ B kinase present at the point where the various NF- ⁇ B activation pathways converge (see, for example, Lee et al . , supra , 1997), or can be the NF- ⁇ B-inducing kinase (NIK) , which reportedly is upstream from IKK in an NF- ⁇ B activation pathway (Regnier et al . , supra, 1997; Malinin et al . , Nature 385:540-544 (1997) ) .
  • NIK NF- ⁇ B-inducing kinase
  • a second protein also can be a regulatory protein, which associates with an IKK catalytic subunit in an IKK complex, either constitutively as part of a 300 kDa or 900 kDa complex or in response to activation of a pathway leading to IKK activation.
  • a regulatory protein can inhibit or activate IKK activity depending, for example, on whether the regulatory protein is associated with IKK and whether the regulatory protein associates with an IKK catalytic subunit in a free form or as part of an IKK complex.
  • the regulatory protein also can be important for "docking" a catalytic IKK subunit to its substrate.
  • a regulatory protein to associate with or dissociate from an IKK subunit or IKK complex can depend, for example, on the relative phosphorylation state of the regulatory protein. It is recognized that an upstream activator of IKK also can interact with such a regulatory protein, thereby indirectly inhibiting or activating the IKK.
  • Example II two copurifying proteins were isolated by ATP and IKB affinity chromatography and identified by SDS-PAGE (Example I) . Partial amino acid sequences were determined and cDNA molecules encoding the proteins were obtained (see Examples I, II and III). One of the proteins has an apparent molecular mass of 85 kDa. Expression in a cell of a cDNA molecule encoding the 85 kDa protein resulted in increased NF- ⁇ B activity following cytokine induction as compared to control cells, whereas expression of the antisense of this cDNA decreased the basal NF- ⁇ B activity in the cells and prevented cytokine induction of NF- ⁇ B activity. ) ⁇ t to ⁇ >
  • I ⁇ B ⁇ can be, for example, an anti-idiotypic antibody as described above, which can inhibit the association of an IKK and IKB .
  • a screening assay of the invention also is useful for identifying agents that directly alter the activity of an IKK. While such an agent can act, for example, by altering the association of an IKK complex or IKK catalytic subunit with a second protein, the agent also can act directly as a specific activator or inhibitor of IKK activity.
  • Specific protein kinase inhibitors include, for example, staurosporin, the heat stable inhibitor of cAMP-dependent protein kinase, and the MLCK inhibitor, which are known in the art and commercially available.
  • a library of molecules based, generally, on such inhibitors or on ATP or adenosine can be screened using an assay of the invention to obtain agents that desirably modulate the activity of an IKK complex or an IKK subunit.
  • IKK activity can be measured by identifying phosphorylation, for example, of I ⁇ B ⁇ , either directly or using an antibody specific for the Ser-32 and Ser-36 phosphorylated form of I ⁇ B ⁇ .
  • An antibody that binds to I ⁇ B ⁇ that is phosphorylated on Ser-32 for example, can be purchased from a commercial source (New England Biolabs; Beverly MA) .
  • Cultured cells can be exposed to various agents suspected of having the ability to directly alter IKK activity, then aliquots of the cells either are collected or are treated with a proinflammatory stimulus such as a cytokine, and collected. The collected cells are lysed and the kinase is immunoprecipitated using an anti-IKK antibody.
  • a substrate such as I ⁇ B ⁇ or I ⁇ B ⁇ is added to the immunocomplex and the ability of the IKK to phosphorylate the substrate is determined as described above.
  • the anti-IKK antibody first can be coated onto a plastic surface such as in 96 well plates, then the cell lysate is added to the wells under conditions that allow binding of IKK by the antibody. Following washing of the wells, IKK activity is measured as described above.
  • Such a method is extremely rapid and provides the additional advantage that it can be automated for high through-put assays .
  • a screening assay of the invention is particularly useful to identify, from among a diverse population of molecules, those agents that modulate the association of an IKK complex or an IKK catalytic subunit and another protein (referred to herein as a "second protein") or that directly alter the activity of IKK.
  • Methods for producing libraries containing diverse populations of molecules including chemical or biological molecules such as simple or complex organic molecules, peptides, proteins, peptidomimetics, glycoproteins, lipoproteins, polynucleotides, and the like, are well known in the art (Huse, U.S. Patent No. 5,264,563, issued November 23, 1993; Blondelle et al . , Trends Anal. Chem. 14:83-92 (1995); York et al .
  • a screening assay of the invention provides a simple means for identifying those agents in the library that can modulate the association of an IKK and a second protein or can alter the activity of an IKK.
  • a screening assay of the invention can be automated, which allows for high through-put screening of randomly designed libraries of agents to identify those particular agents that can modulate the ability of an IKK and a second protein to associate or that alter the activity of the IKK.
  • a drug screening assay of the invention utilizes an IKK complex, which can be isolated as disclosed herein; or an IKK subunit, which can be expressed, for example, from a nucleic acid molecule encoding the amino acid sequence shown in SEQ ID NO: 2 or in SEQ ID NO: 15; or can be purified as disclosed herein; or can utilize an IKK subunit fusion protein such as an IKK ⁇ -glutathione-S-transferase (GST) or IKK ⁇ -histidine 6 (HIS6) fusion protein, wherein the GST or HIS6 is linked to the IKK subunit and comprises a tag (see Example VI) .
  • the IKK or IKK subunit fusion protein is characterized, in part, by having an affinity for a solid substrate as well as having the ability to specifically associate with an appropriate second protein such as an I ⁇ B protein.
  • the solid substrate when an IKK catalytic subunit is used in a screening assay, can contain a covalently attached anti-IKK antibody, provided that the antibody binds the IKK subunit without interfering with the ability of the IKK subunit to associate with the second protein.
  • the solid substrate can contain covalently attached glutathione, which is bound by the GST tag component of the fusion protein.
  • the IKK subunit or IKK subunit fusion protein can be part of an IKK complex in a drug screening assay of the invention.
  • a drug screening assay to identify an agent that alters the association of an IKK complex or an IKK subunit and a second protein can be performed by allowing, for example, the IKK complex or IKK subunit, which can be a fusion protein, to bind to the solid support, then adding the second protein, which can be an IKB such as I ⁇ B ⁇ , and an agent to be tested, under conditions suitable for the association of the IKK and I ⁇ B ⁇ in the absence of a drug (see Example VI) .
  • the IKK can be activated or inactivated as disclosed herein and, typically, the IKK or the second protein is detectably labeled so as to facilitate identification of the association.
  • Control reactions which contain or lack either, the IKK component, or the IKB protein, or the agent, or which substitute the IKB protein with a second protein that is known not to associate specifically with the IKK, also are performed. Following incubation of the reaction mixture, the amount of I ⁇ B ⁇ specifically bound to the IKK in the presence of an agent can be determined and compared to the amount of binding in the absence of the agent so that agents that modulate the association can be identified.
  • An IKK subunit such as IKK ⁇ or IKK ⁇ used in a screening assay can be detectably labeled with a radionuclide, a fluorescent label, an enzyme, a peptide epitope or other such moiety, which facilitates a determination of the amount of association in a reaction.
