WO1998008955A1 - Stimulus-inducible i (kappa)b kinase [ikk] signalsome - Google Patents

Stimulus-inducible i (kappa)b kinase [ikk] signalsome Download PDF

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Publication number
WO1998008955A1
WO1998008955A1 PCT/US1997/015003 US9715003W WO9808955A1 WO 1998008955 A1 WO1998008955 A1 WO 1998008955A1 US 9715003 W US9715003 W US 9715003W WO 9808955 A1 WO9808955 A1 WO 9808955A1
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ikk
leu
glu
signalsome
asp
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PCT/US1997/015003
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French (fr)
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Frank Mercurio
Hengyi Zhu
Miguel Barbosa
Gian Li
Brion W. Murray
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Signal Pharmaceuticals, Inc.
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Priority claimed from US08/697,393 external-priority patent/US5972674A/en
Application filed by Signal Pharmaceuticals, Inc. filed Critical Signal Pharmaceuticals, Inc.
Priority to JP51184098A priority Critical patent/JP4695735B2/en
Priority to EP97938616A priority patent/EP0920518A1/en
Priority to CA2263835A priority patent/CA2263835C/en
Priority to AU40904/97A priority patent/AU726383B2/en
Publication of WO1998008955A1 publication Critical patent/WO1998008955A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates generally to compositions and methods useful for the study of cascades leading to the activation of nuclear factor KB (NF-KB) and for treating diseases associated with such pathways.
  • the invention is more particularly related to a stimulus-inducible I ⁇ B kinase (IKK) signalsome, component I ⁇ B kinases and variants of such kinases.
  • IKK stimulus-inducible I ⁇ B kinase
  • the present invention is also related to the use of a stimulus-inducible IKK signalsome or I ⁇ B kinase to identify antibodies and other agents that inhibit or activate signal transduction via the NF- ⁇ B pathway.
  • Transcription factors of the NF ⁇ B/Rel family are critical regulators of genes involved in inflammation, cell proliferation and apoptosis (for reviews, see Verma et al., Genes Dev. 9:2723-35, 1995; Siebenlist, Biochim. Biophys. Ada 1332:7-13, 1997; Baeuerle and Henkel, Ann. Rev. Immunol. 72: 141-79, 1994; Barnes and Karin, New Engl. J. Med. 336. 1066-71 , 1997; Baeuerle and Baltimore, Cell 87: 13-20, 1996; Grilli et al., NF-kB and Rel: Participants in a multiform transcriptional regulatory system (Academic Press, Inc., 1993), vol.
  • NFKB The prototype member of the family, NFKB, is composed of a dimer of p50 NFKB and p65 RelA (Baeuerle and Baltimore, Cell 55:211-17, 1988; Baeuerle and Baltimore, Genes Dev. 3:1689-98, 1989).
  • NF- ⁇ B plays a pivotal role in the highly specific pattern of gene expression observed for immune, inflammatory and acute phase response genes, including interleukin 1. interleukin 8, tumor necrosis factor and certain cell adhesion molecules.
  • NF- ⁇ B is sequestered in an inactive form in the cytoplasm of most cell types.
  • extracellular stimuli including mitogens, cytokines. antigens, stress inducing agents. UV light and viral proteins initiate a signal transduction pathway that ultimately leads to NFKB release and activation.
  • inhibitors and activators of the signal transduction pathway may be used to alter the level of active NF- ⁇ B, and have potential utility in the treatment of diseases associated with NF- ⁇ B activation.
  • Activation of NFKB in response to each of these stimuli is controlled by an inhibitory subunit, I ⁇ B, which retains NFKB in the cytoplasm.
  • I ⁇ B proteins of which there are six known members, each contain 5-7 ankyrin-like repeats required for association with the NF ⁇ B/Rel dimer and for inhibitory activity (see Beg et al., Genes Dev. 7, 2064-70, 1993; Gilmore and Morin, Trends Genet. 9, 427-33, 1993; Diaz-Meco et al., Mol. Cell. Biol. 73:4770-75, 1993; Haskill et al., Cell (55:1281-89, 1991).
  • I ⁇ B proteins include I ⁇ B ⁇ and I ⁇ B ⁇ .
  • NFKB activation involves the sequential phosphorylation, ubiquitination, and degradation of I ⁇ B.
  • Phosphorylation of I ⁇ B is highly specific for target residues.
  • phosphorylation of the I ⁇ B protein I ⁇ B ⁇ takes place at serine residues S32 and S36, and phosphorylation of I ⁇ B ⁇ occurs at serine residues S19 and S23.
  • the choreographed series of modification and degradation steps results in nuclear import of transcriptionally active NFKB due to the exposure of a nuclear localization signal on NFKB that was previously masked by I ⁇ B (Beg et al., Genes Dev. 6: 1899- 1913, 1992).
  • NFKB activation is mediated by a signal transduction cascade that includes one or more specific I ⁇ B kinases, a linked series of El , E2 and E3 ubiquitin enzymes, the 26S proteasome, and the nuclear import machinery.
  • I ⁇ B kinases a signal transduction cascade that includes one or more specific I ⁇ B kinases, a linked series of El , E2 and E3 ubiquitin enzymes, the 26S proteasome, and the nuclear import machinery.
  • the phosphorylation of I ⁇ B is a critical step in NF- ⁇ B activation, and the identification of an I ⁇ B kinase, as well as proteins that modulate its kinase activity, would further the understanding of the activation process, as well as the development of therapeutic methods.
  • Several protein kinases have been found to phosphorylate
  • I ⁇ B in vitro including protein kinase A (Ghosh and Baltimore, Nature 344:678-82, 1990), protein kinase C (Ghosh and Baltimore, Nature 544:678-82, 1990) and double stranded RNA-dependent protein kinase (Kumar et al., Proc. Natl. Acad. Sci. USA 97:6288-92, 1994).
  • Constitutive phosphorylation of I ⁇ B ⁇ by casein kinase II has also been observed (see Barroga et al., Proc. Nail. Acad. Sci. USA 92:7637-41 , 1995). None of these kinases, however appear to be responsible for in vivo activation of NF- ⁇ B.
  • I ⁇ B kinase activity has been reported, but these proteins also do not appear to be significant activators in vivo.
  • a putative I ⁇ B ⁇ kinase was identified by Kuno et al., J. Biol. Chem. 270:27914-27919, 1995, but that kinase appears to phosphorylate residues in the C-terminal region of I ⁇ B ⁇ . rather than the S32 and S36 residues known to be important for in vivo regulation.
  • Diaz- Meco et al., EMBO J. 73:2842-2848, 1994 also identified a 50 kD I ⁇ B kinase, with uncharacterized phosphorylation sites.
  • the present invention provides compositions and methods employing a large, multi-subunit IKK signalsome, or a component or variant thereof.
  • the present invention provides an IKK signalsome capable of specifically phosphorylating I ⁇ B ⁇ at residues S32 and S36, and I ⁇ B ⁇ at residues 19 and 23, without the addition of exogenous co factors.
  • polypeptide comprising a component of an
  • IKK signalsome or a variant of such a component, is provided, wherein the component has a sequence recited in SEQ ID NO:9.
  • methods for preparing an IKK signalsome comprising combining components of an IKK signalsome in a suitable buffer.
  • methods for phosphorylating a substrate of an IKK signalsome comprising contacting a substrate with an IKK signalsome or a component thereof, and thereby phosphorylating the substrate.
  • the present invention provides a method for screening for an agent that modulates IKK signalsome activity, comprising: (a) contacting a candidate agent with an IKK signalsome, wherein the step of contacting is carried out under conditions and for a time sufficient to allow the candidate agent and the IKK signalsome to interact; and (b) subsequently measuring the ability of the candidate agent to modulate IKK signalsome activity.
  • the present invention provides methods for screening for an agent that modulates IKK signalsome activity, comprising: (a) contacting a candidate agent with a polypeptide comprising a component of an IKK signalsome as described above, wherein the step of contacting is carried out under conditions and for a time sufficient to allow the candidate agent and the polypeptide to interact; and (b) subsequently measuring the ability of the candidate agent to modulate the ability of the polypeptide to phosphorylate an I ⁇ B protein.
  • an antibody that binds to a component (e.g., IKK- 1 and/or IKK-2) of an IKK signalsome, where the component is capable of phosphorylating I ⁇ B ⁇ .
  • a component e.g., IKK- 1 and/or IKK-2
  • the present invention provides methods for modulating NF- ⁇ B activity in a patient, comprising administering to a patient an agent that modulates
  • I ⁇ B kinase activity in combination with a pharmaceutically acceptable carrier.
  • Methods are also provided for treating a patient afflicted with a disorder associated with the activation of IKK signalsome, comprising administering to a patient a therapeutically effective amount of an agent that modulates I ⁇ B kinase activity in combination with a pharmaceutically acceptable carrier.
  • a method for detecting IKK signalsome activity in a sample comprising: (a) contacting a sample with an antibody that binds to an IKK signalsome under conditions and for a time sufficient to allow the antibody to immunoprecipitate an IKK signalsome; (b) separating immunoprecipitated material from the sample; and (c) determining the ability of the immunoprecipitated material to specifically phosphorylate an I ⁇ B protein with in vivo specificity.
  • the ability of the immunoprecipitated material to phosphorylate I ⁇ B ⁇ at residues S32 and/or S36 is determined.
  • kits for detecting IKK signalsome activity in a sample comprising an antibody that binds to an IKK signalsome in combination with a suitable buffer.
  • the present invention provides a method for identifying an upstream kinase in the NF- ⁇ B signal transduction cascade, comprising evaluating the ability of a candidate upstream kinase to phosphorylate an IKK signalsome, a component thereof or a variant of such a component.
  • a method for identifying a component of an IKK signalsome is also provided, comprising: (a) isolating an IKK signalsome; (b) separating the signalsome into components, and (c) obtaining a partial sequence of a component.
  • a method for preparing an IKK signalsome from a biological sample comprising: (a) separating a biological sample into two or more fractions; and (b) monitoring I ⁇ B kinase activity in the fractions.
  • Figures 1A-1C are autoradiograms depicting the results of immunoblot analyses.
  • Figure 1A shows the recruitment of I ⁇ B ⁇ into a high molecular weight complex upon stimulation.
  • Cytoplasmic extracts of either unstimulated or PMA(50 ng/ml)- and PHA(1 ⁇ g/ml)-stimulated (10 min) Jurkat cells were fractionated on a gel filtration column.
  • I ⁇ B ⁇ was visualized by immunoblot analysis.
  • the upper panel shows the elution profile of unstimulated cells, and the lower panel shows the elution profile of PMA/PHA-stimulated cells.
  • Molecular weight standards are indicated by arrows on the top.
  • Figure IB shows that the stimulus-dependent I ⁇ B ⁇ kinase activity chromatographs as a high molecular weight complex, M r 500-700 kDa.
  • Phosphorylation of the GST I ⁇ B ⁇ 1 -54 (wildtype) substrate is indicated by an arrow to the right.
  • Molecular weight standards are indicated by arrows on the top.
  • FIG. 1 C illustrates the identification of proteins that co-chromatograph with the IKK signalsome.
  • IKK signalsome was partially purified from extracts of TNF ⁇ - stimulated HeLa S3 cells by sequential fractionation on a Q Sepharose, Superdex 200, Mono Q, and Phenyl Superose columns. Phenyl Superose fractions containing the peak of IKK signalsome activity were subjected to western blot analysis using several different antibodies as indicated to the left of each respective panel. The level of IKK signalsome activity is indicated in the upper shaded area by increasing number of (+)'s.
  • Figure 2 is a flow chart depicting a representative purification procedure for the preparation of an IKK signalsome.
  • Figures 3A and 3B are autoradiograms that show the results of a Western blot analysis of the levels of I ⁇ B ⁇ in HeLa S3 cytoplasmic extracts following gel filtration. The extracts were prepared from cells that were (Figure 3B) and were not (Figure 3A) exposed to TNF ⁇ .
  • Figures 4A and 4B are autoradiograms depicting the results of an in vitro kinase assay in which the ability of the above cell extracts to phosphorylate the N- terminal portion of I ⁇ B ⁇ was evaluated.
  • Figure 4A shows the results employing an extract from cells that were not treated with TNF ⁇
  • Figure 4B shows the results when the cells were treated with TNF ⁇ .
  • Figures 5A and 5B are autoradiograms depicting the results of an in vitro kinase assay using a cytoplasmic extract of TNF ⁇ -treated HeLa S3 cells, where the extract is subjected to Q Sepharose fractionation.
  • the substrate was the truncated I ⁇ B ⁇ (residues 1 to 54), with Figure 5A showing the results obtained with the wild type I ⁇ B ⁇ sequence and Figure 5B presenting the results obtained using a polypeptide containing threonine substitutions at positions 32 and 36.
  • Figures 6A and 6B are autoradiograms depicting the results of an in vitro kinase assay using a cytoplasmic extract of TNF ⁇ -treated HeLa S3 cells, where the extract was subjected in series to chromatographic fractionation by Q Sepharose, Superdex 200, Mono Q Sepharose and Phenyl Superose.
  • the substrate was the truncated I ⁇ B ⁇ (residues 1 to 54), with Figure 6A showing the results obtained with the wild type I ⁇ B ⁇ sequence and Figure 6B presenting the results obtained using a polypeptide containing threonine substitutions at positions 32 and 36.
  • Figure 7 is an autoradiogram showing the results of immunokinase assays (using anti-MKP-1 antibody) performed using cytoplasmic extracts of TNF ⁇ -treated HeLa S3 cells following gel filtration.
  • the assay was performed using the substrates GST-I ⁇ B ⁇ l -54 wildtype (lane 1) and GST- I ⁇ B ⁇ l -54 S32/36 to T (lane 2).
  • the positions of I ⁇ B ⁇ and GST-I ⁇ B ⁇ 1-54 are indicated on the left.
  • Figures 8A-8C are autoradiograms depicting the results of immunoblot analyses.
  • the upper panel presents a time course for the induction of signalsome activity.
  • Anti MKP-1 immune precipitates from extracts of HeLa S3 cells stimulated with TNF ⁇ (20 ng/ml) for the indicated times, were assayed for IKK signalsome activity by standard immune complex kinase assays. 4 ⁇ g of cither GST I ⁇ B ⁇ 1-54 WT (wildtype) or the GST I ⁇ B ⁇ 1-54 S32/36 to T mutant (S>T) were used as the substrates.
  • HeLa cell extracts prepared as described in the upper panel were examined by western blot analysis for I ⁇ B ⁇ degradation.
  • FIG. 8B illustrates the stimulus-dependent activation of IKK signalsome, which is blocked by TPCK.
  • GST I ⁇ B ⁇ 1-54 WT (4 ⁇ g) was used as a substrate.
  • Figure 8C illustrates the ability of IKK signalsome to specifically phosphorylate serines 32 and 36 of the I ⁇ B ⁇ holoprotein in the context of a RclA:I ⁇ B ⁇ complex.
  • Anti-MKP-1 immunoprecipitates from cell extracts of HeLa S3 cells stimulated with TNF ⁇ (20 ng/ml, 7 min) were examined for their ability to phosphorylate baculoviral expressed RelA:I ⁇ B ⁇ complex containing either the I ⁇ B ⁇ WT (lane 3) or I ⁇ B ⁇ S32/36 to A mutant (lane 4) holoprotein.
  • the specific substrates used are indicated on the top. Positions of the phosphorylatcd substrates are indicated by arrows to the left of the panel.
  • Figure 9A is an autoradiogram depicting the results of an immunokinase assay in which peptides are phosphorylatcd by the IKK signalsome.
  • I ⁇ B ⁇ (21-41) peptides that were unphosphorylated or chemically phosphorylated on either Ser-32 or Ser-36 were incubated with the IKK signalsome in the presence of ⁇ -f 32 P]-ATP.
  • the doubly phosphorylated peptide P32,36 was not phosphorylated by the IKK signalsome, and the unrelated c-Fos(222-241) phosphopeptide with free serine and threonine residues did not function as a signalsome substrate.
  • Figure 9B is a graph illustrating the inhibition of phosphorylation of GST- I ⁇ B ⁇ (1-54) by I ⁇ B ⁇ (21-41) peptides.
  • I ⁇ B ⁇ (21-41) peptide P32,36 inhibits GST- I ⁇ B ⁇ (1-54) as a product inhibitor with a K, value of 14 ⁇ M.
  • the unrelated phosphopeptide c- Fos(222-241) does not function as an inhibitor.
  • This assay only detects precipitated 32 P- labeled proteins, not 32 P-labeled peptides. Addition of the singly- or non-phosphorylated I ⁇ B ⁇ (21-41) peptides results in less phosphorylation of GST-I ⁇ B ⁇ (1-54) and apparent inhibition.
  • Figure 10 is an autoradiogram showing the results of a western blot analysis of the level of ubiquitin within a stimulus-inducible IkB kinase complex.
  • Lane 1 shows the detection of 100 ng ubiquitin
  • Lane 2 shows 10 ng ubiquitin
  • Lane 3 shows 3.4 ⁇ g of IKK signalsome purified through the phenyl superose step (sufficient quantities for 10 kinase reactions).
  • the position of ubiquitin is shown by the arrow on the left.
  • FIG 11A illustrates a procedure for purification of the IKK signalsome.
  • a whole cell extract was prepared from TNF ⁇ -stimulated (20 ng/ml, 7 minute induction) HeLa S3 cells (1.2 g total protein).
  • the IKK signalsome was then immunoprecipitated from the extract using anti-MKP-1 antibodies, washed with buffer containing 3.5 M urea and eluted overnight at 4°C in the presence of excess MKP-1 specific peptide.
  • Eluted IKK signalsome was then fractionated on a Mono Q column, I ⁇ B kinase active fractions were pooled, concentrated and subjected to preparative SDS-PAGE. Individual protein bands were excised and submitted for peptide sequencing.