  • the detectable label can be, for example, ⁇ - 32 P-ATP, and the amount of 32 P-I ⁇ B can be detected as a measure of IKK activity.
  • the drug screening assay provides a rapid and simple method for selecting agents that desirably alter the association of an IKK and a second protein such as an IKB or for ⁇ to to H H o L ⁇ o L ⁇ o L ⁇
  • the invention also provides a method of obtaining an isolated IKK complex or an IKK catalytic subunit.
  • a 300 kDa or a 900 kDa IKK complex, comprising an IKK ⁇ subunit can be isolated from a sample by immunoprecipitation using an anti-IKK ⁇ antibody or by tagging the IKK ⁇ and using an antibody specific for the tag (see Examples III and IV) .
  • an IKK catalytic subunit can be isolated from a sample by 1) incubating the sample containing the IKK subunit with ATP, which is immobilized on a matrix, under conditions suitable for binding of the IKK subunit to the ATP; 2) obtaining from the immobilized ATP a fraction of the sample containing the IKK subunit; 3) incubating the fraction containing the IKK subunit with an IKB, which is immobilized on a matrix, under conditions suitable for binding of the IKK subunit to the IKB; and 4) obtaining from the immobilized IKB an isolated IKK catalytic subunit.
  • Such a method of isolating an IKK subunit is exemplified herein by the use of ATP affinity chromatography and I ⁇ B ⁇ affinity chromatography to isolate IKK ⁇ or IKK ⁇ from a sample of HeLa cells (see Example I) .
  • a ligand such as ATP or an IKB or an anti-IKK antibody also can be immobilized on various other matrices, including, for example, on magnetic beads, which provide a rapid and simple method of obtaining a fraction containing an ATP- or an I ⁇ B-bound IKK complex or IKK subunit or an anti-I ⁇ B kinase-bound IKK from the remainder of the sample.
  • Methods for immobilizing a ligand such as ATP or an IKB or an antibody are well known in the art (Haystead et al., Eur. J. Biochem. 214:459-467 (1993), which is incorporated herein by reference; see, also, Hermanson, supra, 1996) .
  • a sample containing an IKK complex or an IKK subunit can be a cell, tissue or organ sample, which is obtained from an animal, including a mammal such as a human, and prepared as a lysate; or can be a bacterial, insect, yeast or mammalian cell lysate, in which an IKK catalytic subunit is expressed from a recombinant nucleic acid molecule.
  • a recombinantly expressed IKK ⁇ or IKK ⁇ such as a tagged IKK ⁇ or IKK ⁇ associates into an active 300 kDa and 900 kDa IKK complex (see Examples III and IV) .
  • the invention also provides a method of identifying a second protein that associates with an IKK complex, particularly with an IKK subunit.
  • a transcription activation assay such as the yeast two hybrid system is particularly useful for the identification of protein-protein interactions (Fields and Song, Nature 340:245-246 (1989), which is incorporated herein by reference) .
  • the two hybrid assay is useful for the manipulation of protein- protein interaction and, therefore, also is useful in a screening assay to identify agents that modulate the specific interaction.
  • a transcription activation assay such as the two hybrid assay also can be performed in mammalian cells (Fearon et al . , Proc. Natl. Acad. Sci.. USA 89:7958-7962 (1992) , which is incorporated herein by reference) .
  • the yeast two hybrid system provides a particularly useful assay due to the ease of working with yeast and the speed with which the assay can be performed.
  • the invention also provides methods of identifying proteins that can interact with an IKK subunit, including proteins that can act as upstream activators or downstream effectors of IKK activity in a signal transduction pathway mediated by the IKK or LO LO t t H ⁇ >
  • Such second proteins can include additional subunits comprising the 300 kDa or 900 kDa IKK complex.
  • a transcription activation assay such as the yeast two hybrid system also is useful as a screening assay to identify agents that alter association of an IKK subunit and a second protein known to bind the IKK.
  • a transcription activation assay can be used to screen a panel of agents to identify those agents particularly useful for altering the association of an IKK subunit and a second protein in a cell. Such agents can be identified by detecting an altered level of transcription of a reporter gene, as described above, as compared to the level of transcription in the absence of the agent.
  • an agent that increases the interaction between an IKK subunit and IKB can be identified by an increased level of transcription of the reporter gene as compared to the control level of transcription in the absence of the agent.
  • Such a method is particularly useful because it identifies an agent that alters the association of an IKK subunit and a second protein in a living cell.
  • an agent may not be able to cross the yeast cell wall and, therefore, cannot enter the yeast cell to alter a protein-protein interaction.
  • yeast spheroplasts which are yeast cells that lack a cell wall, can circumvent this problem (Smith and Corcoran, In Current Protocols in Molecular Biology (ed. Ausubel et al . ; Green Publ . , NY 1989), which is incorporated herein by reference) .
  • an agent upon entering a cell, may require "activation" by a cellular mechanism that may not be present in yeast. Activation of an agent can include, for example, metabolic processing of the agent or a modification such as phosphorylation of the agent, which can be necessary to confer activity upon the agent.
  • a mammalian cell line can be used to screen a panel of agents (Fearon et al . , supra, 1992).
  • An agent that alters the catalytic activity of an IKK or that alters the association of an IKK subunit or IKK complex and a second protein such as an IKB or an IKK regulatory subunit or an upstream activator of an IKK can be useful as a drug to reduce the severity of a pathology characterized by aberrant NF- ⁇ B activity.
  • a drug that increases the activity of an IKK or that increases the affinity of an IKK catalytic subunit and I ⁇ B ⁇ can increase the amount of I ⁇ B ⁇ phosphorylated on Ser-32 or Ser-36 and, therefore, increase the amount of active NF- ⁇ B and the expression of a gene regulated by NF- ⁇ B, since the drug will increase the level of phosphorylated I ⁇ B ⁇ in the cell, thereby allowing NF- ⁇ B translocation to the nucleus.
  • an antisense IKK subunit molecule of the invention also can be used to decrease IKK activity in a cell by reducing or inhibiting expression of the IKK subunit or by reducing or inhibiting its responsiveness to an inducing agent such as TNF ⁇ , 11-1 or phorbol ester (see Example II) .
  • the invention also provides methods of treating an individual suffering from a pathology characterized by aberrant NF- ⁇ B activity by administering to the individual an agent that modulates the catalytic activity of an IKK or that alters the association of an IKK subunit and a second protein such as IKB or a subunit of a 300 kDa or 900 kDa IKK complex that interacts with the IKK subunit .
  • An agent that decreases the activity of an IKK or otherwise decreases the amount of IKB phosphorylation in a cell can reduce or inhibit NF- ⁇ B mediated gene expression, including, for example, the expression of proinflammatory molecules such as cytokines and other biological effectors involved in an inflammatory, immune or acute phase response.
  • proinflammatory molecules such as cytokines and other biological effectors involved in an inflammatory, immune or acute phase response.
  • the ability to reduce or inhibit such gene expression can be particularly valuable for treating various pathological conditions such as rheumatoid arthritis, asthma and septic shock, which are characterized or exacerbated by the expression of such proinflammatory molecules.