  • FIG. 1 IB is a photograph showing Mono Q fractions containing active
  • IKK signalsome activity following SDS-PAGE and a standard silver stain protocol. Peak activity of IKK signalsome activity is represented in lanes 3, 4, and 5. Protein bands corresponding to IKK-1 and IKK-2 are indicated to the left of the figure. Molecular weight standards (kDa) are indicated to the left of the figure.
  • Figures 12A and 12B are mass spectra obtained during sequencing of
  • FIG. 12A shows part of the mass spectrum of the unseparated mixture of tryptic peptides resulting from in-gel digestion of the IKK-2 band in Figure 1 IB.
  • Figure 12B shows a tandem mass spectrum of the peak at m/z 645.2.
  • Figure 13A illustrates the amino acid sequence of IKK-1 and IKK-2. Symbols: arrows, boundaries of the kinase domain; underlined, peptide sequences identified by nanoelectrospray mass spectrometry; asterisks, indicates leucines comprising the leucine zipper motif; bold face, indicate amino acid identities conserved between IKK-1 and IKK-2; highlighted box, Helix-loop-helix domain; dashes, a gap inserted to optimize alignment.
  • Figure 13B is an autoradiogram depicting the results of Northern blot analysis of IKK-2 mRNA in adult human tissue. The source of the tissue is indicated at the top. Probes spanning the coding region of human IKK-2 and ⁇ -actin cDNA were used and are indicated to the left. Molecular weight standards are indicated to the right.
  • Figure 14A is an autoradiogram depicting the results of kinase assays using IKK-1 and IKK-2. IKK-1 and IKK-2 were immunoprecipitated from rabbit reticulocyte lysates phosphorylate I ⁇ B ⁇ and I ⁇ B ⁇ .
  • IKK-1 (lane 1) undergoes significant autophosphorylation in contrast to IKK-2 (lane 2) which is identified only with longer exposure times.
  • Figures 14B and 14C are micrographs illustrating the results of assays to evaluate the ability of kinase-inactive mutants of IKK-1 and IKK-2 to inhibit RelA translocation in TNF ⁇ -stimulated HeLa cells.
  • HeLa cells were transiently transfected with either HA-tagged IKK-1 K44 to M mutant (14B) or Flag-tagged IKK-2 K44 to M mutant (14C) expression vectors. 36 hours post-transfection cells were either not stimulated (Unstim) or TNF ⁇ -stimulated (20 ng/ml) for 30 min (TNF ⁇ ), as indicated to the right of the figure.
  • FIGS. 15A and 15B are autoradiograms of immunoprecipitated IKK-1 and IKK-2 following in vitro translation.
  • Figure 15 A HA-tagged IKK-1 and Flag- tagged IKK-2 were in vitro translated in wheat germ lysates either separately or in combination, as indicated. The programmed translation mix was then subjected to immunoprecipitation using the indicated antibody.
  • the present invention is generally directed to compositions and methods for modulating (i.e., stimulating or inhibiting) signal transduction leading to NF- ⁇ B activation.
  • the present invention is directed to compositions comprising an I ⁇ B kinase (IKK) signalsome (also referred to herein as a "stimulus-inducible I ⁇ B kinase complex" or "I ⁇ B kinase complex”) that is capable of stimulus-dependent phosphorylation of I ⁇ B ⁇ and I ⁇ B ⁇ on the two N-terminal serine residues critical for the subsequent ubiquitination and degradation in vivo.
  • IKK I ⁇ B kinase
  • Such stimulus-dependent phosphorylation may be achieved without the addition of exogenous cofactors.
  • an IKK signalsome specifically phosphorylates I ⁇ B ⁇ (SEQ ID NO: l ) at residues S32 and S36 and phosphorylates I ⁇ B ⁇ (SEQ ID NO:2) at residues S 19 and S23.
  • the present invention also encompasses compositions that contain one or more components of such an IKK signalsome, or variants of such components.
  • Preferred components referred to herein as “IKK signalsome kinases” "IKB kinases” or IKKs) are kinases that, when incorporated into an IKK signalsome, are capable of phosphorylating I ⁇ B ⁇ at S32 and S36.
  • Particularly preferred components are IKK-1 (SEQ ID NO: 10) and IKK-2 (SEQ ID NO:9).
  • An IKK signalsome and/or IKB kinase may generally be used for phosphorylating a substrate (i.e., an IKB, such as I ⁇ B ⁇ , or a portion or variant thereof that can be phosphorylated at those residues that are phosphorylated in vivo) and for identifying modulators of I ⁇ B kinase activity.
  • a substrate i.e., an IKB, such as I ⁇ B ⁇ , or a portion or variant thereof that can be phosphorylated at those residues that are phosphorylated in vivo
  • modulators and methods employing them for modulating I ⁇ B ⁇ kinase activity, in vivo and/or in vitro are also encompassed by the present invention.
  • compositions that inhibit I ⁇ B kinase activity may inhibit I ⁇ B phosphorylation, or may inhibit the activation of an I ⁇ B kinase and/or IKK signalsome.
  • IKK signalsome has several distinctive properties. Such a complex is stable (i.e., its components remain associated following purification as described herein) and has a high-molecular weight (about 500-700 kD, as determined by gel filtration chromatography). As shown in Figures 3 (A and B) and 4 (A and B), I ⁇ B kinase activity of an IKK signalsome is "stimulus-inducible" in that it is stimulated by TNF ⁇ (i.e..
  • TNF ⁇ results in increased IKB kinase activity and I ⁇ B degradation) and/or by one or more other inducers of NF- ⁇ B, such as IL-1, LPS, TPA, UV irradiation, antigens, viral proteins and stress-inducing agents.
  • inducers of NF- ⁇ B such as IL-1, LPS, TPA, UV irradiation, antigens, viral proteins and stress-inducing agents.
  • the kinetics of stimulation by TNF ⁇ correspond to those found in vivo.
  • IKB kinase activity of an IKK signalsome is also specific for S32 and S36 of I ⁇ B ⁇ .
  • an IKK signalsome is capable of phosphorylating a polypeptide having the N-terminal sequence of I ⁇ B ⁇ (GST-I ⁇ B ⁇ l-54; SEQ ID NO:3), but such phosphorylation cannot be detected in an I ⁇ B ⁇ derivative containing threonine substitutions at positions 32 and 36.
  • I ⁇ B kinase activity is strongly inhibited by a doubly phosphorylated I ⁇ B ⁇ peptide (i.e., phosphorylated at S32 and S36), but not by an unrelated c-fos phosphopeptide that contains a single phosphothreonine.
  • IKK signalsome A further characteristic of an IKK signalsome is its ability to phosphorylate a substrate in vitro in a standard kinase buffer, without the addition of exogenous cofactors. Free ubiquitin is not detectable in preparations of IKK signalsome (see Figure 10), even at very long exposures. Accordingly an IKK signalsome differs from the ubiquitin-dependent I ⁇ B ⁇ kinase activity described by Chen et al., Cell 54:853-62, 1996.
  • An IKK signalsome may be immunoprecipitated by antibodies raised against MKP-1 (MAP kinase phosphatase-1 ; Santa Cruz Biotechnology, Inc., Santa Cruz, CA #SC-1 102), and its activity detected using an in vitro I ⁇ B ⁇ kinase assay.
  • MKP-1 MAP kinase phosphatase-1 ; Santa Cruz Biotechnology, Inc., Santa Cruz, CA #SC-1 102
  • MKP-1 does not appear to be a component of IKB kinase complex.
  • the substrate specificity of the immunoprecipitated IKK signalsome is maintained (i.e., there is strong phosphorylation of wildtype GST-I ⁇ B ⁇ 1 -54 (SEQ ID NO:3) and GST-I ⁇ B ⁇ 1-44 (SEQ ID NO:4), and substantially no detectable phosphorylation of GST-I ⁇ B ⁇ 1-54 in which serines 32 and 36 are replaced by threonines (GST- I ⁇ B ⁇ S32/36 to T; SEQ ID NO:5) or GST-I ⁇ B ⁇ 1-44 in which serines 19 and 23 are replaced by alanines (GST-I ⁇ B ⁇ 1-44 SI 9/23 to A; SEQ ID NO:6)).
  • IKK signalsome may be isolated from human or other cells, and from any of a variety of tissues and/or cell types.
  • cytoplasmic and/or nuclear/membrane extracts may be prepared from HeLa S3 cells following seven minutes induction with 30 ng/mL TNF ⁇ . The extracts may then be subjected to a series of chromatographic steps that includes Q Sepharose, gel filtration (HiLoad 16/60 Superdex 200), Mono Q, Phenyl Superose, gel filtration (Superdex 200 10/30) and Mono Q.
  • This representative purification procedure is illustrated in Figure 2, and results in highly enriched IKK signalsome (compare, for example, Figures 5A and 6A).
  • An alternative purification procedure employs a two-step affinity method, based on recognition of IKK signalsome by the MKP-1 antibody ( Figure 1 1A).
  • Whole cell lysates from TNF ⁇ -stimulated HeLa cells may be immunoprecipitated with an anti- MKP-1 antibody.
  • the IKK signalsome may be eluted with the specific MKP-1 peptide to which the antibody was generated and fractionated further on a Mono Q column.
  • an in vitro kinase assay may be used to monitor the I ⁇ B kinase activity of the IKK signalsome.
  • an appropriate substrate such as I ⁇ B ⁇ (SEQ ID NO:l) or a derivative or variant thereof
  • a substrate may be combined with a chromatographic fraction in a protein kinase buffer containing P ⁇ -ATP, phosphatase inhibitors and protease inhibitors. The mixture may be incubated for 30 minutes at 30°C.
  • Suitable substrates include full length I ⁇ B ⁇ (SEQ ID NO:l), polypeptides comprising the N-terminal 54 amino acids of I ⁇ B ⁇ , full length I ⁇ B ⁇ (SEQ ID NO:2) and polypeptides comprising the N-terminal 44 amino acids of I ⁇ B ⁇ . Any of these substrates may be used with or without an N-terminal tag.
  • One suitable substrate is a protein containing residues 1 -54 of I ⁇ B ⁇ and an N-termina! GST tag (referred to herein as GST-I ⁇ B ⁇ 1-54; SEQ ID NO:3).
  • an IKK signalsome may be prepared from its components which are also encompassed by the present invention. Such components may be produced using well known recombinant techniques, as described in greater detail below.
  • Components of an IKK signalsome may be native, or may be variants of a native component (i e., a component sequence may differ from the native sequence in one or more substitutions and/or modifications, provided that the ability of a complex comprising the component variant to specifically phosphorylate l ⁇ B ⁇ is not substantially diminished). Substitutions and/or modifications may generally be made in non-critical and/or critical regions of the native protein. Variants may generally comprise residues of L-amino acids, D-amino acids, or any combination thereof.
  • Amino acids may be naturally-occurring or may be non-natural, provided that at least one amino group and at least one carboxyl group are present in the molecule; ⁇ - and ⁇ -amino acids are generally preferred.
  • a variant may also contain one or more rare amino acids (such as 4- hydroxyproline or hydroxylysine), organic acids or amides and/or derivatives of common amino acids, such as amino acids having the C-terminal carboxylate esterified (e.g., benzyl, methyl or ethyl ester) or amidated and/or having modifications of the N-terminal amino group (e.g., acetylation or alkoxycarbonylation), with or without any of a wide variety of side-chain modifications and/or substitutions (e.g..).
  • rare amino acids such as 4- hydroxyproline or hydroxylysine
  • variants may also, or alternatively, contain other modifications, including the deletion or addition of amino acids that have minimal influence on the activity of the polypeptide.
  • variants may contain additional amino acid sequences at the amino and/or carboxy termini. Such sequences may be used, for example, to facilitate purification or detection of the component polypeptide. In general, the effect of one or more substitutions and/or modifications may be evaluated using the representative assays provided herein.
  • a component may generally be prepared from a DNA sequence that encodes the component using well known recombinant methods.
  • DNA sequences encoding components of an IKK signalsome may be isolated by, for example, screening a suitable expression library (i.e., a library prepared from a cell line or tissue that expresses IKK signalsome, such as spleen, leukocytes, HeLa cells or Jurkat cells) with antibodies raised against IKK signalsome or against one or more components thereof.
  • Protein components may then be prepared by expression of the identified DNA sequences, using well known recombinant techniques.
  • partial sequences of the components may be obtained using standard biochemical purification and microsequencing techniques. For example, purified complex as described above may be run on an SDS-PAGE gel and individual bands may be isolated and subjected to protein microsequencing. DNA sequences encoding components may then be prepared by amplification from a suitable human cDNA library, using polymerase chain reaction (PCR) and methods well known to those of ordinary skill in the art. For example, an adapter-ligated cDNA library prepared from a cell line or tissue that expresses IKK signalsome (such as HeLa or Jurkat cells) may be screened using a degenerate 5' specific forward primer and an adapter-specific primer.
  • PCR polymerase chain reaction
  • Degenerate oligonucleotides may also be used to screen a cDNA library, using methods well known to those of ordinary skill in the art.
  • known proteins may be identified via Western blot analysis using specific antibodies.
  • Components of an IKK signalsome may also be identified by performing any of a variety of protein-protein interaction assays known to those of ordinary skill in the art. For example, a known component can be used as "bait" in standard two-hybrid screens to identify other regulatory molecules, which may include IKK-1, IKK-2, NF ⁇ Bl , RelA.
  • IKK signalsome Particularly preferred components of IKK signalsome are I ⁇ B kinases.
  • An IKB kinase may be identified based upon its ability to phosphorylate one or more IKB proteins, which may be readily determined using the representative kinase assays described herein.
  • an IKB kinase is incorporated into an IKK signalsome, as described herein, prior to performing such assays, since an IKB kinase that is not complex-associated may not display the same phosphorylation activity as complex- associated IKB kinase.
  • an IKB kinase within an IKK signalsome specifically phosphorylates I ⁇ B ⁇ at residues S32 and S36, and phosphorylates I ⁇ B ⁇ at residues 19 and 23, in response to specific stimuli.
  • IKK-1 and IKK-2 are particularly preferred I ⁇ B kinases.
  • IKK-1 and IKK-2 may be prepared by pooling the fractions from the Mono Q column containing peak I ⁇ B kinase activity and subjecting the pooled fractions to preparative SDS gel electrophoresis. The intensity of two prominent protein bands of -85 and -87 kDa (indicated by silver stain in Figure 1 IB as IKK-1 and IKK-2 respectively) correlates with the profile of I ⁇ B kinase activity.
  • the -85 kDa band corresponding to IKK-1 , has been identified, within the context of the present invention, as CHUK (conserved helix- loop-helix ubiquitous kinase; see Connely and Marcu, Cell. Mol. Biol. Res. 47:537- 49,1995).
  • CHUK conserved helix- loop-helix ubiquitous kinase; see Connely and Marcu, Cell. Mol. Biol. Res. 47:537- 49,1995).
  • the -87 kDa band contains IKK-2.
  • IKK-1 and IKK-2 are related protein serine kinases (51% identity) containing protein interaction motifs ( Figure 13 A). Both IKK-1 and IKK-2 contain the kinase domain at the N-terminus, and a leucine zipper motif and a helix-loop-helix motif in their C-terminal regions.
  • Northern analysis indicates that mRNAs encoding IKK-2 are widely distributed in human tissues, with transcript sizes of -4.5 kb and 6 kb ( Figure 13B).
  • the sequences of IKK-1 and IKK-2 are also provided as SEQ ID NOs: 7 and 8, respectively.
  • IKK signalsome components of an IKK signalsome are also contemplated by the present invention.
  • Such components may include, but are not limited to, upstream kinases such as MEKK-1 (Lee et al., Cell 88,:2 ⁇ 3-22, 1997; Hirano et al., J. Biol. Chem.
  • a component of an IKK signalsome may generally be prepared from DNA encoding the component by expression of the DNA in cultured host cells, which may be stable cell lines or transiently transfected cells.
  • the host cells are bacteria, yeast, baculo virus-infected insect cells or mammalian cells.
  • the recombinant DNA may be cloned into any expression vector suitable for use within the host cell, using techniques well known to those of ordinary skill in the art.
  • An expression vector may, but need not, include DNA encoding an epitope, such that the recombinant protein contains the epitope at the N- or C-terminus.
  • Epitopes such as glutathione-S transferase protein (GST), HA (hemagglutinin)-tag, FLAG and Histidine-tag may be added using techniques well known to those of ordinary skill in the art.
  • DNA sequences expressed in this manner may encode native components of an IKK signalsome, or may encode portions or variants of native components, as described above.
  • DNA molecules encoding variants may generally be prepared using standard mutagenesis techniques, such as oligonucleotide-directed site- specific mutagenesis. Sections of the DNA sequence may also, or alternatively, be removed to permit preparation of truncated polypeptides and DNA encoding additional sequences such as "tags" may be added to the 5' or 3' end of the DNA molecule.
  • IKK signalsome components may generally be used to reconstitute IKK signalsome. Such reconstitution may be achieved in vitro by combining IKK signalsome components in a suitable buffer. Alternatively, reconstitution may be achieved in vivo by expressing components in a suitable host cell, such as HeLa or HUVEC, as described herein.
  • IKK signalsome may be isolated in substantially pure form.
  • IKK signalsome or a component is isolated to a purity of at least 80% by weight, more preferably to a purity of at least 95% by weight, and most preferably to a purity of at least 99% by weight.
  • purification may be achieved using, for example, the representative purification methods described herein or the standard techniques of ammonium sulfate fractionation, SDS-PAGE electrophoresis, and affinity chromatography.
  • IKK signalsome and components for use in the methods of the present invention may be native, purified or recombinant.
  • an IKK signalsome and/or one or more components thereof may be used to identify modulating agents, which may be antibodies (e.g., monoclonal), polynucleotides or other drugs, that inhibit or stimulate signal transduction via the NF- ⁇ B cascade.