  • Glucocorticoids are potent anti-inflammatory and immunosuppressive agents that are used clinically to treat various pathologic conditions, including autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosis and asthma. Glucocorticoids suppress the immune and inflammatory responses, at least in part, by increasing the rate of I ⁇ B ⁇ synthesis, resulting in increased cellular levels of I ⁇ B ⁇ , which bind to and inactivate NF- ⁇ B (Scheinman et al . , Science 270:283-286 (1995); Auphan et al . , Science 270:286-290 (1995)).
  • glucocorticoids suppress NF- ⁇ B mediated expression of genes encoding, for example, cytokines, thereby suppressing the immune, inflammatory and acute phase responses.
  • glucocorticoids and glucocorticoid- like steroids also are produced physiologically and are required for normal growth and development.
  • prolonged treatment of an individual with higher than physiological amounts of glucocorticoids produces clinically undesirable side effects.
  • an agent that alters the activity of an IKK or that alters the association of an IKK complex or IKK subunit and a second protein can provide a means for selectively altering NF- ⁇ B activity without producing some of the undesirable side effects associated with glucocorticoid treatment.
  • HIV-1 human immunodeficiency virus-1
  • HSV-1 herpes simplex virus-1
  • CMV cytomegalovirus
  • Tat-mediated transcription from the HIV-1 enhancer is decreased if the NF- ⁇ B and SP1 binding sites are deleted from the enhancer/promotor region, indicating that Tat interacts with NF- ⁇ B, SP1 or other transcription factors bound at this site to stimulate transcription (Roulston et al., Microbiol. Rev. 59:481-505 (1995)).
  • chronic HIV-1 infection, and progression to AIDS is associated with the development of constitutive NF- ⁇ B DNA binding activity in myeloid cells (Roulston et al . , supra, 1995) .
  • a positive autoregulatory loop is formed, whereby HIV-1 infection results in constitutively active NF- ⁇ B, which induces expression of HIV-1 genes (Baeuerle and Baltimore, Cell 87:13-20 (1996).
  • Constitutive NF- ⁇ B activation also may protect cells against apoptosis, preventing clearance of virus-infected cells by the immune system (Liu et al . , supra, 1996) .
  • An agent that decreases the activity of an IKK or that alters the association of an IKK and a second protein such that IKB phosphorylation is decreased can be useful for reducing the severity of a viral infection such as HIV-1 infection in an individual by providing increased levels of unphosphorylated IKB in virus- infected cells.
  • the unphosphorylated IKB then can bind to NF- ⁇ B in the cell, thereby preventing nuclear translocation of the NF- ⁇ B and viral gene expression. In this way, the rate of expansion of the virus population can be limited, thereby providing a therapeutic advantage to the individual.
  • the decreased level of NF- ⁇ B activity may allow the virus-infected cell to undergo apoptosis, resulting in a decrease in the viral load in the individual.
  • it can be particularly useful to treat virus-infected cells ex vivo with an agent identified using a method of the invention.
  • peripheral blood mononuclear cells PBMCs
  • PBMCs peripheral blood mononuclear cells
  • Such a treatment can be useful to purge the PBMCs of the virus-infected cells by allowing apoptosis to proceed.
  • the purged population of PBMCs then can be expanded, if desired, and readministered to the individual.
  • Rel/NF- ⁇ B proteins also are involved in a number of different types of cancer.
  • the adhesion of cancer cells to endothelial cells is increased due to treatment of the cancer cells with IL-1, suggesting that NF- ⁇ B induced the expression of cell adhesion molecules, which mediated adherence of the tumor cells to the endothelial cells; agents such as aspirin, which decrease NF- ⁇ B activity, blocked the adhesion by inhibiting expression of the cell adhesion molecules (Tozawa et al . , Cancer Res. 55:4162-4167 (1995)).
  • an agent that decreases the activity of an IKK or that decrease the association of an IKK and I ⁇ B or of an IKK subunit and a second protein, for example, a second protein present in an IKK complex can be useful for reducing the likelihood of metastasis of a tumor in an individual .
  • constitutive NF- ⁇ B activation also may protect tumor cells against programmed cell death as well as apoptosis induced by chemotherapeutic agents (Liu et al . , supra, 1996; Baeuerle and Baltimore, Cell 87:13-20 (1996)).
  • an agent that decreases IKK activity or that decreases the association of IKK and IKB also can be useful for allowing programmed cell death to occur in a tumor cell by increasing the level of unphosphorylated IKB, which can bind NF- ⁇ B and decrease the level of active NF- ⁇ B in the tumor cell.
  • This example provides a method for identifying and isolating a cytokine responsive protein kinase complex that phosphorylates IKB, which regulates NF- ⁇ B activity, and catalytic subunits of the protein kinase complex.
  • the fusion proteins were linked to glutathione SEPHAROSE and the beads were used directly in the assays. At earlier stages in the purification of the IKK activity, the beads were washed prior to loading onto the gel to minimize contributions from other proteins. In some of the later characterization of highly purified material, soluble fusion protein was used.
  • substrates for the IKK activity were used: 1) substrate “WT” contained amino acid residues 1 to 54 of I ⁇ B ⁇ ; 2) substrate “AA” contained amino acid residues 1 to 54 of I ⁇ B ⁇ , except that Ser-32 (S32) and S36 were replaced with Ala-32 (A32) and A36, respectively; and 3) substrate "TT” contained amino acid residues 1 to 54 of I ⁇ B ⁇ , except that S32 and S36 were replaced with Thr-32 (T32) and T36, respectively (DiDonato et al . , Mol. Cell. Biol. 16:1295-1304 (1996)). Each substrate was expressed as a GST fusion protein.
  • the physiologic, inducible I ⁇ B kinase is specific for S32 and S36 (WT) in I ⁇ B ⁇ , but does not recognize the TT or AA mutants (DiDonato et al . , Mol. Cell. Biol. 16:1295-1304 (1996) ) .
  • the protein purification buffer (Buffer A) consisted of 20 mM Tris (pH 7.6, measured at RT) , 20 mM NaF, 20 mM ⁇ -GP, 1 mM PNPP, 500 ⁇ M Na 3 V0 4 , 2 mM DTT,
  • Cell lysis buffer was Buffer A containing an additional 19 mM PNPP, 20 mM ⁇ -GP and 500 ⁇ M Na 3 V0 4 , and 20 ⁇ g/ml aprotinin, 2.5 ⁇ g/ml leupeptin, 8.3 ⁇ g/ml bestatin, 1.7 ⁇ g/ml pepstatin.
  • TNF ⁇ was either recombinant TNF ⁇ , which was purchased from R&D
  • HIS6 -tagged TNF ⁇ which was expressed and partially purified from E. coli and used at 5 ⁇ g/ml.
  • TNF ⁇ -induced HeLa S3 cell killing activity assays were performed in the presence of cycloheximide and indicated that the partially purified HIS6 -tagged TNF ⁇ had approximately one-tenth the activity of the commercial TNF ⁇ .
  • HeLa S3 cells Fifteen liters of HeLa S3 cells were grown in suspension in high glucose Dulbecco's modified Eagle's medium supplemented with 10% calf serum, 2 mg/ml
  • L-glutamine 100 U/ml penicillin/streptomycin, 0.11 mg/ml sodium pyruvate, and IX nonessential amino acids (Irvine Scientific; Irvine CA) .
  • Cell density was approximately 5 x 10 5 cells/ml at the time of collection. Cells were concentrated 10 -fold by centrifugation. stimulated for
  • IKB kinase For purification of IKB kinase, cells were thawed and cytoplasmic extract prepared. Lysis was achieved by 40 strokes in an all glass Dounce homogenizer (pestle A) in lysis buffer containing 0.05% NP-40 on ice. The homogenate was centrifuged at 12,000 rpm for 19 min in a Beckman SS34 rotor at 4°C.
  • S100 fraction was quick frozen in liquid nitrogen and stored at -80°C.
  • Small aliquots of S100 material prepared from either unstimulated HeLa cells or from TNF ⁇ stimulated cells, were purified ih a single passage over a SUPEROSE 6 gel filtration column (1.0 x 30 cm; Pharmacia; Uppsalla Sweden) equilibrated in Buffer A containing 0.1% Brij-35 and 300 mM NaCl and eluted at a flow rate of 0.3 ml/min. 0.6 ml fractions were collected and kinase assays were performed on an aliquot of each fraction.
  • the high molecular weight material (fractions 16-20) contained TNF ⁇ -inducible IKK activity, which is specific for the WT substrate.
  • the pooled material was diluted to 390 ml by addition of Buffer A containing 0.1 % Brij-35 and loaded onto a pre-equilibrated 5 ml HITRAP Q column (Pharmacia) at a flow rate of 4 ml/min. Following sample loading, the column was washed with 20 ml of Buffer A containing 0.1 % Brij-35. The protein was eluted at 1 ml/min isocratically in Buffer A containing 0.1 % Brij-35 and 300 mM NaCl and 1 ml fractions were collected. Protein- containing fractions were identified using the BioRad assay and were collected and pooled to yield 4 ml of solution. Previously performed control experiments demonstrated that the IKK activity directly correlated with protein concentration.
  • the pooled material was diluted 1:1 with ATP column buffer (20 mM HEPES (pH 7.3), 50 mM ⁇ -GP, 60 mM MgCl 2 , 1 mM Na 2 V0 4 , 1.5 mM EGTA, 1 mM DTT, 10 ⁇ g/ml aprotinin) , then passed 4 times over a ⁇ -ATP affinity column having 4 ml bed volume (Haystead et al . , supra, 1993); the column had been prewashed with 2 M NaCl, 0.25% Brij-35 and equilibrated with 10 bed volumes of ATP column buffer containing 0.05% Brij-35 at a flow rate of 0.5 ml/min. Following loading of the sample, the column was washed with 10 ml of ATP column buffer containing 0.05% Brij-35, then with 10 ml ATP column buffer containing 0.05% Brij-35 and 250 mM NaCl.
  • ATP column buffer 20
  • Bound material was eluted in 10 ml of ATP column buffer containing 0.05 % Brij-35, 250 mM NaCl and 10 mM ATP (elution buffer) . Elution was performed by passing 5 ml of elution buffer through the column, allowing the column to incubate, capped, for 20 min, then passing an additional 5 ml of elution buffer through the column. The samples were pooled to yield 10 ml.
  • the 10 ml pooled sample from the ATP column was diluted with 30 ml Buffer A containing 0.1 % Brij-35 and loaded onto a 1 ml HITRAP Q column (Pharmacia) at 1 ml/min.
  • the column was eluted at 0.4 ml/min with Buffer A containing 0.1 % Brij-35 and 300 mM NaCl.
  • 0.2 ml fractions were collected and the four protein- containing fractions were pooled (0.5 mg) .
  • the pooled material was concentrated to 200 ⁇ l on a 10K NANOSEP concentrator (Pall/Filtron) and loaded onto a SUPEROSE 6 gel filtration column (1.0 x 30 cm) .
  • the SUPEROSE 6 column was equilibrated in Buffer A containing 0.1 % Brij-35 and 300 mM NaCl and run at a flow rate of 0.3 ml/min; 0.6 ml fractions were collected. Fractions 17, 18 and 19 contained kinase activity.
  • the final purified material consisted of approximately 20 ⁇ g to 40 ⁇ g of total protein, of which approximately 2 ⁇ g corresponded to the 85 kDa band, later designated IKK ⁇ (see Example II) .
  • a second band migrating at 87 kDa was later designated IKK ⁇ (see Example III) .
  • the total time from the thawing of the S100 material until the collection of fractions from the gel filtration column was 24 hours.
  • the 85 kDa IKK ⁇ band identified by the kinase assay following the above procedure contained only about 10% of the total purified protein, three additional criteria were used to confirm that the identified band was an intrinsic component of the IKK complex.
  • the elution profile of the SUPEROSE 6 column was analyzed by silver stained 8% SDS-PAGE gels, then compared to the kinase activity profile. For this analysis, 0.3 ml fractions were collected from the SUPEROSE 6 column, then separated by
  • IKK-containing material was diluted into Buffer A to yield a final concentration of 70 mM NaCl, 0.025% Brij-35, then added to the substrate affinity resin at a ratio of 4:1 (solution: swollen beads).
  • the resin was suspended and the mixture rotated gently overnight in a small column at 4°C. The resin was allowed to settle for 30 min, then the column was eluted by gravity. The column was washed with 4 bed volumes Buffer A containing 0.02% Brij-35, then the resin was suspended with 1.1 bed volumes of Buffer A containing 600 mM NaCl and 0.1 % Brij-35. The resin was allowed to settle for 40 min, then gravity elution was performed. The column was washed with an additional 1.1 bed volumes of Buffer A containing 600 mM NaCl and 0.1 % Brij-35 and the two fractions were pooled.
  • the I ⁇ B ⁇ substrate affinity column was used for two separate experiments. In one experiment, the material that eluted from the final SUPEROSE 6 column was further purified on the I ⁇ B ⁇ substrate affinity column. In the second experiment, material obtained after the initial Q-SEPHAROSE column was purified on the I ⁇ B ⁇ substrate affinity column. The Q-SEPHAROSE bound fraction then was further purified on the ATP column and the SUPEROSE 6 column (see above) .
  • Cytoplasmic extract was prepared using HeLa S3 cells. The cells were stimulated with TNF ⁇ for 5 min, then harvested in lysis buffer containing 0.1 % NP-40 and 0.15 M NaCl. Reactivation was performed at 30°C in kinase buffer for 60 min in the absence of ( ⁇ - 32 P)ATP. Samples containing only cold ATP were used for kinase activity assays. Reactivation by the HeLa cell extract was performed in the presence of ( ⁇ - 32 P)ATP, then the sample was separated by 8% SDS-PAGE and examined by autoradiography . A band of approximately 86 kDa was phosphorylated in the reactivated material and, associated with the reactivation procedure, was restoration of the IKK activity. D. Partial amino acid sequences of IKK ⁇ and IKK ⁇
  • the 85 kDa IKK ⁇ and 87 kDa IKK ⁇ bands were excised from the gel and submitted for internal peptide sequencing analysis. From the IKK ⁇ polypeptide, the sequences of two proteolytic fragments were identified, as follows: KIIDLLPK (SEQ ID NO: 3) and KHR (D/A) LKPENIVLQDVG (P/G) K (SEQ ID NO: 4) . Where a residue could not be unambiguously determined, an "X" was used to indicate no amino acid could be determined and parentheses were used to delimit amino acids that could not be distinguished.