  • modulating agents which may be antibodies (e.g., monoclonal), polynucleotides or other drugs, that inhibit or stimulate signal transduction via the NF- ⁇ B cascade.
  • Modulation includes the suppression or enhancement of NF- ⁇ B activity. Modulation may also include suppression or enhancement of IKB phosphorylation or the stimulation or inhibition of the ability of activated (i.e., phosphorylated) IKK signalsome to phosphorylate a substrate.
  • Compositions that inhibit NF- ⁇ B activity by inhibiting IKB phosphorylation may include one or more agents that inhibit or block I ⁇ B ⁇ kinase activity, such as an antibody that neutralizes IKK signalsome, a competing peptide that represents the substrate binding domain of IKB kinase or a phosphorylation motif of IKB, an antisense polynucleotide or ribozyme that interferes with transcription and/or translation of IKB kinase, a molecule that inactivates IKK signalsome by binding to the complex, a molecule that binds to IKB and prevents phosphorylation by IKK signalsome or a molecule that prevents transfer of phosphate groups from the kinase to the substrate.
  • agents that inhibit or block I ⁇ B ⁇ kinase activity such as an antibody that neutralizes IKK signalsome, a competing peptide that represents the substrate binding domain of IKB kinase or a phosphorylation motif of IKB, an antisense poly
  • a modulating agent inhibits or enhances the expression or activity of IKK-1 and/or IKK-2.
  • modulating agents may be identified by combining a test compound with an IKK signalsome, IKB kinase or a polynucleotide encoding an IKB kinase in vitro or in vivo, and evaluating the effect of the test compound on the IKB kinase activity using, for example, a representative assay described herein.
  • An increase or decrease in kinase activity can be measured by adding a radioactive compound, such as 32 P-ATP and observing radioactive incorporation into a suitable substrate for IKK signalsome, thereby determining whether the compound inhibits or stimulates kinase activity.
  • a candidate agent may be included in a reaction mixture containing compounds necessary for the kinase reaction (as described herein) and IKB substrate, along with IKK signalsome, I ⁇ B kinase or a polynucleotide encoding an I ⁇ B kinase.
  • a suitable amount of antibody or other agent for use in such an assay ranges from about 0.01 ⁇ M to about 10 ⁇ M.
  • the effect of the agent on I ⁇ B kinase activity may then be evaluated by quantitating the incorporation of f Pjphosphate into an I ⁇ B such as I ⁇ B ⁇ (or a derivative or variant thereof), and comparing the level of incorporation with that achieved using I ⁇ B kinase without the addition of a candidate agent.
  • the effect of a candidate modulating agent on transcription of an I ⁇ B kinase may be measured, for example, by Northern blot analysis or a promoter/reporter- based whole cell assay.
  • an IKK signalsome also displays p65 kinase activity and IKK phosphatase activity.
  • Assays to evaluate the effect of a test compound on such activities may be performed using well known techniques. For example, assays for p65 kinase activity may generally be performed as described by Zhong et al., Cell 59:413-24, 1997. For phosphatase activity, an assay may generally be performed as described by Sullivan et al., J.
  • IKK signalsome or I ⁇ B kinase may be used for phosphorylating an I ⁇ B such as I ⁇ B ⁇ (or a derivative or variant thereof) so as to render it a target for ubiquitination and subsequent degradation.
  • IKB may be phosphorylated in vitro by incubating IKK signalsome or IKB kinase with IKB in a suitable buffer for 30 minutes at 30°C.
  • IKB kinase complex In general, about 0.01 ⁇ g to about 9 ⁇ g of IKB kinase complex is sufficient to phosphorylate from about 0.5 ⁇ g to about 2 ⁇ g of IKB.
  • Phosphorylated substrate may then be purified by binding to GSH-sepharose and washing. The extent of substrate phosphorylation may generally be monitored by adding [ ⁇ - 32 P]ATP to a test aliquot, and evaluating the level of substrate phosphorylation as described herein.
  • An IKK signalsome, component thereof, modulating agent and/or polynucleotide encoding a component and/or modulating agent may also be used to modulate NF- ⁇ B activity in a patient.
  • Such modulation may occur by any of a variety of mechanisms including, but not limited to, direct inhibition or enhancement of I ⁇ B phosphorylation using a component or modulating agent; or inhibiting upstream activators, such as NIK or MEK, with IKK signalsome or a component thereof.
  • a "patient” may be any mammal, including a human, and may be afflicted with a disease associated with I ⁇ B kinase activation and the NF- ⁇ B cascade, or may be free of detectable disease. Accordingly, the treatment may be of an existing disease or may be prophylactic. Diseases associated with the NF- ⁇ B cascade include inflammatory diseases, neurodegenerative diseases, autoimmune diseases, cancer and viral infection.
  • Treatment may include administration of an IKK signalsome, a component thereof and/or an agent which modulates I ⁇ B kinase activity.
  • one or more such compounds are generally formulated as a pharmaceutical composition.
  • a pharmaceutical composition may be a sterile aqueous or non-aqueous solution, suspension or emulsion, which additionally comprises a physiologically acceptable carrier (i.e., a non-toxic material that does not interfere with the activity of the active ingredient). Any suitable carrier known to those of ordinary skill in the art may be employed in the pharmaceutical compositions of the present invention.
  • Representative carriers include physiological saline solutions, gelatin, water, alcohols, natural or synthetic oils, saccharide solutions, glycols, injectable organic esters such as ethyl oleate or a combination of such materials.
  • a pharmaceutical composition may additionally contain preservatives and/or other additives such as, for example, antimicrobial agents, anti-oxidants, chelating agents and/or inert gases, and/or other active ingredients.
  • a pharmaceutical composition may comprise a polynucleotide encoding a component of an IKK signalsome and/or a modulating agent (such that the component and/or modulating agent is generated in situ) in combination with a physiologically acceptable carrier.
  • the polynucleotide may be present within any of a variety of delivery systems known to those of ordinary skill in the art, including nucleic acid, bacterial and viral expression systems, as well as colloidal dispersion systems, including liposomes.
  • Appropriate nucleic acid expression systems contain the necessary polynucleotide sequences for expression in the patient (such as a suitable promoter and terminating signal). DNA may also be "naked," as described, for example, in Ulmer et al., Science 259: 1745-49, 1993.
  • the retroviral vector is a derivative of a murine or avian retrovirus including, but not limited to, Moloney murine leukemia virus (MoMuLV), Harvey murine sarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), and Rous Sarcoma Virus (RSV).
  • MoMuLV Moloney murine leukemia virus
  • HaMuSV Harvey murine sarcoma virus
  • MuMTV murine mammary tumor virus
  • RSV Rous Sarcoma Virus
  • a retroviral vector may additionally transfer or incorporate a gene for a selectable marker (to aid in the identification or selection of transduced cells) and/or a gene that encodes the ligand for a receptor on a specific target cell (to render the vector target specific).
  • retroviral vectors can be made target specific by inserting a nucleotide sequence encoding a sugar, a glycolipid, or a protein. Targeting may also be accomplished using an antibody, by methods known to those of ordinary skill in the art.
  • Viral vectors are typically non-pathogenic (defective), replication competent viruses, which require assistance in order to produce infectious vector particles.
  • helper cell lines that contain plasmids that encode all of the structural genes of the retrovirus under the control of regulatory sequences within the LTR, but that are missing a nucleotide sequence which enables the packaging mechanism to recognize an RNA transcript for encapsulation.
  • helper cell lines include (but are not limited to) ⁇ 2, PA317 and PA 12.
  • a retroviral vector introduced into such cells can be packaged and vector virion produced.
  • the vector virions produced by this method can then be used to infect a tissue cell line, such as NIFI 3T3 cells, to produce large quantities of chimeric retroviral virions.
  • colloidal dispersion systems include macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes.
  • a preferred colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (i.e., an artificial membrane vesicle). It has been shown that large unilamellar vesicles (LUV), which range in size from 0.2-4.0 ⁇ m can encapsulate a substantial percentage of an aqueous buffer containing large macromolecules.
  • LUV large unilamellar vesicles
  • RNA, DNA and intact virions can be encapsulated within the aqueous interior and be delivered to cells in a biologically active form (Fraley, et al., Trends Biochem. Sci. 6:77, 1981).
  • liposomes In addition to mammalian cells, liposomes have been used for delivery of polynucleotides in plant, yeast and bacterial cells.
  • a liposome In order for a liposome to be an efficient gene transfer vehicle, the following characteristics should be present: (1) encapsulation of the genes of interest at high efficiency while not compromising their biological activity; (2) preferential and substantial binding to a target cell in comparison to non-target cells; (3) delivery of the aqueous contents of the vesicle to the target cell cytoplasm at high efficiency; and (4) accurate and effective expression of genetic information (Mannino, et al., Biotechniques 6:882, 1988).
  • the targeting of liposomes can be classified based on anatomical and mechanistic factors.
  • Anatomical classification is based on the level of selectivity and may be, for example, organ-specific, cell-specific, and/or organelle-specific.
  • Mechanistic targeting can be distinguished based upon whether it is passive or active. Passive targeting utilizes the natural tendency of liposomes to distribute to cells of the reticuloendothelial system (RES) in organs which contain sinusoidal capillaries.
  • RES reticuloendothelial system
  • Active targeting involves alteration of the liposome by coupling the liposome to a specific ligand such as a monoclonal antibody, sugar, glycolipid, or protein, or by changing the composition or size of the liposome in order to achieve targeting to organs and cell types other than the naturally occurring sites of localization.
  • a specific ligand such as a monoclonal antibody, sugar, glycolipid, or protein
  • the pharmaceutical compositions may be administered intravenously, intraperitoneally, intramuscularly, subcutaneously, intracavity or transdermally. Between 1 and 6 doses may be administered daily.
  • a suitable dose is an amount that is sufficient to show improvement in the symptoms of a patient afflicted with a disease associated with the NF- ⁇ B cascade. Such improvement may be detected by monitoring inflammatory responses (e.g., edema, transplant rejection, hyperscnsitivity) or through an improvement in clinical symptoms associated with the disease.
  • the dosage may generally vary depending on the nature of the modulating agent and the disease to be treated.
  • the amount of modulating agent present in a dose, or produced in situ by DNA present in a dose ranges from about 1 ⁇ g to about 200 mg per kg of host. Suitable dose sizes will vary with the size of the patient, but will typically range from about 10 mL to about 500 mL for 10-60 kg animal.
  • the present invention provides methods for detecting the level of stimulus-inducible I ⁇ B kinase activity in a sample.
  • the level of IKB kinase activity may generally be determined via an immunokinase assay, in which IKK signalsome is first immunoprecipitated with an antibody that binds to the complex. The immunoprecipitated material is then subjected to a kinase assay as described herein. Substrate specificity may be further evaluated as described herein to distinguish the activity of a stimulus-inducible IKB kinase complex from other kinase activities.
  • the present invention also provides methods for detecting the level of IKK signalsome, or a component thereof, in a sample.
  • the amount of IKK signalsome, IKB kinase or nucleic acid encoding IKB kinase may generally be determined using a reagent that binds to IKB kinase, or to DNA or RNA encoding a component thereof.
  • a reagent that binds to IKB kinase or to DNA or RNA encoding a component thereof.
  • standard hybridization and/or PCR techniques may be employed using a nucleic acid probe or a PCR primer. Suitable probes and primers may be designed by those of ordinary skill in the art based on the component sequence.
  • the reagent is typically an antibody, which may be prepared as described below.
  • the antibody may be immobilized on a solid support such that it can bind to and remove the protein from the sample.
  • the bound protein may then be detected using a second antibody that binds to the antibody/protein complex and contains a detectable reporter group.
  • a competitive assay may be utilized, in which protein that binds to the immobilized antibody is labeled with a reporter group and allowed to bind to the immobilized antibody after incubation of the antibody with the sample.
  • reporter groups for use in these methods include, but are not limited to, enzymes (e.g., horseradish peroxidase), substrates, cofactors, inhibitors, dyes, radionuclides, luminescent groups, fluorescent groups and biotin.
  • enzymes e.g., horseradish peroxidase
  • substrates e.g., cofactors, inhibitors, dyes, radionuclides, luminescent groups, fluorescent groups and biotin.
  • Antibodies encompassed by the present invention may be polyclonal or monoclonal, and may bind to IKK signalsome and/or one or more components thereof (e.g., IKK-1 and/or IKK-2).
  • Preferred antibodies are those antibodies that inhibit or block IKB kinase activity in vivo and within an in vitro assay, as described above.
  • Other preferred antibodies are those that bind to one or more IKB proteins.
  • antibodies and other agents having a desired effect on IKB kinase activity may be administered to a patient (either prophylactically or for treatment of an existing disease) to modulate the phosphorylation of an IKB, such as I ⁇ B ⁇ , in vivo.
  • Antibodies may be prepared by any of a variety of techniques known to those of ordinary skill in the art (see, e.g., Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988).
  • an immunogen comprising the protein of interest is initially injected into a suitable animal (e.g., mice, rats, rabbits, sheep and goats), preferably according to a predetermined schedule incorporating one or more booster immunizations, and the animals are bled periodically.
  • Polyclonal antibodies specific for the protein may then be purified from such antisera by, for example, affinity chromatography using the protein coupled to a suitable solid support.
  • Monoclonal antibodies specific for an IKK signalsome or a component thereof may be prepared, for example, using the technique of Kohler and Milstein, Eur. J. Immunol. 6:51 1 -519, 1976, and improvements thereto. Briefly, these methods involve the preparation of immortal cell lines capable of producing antibodies having the desired specificity (i.e., reactivity with the complex and/or component of interest). Such cell lines may be produced, for example, from spleen cells obtained from an animal immunized as described above. The spleen cells are then immortalized by, for example, fusion with a myeloma cell fusion partner, preferably one that is syngeneic with the immunized animal.
  • the spleen cells and myeloma cells may be combined with a nonionic detergent for a few minutes and then plated at low density on a selective medium that supports the growth of hybrid cells, but not myeloma cells.
  • a preferred selection technique uses HAT (hypoxanthine, aminopterin, thymidine) selection. After a sufficient time, usually about 1 to 2 weeks, colonies of hybrids are observed. Single colonies are selected and tested for binding activity against the polypeptide. Hybridomas having high reactivity and specificity are preferred. Monoclonal antibodies may be isolated from the supernatants of growing hybridoma colonies.
  • various techniques may be employed to enhance the yield, such as injection of the hybridoma cell line into the peritoneal cavity of a suitable vertebrate host, such as a mouse.
  • Monoclonal antibodies may then be harvested from the ascites fluid or the blood.
  • Contaminants may be removed from the antibodies by conventional techniques, such as chromatography, gel filtration, precipitation, and extraction.
  • kits for detecting the level of IKK signalsome comprising IKB kinase, nucleic acid encoding I ⁇ B kinase and/or IKB kinase activity in a sample are provided. Any of a variety of samples may be used in such assays, including eukaryotic cells, bacteria, viruses, extracts prepared from such organisms and fluids found within living organisms.
  • the kits of the present invention comprise one or more containers enclosing elements, such as reagents or buffers, to be used in the assay.
  • a kit for detecting the level of IKK signalsome, I ⁇ B kinase or nucleic acid encoding IKB kinase typically contains a reagent that binds to the compound of interest.
  • the reagent may be a nucleic acid probe or a PCR primer.
  • the reagent is typically an antibody.
  • kits also contain a reporter group suitable for direct or indirect detection of the reagent (i.e., the reporter group may be covalently bound to the reagent or may be bound to a second molecule, such as Protein A, Protein G, immunoglobulin or lectin, which is itself capable of binding to the reagent).
  • Suitable reporter groups include, but are not limited to, enzymes (e.g., horseradish peroxidase), substrates, cofactors, inhibitors, dyes, radionuclides, luminescent groups, fluorescent groups and biotin.
  • enzymes e.g., horseradish peroxidase
  • substrates e.g., cofactors, inhibitors, dyes, radionuclides, luminescent groups, fluorescent groups and biotin.
  • reporter groups may be used to directly or indirectly detect binding of the reagent to a sample component using standard methods known to those of ordinary skill in the art.
  • IKK signalsome may be used to identify one or more native upstream kinases (i.e., kinases that phosphorylate and activate IKK signalsome in vivo) or other regulatory molecules that affect IKB kinase activity (such as phosphatases or molecules involved in ubiquitination), using methods well known to those of ordinary skill in the art.
  • IKK signalsome components may be used in a yeast two- hybrid system to identify proteins that interact with such components.
  • an expression library may be screened for cDNAs that phosphorylate IKK signalsome or a component thereof.
  • This example illustrates the recruitment of NFKB into a protein complex (the IKK signalsome) containing I ⁇ B kinase and other signaling proteins.
  • Cytoplasmic extracts of either unstimulated or stimulated Jurkat cells were fractionated on a Superdex 200 gel filtration column, and I ⁇ B ⁇ was visualized by immunoblot analysis.
  • Jurkat cells were grown to a cell density of 1.5 X 10 6 cells/ml and either not stimulated or induced for 10 minutes with PMA (50 ng/ml)/PHA (1 ⁇ g/ml).
  • HLB buffer (20 mM Tris pH 8.0, 2 mM EDTA, 1 mM EGTA, 10 mM ⁇ -glycerophosphate, 10 mM NaF, 10 mM PNPP, 300 ⁇ M Na 3 VO 4 , 1 mM benzamidine, 2 mM PMSF, 10 ⁇ g/ml aprotonin, 1 ⁇ g/ml leupeptin, 1 ⁇ g/ml pepstatin, 1 mM DTT), made 0.1% NP40 and left on ice for 15 minutes, and lysed with a glass Dounce homogenizer.
  • the nuclei were pelleted at 10,000 rpm for 20 minutes in a Sorval SS34 rotor. The supernatant was further centrifuged at 40,000 rpm for 60 min in a Ti50.1 rotor. All procedures were carried out at 4°C.
  • the S-100 fraction was concentrated and chromatographed on Hi Load 16/60 Superdex 200 prep grade gel filtration column that was equilibrated in GF buffer (20 mM Tris HCl pH 8.0, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 5% glycerol, 0.025% Brij 35, 1 mM benzamidine, 2 mM PMSF, 10 mM ⁇ -glycerophosphate, 10 mM NaF, 10 mM PNPP, 300 ⁇ M Na 3 VO 4 , 10 ⁇ g/ml aprotonin, 1 ⁇ g/ml leupeptin, 1 ⁇ g/ml pepstatin, 1 mM DTT). Isolated fractions were analyzed by western blot analysis using either anti-I ⁇ B ⁇ or anti-JNK antibodies (Santa Cruz, Inc., Santa Cruz, CA).
  • I ⁇ B ⁇ in cell extracts from unstimulated cells eluted with an apparent molecular weight of -300 kDa (lanes 5-7), consistent with the chromatographic properties of the inactive NF ⁇ B-I ⁇ B complex (Baeuerle and Baltimore, Genes Dev. 5:1689-98, 1989).
  • phosphorylatcd I ⁇ B ⁇ from cells stimulated for periods too short to permit complete degradation of the protein
  • Example 2 Partial Purification of IKK Signalsome and Identification of Co-purifying Components This Example illustrates the fractionation of extracts containing IKB kinase.
  • Whole cell extracts from TNF ⁇ -stimulated cells were fractionated by gel filtration, ion exchange, and other chromatographic methods, as described above.
  • IKB kinase activity in the fractions was assayed by phosphorylation of GST-I ⁇ B ⁇ (1-54) (SEQ ID NO:3) or GST-I ⁇ B ⁇ (1-44) (SEQ ID NO:4).
  • Kinase assays were performed in 20 mM HEPES pH 7.7, 2 mM MgCl 2 , 2 mM MnCl 2 , 10 ⁇ M ATP, 1-3 ⁇ Ci ⁇ -[ 32 P]-ATP, 10 mM ⁇ -glycerophosphate, 10 mM NaF, 10 mM PNPP, 300 ⁇ M Na 3 VO 4 , 1 mM benzamidine, 2 ⁇ M PMSF, 10 ⁇ g/ml aprotonin, 1 ⁇ g/ml leupeptin, 1 ⁇ g/ml pepstatin, 1 mM DTT) at 30°C for 30 to 60 minutes in the presence of the indicated substrate.
  • the kinase reaction was stopped by the addition of 6X SDS-PAGE sample buffer, subjected to SDS-PAGE analysis and visualized using autoradiography.
  • GST-I ⁇ B substrates for use in the above assay were prepared using standard techniques for bacterially expressed GST-protein (see Current Protocols in Molecular Biology 2:16.7.1-16.7.7, 1996). Bacterial cells were lysed, GST proteins were purified via binding to GST-agarose beads, washed several times, eluted from the beads with glutathione, dialyzed against kinase assay buffer and stored at -80°C.
  • the specificity of the kinase was established by using mutant GST-I ⁇ B ⁇ (1-54) in which serines 32, 36 had been mutated to threonine (SEQ ID NO:5). and GST- I ⁇ B ⁇ ( 1 -44) in which serines 19, 23 had been mutated to alanine (SEQ ID NO:6). IKB kinase activity was not observed in extracts from unstimulated cells, while stimulation with TNF ⁇ for 5-7 minutes resulted in strong induction of kinase activity.
  • NFKB activation is known to occur under conditions that also stimulate MAP kinase pathways (Lee et al., Cell 55:213-22, 1997; Hirano, et al., J. Biol. Chem. 277: 13234-38, 1996). Accordingly, further experiments were performed to detect proteins associated with MAP kinase and phosphatasc cascades at various stages of purification of the IKK signalsome. In addition to RelA and I ⁇ B ⁇ , MEKK- 1 and two tyrosine-phosphorylated proteins of -55 and -40 kDa copurified with IKB kinase activity (Figure IC). Antibodies to Rel A and I ⁇ B ⁇ were obtained from Santa Cruz Biotechnology, Inc.
  • This Example illustrates an alternate preparation of an IKK signalsome, and the characterization of the complex.
  • HeLa S3 cells were grown to a cell density of approximately 0.6 x 10 6 /mL, concentrated 10 fold and induced with TNF ⁇ (30 ng/mL) for seven minutes. Two volumes of ice-cold PBS containing phosphatase inhibitors (10 mM sodium fluoride, 0.3 mM sodium orthovanadate and 20 mM ⁇ -glycerophosphate) were then added. The cells were spun down, washed once with ice-cold PBS containing phosphatase inhibitors and snap frozen.
  • phosphatase inhibitors 10 mM sodium fluoride, 0.3 mM sodium orthovanadate and 20 mM ⁇ -glycerophosphate
  • the supernatant was clarified via ultracentrifugation (50,000 for 1 hour at 4°C) to generate the final cytoplasmic extract.
  • the nuclear/membrane pellet was resuspended in an equal volume of High Salt Extraction Buffer (20mM Tris pH 8.0, 0.5M NaCl, ImM EDTA, ImM EGTA, 0.25% Triton X-100, 20mM ⁇ -glycerophosphate, lOmM NaF, lOmM PNPP, 0.3mM Na 2 VO 4 , ImM benzamidine, ImM PMSF, ImM DTT, lO ⁇ g/mL aprotinin, l ⁇ g/mL leupeptin and l ⁇ g/mL pepstatin) and allowed to rotate at 4°C for 30 minutes. Cell debris was removed via centrifugation at 12,500 ⁇ m for 30 minutes at 4°C and the resulting supernatant was saved as the nuclear/membrane extract.
  • I ⁇ B kinase activity was monitored using an in vitro assay.
  • the assay was performed by combining 2 ⁇ g of the respective IKB substrate (GST-I ⁇ B ⁇ 1-54 (wildtype) or GST-I ⁇ B ⁇ (S32/36 to T), as described below) with 3-5 ⁇ L chromatographic fraction and 20 ⁇ L of Kinase Assay Buffer (20 mM HEPES pH 7.4, 10 mM MgCl 2 , 10 mM MnCl 2 , 20 mM NaCl, ImM DTT, 20mM PNPP, 20 ⁇ M ATP, 20mM ⁇ -glycerophosphate, l OmM NaF, O.
  • IKB substrates for use in the above assay were prepared using standard techniques (see Haskill et al., Cell 65:1281-1289, 1991).
  • the GST-I ⁇ B ⁇ 1-54 (wildtype) or GST-I ⁇ B ⁇ (S32/36 to T) substrates were prepared using standard techniques for bacterially expressed GST-protein.
  • I ⁇ B ⁇ was assayed by running 18 ⁇ L of the gel filtration fractions on 10% SDS PAGE, transferring to Nitrocellulose Membrane (Hybond-ECL, Amersham Life Sciences, Arlington Height, IL) using standard blotting techniques and probing with anti-I ⁇ B ⁇ antibodies (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). TNF ⁇ -inducibility was evaluated by comparing the level of I ⁇ B ⁇ in cells that were ( Figure 3B) and were not ( Figure 3A) exposed to TNF ⁇ (30 ng/mL for seven minutes, as described above). The IKB kinase activity of these cytoplasmic extracts was evaluated using the kinase assay described above. As shown in Figure 4B, the extract of TNF ⁇ -treated cells phosphorylated GST-I ⁇ B ⁇ 1-54 (wildtype), while the untreated cell extract showed significantly lower levels of I ⁇ B ⁇ kinase activity (Figure 4A).
  • Cytoplasmic extracts of TNF ⁇ -treated HeLa S3 cells were also subjected to in vitro kinase assays, using the N- terminal portion of I ⁇ B ⁇ (residues 1 to 54) as substrate.
  • I ⁇ B ⁇ phosphorylation of GST-I ⁇ B ⁇ 1-54
  • Figure 5A substrate containing threonine substitutions at positions 32 and 36 was not phosphorylated
  • Figure 5B substrate containing threonine substitutions at positions 32 and 36 was not phosphorylated
  • This Example illustrates the immunoprecipitation of I ⁇ B kinase activity from cytoplasmic extracts prepared from stimulated cells.
  • HeLa cells were TNF- ⁇ -treated (30 ⁇ g/mL, 7 minutes) and fractionated by gel filtration as described in Example 3. Twenty ⁇ L of gel filtration fraction #6 (corresponding to about 700 kD molecular weight) and l ⁇ g purified antibodies raised against MKP-1 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) were added to 400 ⁇ L of ice cold lx Pull Down Buffer (20mM Tris pH 8.0, 250 mM NaCl, 0.05% NP-40, 3mM EGTA, 5 mM EDTA, 10 mM ⁇ -glycerophosphate, 10 mM NaF, 10 mM PNPP, 300 ⁇ M Na 3 VO 4 , 1 mM benzamidine, 2 ⁇ M PMSF, 10 ⁇ g/ml aprotonin, 1 ⁇ g/ml leupeptin, 1 ⁇ g/ml pepstatin, 1 mM DTT).
  • gel filtration fraction #6
  • the sample was gently rotated for 1 hour at 4°C, at which time 40 ⁇ L of protein A-agarose beads (50:50 slurry. Santa Cruz Biotechnology, Inc., Santa Cruz, CA) was added. The sample was then rotated for an additional 1.5 hours at 4°C.
  • the protein A-agarose beads were pelleted at 3,000 ⁇ m for 2 minutes at 4°C and the pellet was washed three times with ice cold Pull Down Buffer (800 ⁇ L per wash).
  • the pellet was subjected to the standard in vitro I ⁇ B ⁇ kinase assay (as described above) using either 2 ⁇ g GST-I ⁇ B l-54 (wildtype) or 2 ⁇ g GST-I ⁇ B ⁇ l-54 (S32/36 to T) as the substrate.
  • Lysates were incubated on ice for 1 -2 hours, 10 ⁇ l of Protein A or G beads were added, and lysates were left to incubate with gentle rotation for an additional 1 hour at 4°C. The immunoprecipitate was then washed 3 times with 2x Pull-Down Buffer, IX with kinase buffer without ATP and subjected to a kinase assay as described in Example 2.
  • I ⁇ B kinase activity there was no noticeable loss in I ⁇ B kinase activity when the immunoprecipitate was subjected to more rigorous washing, such as in RIPA buffer (20 mM Tris, 250 mM NaCl, 1% NP-40, 1% DOC, 0.1% SDS, 3mM EDTA, 3mM EGTA, 10 mM ⁇ -glycerophosphate, 10 mM NaF, 10 mM PNPP, 300 ⁇ M Na 3 VO 4 , 1 mM benzamidine, 2 ⁇ M PMSF, 10 ⁇ g/ml aprotonin, 1 ⁇ g/ml leupeptin, 1 ⁇ g/ml pepstatin, 1 mM DTT) or washes up to 3.5 M urea.
  • RIPA buffer 20 mM Tris, 250 mM NaCl, 1% NP-40, 1% DOC, 0.1% SDS, 3mM EDTA, 3mM EGTA,
  • IKK signalsome kinase was inactive in unstimulated HeLa cells, but was rapidly activated within minutes of TNF ⁇ stimulation.
  • the IKK signalsome kinase did not phosphorylate a mutant GST-I ⁇ B ⁇ protein in which serine residues 32 and 36 had been mutated to threonine ( Figure 8A top panel, even-numbered lanes).
  • the IKK signalsome kinase specifically phosphorylated full-length wild-type I ⁇ B ⁇ , but not a mutant I ⁇ B ⁇ bearing the serine 32, 36 to alanine mutations, in the context of a physiological RelA-I ⁇ B ⁇ complex ( Figure 8C, lanes 3, 4). Together these results indicate that the anti-MKP-1 antibody co-immunoprecipitated the IKK signalsome.
  • the signalsome-associated IKB kinase met all the criteria expected of the authentic I ⁇ B kinase and had no detectable I ⁇ B ⁇ C-terminal kinase activity.
  • IKK signalsome kinase The specificity of the IKK signalsome kinase was further established by kinetic analysis and by examining its activity on various peptides and recombinant protein substrates ( Figure 9A). For these studies, synthetic peptides (Alpha Diagnostics International, San Antonio, TX) were prepared with the following sequences:
  • Inhibition of immunopurified IKK signalsome activity was measured by 32 P inco ⁇ oration into GST-I ⁇ B ⁇ (l-54) in a discontinuous assay using the reaction conditions described above.
  • concentrations of GST-I ⁇ B ⁇ (l-54) and ATP used in the inhibition studies were 0.56 ⁇ M and 3 ⁇ M, respectively.
  • Enzymatic reactions 32 ⁇ L were carried out in wells of a 96 well assay plate for one hour at room temperature and terminated with the addition of trichloroacetic acid (TCA) (150 ⁇ L/well of 12.5% w/v). The subsequent 20 minute incubation with TCA precipitated the proteins but not peptides from solution.
  • TCA trichloroacetic acid
  • the TCA precipitate was collected on 96-well glass fiber plates (Packard) and washed 10 times with approximately 0.3 mL per well of Dulbecco's phosphate buffered saline pH 7.4 (Sigma) using a Packard Filtermate 196. Scintillation fluid (0.50 mL, Micro Scint, Packard) was added to each well and the plate was analyzed for 32 P using a Packard TopCount scintillation counter. Less than 10% of ATP was turned over in the course of the assay reaction, ensuring that the kinetic data represented initial rate data. The inhibition constant of the P32, 36 peptide was determined by Dixon analysis (Dixon and Webb, In Enzymes (Academic Press: New York, ed.
  • the kinase displayed normal Michaelis-Menten kinetics, suggesting that it was not a mixture of diverse unrelated kinases.
  • the kinase was capable of phosphorylating an l ⁇ B ⁇ (21-41) peptide ( Figures 9A and 9B)) as well as two different I ⁇ B ⁇ (21-41) peptides, each bearing a free serine at either position 32 or 36 and phosphoserine at the other position ( Figures 9A and 9B, lanes 2, 3).
  • a peptide with phosphoserines at both positions was not phosphorylated at all ( Figure 9B, top), indicating that there was no significant turnover of the phosphates under our reaction conditions.
  • IKK signalsome kinase efficiently phosphorylated I ⁇ B peptides, it did not phosphorylate the c-Fos phosphopeptide containing a free serine and a free threonine (Figure 9B, top), two c-Jun peptides containing serine 63 and 73, respectively, ( Figure 9A, top panel, lanes 4, 5), or an MKP-1 peptide containing four serines and three threonines ( Figure 9A, lane 6). The latter peptides were substrates for JNK2 ( Figure 9A, bottom panel, lanes 4-6).
  • I ⁇ B ⁇ (21-41) peptide in which serines 32 and 36 were replaced by threonines was phosphorylated by the signalsome at least 10-fold less well than the wild-type serine-containing peptide, consistent with the slower phosphorylation and degradation kinetics of I ⁇ B ⁇ (S32/36 to T) in cells (DiDonato et al., Mol Cell. Biol. 76: 1295-1304. 1996), and the preference of the kinase for serine over threonine at positions 32, 36 in both full-length I ⁇ B ⁇ and GST-I ⁇ B ⁇ (1 -54) ( Figures 8 A and C). In addition, the kinase phosphorylated GST-I ⁇ B ⁇ (1-54), albeit with lower affinity.
  • I ⁇ B kinase activity was also associated with strong RelA kinase activity (Figure 8C, lanes 3, 4), but no activity was observed towards several other substrates including myelin basic protein (MBP), GST-ATF2 (1 -1 12), GST-cJun (1-79), GST-ERK3, GST-Elk (307-428), GST-p38, and a GST fusion protein containing the C- terminal region of I ⁇ B ⁇ (242-314).
  • MBP myelin basic protein
  • GST-ATF2 (1 -1 12
  • GST-cJun (1-79
  • GST-ERK3 GST-Elk
  • GST-p38 GST-p38
  • the specificity of the IKK signalsome kinase was further emphasized by its susceptibility to product inhibition (Figure 9B, bottom).
  • the kinase was strongly inhibited by a doubly-phosphorylated I ⁇ B ⁇ peptide bearing phosphoserines at both positions 32 and 36, but not by the unrelated c-Fos phosphopeptide that contained a single phospho threonine.
  • the singly-phosphorylated and the unphosphorylated I ⁇ B ⁇ peptides were less effective inhibitors.
  • This example illustrates the absence of detectable free ubiquitin with a IKK signalsome prepared as in Example 3.
  • Standard western blot procedures were performed (Amersham Life Science protocol, Arlington Heights, IL). 100 ng ubiquitin, 10 ng ubiquitin and 20 ul purified IkB ⁇ kinase complex was subjected to 16% Tricine SDS-PAGE (Novex, San Diego, CA), transferred to Hybond ECL Nitrocellulose membrane (Amersham Life Science, Arlington Heights, IL), and probed with antibodies directed against ubiquitin (MAB1510; Chemicon, Temecula, CA). The results are shown in Figure 10. Free ubiquitin could not be detected in the purified IkB ⁇ kinase preparation (even at very long exposures).
  • This Example illustrates a two-step affinity method for purification of the IKK signalsome, based on its recognition by the MKP-1 antibody (depicted in Figure 1 1 A) and the identification of I ⁇ B kinases.
  • HeLa S3 cells were stimulated for 7 minutes with 20 ng/ml TNF ⁇ (R&D Systems, Minneapolis, MN), harvested, whole cell lysates were prepared (1.2 g total protein) and approximately 5 mg of anti-MKP-1 antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) was added and incubated at 4°C for 2 hours with gentle rotation. Subsequently, 50 ml of Protein A agarose (Calbiochem, San Diego, CA) was added and the mixture was incubated for an additional 2 hours.
  • the immunoprecipitate was then sequentially washed 4X Pull-Down Buffer, 2X RIPA buffer, 2X Pull-Down Buffer, IX 3.5 M urea-Pull-Down Buffer and 3X Pull -Down Buffer.