  • Lys-C protease was used to digest the protein, the presence of lysine residues at the N-termini of the peptides was inferred. From the 87 kDa IKK ⁇ band, the sequences of five proteolytic fragments were determined (see Figure 3, underlined; see, also, Example III).
  • This example provides methods for isolating a nucleic acid molecule encoding the IKK ⁇ subunit and for characterizing the functional activity of the subunit.
  • oligonucleotide (length) sequences of the amino acid sequences of two peptide fragments (SEQ ID NOS: 3 and 4) of the IKK ⁇ were searched in the GenBank DNA sequence database. This search revealed that nucleotide sequences encoding both peptide fragments were present in a partial cDNA encoding a portion of a protein designated human CHUK (GenBank Accession #U22512; Connelly and Marcu, supra, 1995).
  • PCR primers were prepared corresponding to the 5 ' -terminus (5' -CCCCATATGTACCAGCATCGGGAA-3 ' ; SEQ ID NO: 5) and 3 '-terminus (3 ' -CCCCTCGAGTTCTGTTAACCAACT-5 ' ; SEQ ID NO: 6) .
  • SEQ ID NO: 5 also contains a Nde I restriction endonuclease site (underlined) and an ATG (AUG) methionine codon (bold) and SEQ ID NO: 6 also contains an Xho I site.
  • RNA was isolated from HeLa cells and first strand cDNA was prepared and used for a template by PCR using SEQ ID NOS: 5 and 6 as primers. The resulting
  • kilobase (kb) fragment was gel purified, 32 P-labeled using oligo-dT and random primers, and used to screen a human fetal brain library (Clontech; Palo Alto CA) under high stringency conditions (50% formamide, 42°C; Sambrook et al., supra, 1989).
  • mouse CHUK N-terminus as compared to human CHUK.
  • the human IKK ⁇ shares a high amount of sequence identity with a protein designated mouse CHUK (GenBank Accession #U12473; Connelly and Marcu, supra, 1995) .
  • mouse CHUK contains a domain having characteristics of a serine-threonine protein kinase, no functional activity of the protein was reported and no potential substrates were identified.
  • the putative serine-threonine protein kinase domain of human CHUK was truncated at the N-terminus.
  • the full length IKK ⁇ cDNA and a cDNA encoding the ⁇ 31 human CHUK protein were subcloned into the Nde I and Xho I sites of a bacterial expression vector encoding a carboxy terminal FLAG epitope and HIS6 tag.
  • Mammalian cell expression vectors were constructed by cleaving the bacterial expression vector with Nde I and Hind III, to release the cDNA inserts, converting the ends of the inserts to blunt ends using Klenow polymerase, and ligating the cDNA inserts encoding the full length IKK ⁇ or the ⁇ 31 human CHUK into pCDNA3 (Invitrogen) .
  • the IKK ⁇ cDNA and ⁇ 31 cDNA were subcloned into the Bst XI site of the pRc ⁇ actin vector (DiDonato et al . , supra, 1996) .
  • Orientation of the inserts was determined by restriction endonuclease mapping and partial sequence using vector-specific primers.
  • Vector containing the cDNA's inserted in the sense orientation were examined for expression of the encoded product by immunoblot analysis using an antibody specific for the FLAG epitope.
  • Transfection experiments were performed to determine the effect of expressing the cloned IKK ⁇ in HeLa cells or of expressing the cloned IKK ⁇ cDNA in the antisense orientation.
  • HeLa cells were split into 35 mm dishes to approximately 50% confluency.
  • Cells were transfected with 0.25 ⁇ g of a luciferase reporter gene containing an IL-8 promotor (Eckman et al . , Amer. Soc. Clin. Invest.
  • Transfected cells were incubated in DMEM containing 10% FBS for 24 hr. The cells then were washed and the growth medium was replaced with DMEM containing 0.1% FBS. Cells either were left untreated, or were treated with 20 ng/ml TNF ⁇ , 20 ng/ml IL-l ⁇ , or 100 ng/ml TPA (phorbol ester) for 3.5 hr. Cells were harvested by scraping and washed once with PBS, then lysed in 100 ⁇ l PBS containing 1% TRITON-X100. Luciferase assays were performed using 20 ⁇ l of lysate (DiDonato et al . , supra, 1995) . The protein concentration of each extract was determined using the BIORAD protein assay kit and luciferase activity was normalized according to the protein concentrations.
  • NF-KB is known to induce expression for the IL-8 promotor.
  • treatment of the vector transfected control cells with TNF ⁇ , IL-l ⁇ or TPA resulted in a 3- to 5-fold increase in normalized luciferase activity.
  • treatment with TNF ⁇ , IL-l ⁇ or TPA potentiated induction of luciferase activity 5- to 6-fold above the level of induction observed in the vector transfected cells.
  • This example provides methods for isolating a nucleic acid molecule encoding an IKK ⁇ catalytic subunit of IKK and characterizing the activity of the IKK ⁇ subunit .
  • IKK ⁇ was purified following SDS-PAGE and subjected to internal peptide sequencing (Example I) .
  • KXXIQQD(T/A)GIP SEQ ID NO: 11
  • KXRVIYTQL SEQ ID NO: 12
  • KXEEWSLMNEDEK SEQ ID NO: 13
  • amino acid residues that could not be unambiguously determined are indicated by an "X” and where amino acids that could not be distinguished are shown in parentheses.
  • These peptide sequences were used to screen the NCBI EST database and a 336 base pair EST (EST29518; Accession No. AA326115) encoding SEQ ID NOS: 12 and 13 was identified. This EST was determined to correspond to amino acid residues 551 to 661 of SEQ ID NO: 15.
  • cDNA corresponding to the EST was obtained by PCR using first strand HeLa cDNA as a template and used to probe a human fetal brain library (Clontech) .
  • a 1 kb fragment was identified and used as a probe to screen a plasmid based B cell library (Invitrogen) .
  • a 3 kb cDNA insert was isolated and sequenced ( Figure 2; SEQ ID NO: 14) and encoded the full length IKK ⁇ (SEQ ID NO: 15), including all five proteolytic fragments (see Figure 3) .
  • SEQ ID NO: 15 contains a kinase domain, which shares 65% amino acid identity with IKK ⁇ , a leucine zipper and a helix-loop-helix domain. Based on the sequence homology and domain structure, the polypeptide (SEQ ID NO: 15) was determined to be a member of the IKK catalytic subunit family of proteins with IKK ⁇ and, therefore, was designated IKK ⁇ .
  • the kinase activity associated with IKK ⁇ was characterized using HeLa or 293 cells transiently transfected with an HA-tagged IKK ⁇ expression vector. Transfected cells were stimulated with 20 ng/ml TNF for 10 min and HA- IKK ⁇ was isolated by immunoprecipitation using anti-HA antibody (Kolodziej and Young, Meth. Enzymol . 194:508-519 (1991)). The immune complexes were tested for the ability to phosphorylate wild type (wt) and mutant forms of I ⁇ B ⁇ and I ⁇ B ⁇ (see Example I) .