  • the immunoprecipitate was then made into a thick slurry by the addition of 10 ml of Pull-Down Buffer, 25 mg of the specific MKP-1 peptide to which the antibody was generated (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) was added, and the mixture was incubated overnight at 4°C with gentle rotation.
  • the eluted IKK signalsome was then desalted on PD 10 desalting columns (Pharmacia Biotech, Piscataway, NJ) equilibrated with 50 mM Q buffer and chromatographed on a Mono Q column (Pharmacia Biotech, Piscataway, NJ). Fractions containing peak IkB kinase activity were pooled, concentrated and subjected to preparative SDS-PAGE. The intensity of two prominent protein bands of -85 and -87 kDa (indicated by silver stain in Figure 1 1B as IKK-1 and IKK-2 respectively) correlated with the profile of IKB kinase activity.
  • Coomassie stained -85 and -87 kDa bands were excised, in-gel digested with trypsin (Wil et al., Nature 57:466-69, 1 96) and a small aliquot of the supernatant was analyzed by high mass accuracy MALDI peptide mass mapping, as described by Shevchenko et al., Proc. Nail. Acad. Sci. USA 95:14440-45, 1996.
  • the peptide mass map from the IKK-1 band was searched against a comprehensive protein sequence database using the program PeptideSearch developed in house at EMBL Heidelberg.
  • the peptides were step-eluted with 0.5 ⁇ l of 50% MeOH in 5% formic acid into a nanoelectrospray needle. This needle was transferred to an APIII mass spectrometer (Perkin-Elmer, Sciex, Toronto, Canada) and the sample sprayed for approximately 20 minutes. During this time, peptide ions apparent from the mass spectrum were selected and isolated in turn and fragmented in the collision chamber of the mass spectrometer. From the tandem mass spectra, short stretches of sequence were assembled into peptide sequence tags (Mann and Wilm, Anal. Chem. 66:4390-99, 1994) and searched against a protein sequence database or against dbEST using PeptideSearch.
  • FIGS. 12A and 12B Representative mass spectra are shown in Figures 12A and 12B.
  • Figure 12A peaks labeled A were matched to the tryptic peptides of IKK-1 upon fragmentation followed by database searching with peptide sequence tags. Peaks labeled B2. B4, B6 were not found in protein databases but instead matched human EST sequences. One more peptide (Bl) matching a human EST clone was observed at m/z 392.2 and is not shown in panel A.
  • Figure 12B a continuous series of C-terminal-containing fragments (Y"- ions) was used to construct a peptide sequence tag as shown by boxed letters.
  • Full-length human IKK-1 and IKK-2 cDNAs were cloned based on the. human EST clones, which were obtained from Genome Systems, Inc. (St. Louis, MO). The precise nucleotide sequences were determined and used to design primers to PCR clone human IKK-2 from a human HeLa cell cDNA library (Clontech, Inc., Palo Alto, CA). Several IKK-2 cDNA clones were isolated and sequenced. Full-length mouse IKK- 1 and a partial human IKK-1 nucleotide sequence was available in the comprehensive database, primers were designed to PCR clone the respective human and mouse IKK-1 cDNAs. The partial human IKK-1 coding region was used to probe a HeLa cDNA phage library (Stratagene, Inc., La Jolla, CA) to obtain the full-length human IKK-1 cDNA clone using standard procedures.
  • IKK-1 and IKK-2 were related protein serine kinases (51% identity) containing protein interaction motifs ( Figure 13 A). Both IKK-1 and IKK-2 contain the kinase domain at the N-terminus. and a leucine zipper motif and a helix-loop-helix motif in their C-terminal regions ( Figure 13 A).
  • Northern analysis indicated that mRNAs encoding IKK-2 were widely distributed in human tissues, with transcript sizes of -4.5 kb and 6 kb ( Figure 13B). The distribution of IKK-1 (CHUK) transcripts has been reported previously (Connely et al., Cell. Mol. Biol. Res. 47:537-49, 1995).
  • IKK-1 and IKK-2 mRNAs are constitutively expressed in Jurkat. HeLa and HUVEC cell lines, and their levels are not altered for up to 8 hours following stimulation with NFKB inducers such as TNF ⁇ (HeLa, HUVEC) or anti-CD28 plus PMA (Jurkat).
  • NFKB inducers such as TNF ⁇ (HeLa, HUVEC) or anti-CD28 plus PMA (Jurkat).
  • IKK-1 and IKK-2 recombinant HA-tagged IKK-1 and Flag-tagged IKK-2, either separately or alone, were in vitro transcribed and translated in wheat germ or rabbit reticulocyte lysate (Promega. Madison. WI). The reactions were performed exactly as described in the manufacturer's protocol. Epitope-tagged IKK-1 and IKK-2 then immunoprecipitated with the appropriate anti-tag antibody.
  • Immunoprecipitates containing these proteins phosphorylated I ⁇ B ⁇ and I ⁇ B ⁇ with the correct substrate specificity i.e., immunoprecipitates of IKK-1 and IKK-2 phosphorylated both GST-I ⁇ B ⁇ ( Figure 14A, panel 3) and GST-I ⁇ B ⁇ (panel 4), but did not phosphorylate the corresponding S32/36 to T mutant protein).
  • IKK-1 expressed in rabbit reticulocyte lysates was also capable of autophosphorylation ( Figure 14A, panel 2, lane 1), whereas a kinase-inactive version of IKK-1 , in which the conserved lysine 44 had been mutated to methionine, showed no autophosphorylation.
  • IKK-2 although expressed at equivalent levels in the lysates (panel 1), showed very weak autophosphorylation (panel 2, lane 2).
  • the cells were incubated sequentially with primary antibody in PBS containing 10% donkey serum and 0.25% Triton X-100 for 2 hours followed by fluorescein-conjugated or Texas red- conjugated secondary antibody (Jackson Immunoresearch Laboratories, Inc., West Grove, PA; used at 1 :500 dilution) for 1 hour at room temperature.
  • the coverslips were rinsed and coverslipped with Vectashield (Vector Laboratories, Burlingame, CA) before scoring and photographing representative fields.
  • Primary antibodies used for immunofluorescence staining included antibodies against Rel A (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), HA tag (Babco, Berkeley, CA) and Flag tag (IBI- Kodak, New Haven, CT).
  • IKK-1 and IKK-2 The presence of the leucine zipper and helix-loop-helix motif in IKK-1 and IKK-2 suggested that they interacted functionally with other proteins in the signalsome. An obvious possibility was that the proteins formed hetero- or homodimers with one another.
  • HA-tagged IKK-1 and FLAG-tagged IKK-2 were translated in rabbit reticulocyte lysates, either alone or together, and then immunoprecipitated with antibodies to the appropriate epitope tags. This experiment demonstrated clearly that IKK-2 was present in IKK-1 immunoprecipitates ( Figure 15A, lane 3) and vice versa (lane 4), suggesting that these proteins either associated directly or via adapter proteins/IKK signalsome components present in the rabbit reticulocyte lysates.
  • IKK-1 and IKK-2 kinases were active when expressed in wheat germ extracts, since they were capable of autophosphorylation, but they were inactive with respect to phosphorylation of I ⁇ B substrates. Since both autophosphorylation and substrate phosphorylation were intact in rabbit reticulocyte lysates, there appeared to be a direct correlation between the association of IKK-1 and IKK-2 into a higher order protein complex and the presence of specific I ⁇ B kinase activity in IKK-1 and IKK-2 immunoprecipitates. This higher order complex is most likely the IKK signalsome itself.
  • IKK signalsome contains multiple protein components in addition to IKK-1 and IKK-2 ( Figure 1 IB). Some of these may be upstream kinases such as MEKK-1 (Chen et al., Cell 54:853-62, 1996) or NIK (Malinin, et al., Nature 555:540-44, 1997); others may be adapter proteins that mediate the IKK-1 :IKK-2 interaction. Indeed MEKK-1 copurifies with IKK signalsome activity ( Figure IC), and two other signalsome proteins have been functionally identified.
  • the protein crossreactive with anti-MKP-1 is an intrinsic component of the IKK signalsome kinases, since the I ⁇ B kinase activity coprecipitated with this antibody is stable to washes with 2-4 M urea.
  • both IKK-1 immunoprecipitates and MKP-1 immunoprecipitates containing the IKK signalsome ( Figure 8C) contain an inducible RelA kinase whose kinetics of activation parallel those of the IKB kinase in the same immunoprecipitates.
  • Another strong candidate for a protein in the signalsome complex is the E3 ubiquitin ligase that transfers multiubiquitin chains to phosphorylated IKB (Hershko et al., Annu. Rev. Biochem. 67:761-807, 1992).
  • IKK- 1 and IKK-2 are functional kinases within the IKK signalsome, which mediate IKB phosphorylation and NFKB activation.
  • Appropriate regulation of IKK-1 and IKK-2 may require their assembly into a higher order protein complex, which may be a heterodimer facilitated by adapter proteins, the complete IKK signalsome, or some intermediate subcomplex that contains both IKK-1 and IKK-2.
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  • TCTGTGTACA TCTTTTGTGG GAACACTGCA GTATCTGGCC CCAGAGCTCT TTGAGAATAA 600
  • AAAGGCCATC CACTATGCTG AGGTTGGTGT CATTGGA AC CTGGAGGATC AGATTATGTC 1620
  • CTTAGCCAGC CTGGGCAGCT GATGTCTCAG CCCTCCACGG CCTCCAACAG CTTACCTGAG 2100
  • AATGCCAT ⁇ C AGGACACTGT GAGGGAACA ⁇ GACCAGAGTT TCACGGCCCT AGACTGGAGC 2220
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Abstract

Compositions and methods are provided for treating NF-λB-related conditions. In particular, the invention provides a stimulus-inducible IKK signalsome, and components and variants thereof. An IKK signalsome or component thereof may be used, for example, to identify antibodies and other modulating agents that inhibit or activate signal transduction via the NF-λB cascade. IKK signalsome, components thereof and/or modulating agents may also be used for the treatment of diseases associated with NF-λB activation.

Description

Description
STIMULUS-INDUCIBIJE I (KAPPA)B KINASE [KKJ SIGNALSOME
Technical Field
The present invention relates generally to compositions and methods useful for the study of cascades leading to the activation of nuclear factor KB (NF-KB) and for treating diseases associated with such pathways. The invention is more particularly related to a stimulus-inducible IκB kinase (IKK) signalsome, component IκB kinases and variants of such kinases. The present invention is also related to the use of a stimulus-inducible IKK signalsome or IκB kinase to identify antibodies and other agents that inhibit or activate signal transduction via the NF-κB pathway.
Background of the Invention
Transcription factors of the NFκB/Rel family are critical regulators of genes involved in inflammation, cell proliferation and apoptosis (for reviews, see Verma et al., Genes Dev. 9:2723-35, 1995; Siebenlist, Biochim. Biophys. Ada 1332:7-13, 1997; Baeuerle and Henkel, Ann. Rev. Immunol. 72: 141-79, 1994; Barnes and Karin, New Engl. J. Med. 336. 1066-71 , 1997; Baeuerle and Baltimore, Cell 87: 13-20, 1996; Grilli et al., NF-kB and Rel: Participants in a multiform transcriptional regulatory system (Academic Press, Inc., 1993), vol. 143; Baichwal and Baeuerle, Curr. Biol. 7:94-96, 1997). The prototype member of the family, NFKB, is composed of a dimer of p50 NFKB and p65 RelA (Baeuerle and Baltimore, Cell 55:211-17, 1988; Baeuerle and Baltimore, Genes Dev. 3:1689-98, 1989). NF-κB plays a pivotal role in the highly specific pattern of gene expression observed for immune, inflammatory and acute phase response genes, including interleukin 1. interleukin 8, tumor necrosis factor and certain cell adhesion molecules.
Like other members of the Rel family of transcriptional activators, NF-κB is sequestered in an inactive form in the cytoplasm of most cell types. A variety of extracellular stimuli including mitogens, cytokines. antigens, stress inducing agents. UV light and viral proteins initiate a signal transduction pathway that ultimately leads to NFKB release and activation. Thus, inhibitors and activators of the signal transduction pathway may be used to alter the level of active NF-κB, and have potential utility in the treatment of diseases associated with NF-κB activation. Activation of NFKB in response to each of these stimuli is controlled by an inhibitory subunit, IκB, which retains NFKB in the cytoplasm. IκB proteins, of which there are six known members, each contain 5-7 ankyrin-like repeats required for association with the NFκB/Rel dimer and for inhibitory activity (see Beg et al., Genes Dev. 7, 2064-70, 1993; Gilmore and Morin, Trends Genet. 9, 427-33, 1993; Diaz-Meco et al., Mol. Cell. Biol. 73:4770-75, 1993; Haskill et al., Cell (55:1281-89, 1991). IκB proteins include IκBα and IκBβ.
NFKB activation involves the sequential phosphorylation, ubiquitination, and degradation of IκB. Phosphorylation of IκB is highly specific for target residues. For example, phosphorylation of the IκB protein IκBα takes place at serine residues S32 and S36, and phosphorylation of IκBβ occurs at serine residues S19 and S23. The choreographed series of modification and degradation steps results in nuclear import of transcriptionally active NFKB due to the exposure of a nuclear localization signal on NFKB that was previously masked by IκB (Beg et al., Genes Dev. 6: 1899- 1913, 1992). Thus, NFKB activation is mediated by a signal transduction cascade that includes one or more specific IκB kinases, a linked series of El , E2 and E3 ubiquitin enzymes, the 26S proteasome, and the nuclear import machinery. The phosphorylation of IκB is a critical step in NF-κB activation, and the identification of an IκB kinase, as well as proteins that modulate its kinase activity, would further the understanding of the activation process, as well as the development of therapeutic methods. Several protein kinases have been found to phosphorylate
IκB in vitro, including protein kinase A (Ghosh and Baltimore, Nature 344:678-82, 1990), protein kinase C (Ghosh and Baltimore, Nature 544:678-82, 1990) and double stranded RNA-dependent protein kinase (Kumar et al., Proc. Natl. Acad. Sci. USA 97:6288-92, 1994). Constitutive phosphorylation of IκBα by casein kinase II has also been observed (see Barroga et al., Proc. Nail. Acad. Sci. USA 92:7637-41 , 1995). None of these kinases, however appear to be responsible for in vivo activation of NF-κB. For example, phosphorylation of IκBα in vitro by protein kinase A and protein kinase C prevent its association with NF-κB, and phosphorylation by double-stranded RNA- dependent protein kinase results in dissociation of NF-κB. Neither of these conform to the effect of phosphorylation in vivo, where IκBα phosphorylation at S32 and S36 does not result in dissociation from NF-κB.
Other previously unknown proteins with IκB kinase activity have been reported, but these proteins also do not appear to be significant activators in vivo. A putative IκBα kinase was identified by Kuno et al., J. Biol. Chem. 270:27914-27919, 1995, but that kinase appears to phosphorylate residues in the C-terminal region of IκBα. rather than the S32 and S36 residues known to be important for in vivo regulation. Diaz- Meco et al., EMBO J. 73:2842-2848, 1994 also identified a 50 kD IκB kinase, with uncharacterized phosphorylation sites. Schouten et al., EMBO J 76:3133-44, 1997 identified p90rskl as a putative IκBα kinase; however, p90rskl is only activated by TPA and phosphorylates IκBα only on Ser32, which is insufficient to render IκBα a target for ubiquitination. Finally, Chen et al, Cell #4:853-862, 1996 identified a kinase that phosphorylates IκBα, but that kinase was identified using a non-physiological inducer of IκBα kinase activity and requires the addition of exogenous factors for in vitro phosphorylation. Accordingly, there is a need in the art for an IκB kinase that possesses the substrate specificity and other properties of the in vivo kinase. There is also a need for improved methods for modulating the activity of proteins involved in activation of NFKB, and for treating diseases associated with NF-κB activation. The present invention fulfills these needs and further provides other related advantages.
Summary of the Invention
Briefly stated, the present invention provides compositions and methods employing a large, multi-subunit IKK signalsome, or a component or variant thereof. In one aspect, the present invention provides an IKK signalsome capable of specifically phosphorylating IκBα at residues S32 and S36, and IκBβ at residues 19 and 23, without the addition of exogenous co factors.
In a further related aspect, a polypeptide comprising a component of an
IKK signalsome, or a variant of such a component, is provided, wherein the component has a sequence recited in SEQ ID NO:9. An isolated DNA molecule and recombinant expression vector encoding such a polypeptide, as well as a transfected host cell, are also provided.
In another aspect, methods for preparing an IKK signalsome are provided, comprising combining components of an IKK signalsome in a suitable buffer. In yet another aspect, methods are provided for phosphorylating a substrate of an IKK signalsome, comprising contacting a substrate with an IKK signalsome or a component thereof, and thereby phosphorylating the substrate.
In a further aspect, the present invention provides a method for screening for an agent that modulates IKK signalsome activity, comprising: (a) contacting a candidate agent with an IKK signalsome, wherein the step of contacting is carried out under conditions and for a time sufficient to allow the candidate agent and the IKK signalsome to interact; and (b) subsequently measuring the ability of the candidate agent to modulate IKK signalsome activity.
Within a related aspect, the present invention provides methods for screening for an agent that modulates IKK signalsome activity, comprising: (a) contacting a candidate agent with a polypeptide comprising a component of an IKK signalsome as described above, wherein the step of contacting is carried out under conditions and for a time sufficient to allow the candidate agent and the polypeptide to interact; and (b) subsequently measuring the ability of the candidate agent to modulate the ability of the polypeptide to phosphorylate an IκB protein.
In another aspect, an antibody is provided that binds to a component (e.g., IKK- 1 and/or IKK-2) of an IKK signalsome, where the component is capable of phosphorylating IκBα.