  • the IKK ⁇ immune complex phosphorylated wt I ⁇ B ⁇ and I ⁇ B ⁇ , but not mutants in which the inducible phosphorylation sites (Ser-32 and Ser-36 for I ⁇ B ⁇ and Ser-19 and Ser-23 for I ⁇ B ⁇ ) were replaced with either alanines or threonines.
  • Ser-32 and Ser-36 for I ⁇ B ⁇ and Ser-19 and Ser-23 for I ⁇ B ⁇ were replaced with either alanines or threonines.
  • a low level of residual phosphorylation of full length I ⁇ B ⁇ was observed due to phosphorylation of sites in the C-terminal portion of the protein (DiDonato et al . , supra, 1997) .
  • the response of IKK ⁇ -associated kinase activity to various stimuli also was examined in HeLa cells transiently transfected with the HA- IKK ⁇ expression vector. After 24 hr, the cells were stimulated with either 10 ng/ml IL-1, 20 ng/ml TNF or 100 ng/ml TPA, then HA- IKK ⁇ immune complexes were isolated by immunoprecipitation and IKK activity was measured. TNF and IL-1 potently stimulated IKK ⁇ -associated kinase activity, whereas the response to TPA was weaker.
  • the kinetics of IKK ⁇ activation by either TNF or IL-1 essentially were identical to the kinetics of activation of the IKK ⁇ -associated IKB kinase measured by a similar protocol .
  • IKK ⁇ and IKK ⁇ copurified in about a 1:1 ratio through several chromatographic steps, suggesting that the two proteins interact with each other.
  • the ability of the IKK subunits to interact in a functional complex and the effect of each subunit on the activity of the other subunit was examined using 293 cells transfected with expression vectors encoding Flag (M2) -IKK ⁇ or M2-IKK ⁇ and HA- IKK ⁇ , either alone or in combination (see Hopp et al . , BioTechnology 6:1204-1210 (1988)) .
  • samples of the cells were stimulated with TNF, lysates were prepared from stimulated and unstimulated cells, and one portion of the lysates was precipitated with anti-Flag antibodies (Eastman Kodak Co.; New Haven CT) and another portion was precipitated with anti-HA antibodies.
  • anti-Flag antibodies Eastman Kodak Co.; New Haven CT
  • the levels of IKK activities associated with IKK ⁇ and IKK ⁇ were compared more precisely by transfecting 293 cells with increasing amounts of HA- IKK ⁇ or HA-IKK ⁇ expression vectors (0.1 to 0.5 ⁇ g/10 6 cells) and determining the kinase activities associated with the two proteins in cell lysates prepared before or after TNF stimulation (20 ng/ml, 5 min); GST-I ⁇ B ⁇ (1-54) was used as substrate.
  • the level of expression of each protein was determined by immunoblot analysis and used to calculate the relative levels of specific IKK activity.
  • the HA-IKK ⁇ -associated IKK had a low level of basal specific activity, whereas expression of HA- IKK ⁇ resulted in high basal specific activity that was increased when higher amounts of HA- IKK ⁇ were expressed.
  • the specific IKK activity associated with either IKK ⁇ or IKK ⁇ isolated from TNF-stimulated cells was very similar and was not considerably affected by their expression level.
  • IKK ⁇ and IKK ⁇ were further examined by transfecting HeLa cells with various amounts (0.1 to 1.0 ⁇ g/10 6 cells) of the HA-IKK ⁇ vector. After 24 hr, the cells were incubated for 5 min in the absence or presence of 20 ng/ml TNF, then lysed. The lysates were examined for IKK activity and for the amount of HA-IKK ⁇ and endogenous IKK ⁇ . Expression of increasing amounts of HA-IKK ⁇ resulted in higher basal levels of IKK activity and increasing amounts of coprecipitated IKK ⁇ . The level of TNF stimulated IKK activity increased only marginally in response to IKK ⁇ overexpression and TNF had no effect on the association of IKK ⁇ and IKK ⁇ .
  • each column fraction was immunoprecipitated with a polyclonal antibody specific for IKK ⁇ and assayed for IKK ⁇ -associated IKK activity, while a second portion was precipitated with anti-HA antibody and examined for HA-IKK ⁇ - or HA-IKK ⁇ -associated IKK activity.
  • Relative specific activity was determined by immunoprecipitating the complexes, separating the proteins by SDS-PAGE, blotting the proteins onto IMOBILON membranes (Millipore; Bedford MA) , immunoblotting with anti-HA antibody and quantitating the levels of IKB phosphorylation and HA- tagged proteins by phosphoimaging.
  • HA- IKK ⁇ -associated IKK activity had exactly the same distribution as the IKK ⁇ -associated activity, eluting at 900 kDa and 300 kDa and, again, the extent of TNF responsiveness was considerably greater for the 900 kDa complex.
  • Comparison to the IKK ⁇ -associated activity in cells transfected with the empty vector indicated that HA-IKK ⁇ expression produced a modest, approximately 2 -fold increase in the relative amount of IKK activity associated with the smaller 300 kDa complex.
  • the 300 kDa IKK complex like the 900 kDa complex, contains both IKK ⁇ and IKK ⁇ .
  • the 300 kDa lacks other subunits present in the 900 kDa complex.
  • IKK ⁇ was overexpressed, the relative amount of the smaller complex increased, indicating that some of the subunits that are unique to the larger complex are present in a limited amount.
  • IKK activity was examined by constructing mutant subunits in which the lysine (K) codon present at position 44 of each subunit was substituted with a codon for either methionine (M) or alanine (A) codon, respectively. Similar mutations in other protein kinases render the enzymes defective in binding ATP and, therefore, catalytically inactive (Taylor et al . , Ann. Rev. Cell Biol. 8:429-462 (1992)) . The activity of the IKK mutants was compared to the activity of their wild type (wt) counterparts by cell-free translation in reticulocyte lysates using GST-I ⁇ B (1-54) as a substrate.
  • HA-IKK ⁇ (KM) resultsed in isolation of cytokine stimulated IKK activity that, after TNF stimulation, was 2-to 3-fold lower than the activity of IKK formed by wt HA- IKK ⁇ isolated from TNF-stimulated cells.
  • expression and immunoprecipitation of HA-IKK ⁇ resulted in formation of a cytokine responsive IKK activity that, after TNF stimulation, was 3- to 5-fold lower than the activity of IKK generated by wt HA-IKK ⁇ isolated from TNF stimulated cells.
  • IKK ⁇ and IKK ⁇ both contain leucine zipper (LZ) and helix- loop-helix (HLH) motifs, which are known to mediate protein-protein interactions through their hydrophobic surfaces.
  • LZ leucine zipper
  • HLH helix- loop-helix
  • the role of the LZ motif in the IKK subunit interaction was examined using an IKK ⁇ mutant in which the L462 and L469 residues within the LZ region were substituted with serine residues.
  • the role of the HLH motif was examined using an HLH mutant of IKK ⁇ containing a substitution of L605 with arginine (R) and of F606 with proline (P) .
  • R arginine
  • P proline
  • the activity of the IKK ⁇ LZ " and HLH " mutants was examined by transient transfection in 293 cells, either alone or in the presence of cotransfected Flag- IKK ⁇ .