In further aspects, the present invention provides methods for modulating NF-κB activity in a patient, comprising administering to a patient an agent that modulates
IκB kinase activity in combination with a pharmaceutically acceptable carrier. Methods are also provided for treating a patient afflicted with a disorder associated with the activation of IKK signalsome, comprising administering to a patient a therapeutically effective amount of an agent that modulates IκB kinase activity in combination with a pharmaceutically acceptable carrier. In yet another aspect, a method for detecting IKK signalsome activity in a sample is provided, comprising: (a) contacting a sample with an antibody that binds to an IKK signalsome under conditions and for a time sufficient to allow the antibody to immunoprecipitate an IKK signalsome; (b) separating immunoprecipitated material from the sample; and (c) determining the ability of the immunoprecipitated material to specifically phosphorylate an IκB protein with in vivo specificity. Within one such embodiment, the ability of the immunoprecipitated material to phosphorylate IκBα at residues S32 and/or S36 is determined.
In a related aspect, a kit for detecting IKK signalsome activity in a sample is provided, comprising an antibody that binds to an IKK signalsome in combination with a suitable buffer.
In a further aspect, the present invention provides a method for identifying an upstream kinase in the NF-κB signal transduction cascade, comprising evaluating the ability of a candidate upstream kinase to phosphorylate an IKK signalsome, a component thereof or a variant of such a component. A method for identifying a component of an IKK signalsome is also provided, comprising: (a) isolating an IKK signalsome; (b) separating the signalsome into components, and (c) obtaining a partial sequence of a component.
In yet another aspect, a method is provided for preparing an IKK signalsome from a biological sample, comprising: (a) separating a biological sample into two or more fractions; and (b) monitoring IκB kinase activity in the fractions.
These and other aspects of the present invention will become apparent upon reference to the following detailed description and attached drawings. All references disclosed herein are hereby incorporated by reference in their entirety as if each was incorporated individually. Brief Description of the Drawings
Figures 1A-1C are autoradiograms depicting the results of immunoblot analyses. Figure 1A shows the recruitment of IκBα into a high molecular weight complex upon stimulation. Cytoplasmic extracts of either unstimulated or PMA(50 ng/ml)- and PHA(1 μg/ml)-stimulated (10 min) Jurkat cells were fractionated on a gel filtration column. IκBα was visualized by immunoblot analysis. The upper panel shows the elution profile of unstimulated cells, and the lower panel shows the elution profile of PMA/PHA-stimulated cells. Molecular weight standards are indicated by arrows on the top. Figure IB shows that the stimulus-dependent IκBα kinase activity chromatographs as a high molecular weight complex, Mr 500-700 kDa. Whole cell extract of TNFα-stimulated (20 ng/ml, 7 min) HeLa S3 cells was fractionated on a Superdex 200 gel filtration column and monitored for IκBα kinase activity. Phosphorylation of the GST IκBα 1 -54 (wildtype) substrate is indicated by an arrow to the right. Molecular weight standards are indicated by arrows on the top.
Figure 1 C illustrates the identification of proteins that co-chromatograph with the IKK signalsome. IKK signalsome was partially purified from extracts of TNFα- stimulated HeLa S3 cells by sequential fractionation on a Q Sepharose, Superdex 200, Mono Q, and Phenyl Superose columns. Phenyl Superose fractions containing the peak of IKK signalsome activity were subjected to western blot analysis using several different antibodies as indicated to the left of each respective panel. The level of IKK signalsome activity is indicated in the upper shaded area by increasing number of (+)'s.
Figure 2 is a flow chart depicting a representative purification procedure for the preparation of an IKK signalsome. Figures 3A and 3B are autoradiograms that show the results of a Western blot analysis of the levels of IκBα in HeLa S3 cytoplasmic extracts following gel filtration. The extracts were prepared from cells that were (Figure 3B) and were not (Figure 3A) exposed to TNFα.
Figures 4A and 4B are autoradiograms depicting the results of an in vitro kinase assay in which the ability of the above cell extracts to phosphorylate the N- terminal portion of IκBα was evaluated. Figure 4A shows the results employing an extract from cells that were not treated with TNFα, and Figure 4B shows the results when the cells were treated with TNFα.
Figures 5A and 5B are autoradiograms depicting the results of an in vitro kinase assay using a cytoplasmic extract of TNFα-treated HeLa S3 cells, where the extract is subjected to Q Sepharose fractionation. The substrate was the truncated IκBα (residues 1 to 54), with Figure 5A showing the results obtained with the wild type IκBα sequence and Figure 5B presenting the results obtained using a polypeptide containing threonine substitutions at positions 32 and 36.
Figures 6A and 6B are autoradiograms depicting the results of an in vitro kinase assay using a cytoplasmic extract of TNFα-treated HeLa S3 cells, where the extract was subjected in series to chromatographic fractionation by Q Sepharose, Superdex 200, Mono Q Sepharose and Phenyl Superose. The substrate was the truncated IκBα (residues 1 to 54), with Figure 6A showing the results obtained with the wild type IκBα sequence and Figure 6B presenting the results obtained using a polypeptide containing threonine substitutions at positions 32 and 36.
Figure 7 is an autoradiogram showing the results of immunokinase assays (using anti-MKP-1 antibody) performed using cytoplasmic extracts of TNFα-treated HeLa S3 cells following gel filtration. The assay was performed using the substrates GST-IκBαl -54 wildtype (lane 1) and GST- IκBαl -54 S32/36 to T (lane 2). The positions of IκBα and GST-IκBα 1-54 are indicated on the left.
Figures 8A-8C are autoradiograms depicting the results of immunoblot analyses. In Figure 8A, the upper panel presents a time course for the induction of signalsome activity. Anti MKP-1 immune precipitates from extracts of HeLa S3 cells stimulated with TNFα (20 ng/ml) for the indicated times, were assayed for IKK signalsome activity by standard immune complex kinase assays. 4 μg of cither GST IκBα 1-54 WT (wildtype) or the GST IκBα 1-54 S32/36 to T mutant (S>T) were used as the substrates. In the lower panel, HeLa cell extracts prepared as described in the upper panel were examined by western blot analysis for IκBα degradation. IκBα supershifting phosphorylation can be seen after 3 and 5 minutes of stimulation followed by the disappearance of IκBα. Figure 8B illustrates the stimulus-dependent activation of IKK signalsome, which is blocked by TPCK. Anti-MKP-1 immunoprecipitates from cell extracts of HeLa S3 cells either stimulated for 7 min with TNFα (20 ng/ml, lane 2 and 6), IL-1 (10 ng/ml, lane 3), PMA (50 ng/ml, lane 4) or pretreated for 30 min with TPCK (15 μM, lane 7) prior to TNFα-induction were examined for IKK signalsome activity. GST IκBα 1-54 WT (4 μg) was used as a substrate.
Figure 8C illustrates the ability of IKK signalsome to specifically phosphorylate serines 32 and 36 of the IκBα holoprotein in the context of a RclA:IκBα complex. Anti-MKP-1 immunoprecipitates from cell extracts of HeLa S3 cells stimulated with TNFα (20 ng/ml, 7 min) were examined for their ability to phosphorylate baculoviral expressed RelA:IκBα complex containing either the IκBα WT (lane 3) or IκBα S32/36 to A mutant (lane 4) holoprotein. The specific substrates used are indicated on the top. Positions of the phosphorylatcd substrates are indicated by arrows to the left of the panel. Figure 9A is an autoradiogram depicting the results of an immunokinase assay in which peptides are phosphorylatcd by the IKK signalsome. In the top panel, IκBα(21-41) peptides that were unphosphorylated or chemically phosphorylated on either Ser-32 or Ser-36 were incubated with the IKK signalsome in the presence of γ-f32P]-ATP. The doubly phosphorylated peptide P32,36 was not phosphorylated by the IKK signalsome, and the unrelated c-Fos(222-241) phosphopeptide with free serine and threonine residues did not function as a signalsome substrate.
Figure 9B is a graph illustrating the inhibition of phosphorylation of GST- IκBα (1-54) by IκBα(21-41) peptides. IκBα(21-41) peptide P32,36 inhibits GST- IκBα (1-54) as a product inhibitor with a K, value of 14 μM. The unrelated phosphopeptide c- Fos(222-241) does not function as an inhibitor. This assay only detects precipitated 32P- labeled proteins, not 32P-labeled peptides. Addition of the singly- or non-phosphorylated IκBα(21-41) peptides results in less phosphorylation of GST-IκBα (1-54) and apparent inhibition.
Figure 10 is an autoradiogram showing the results of a western blot analysis of the level of ubiquitin within a stimulus-inducible IkB kinase complex. Lane 1 shows the detection of 100 ng ubiquitin, Lane 2 shows 10 ng ubiquitin and Lane 3 shows 3.4 μg of IKK signalsome purified through the phenyl superose step (sufficient quantities for 10 kinase reactions). The position of ubiquitin is shown by the arrow on the left.
Figure 11A illustrates a procedure for purification of the IKK signalsome. A whole cell extract was prepared from TNFα-stimulated (20 ng/ml, 7 minute induction) HeLa S3 cells (1.2 g total protein). The IKK signalsome was then immunoprecipitated from the extract using anti-MKP-1 antibodies, washed with buffer containing 3.5 M urea and eluted overnight at 4°C in the presence of excess MKP-1 specific peptide. Eluted IKK signalsome was then fractionated on a Mono Q column, IκB kinase active fractions were pooled, concentrated and subjected to preparative SDS-PAGE. Individual protein bands were excised and submitted for peptide sequencing.
Figure 1 IB is a photograph showing Mono Q fractions containing active
IKK signalsome activity following SDS-PAGE and a standard silver stain protocol. Peak activity of IKK signalsome activity is represented in lanes 3, 4, and 5. Protein bands corresponding to IKK-1 and IKK-2 are indicated to the left of the figure. Molecular weight standards (kDa) are indicated to the left of the figure.
Figures 12A and 12B are mass spectra obtained during sequencing of
IKK-2 by nanoelectrospray mass spectrometry. Figure 12A shows part of the mass spectrum of the unseparated mixture of tryptic peptides resulting from in-gel digestion of the IKK-2 band in Figure 1 IB. Figure 12B shows a tandem mass spectrum of the peak at m/z 645.2.
Figure 13A illustrates the amino acid sequence of IKK-1 and IKK-2. Symbols: arrows, boundaries of the kinase domain; underlined, peptide sequences identified by nanoelectrospray mass spectrometry; asterisks, indicates leucines comprising the leucine zipper motif; bold face, indicate amino acid identities conserved between IKK-1 and IKK-2; highlighted box, Helix-loop-helix domain; dashes, a gap inserted to optimize alignment.
Figure 13B is an autoradiogram depicting the results of Northern blot analysis of IKK-2 mRNA in adult human tissue. The source of the tissue is indicated at the top. Probes spanning the coding region of human IKK-2 and β-actin cDNA were used and are indicated to the left. Molecular weight standards are indicated to the right. Figure 14A is an autoradiogram depicting the results of kinase assays using IKK-1 and IKK-2. IKK-1 and IKK-2 were immunoprecipitated from rabbit reticulocyte lysates phosphorylate IκBα and IκBβ. Either HA-tagged IKK-1 (lane 1 ) or Flag-tagged IKK-2 (lane 2) were translated in rabbit reticulocyte lysates, immunoprecipitated, and examined for their ability to phosphorylate GST IκBα 1 -54 WT and GST lκBβ 1-44 as indicated by an arrow to the left. IKK-1 (lane 1) undergoes significant autophosphorylation in contrast to IKK-2 (lane 2) which is identified only with longer exposure times.
Figures 14B and 14C are micrographs illustrating the results of assays to evaluate the ability of kinase-inactive mutants of IKK-1 and IKK-2 to inhibit RelA translocation in TNFα-stimulated HeLa cells. HeLa cells were transiently transfected with either HA-tagged IKK-1 K44 to M mutant (14B) or Flag-tagged IKK-2 K44 to M mutant (14C) expression vectors. 36 hours post-transfection cells were either not stimulated (Unstim) or TNFα-stimulated (20 ng/ml) for 30 min (TNFα), as indicated to the right of the figure. Cells were then subjected to immunofluorescence staining using anti-HA of anti-Flag antibodies to visualize expression of IKK-1 K44 to M or IKK-2 K44 to M, respectively. Stimulus-dependent translocation of Rel A was monitored using anti- Rel A antibodies. Antibodies used are indicated to the top of the figure. IKK mutant transfected is indicated to the left of the figure. Figures 15A and 15B are autoradiograms of immunoprecipitated IKK-1 and IKK-2 following in vitro translation. In Figure 15 A, HA-tagged IKK-1 and Flag- tagged IKK-2 were in vitro translated in wheat germ lysates either separately or in combination, as indicated. The programmed translation mix was then subjected to immunoprecipitation using the indicated antibody. The samples were run on SDS-PAGE and subjected to autoradiography. In Figure 15B, HA-tagged IKK-1 and Flag-tagged IKK-2 were in vitro translated in rabbit reticulocyte lysates either separately or in combination, as indicated. The programmed translation mix was then subjected to immunoprecipitation using the indicated antibody. The samples were run on SDS-PAGE and subjected to autoradiography. The results show that IKK-1 and IKK-2 coprecipitate when translated in rabbit reticulocyte lysates. Detailed Description of the Invention
As noted above, the present invention is generally directed to compositions and methods for modulating (i.e., stimulating or inhibiting) signal transduction leading to NF-κB activation. In particular, the present invention is directed to compositions comprising an IκB kinase (IKK) signalsome (also referred to herein as a "stimulus-inducible IκB kinase complex" or "IκB kinase complex") that is capable of stimulus-dependent phosphorylation of IκBα and IκBβ on the two N-terminal serine residues critical for the subsequent ubiquitination and degradation in vivo. Such stimulus-dependent phosphorylation may be achieved without the addition of exogenous cofactors. In particular, an IKK signalsome specifically phosphorylates IκBα (SEQ ID NO: l ) at residues S32 and S36 and phosphorylates IκBβ (SEQ ID NO:2) at residues S 19 and S23. The present invention also encompasses compositions that contain one or more components of such an IKK signalsome, or variants of such components. Preferred components, referred to herein as "IKK signalsome kinases" "IKB kinases" or IKKs) are kinases that, when incorporated into an IKK signalsome, are capable of phosphorylating IκBα at S32 and S36. Particularly preferred components are IKK-1 (SEQ ID NO: 10) and IKK-2 (SEQ ID NO:9).
An IKK signalsome and/or IKB kinase may generally be used for phosphorylating a substrate (i.e., an IKB, such as IκBα, or a portion or variant thereof that can be phosphorylated at those residues that are phosphorylated in vivo) and for identifying modulators of IκB kinase activity. Such modulators and methods employing them for modulating IκBα kinase activity, in vivo and/or in vitro, are also encompassed by the present invention. In general, compositions that inhibit IκB kinase activity may inhibit IκB phosphorylation, or may inhibit the activation of an IκB kinase and/or IKK signalsome.
An IKK signalsome has several distinctive properties. Such a complex is stable (i.e., its components remain associated following purification as described herein) and has a high-molecular weight (about 500-700 kD, as determined by gel filtration chromatography). As shown in Figures 3 (A and B) and 4 (A and B), IκB kinase activity of an IKK signalsome is "stimulus-inducible" in that it is stimulated by TNFα (i.e.. treatment of cells with TNFα results in increased IKB kinase activity and IκB degradation) and/or by one or more other inducers of NF-κB, such as IL-1, LPS, TPA, UV irradiation, antigens, viral proteins and stress-inducing agents. The kinetics of stimulation by TNFα correspond to those found in vivo. IKB kinase activity of an IKK signalsome is also specific for S32 and S36 of IκBα. As shown in Figures 5 (A and B) and 6 (A and B), an IKK signalsome is capable of phosphorylating a polypeptide having the N-terminal sequence of IκBα (GST-IκBαl-54; SEQ ID NO:3), but such phosphorylation cannot be detected in an IκBα derivative containing threonine substitutions at positions 32 and 36. In addition, IκB kinase activity is strongly inhibited by a doubly phosphorylated IκBα peptide (i.e., phosphorylated at S32 and S36), but not by an unrelated c-fos phosphopeptide that contains a single phosphothreonine. A further characteristic of an IKK signalsome is its ability to phosphorylate a substrate in vitro in a standard kinase buffer, without the addition of exogenous cofactors. Free ubiquitin is not detectable in preparations of IKK signalsome (see Figure 10), even at very long exposures. Accordingly an IKK signalsome differs from the ubiquitin-dependent IκBα kinase activity described by Chen et al., Cell 54:853-62, 1996.
An IKK signalsome may be immunoprecipitated by antibodies raised against MKP-1 (MAP kinase phosphatase-1 ; Santa Cruz Biotechnology, Inc., Santa Cruz, CA #SC-1 102), and its activity detected using an in vitro IκBα kinase assay. However, as discussed further below, MKP-1 does not appear to be a component of IKB kinase complex. The substrate specificity of the immunoprecipitated IKK signalsome is maintained (i.e., there is strong phosphorylation of wildtype GST-IκBα 1 -54 (SEQ ID NO:3) and GST-IκBβ 1-44 (SEQ ID NO:4), and substantially no detectable phosphorylation of GST-IκBα 1-54 in which serines 32 and 36 are replaced by threonines (GST- IκBα S32/36 to T; SEQ ID NO:5) or GST-IκBβ 1-44 in which serines 19 and 23 are replaced by alanines (GST-IκBβ 1-44 SI 9/23 to A; SEQ ID NO:6)).
An IKK signalsome may be isolated from human or other cells, and from any of a variety of tissues and/or cell types. For example, using standard protocols, cytoplasmic and/or nuclear/membrane extracts may be prepared from HeLa S3 cells following seven minutes induction with 30 ng/mL TNFα. The extracts may then be subjected to a series of chromatographic steps that includes Q Sepharose, gel filtration (HiLoad 16/60 Superdex 200), Mono Q, Phenyl Superose, gel filtration (Superdex 200 10/30) and Mono Q. This representative purification procedure is illustrated in Figure 2, and results in highly enriched IKK signalsome (compare, for example, Figures 5A and 6A).