  • IKK ⁇ (LZ) " mutant is due to a defect in its ability to interact with IKK ⁇ .
  • the lower IKB kinase activity of the IKK ⁇ (HLH) " mutant likely is due to a defect in the ability to interact with a second, undefined protein, since the HLH mutant can interact with IKK ⁇ .
  • IKK ⁇ and IKK ⁇ were examined using HeLa cells transfected with expression vectors encoding HA-tagged wt IKK ⁇ , IKK ⁇ (KM), wt IKK ⁇ and IKK ⁇ (KA) ; an HA-JNK1 vector was used as a control.
  • NF- ⁇ B activation was assessed by examining the subcellular distribution of RelA(p65) by indirect immunofluorescence .
  • HeLa cells were grown on glass cover slips in growth medium, then transfected with 1 ⁇ g plasmid DNA by the lipofectamine method. After 24 hr, samples of cells were stimulated with 20 ng/ml TNF for 30 min, then stimulated or unstimulated cells were washed with PBS and fixed with 3.5% formaldehyde in PBS for 15 min at room temperature (RT) . The fixed cells were permeablized with 0.02% NP-40 in PBS for 1 min, then incubated with 100% goat serum at 4°C for 12 hr.
  • the cells then were washed 3 times with PBS and incubated with a mixture of a rabbit anti-NF-KB p65 (RelA) antibody (1:100 dilution; Santa Cruz Biotech) and a mouse monoclonal anti-HA antibody in PBS containing 1% BSA and 0.2% TRITON X-100 at 37 °C for 2 hr.
  • Cells then were washed 3 times with PBS containing 0.2% TRITON X-100 and incubated for 2 hr at RT with secondary antibodies, fluorescein-conjugated goat affinity purified anti-mouse IgG-IgM and rhodamine- conjugated IgG fraction goat anti-rabbit IgG (1:200 dilution; Cappel) .
  • the wt IKK proteins did not interfere with the nuclear translocation of RelA induced by TNF treatment.
  • This example demonstrates a method for isolating the 900 kDa IKB kinase complex comprising an IKK ⁇ polypeptide.
  • HIS6-FLAG- IKK ⁇ (HF-IKK ⁇ ) encoding construct was prepared using a double stranded oligonucleotide, 5 ' -AGCTTGCGCGTATGGCTTCGGGTCATCACCATCACCA TCACGGTGACTACAAGGACGACGATGACAAAGGTGACATCGAAGGTAGAGGTCA-3 ' (SEQ ID NO: 16) , which encodes six histidine residues (HIS6) , the FLAG epitope and the factor Xa site in tandem.
  • the oligonucleotide was inserted using Hindlll- Ndel site in frame with the N-terminus of the IKK ⁇ coding sequence in the BLUESCRIPT KS plasmid (Stratagene;
  • a 293 cell line that expresses HF-IKK ⁇ was selected and expanded to approximately 4 x 10 8 cells.
  • the cells were treated with 10 ng/ml TNF ⁇ for 5 min, then harvested in ice cold PBS by centrifugation at 2500 x g.
  • the cell pellet was washed with ice cold PBS, resuspended in lysis buffer (20 mM Tris, pH 7.6), 150 mM NaCl,
  • the homogenate was centrifuged at 15,000 rpm in a Beckman SS34 rotor for 30 min at 4°C. The supernatant was collected, supplemented with 20 mM imidazole and 300 mM NaCl, then mixed with 0.5 ml of a 50% slurry of Ni-NTA (nickel nitrilotriacetic acid; Qiagen, Inc.;
  • Proteins bound to the resin were eluted in 2 ml binding buffer containing 150 mM imidazole and 20 mM DTT. The eluate was mixed with 100 ⁇ l of a 50% slurry of anti-FLAG antibody coupled to SEPHAROSE resin using the AMINOLINK PLUS immobilization kit (Pierce Chem. Co.; Rockford IL) and stirred for 4 hr at 4°C. The resin was pelleted at 1000 x g, the supernatant was removed, and the resin was washed with 10 ml binding buffer (without imidazole) . Proteins bound to the resin then were eluted with 1% SDS or with FLAG peptide and examined by 10% SDS- PAGE.
  • This example provides a method of producing anti-IKK antisera.
  • Anti-IKK ⁇ antibodies were raised in rabbits using either His-tagged IKK ⁇ expressed in E. coli or the IKK ⁇ peptide ERPPGLRPGAGGPWE (SEQ ID NO: 17) or TIIHEAWEEQGNS (SEQ ID NO: 18) as an immunogen.
  • Anti-IKK ⁇ antibodies were raised using the peptide SKVRGPVSGSPDS (SEQ ID NO: 19) .
  • the peptides were conjugated to keyhole limpet hemocyanin (Sigma Chemical Co.; St. Louis MO). Rabbits were immunized with 250 to 500 ⁇ g conjugated peptide in complete Freund's adjuvant.
  • booster immunizations were performed using 50 to 100 ⁇ g immunogen and were repeated three times, at 3 to 4 week intervals. Rabbits were bled one week after the final booster and antisera were collected. Anti-IKK ⁇ antiserum was specific for IKK ⁇ and did not cross react with IKK ⁇ .
  • This example describes an assay for screening for agents such as drugs that alter the association of an IKK subunit and a second protein that specifically associates with the IKK subunit.
  • a GST-IKK subunit fusion protein or HIS6-IKK subunit fusion protein can be prepared using methods as described above and purified using glutathione- or metal- chelation chromatography, respectively (Smith and Johnson, Gene 67:31-40 (1988), which is incorporated herein by reference; see, also, Example IV) .
  • the fusion protein is immobilized to a solid support taking advantage of the ability of the GST protein to specifically bind glutathione or of the HIS6 peptide region to chelate a metal ion such as nickel (Ni) ion or cobalt (Co) ion (Clontech) by immobilized metal affinity chromatography.
  • a metal ion such as nickel (Ni) ion or cobalt (Co) ion (Clontech) by immobilized metal affinity chromatography.
  • an anti-IKK antibody can be immobilized on a matrix and the IKK- ⁇ can be allowed to bind to the antibody.
  • the second protein which can be IKB or a protein that copurifies with IKK subunit as part of the 900 kDa IKB kinase, for example, can be detectably labeled with a moiety such as a fluorescent molecule or a radiolabel (Her anson, supra , 1996) , then contacted in solution with the immobilized IKK subunit under conditions as described in Example I, which allow IKB to specifically associate with the IKK subunit.
  • the reactions are performed in 96 well plates, which allow automated reading of the reactions.
  • Various agents such as drugs then are screened for the ability to alter the association of the IKK subunit and IKB.
  • the agent and labeled IKB can be added together to the immobilized IKK subunit, incubated to allow binding, then washed to remove unbound labeled IKB.
  • the relative amount of binding of labeled IKB in the absence as compared to the presence of the agent being screened is determined by detecting the amount of label remaining in the plate. Appropriate controls are performed to account, for example, for nonspecific binding of the labeled IKB to the matrix.
  • Such a method allows the identification of an agent that alter the association of an IKK subunit and a second protein such as IKB.
  • the labeled IKB or other appropriate second protein can be added to the immobilized IKK subunit and allowed to associate, then the agent can be added.
  • the agent can be added.
  • a method allows the identification of agents that can induce the dissociation of a bound complex comprising the IKK subunit and IKB.
  • a screening assay of the invention can be performed using the 900 kDa IKK complex, comprising an IKK subunit.

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Abstract

La présente invention concerne des molécules d'acides nucléiques isolées, codant des polypeptides de la sous-unité catalytique de la kinase IλB (IKK), qui sont associées à une protéine kinase sérine phosphorylant une protéine (IλB) qui inhibe l'activité du facteur de transcription NF-λB; des vecteurs contenant ces molécules d'acides nucléiques; et des cellules hôtes contenant ces vecteurs. En outre, l'invention concerne des séquences nucléotidiques qui peuvent se fixer sur une molécule d'acides nucléiques de l'invention, ces séquences nucléotidiques étant utilisées comme sondes ou comme molécules anti-sens. L'invention concerne également des sous-unités catalytiques isolées de l'IKK qui peuvent phosphoryler une protéine IλB et des parties peptidiques de cette sous-unité de l'IKK. En outre, l'invention traite d'anticorps anti-IKK, qui se fixent de manière spécifique à un complexe de l'IKK ou à une sous-unité catalytique de l'IKK, ainsi que des fragments se fixant sur l'IKK de ces anticorps. L'invention concerne par ailleurs des procédés de purification sensible d'un complexe de l'IKK, des procédés d'identification d'un agent qui peut modifier l'association d'un complexe de l'IKK, ou d'une sous-unité catalytique de l'IKK, avec une seconde protéine, et des procédés d'identification de protéines qui peuvent interagir avec un complexe de l'IKK ou une sous-unité catalytique de l'IKK.
EP98908673A 1997-02-25 1998-02-23 Kinase inhibitrice de nf-kappa b kappa b, sous-unites de la kinase kappa b et procedes d'utilisation Ceased EP0981642A4 (fr)

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PCT/US1998/003511 WO1998037228A1 (fr) 1997-02-25 1998-02-23 KINASE INHIBITRICE DE NF-λB (IλB), SOUS-UNITES DE LA KINASE IλB ET PROCEDES D'UTILISATION

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Families Citing this family (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2249551A1 (fr) 1996-03-19 1997-09-25 Proscript, Inc. Kinase permettant la phosphorylation stereospecifique de la i.kappa.b.alpha.kinase
US6235512B1 (en) * 1997-07-01 2001-05-22 Tularik Inc. IKK-α proteins, nucleic acids and methods
US5851812A (en) * 1997-07-01 1998-12-22 Tularik Inc. IKK-β proteins, nucleic acids and methods
US6087169A (en) 1997-08-04 2000-07-11 Smithkline Beecham Plc HKABY60: polynucleotide encoding a helix-loop-helix ubiquitous kinase family polypeptide
EP0897009A3 (fr) * 1997-08-04 1999-06-09 Smithkline Beecham Plc HKABY60 polypeptides
US6030834A (en) * 1997-12-30 2000-02-29 Chiron Corporation Human IKK-beta DNA constructs and cells
AU6189999A (en) * 1998-10-15 2000-05-08 Bioimage A/S Specific therapeutic interventions obtained by interference with redistribution and/or targetting
US5962673A (en) * 1998-11-20 1999-10-05 Isis Pharmaceuticals Inc. Antisense modulation of inhibitor-kappa B kinase-alpha expression
US5977341A (en) * 1998-11-20 1999-11-02 Isis Pharmaceuticals Inc. Antisense modulation of inhibitor-kappa B kinase-beta expression
FR2787119A1 (fr) * 1998-12-09 2000-06-16 Inst Nat Sante Rech Med Procede de criblage en levure d'inhibiteurs de proteines kinases specifiques de cellules humaines et de mammiferes
US6468755B1 (en) 1999-08-10 2002-10-22 Joslin Diabetes Center, Inc. Method for identifying compounds for treatment of insulin resistance
US6630312B2 (en) 1999-08-10 2003-10-07 Joslin Diabetes Center, Inc. Method for identifying compounds for treatment of insulin resistance
US6642215B2 (en) * 2001-05-24 2003-11-04 Leo Pharma A/S Method of modulating NF-kB activity
KR101889140B1 (ko) * 2015-10-12 2018-08-17 연세대학교 산학협력단 p65의 전사 조절 도메인과 단백질 운반 도메인을 포함하는 신규 융합 단백질 및 이의 용도
CN115819603A (zh) * 2022-12-26 2023-03-21 广西大学 一种猪IKKα多克隆抗体的制备方法和应用

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998008955A1 (fr) * 1996-08-26 1998-03-05 Signal Pharmaceuticals, Inc. Agent caracterise par un signal i(kappa)b kinase [ikk] inductible par stimulus
WO1999001542A1 (fr) * 1997-07-01 1999-01-14 Tularik Inc. PROTEINES IKK-β, ACIDES NUCLEIQUES ET PROCEDES
EP0897009A2 (fr) * 1997-08-04 1999-02-17 Smithkline Beecham Plc HKABY60 polypeptides

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998008955A1 (fr) * 1996-08-26 1998-03-05 Signal Pharmaceuticals, Inc. Agent caracterise par un signal i(kappa)b kinase [ikk] inductible par stimulus
WO1999001542A1 (fr) * 1997-07-01 1999-01-14 Tularik Inc. PROTEINES IKK-β, ACIDES NUCLEIQUES ET PROCEDES
EP0897009A2 (fr) * 1997-08-04 1999-02-17 Smithkline Beecham Plc HKABY60 polypeptides

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
CHEN Z J ET AL: "SITE-SPECIFIC PHOSPHORYLATION OF IKAPPABALPHA BY A NOVEL UBIQUITINATION-DEPENDENT PROTEIN KINASE ACTIVITY" CELL, CELL PRESS, CAMBRIDGE, NA, US, vol. 84, 22 March 1996 (1996-03-22), pages 853-862, XP002037788 ISSN: 0092-8674 *
CONNELLY M A ET AL: "CHUK, A NEW MEMBER OF THE HELIX-LOOP-HELIX AND LEUCINE ZIPPER FAMILIES OF INTERACTING PROTEINS, CONTAINS A SERINE-THREONINE KINASE CATALYTIC DOMAIN" CELLULAR AND MOLECULAR BIOLOGY RESEARCH, vol. 41, no. 6, 1995, pages 537-549, XP002914743 *
REGNIER C H ET AL: "IDENTIFICATION AND CHARACTERIZATION OF AN IKAPPAB KINASE" CELL, CELL PRESS, CAMBRIDGE, NA, US, vol. 90, no. 2, 25 July 1997 (1997-07-25), pages 373-383, XP002053258 ISSN: 0092-8674 *
See also references of WO9837228A1 *

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CA2281955A1 (fr) 1998-08-27
AU6664698A (en) 1998-09-09
JP2001524813A (ja) 2001-12-04
WO1998037228A1 (fr) 1998-08-27
JP2008115164A (ja) 2008-05-22
AU740622B2 (en) 2001-11-08
EP0981642A4 (fr) 2003-03-19
JP4125379B2 (ja) 2008-07-30
CA2281955C (fr) 2009-09-08

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