An alternative purification procedure employs a two-step affinity method, based on recognition of IKK signalsome by the MKP-1 antibody (Figure 1 1A). Whole cell lysates from TNFα-stimulated HeLa cells may be immunoprecipitated with an anti- MKP-1 antibody. The IKK signalsome may be eluted with the specific MKP-1 peptide to which the antibody was generated and fractionated further on a Mono Q column.
Throughout the fractionation, an in vitro kinase assay may be used to monitor the IκB kinase activity of the IKK signalsome. In such an assay, the ability of a fraction to phosphorylate an appropriate substrate (such as IκBα (SEQ ID NO:l) or a derivative or variant thereof) is evaluated by any of a variety of means that will be apparent to those of ordinary skill in the art. For example, a substrate may be combined with a chromatographic fraction in a protein kinase buffer containing P γ-ATP, phosphatase inhibitors and protease inhibitors. The mixture may be incubated for 30 minutes at 30°C. The reaction may then be stopped by the addition of SDS sample buffer and analyzed using SDS-PAGE with subsequent autoradiography. Suitable substrates include full length IκBα (SEQ ID NO:l), polypeptides comprising the N-terminal 54 amino acids of IκBα, full length IκBβ (SEQ ID NO:2) and polypeptides comprising the N-terminal 44 amino acids of IκBβ. Any of these substrates may be used with or without an N-terminal tag. One suitable substrate is a protein containing residues 1 -54 of IκBα and an N-termina! GST tag (referred to herein as GST-IκBα 1-54; SEQ ID NO:3). To evaluate the specificity of an IκB kinase complex, IκBα mutants containing threonine or alanine residues at positions 32 and 36, and/or other modifications, may be employed.
Alternatively, an IKK signalsome may be prepared from its components which are also encompassed by the present invention. Such components may be produced using well known recombinant techniques, as described in greater detail below. Components of an IKK signalsome may be native, or may be variants of a native component (i e., a component sequence may differ from the native sequence in one or more substitutions and/or modifications, provided that the ability of a complex comprising the component variant to specifically phosphorylate lκBα is not substantially diminished). Substitutions and/or modifications may generally be made in non-critical and/or critical regions of the native protein. Variants may generally comprise residues of L-amino acids, D-amino acids, or any combination thereof. Amino acids may be naturally-occurring or may be non-natural, provided that at least one amino group and at least one carboxyl group are present in the molecule; α- and β-amino acids are generally preferred. A variant may also contain one or more rare amino acids (such as 4- hydroxyproline or hydroxylysine), organic acids or amides and/or derivatives of common amino acids, such as amino acids having the C-terminal carboxylate esterified (e.g., benzyl, methyl or ethyl ester) or amidated and/or having modifications of the N-terminal amino group (e.g., acetylation or alkoxycarbonylation), with or without any of a wide variety of side-chain modifications and/or substitutions (e.g.. methylation, benzylation, t- butylation, tosylation, alkoxycarbonylation, and the like). Component variants may also, or alternatively, contain other modifications, including the deletion or addition of amino acids that have minimal influence on the activity of the polypeptide. In particular, variants may contain additional amino acid sequences at the amino and/or carboxy termini. Such sequences may be used, for example, to facilitate purification or detection of the component polypeptide. In general, the effect of one or more substitutions and/or modifications may be evaluated using the representative assays provided herein.
A component may generally be prepared from a DNA sequence that encodes the component using well known recombinant methods. DNA sequences encoding components of an IKK signalsome may be isolated by, for example, screening a suitable expression library (i.e., a library prepared from a cell line or tissue that expresses IKK signalsome, such as spleen, leukocytes, HeLa cells or Jurkat cells) with antibodies raised against IKK signalsome or against one or more components thereof. Protein components may then be prepared by expression of the identified DNA sequences, using well known recombinant techniques.
Alternatively, partial sequences of the components may be obtained using standard biochemical purification and microsequencing techniques. For example, purified complex as described above may be run on an SDS-PAGE gel and individual bands may be isolated and subjected to protein microsequencing. DNA sequences encoding components may then be prepared by amplification from a suitable human cDNA library, using polymerase chain reaction (PCR) and methods well known to those of ordinary skill in the art. For example, an adapter-ligated cDNA library prepared from a cell line or tissue that expresses IKK signalsome (such as HeLa or Jurkat cells) may be screened using a degenerate 5' specific forward primer and an adapter-specific primer. Degenerate oligonucleotides may also be used to screen a cDNA library, using methods well known to those of ordinary skill in the art. In addition, known proteins may be identified via Western blot analysis using specific antibodies. Components of an IKK signalsome may also be identified by performing any of a variety of protein-protein interaction assays known to those of ordinary skill in the art. For example, a known component can be used as "bait" in standard two-hybrid screens to identify other regulatory molecules, which may include IKK-1, IKK-2, NFκBl , RelA. IκBβ and/or p70 S6 kinase (Kieran et al., Cell 62:1007-1018, 1990; Nolan et al., Cell 64:961-69, 1991 ; Thompson et al., Cell 80:573-82, 1995; Grove et al., Mol. Cell. Biol. 77:5541 -50, 1991).
Particularly preferred components of IKK signalsome are IκB kinases. An IKB kinase may be identified based upon its ability to phosphorylate one or more IKB proteins, which may be readily determined using the representative kinase assays described herein. In general, an IKB kinase is incorporated into an IKK signalsome, as described herein, prior to performing such assays, since an IKB kinase that is not complex-associated may not display the same phosphorylation activity as complex- associated IKB kinase. As noted above, an IKB kinase within an IKK signalsome specifically phosphorylates IκBα at residues S32 and S36, and phosphorylates IκBβ at residues 19 and 23, in response to specific stimuli.
As noted above, IKK-1 and IKK-2 are particularly preferred IκB kinases. IKK-1 and IKK-2 may be prepared by pooling the fractions from the Mono Q column containing peak IκB kinase activity and subjecting the pooled fractions to preparative SDS gel electrophoresis. The intensity of two prominent protein bands of -85 and -87 kDa (indicated by silver stain in Figure 1 IB as IKK-1 and IKK-2 respectively) correlates with the profile of IκB kinase activity. The -85 kDa band, corresponding to IKK-1 , has been identified, within the context of the present invention, as CHUK (conserved helix- loop-helix ubiquitous kinase; see Connely and Marcu, Cell. Mol. Biol. Res. 47:537- 49,1995). The -87 kDa band contains IKK-2.
Sequence analysis reveals that IKK-1 and IKK-2 are related protein serine kinases (51% identity) containing protein interaction motifs (Figure 13 A). Both IKK-1 and IKK-2 contain the kinase domain at the N-terminus, and a leucine zipper motif and a helix-loop-helix motif in their C-terminal regions. Northern analysis indicates that mRNAs encoding IKK-2 are widely distributed in human tissues, with transcript sizes of -4.5 kb and 6 kb (Figure 13B). The sequences of IKK-1 and IKK-2 are also provided as SEQ ID NOs: 7 and 8, respectively.
It has been found, within the context of the present invention, that rabbit reticulocyte lysate immunoprecipitates that contain IKK- 1 or IKK-2 phosphorylate IκBα and IκBβ with the correct substrate specificity (see Figure 14A). Altered versions of these kinases interfere with translocation of RelA to the nucleus of TNFα-stimulated HeLa cells. Accordingly, IKK-1 and IKK-2 appear to control a significant early step of NFKB activation.
Other components of an IKK signalsome are also contemplated by the present invention. Such components may include, but are not limited to, upstream kinases such as MEKK-1 (Lee et al., Cell 88,:2\ 3-22, 1997; Hirano et al., J. Biol. Chem. 277: 13234-38, 1996) or NIK (Malinin et al., Nature 555:540-44, 1997); adapter proteins that mediate an IKK-1 :IKK-2 interaction; a component that crossreacts with anti-MKP-1 ; an inducible RelA kinase; and/or the E3 ubiquitin ligase that transfers multiubiquitin chains to phosphorylated IKB (Hershko and Ciechanover, Annu. Rev. Biochem. 67:761 - 807, 1992). A component of an IKK signalsome may generally be prepared from DNA encoding the component by expression of the DNA in cultured host cells, which may be stable cell lines or transiently transfected cells. Preferably, the host cells are bacteria, yeast, baculo virus-infected insect cells or mammalian cells. The recombinant DNA may be cloned into any expression vector suitable for use within the host cell, using techniques well known to those of ordinary skill in the art. An expression vector may, but need not, include DNA encoding an epitope, such that the recombinant protein contains the epitope at the N- or C-terminus. Epitopes such as glutathione-S transferase protein (GST), HA (hemagglutinin)-tag, FLAG and Histidine-tag may be added using techniques well known to those of ordinary skill in the art.
The DNA sequences expressed in this manner may encode native components of an IKK signalsome, or may encode portions or variants of native components, as described above. DNA molecules encoding variants may generally be prepared using standard mutagenesis techniques, such as oligonucleotide-directed site- specific mutagenesis. Sections of the DNA sequence may also, or alternatively, be removed to permit preparation of truncated polypeptides and DNA encoding additional sequences such as "tags" may be added to the 5' or 3' end of the DNA molecule.
IKK signalsome components may generally be used to reconstitute IKK signalsome. Such reconstitution may be achieved in vitro by combining IKK signalsome components in a suitable buffer. Alternatively, reconstitution may be achieved in vivo by expressing components in a suitable host cell, such as HeLa or HUVEC, as described herein.
Expressed IKK signalsome, or a component thereof, may be isolated in substantially pure form. Preferably, IKK signalsome or a component is isolated to a purity of at least 80% by weight, more preferably to a purity of at least 95% by weight, and most preferably to a purity of at least 99% by weight. In general, such purification may be achieved using, for example, the representative purification methods described herein or the standard techniques of ammonium sulfate fractionation, SDS-PAGE electrophoresis, and affinity chromatography. IKK signalsome and components for use in the methods of the present invention may be native, purified or recombinant.
In one aspect of the present invention, an IKK signalsome and/or one or more components thereof may be used to identify modulating agents, which may be antibodies (e.g., monoclonal), polynucleotides or other drugs, that inhibit or stimulate signal transduction via the NF-κB cascade. Modulation includes the suppression or enhancement of NF-κB activity. Modulation may also include suppression or enhancement of IKB phosphorylation or the stimulation or inhibition of the ability of activated (i.e., phosphorylated) IKK signalsome to phosphorylate a substrate. Compositions that inhibit NF-κB activity by inhibiting IKB phosphorylation may include one or more agents that inhibit or block IκBα kinase activity, such as an antibody that neutralizes IKK signalsome, a competing peptide that represents the substrate binding domain of IKB kinase or a phosphorylation motif of IKB, an antisense polynucleotide or ribozyme that interferes with transcription and/or translation of IKB kinase, a molecule that inactivates IKK signalsome by binding to the complex, a molecule that binds to IKB and prevents phosphorylation by IKK signalsome or a molecule that prevents transfer of phosphate groups from the kinase to the substrate. Within certain embodiments, a modulating agent inhibits or enhances the expression or activity of IKK-1 and/or IKK-2. In general, modulating agents may be identified by combining a test compound with an IKK signalsome, IKB kinase or a polynucleotide encoding an IKB kinase in vitro or in vivo, and evaluating the effect of the test compound on the IKB kinase activity using, for example, a representative assay described herein. An increase or decrease in kinase activity can be measured by adding a radioactive compound, such as 32P-ATP and observing radioactive incorporation into a suitable substrate for IKK signalsome, thereby determining whether the compound inhibits or stimulates kinase activity. Briefly, a candidate agent may be included in a reaction mixture containing compounds necessary for the kinase reaction (as described herein) and IKB substrate, along with IKK signalsome, IκB kinase or a polynucleotide encoding an IκB kinase. In general, a suitable amount of antibody or other agent for use in such an assay ranges from about 0.01 μM to about 10 μM. The effect of the agent on IκB kinase activity may then be evaluated by quantitating the incorporation of f Pjphosphate into an IκB such as IκBα (or a derivative or variant thereof), and comparing the level of incorporation with that achieved using IκB kinase without the addition of a candidate agent. Alternatively, the effect of a candidate modulating agent on transcription of an IκB kinase may be measured, for example, by Northern blot analysis or a promoter/reporter- based whole cell assay.
Alternatively, for assays in which the test compound is combined with an IKK signalsome, the effect on a different IKK signalsome activity may be assayed. For example, an IKK signalsome also displays p65 kinase activity and IKK phosphatase activity. Assays to evaluate the effect of a test compound on such activities may be performed using well known techniques. For example, assays for p65 kinase activity may generally be performed as described by Zhong et al., Cell 59:413-24, 1997. For phosphatase activity, an assay may generally be performed as described by Sullivan et al., J. Biomolecular Screening 2:19-24, 1997, using a recombinant phosphorylated IκB kinase as a substrate. In another aspect of the present invention, IKK signalsome or IκB kinase may be used for phosphorylating an IκB such as IκBα (or a derivative or variant thereof) so as to render it a target for ubiquitination and subsequent degradation. IKB may be phosphorylated in vitro by incubating IKK signalsome or IKB kinase with IKB in a suitable buffer for 30 minutes at 30°C. In general, about 0.01 μg to about 9 μg of IKB kinase complex is sufficient to phosphorylate from about 0.5 μg to about 2 μg of IKB. Phosphorylated substrate may then be purified by binding to GSH-sepharose and washing. The extent of substrate phosphorylation may generally be monitored by adding [γ-32P]ATP to a test aliquot, and evaluating the level of substrate phosphorylation as described herein. An IKK signalsome, component thereof, modulating agent and/or polynucleotide encoding a component and/or modulating agent may also be used to modulate NF-κB activity in a patient. Such modulation may occur by any of a variety of mechanisms including, but not limited to, direct inhibition or enhancement of IκB phosphorylation using a component or modulating agent; or inhibiting upstream activators, such as NIK or MEK, with IKK signalsome or a component thereof. As used herein, a "patient" may be any mammal, including a human, and may be afflicted with a disease associated with IκB kinase activation and the NF-κB cascade, or may be free of detectable disease. Accordingly, the treatment may be of an existing disease or may be prophylactic. Diseases associated with the NF-κB cascade include inflammatory diseases, neurodegenerative diseases, autoimmune diseases, cancer and viral infection.
Treatment may include administration of an IKK signalsome, a component thereof and/or an agent which modulates IκB kinase activity. For administration to a patient, one or more such compounds are generally formulated as a pharmaceutical composition. A pharmaceutical composition may be a sterile aqueous or non-aqueous solution, suspension or emulsion, which additionally comprises a physiologically acceptable carrier (i.e., a non-toxic material that does not interfere with the activity of the active ingredient). Any suitable carrier known to those of ordinary skill in the art may be employed in the pharmaceutical compositions of the present invention. Representative carriers include physiological saline solutions, gelatin, water, alcohols, natural or synthetic oils, saccharide solutions, glycols, injectable organic esters such as ethyl oleate or a combination of such materials. Optionally, a pharmaceutical composition may additionally contain preservatives and/or other additives such as, for example, antimicrobial agents, anti-oxidants, chelating agents and/or inert gases, and/or other active ingredients.
Alternatively, a pharmaceutical composition may comprise a polynucleotide encoding a component of an IKK signalsome and/or a modulating agent (such that the component and/or modulating agent is generated in situ) in combination with a physiologically acceptable carrier. In such pharmaceutical compositions, the polynucleotide may be present within any of a variety of delivery systems known to those of ordinary skill in the art, including nucleic acid, bacterial and viral expression systems, as well as colloidal dispersion systems, including liposomes. Appropriate nucleic acid expression systems contain the necessary polynucleotide sequences for expression in the patient (such as a suitable promoter and terminating signal). DNA may also be "naked," as described, for example, in Ulmer et al., Science 259: 1745-49, 1993.
Various viral vectors that can be used to introduce a nucleic acid sequence into the targeted patient's cells include, but are not limited to, vaccinia or other pox virus, herpes virus, retrovirus, or adenovirus. Techniques for incorporating DNA into such vectors are well known to those of ordinary skill in the art. Preferably, the retroviral vector is a derivative of a murine or avian retrovirus including, but not limited to, Moloney murine leukemia virus (MoMuLV), Harvey murine sarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), and Rous Sarcoma Virus (RSV). A retroviral vector may additionally transfer or incorporate a gene for a selectable marker (to aid in the identification or selection of transduced cells) and/or a gene that encodes the ligand for a receptor on a specific target cell (to render the vector target specific). For example, retroviral vectors can be made target specific by inserting a nucleotide sequence encoding a sugar, a glycolipid, or a protein. Targeting may also be accomplished using an antibody, by methods known to those of ordinary skill in the art. Viral vectors are typically non-pathogenic (defective), replication competent viruses, which require assistance in order to produce infectious vector particles. This assistance can be provided, for example, by using helper cell lines that contain plasmids that encode all of the structural genes of the retrovirus under the control of regulatory sequences within the LTR, but that are missing a nucleotide sequence which enables the packaging mechanism to recognize an RNA transcript for encapsulation. Such helper cell lines include (but are not limited to) Ψ2, PA317 and PA 12. A retroviral vector introduced into such cells can be packaged and vector virion produced. The vector virions produced by this method can then be used to infect a tissue cell line, such as NIFI 3T3 cells, to produce large quantities of chimeric retroviral virions.
Another targeted delivery system for polynucleotides is a colloidal dispersion system. Colloidal dispersion systems include macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. A preferred colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (i.e., an artificial membrane vesicle). It has been shown that large unilamellar vesicles (LUV), which range in size from 0.2-4.0 μm can encapsulate a substantial percentage of an aqueous buffer containing large macromolecules. RNA, DNA and intact virions can be encapsulated within the aqueous interior and be delivered to cells in a biologically active form (Fraley, et al., Trends Biochem. Sci. 6:77, 1981). In addition to mammalian cells, liposomes have been used for delivery of polynucleotides in plant, yeast and bacterial cells. In order for a liposome to be an efficient gene transfer vehicle, the following characteristics should be present: (1) encapsulation of the genes of interest at high efficiency while not compromising their biological activity; (2) preferential and substantial binding to a target cell in comparison to non-target cells; (3) delivery of the aqueous contents of the vesicle to the target cell cytoplasm at high efficiency; and (4) accurate and effective expression of genetic information (Mannino, et al., Biotechniques 6:882, 1988).
The targeting of liposomes can be classified based on anatomical and mechanistic factors. Anatomical classification is based on the level of selectivity and may be, for example, organ-specific, cell-specific, and/or organelle-specific. Mechanistic targeting can be distinguished based upon whether it is passive or active. Passive targeting utilizes the natural tendency of liposomes to distribute to cells of the reticuloendothelial system (RES) in organs which contain sinusoidal capillaries. Active targeting, on the other hand, involves alteration of the liposome by coupling the liposome to a specific ligand such as a monoclonal antibody, sugar, glycolipid, or protein, or by changing the composition or size of the liposome in order to achieve targeting to organs and cell types other than the naturally occurring sites of localization.
Routes and frequency of administration, as well doses, will vary from patient to patient. In general, the pharmaceutical compositions may be administered intravenously, intraperitoneally, intramuscularly, subcutaneously, intracavity or transdermally. Between 1 and 6 doses may be administered daily. A suitable dose is an amount that is sufficient to show improvement in the symptoms of a patient afflicted with a disease associated with the NF-κB cascade. Such improvement may be detected by monitoring inflammatory responses (e.g., edema, transplant rejection, hyperscnsitivity) or through an improvement in clinical symptoms associated with the disease. The dosage may generally vary depending on the nature of the modulating agent and the disease to be treated. Typically, the amount of modulating agent present in a dose, or produced in situ by DNA present in a dose, ranges from about 1 μg to about 200 mg per kg of host. Suitable dose sizes will vary with the size of the patient, but will typically range from about 10 mL to about 500 mL for 10-60 kg animal. In another aspect, the present invention provides methods for detecting the level of stimulus-inducible IκB kinase activity in a sample. The level of IKB kinase activity may generally be determined via an immunokinase assay, in which IKK signalsome is first immunoprecipitated with an antibody that binds to the complex. The immunoprecipitated material is then subjected to a kinase assay as described herein. Substrate specificity may be further evaluated as described herein to distinguish the activity of a stimulus-inducible IKB kinase complex from other kinase activities.
The present invention also provides methods for detecting the level of IKK signalsome, or a component thereof, in a sample. The amount of IKK signalsome, IKB kinase or nucleic acid encoding IKB kinase, may generally be determined using a reagent that binds to IKB kinase, or to DNA or RNA encoding a component thereof. To detect nucleic acid encoding a component, standard hybridization and/or PCR techniques may be employed using a nucleic acid probe or a PCR primer. Suitable probes and primers may be designed by those of ordinary skill in the art based on the component sequence. To detect IKK signalsome or a component thereof, the reagent is typically an antibody, which may be prepared as described below. There are a variety of assay formats known to those of ordinary skill in the art for using an antibody to detect a protein in a sample. See, e.g., Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988. For example, the antibody may be immobilized on a solid support such that it can bind to and remove the protein from the sample. The bound protein may then be detected using a second antibody that binds to the antibody/protein complex and contains a detectable reporter group. Alternatively, a competitive assay may be utilized, in which protein that binds to the immobilized antibody is labeled with a reporter group and allowed to bind to the immobilized antibody after incubation of the antibody with the sample. The extent to which components of the sample inhibit the binding of the labeled protein to the antibody is indicative of the level of protein within the sample. Suitable reporter groups for use in these methods include, but are not limited to, enzymes (e.g., horseradish peroxidase), substrates, cofactors, inhibitors, dyes, radionuclides, luminescent groups, fluorescent groups and biotin.
Antibodies encompassed by the present invention may be polyclonal or monoclonal, and may bind to IKK signalsome and/or one or more components thereof (e.g., IKK-1 and/or IKK-2). Preferred antibodies are those antibodies that inhibit or block IKB kinase activity in vivo and within an in vitro assay, as described above. Other preferred antibodies are those that bind to one or more IKB proteins. As noted above, antibodies and other agents having a desired effect on IKB kinase activity may be administered to a patient (either prophylactically or for treatment of an existing disease) to modulate the phosphorylation of an IKB, such as IκBα, in vivo.
Antibodies may be prepared by any of a variety of techniques known to those of ordinary skill in the art (see, e.g., Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988). In one such technique, an immunogen comprising the protein of interest is initially injected into a suitable animal (e.g., mice, rats, rabbits, sheep and goats), preferably according to a predetermined schedule incorporating one or more booster immunizations, and the animals are bled periodically. Polyclonal antibodies specific for the protein may then be purified from such antisera by, for example, affinity chromatography using the protein coupled to a suitable solid support. Monoclonal antibodies specific for an IKK signalsome or a component thereof may be prepared, for example, using the technique of Kohler and Milstein, Eur. J. Immunol. 6:51 1 -519, 1976, and improvements thereto. Briefly, these methods involve the preparation of immortal cell lines capable of producing antibodies having the desired specificity (i.e., reactivity with the complex and/or component of interest). Such cell lines may be produced, for example, from spleen cells obtained from an animal immunized as described above. The spleen cells are then immortalized by, for example, fusion with a myeloma cell fusion partner, preferably one that is syngeneic with the immunized animal. For example, the spleen cells and myeloma cells may be combined with a nonionic detergent for a few minutes and then plated at low density on a selective medium that supports the growth of hybrid cells, but not myeloma cells. A preferred selection technique uses HAT (hypoxanthine, aminopterin, thymidine) selection. After a sufficient time, usually about 1 to 2 weeks, colonies of hybrids are observed. Single colonies are selected and tested for binding activity against the polypeptide. Hybridomas having high reactivity and specificity are preferred. Monoclonal antibodies may be isolated from the supernatants of growing hybridoma colonies. In addition, various techniques may be employed to enhance the yield, such as injection of the hybridoma cell line into the peritoneal cavity of a suitable vertebrate host, such as a mouse. Monoclonal antibodies may then be harvested from the ascites fluid or the blood. Contaminants may be removed from the antibodies by conventional techniques, such as chromatography, gel filtration, precipitation, and extraction.
In a related aspect of the present invention, kits for detecting the level of IKK signalsome. IKB kinase, nucleic acid encoding IκB kinase and/or IKB kinase activity in a sample are provided. Any of a variety of samples may be used in such assays, including eukaryotic cells, bacteria, viruses, extracts prepared from such organisms and fluids found within living organisms. In general, the kits of the present invention comprise one or more containers enclosing elements, such as reagents or buffers, to be used in the assay.
A kit for detecting the level of IKK signalsome, IκB kinase or nucleic acid encoding IKB kinase typically contains a reagent that binds to the compound of interest. To detect nucleic acid encoding IKB kinase, the reagent may be a nucleic acid probe or a PCR primer. To detect IKK signalsome or IKB kinase, the reagent is typically an antibody. Such kits also contain a reporter group suitable for direct or indirect detection of the reagent (i.e., the reporter group may be covalently bound to the reagent or may be bound to a second molecule, such as Protein A, Protein G, immunoglobulin or lectin, which is itself capable of binding to the reagent). Suitable reporter groups include, but are not limited to, enzymes (e.g., horseradish peroxidase), substrates, cofactors, inhibitors, dyes, radionuclides, luminescent groups, fluorescent groups and biotin. Such reporter groups may be used to directly or indirectly detect binding of the reagent to a sample component using standard methods known to those of ordinary skill in the art. In yet another aspect, IKK signalsome may be used to identify one or more native upstream kinases (i.e., kinases that phosphorylate and activate IKK signalsome in vivo) or other regulatory molecules that affect IKB kinase activity (such as phosphatases or molecules involved in ubiquitination), using methods well known to those of ordinary skill in the art. For example, IKK signalsome components may be used in a yeast two- hybrid system to identify proteins that interact with such components. Alternatively, an expression library may be screened for cDNAs that phosphorylate IKK signalsome or a component thereof.
The following Examples are offered by way of illustration and not by way of limitation. EXAMPLES
Example 1 Recruitment of NFKB into IKK Signalsome during Activation
This example illustrates the recruitment of NFKB into a protein complex (the IKK signalsome) containing IκB kinase and other signaling proteins.
Cytoplasmic extracts of either unstimulated or stimulated Jurkat cells were fractionated on a Superdex 200 gel filtration column, and IκBα was visualized by immunoblot analysis. Jurkat cells were grown to a cell density of 1.5 X 106 cells/ml and either not stimulated or induced for 10 minutes with PMA (50 ng/ml)/PHA (1 μg/ml). Cells were harvested and resuspended in two volumes HLB buffer (20 mM Tris pH 8.0, 2 mM EDTA, 1 mM EGTA, 10 mM β-glycerophosphate, 10 mM NaF, 10 mM PNPP, 300 μM Na3VO4, 1 mM benzamidine, 2 mM PMSF, 10 μg/ml aprotonin, 1 μg/ml leupeptin, 1 μg/ml pepstatin, 1 mM DTT), made 0.1% NP40 and left on ice for 15 minutes, and lysed with a glass Dounce homogenizer. The nuclei were pelleted at 10,000 rpm for 20 minutes in a Sorval SS34 rotor. The supernatant was further centrifuged at 40,000 rpm for 60 min in a Ti50.1 rotor. All procedures were carried out at 4°C. The S-100 fraction was concentrated and chromatographed on Hi Load 16/60 Superdex 200 prep grade gel filtration column that was equilibrated in GF buffer (20 mM Tris HCl pH 8.0, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 5% glycerol, 0.025% Brij 35, 1 mM benzamidine, 2 mM PMSF, 10 mM β-glycerophosphate, 10 mM NaF, 10 mM PNPP, 300 μM Na3VO4, 10 μg/ml aprotonin, 1 μg/ml leupeptin, 1 μg/ml pepstatin, 1 mM DTT). Isolated fractions were analyzed by western blot analysis using either anti-IκBα or anti-JNK antibodies (Santa Cruz, Inc., Santa Cruz, CA).
As shown in Figure 1A, IκBα in cell extracts from unstimulated cells eluted with an apparent molecular weight of -300 kDa (lanes 5-7), consistent with the chromatographic properties of the inactive NFκB-IκB complex (Baeuerle and Baltimore, Genes Dev. 5:1689-98, 1989). In contrast, phosphorylatcd IκBα (from cells stimulated for periods too short to permit complete degradation of the protein) migrated at -600 kDa on the same chromatography columns (lanes 2, 3). This difference in migration was specific for IKB, since analysis of the same fractions indicated that the Jun N-terminal kinases JNK1 and JNK2 migrated with low apparent molecular weight and showed no difference in chromatographic behavior between stimulated and unstimulated cells. Stimulation-dependent recruitment of IKB into this larger protein complex corresponded with the appearance of phosphorylated IKB, suggesting that the complex contained the specific IKB kinases that mediate IKB phosphorylation. These results demonstrate that that NFKB activation involves recruitment into a protein complex (the IKK signalsome) containing IKB kinase and other signaling proteins.
Example 2 Partial Purification of IKK Signalsome and Identification of Co-purifying Components This Example illustrates the fractionation of extracts containing IKB kinase. Whole cell extracts from TNFα-stimulated cells were fractionated by gel filtration, ion exchange, and other chromatographic methods, as described above. IKB kinase activity in the fractions was assayed by phosphorylation of GST-IκBα (1-54) (SEQ ID NO:3) or GST-IκBβ (1-44) (SEQ ID NO:4). Kinase assays were performed in 20 mM HEPES pH 7.7, 2 mM MgCl2, 2 mM MnCl2, 10 μM ATP, 1-3 μCi γ-[32P]-ATP, 10 mM β-glycerophosphate, 10 mM NaF, 10 mM PNPP, 300 μM Na3VO4, 1 mM benzamidine, 2 μM PMSF, 10 μg/ml aprotonin, 1 μg/ml leupeptin, 1 μg/ml pepstatin, 1 mM DTT) at 30°C for 30 to 60 minutes in the presence of the indicated substrate. The kinase reaction was stopped by the addition of 6X SDS-PAGE sample buffer, subjected to SDS-PAGE analysis and visualized using autoradiography. GST-IκB substrates for use in the above assay were prepared using standard techniques for bacterially expressed GST-protein (see Current Protocols in Molecular Biology 2:16.7.1-16.7.7, 1996). Bacterial cells were lysed, GST proteins were purified via binding to GST-agarose beads, washed several times, eluted from the beads with glutathione, dialyzed against kinase assay buffer and stored at -80°C. The specificity of the kinase was established by using mutant GST-IκBα (1-54) in which serines 32, 36 had been mutated to threonine (SEQ ID NO:5). and GST- IκBβ ( 1 -44) in which serines 19, 23 had been mutated to alanine (SEQ ID NO:6). IKB kinase activity was not observed in extracts from unstimulated cells, while stimulation with TNFα for 5-7 minutes resulted in strong induction of kinase activity. As shown in Figure IB, the IKB kinase activity from stimulated cells chromatographed on gel filtration as a broad peak of -500-700 kDa, consistent with its presence in a large protein complex potentially containing other components required for NFKB activation.
NFKB activation is known to occur under conditions that also stimulate MAP kinase pathways (Lee et al., Cell 55:213-22, 1997; Hirano, et al., J. Biol. Chem. 277: 13234-38, 1996). Accordingly, further experiments were performed to detect proteins associated with MAP kinase and phosphatasc cascades at various stages of purification of the IKK signalsome. In addition to RelA and IκBβ, MEKK- 1 and two tyrosine-phosphorylated proteins of -55 and -40 kDa copurified with IKB kinase activity (Figure IC). Antibodies to Rel A and IκBβ were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), and antibodies to MEKK-1 were obtained from Upstate Biotechnology (Lake Placid, NY). Other signaling components, including PKCζ, PP1 and PP2A, were detected in the same fractions as the IKB kinase in early chromatographic steps but did not copurify al later chromatographic steps (data not shown). Most interestingly, an unidentified protein of -50 kDa, detected by its crossreaction with an antibody to MKP-1, copurified with IKB kinase through several purification steps (Figure IC). This protein is unlikely to be MKP-1 itself, since the molecular weight of authentic MKP-1 is 38 kDa.
Example 3 Preparation of IKK Signalsome from HeLa S3 Cell Extracts
This Example illustrates an alternate preparation of an IKK signalsome, and the characterization of the complex.
HeLa S3 cells were grown to a cell density of approximately 0.6 x 106/mL, concentrated 10 fold and induced with TNFα (30 ng/mL) for seven minutes. Two volumes of ice-cold PBS containing phosphatase inhibitors (10 mM sodium fluoride, 0.3 mM sodium orthovanadate and 20 mM β-glycerophosphate) were then added. The cells were spun down, washed once with ice-cold PBS containing phosphatase inhibitors and snap frozen.
Standard protocols were then used to prepare cytoplasmic and nuclear extracts. More specifically, the frozen HeLa S3 cell pellet was quick-thawed at 37°C, resuspended in 2 volumes of ice-cold Hypotonic Lysis Buffer (20mM Tris pH 8.0, 2mM EDTA. ImM EGTA, lOmM β-glycerophosphate, lOmM NaF, lOmM PNPP, 0.3mM Na2V04, 5mM sodium pyrophosphate, ImM benzamidine, 2mM PMSF, lOμg/mL aprotinin, lμg/mL leupeptin and lμg/mL pepstatin), and left to incubate on ice for 30 min. The swollen cells were then dounced 30 times using a tight pestle and the nuclei were pelleted at 10,000 m for 15 minutes at 4°C. The supernatant was clarified via ultracentrifugation (50,000 for 1 hour at 4°C) to generate the final cytoplasmic extract. The nuclear/membrane pellet was resuspended in an equal volume of High Salt Extraction Buffer (20mM Tris pH 8.0, 0.5M NaCl, ImM EDTA, ImM EGTA, 0.25% Triton X-100, 20mM β-glycerophosphate, lOmM NaF, lOmM PNPP, 0.3mM Na2VO4, ImM benzamidine, ImM PMSF, ImM DTT, lOμg/mL aprotinin, lμg/mL leupeptin and lμg/mL pepstatin) and allowed to rotate at 4°C for 30 minutes. Cell debris was removed via centrifugation at 12,500 φm for 30 minutes at 4°C and the resulting supernatant was saved as the nuclear/membrane extract.
These extracts were then independently subjected to a series of chromatographic steps (shown in Figure 2) using a Pharmacia FPLC system (Pharmacia Biotech, Piscataway, NJ):
(1) Q Sepharose (Pharmacia Biotech, Piscataway, NJ) - the column was run with a linear gradient starting with 0.0M NaCl Q Buffer (20mM Tris pH 8.0, 0.5mM EDTA, 0.5mM EGTA, 0.025% Brij 35, 20mM β-glycerophosphate. lOmM NaF. 0.3mM Na2VO4, ImM benzamidine, ImM PMSF, 2mM DTT, lOμg/mL aprotinin, lμg/mL leupeptin and lμg/mL pepstatin) and ending with 0.5M NaCl Q Buffer. The IκBα kinase activity eluted between 0.25 and 0.4 M NaCl.
(2) Gel Filtration HiLoad 16/60 Superdex 200) (Pharmacia Biotech, Piscataway, NJ) - the column was run with Gel Filtration Buffer (20mM Tris pll 8.0, 150mM NaCl. ImM EDTA, ImM EGTA, 0.05% Brij 35, 20mM β- glycerophosphate, l OmM NaF, 0.3mM Na2VO , ImM benzamidine, ImM PMSF. ImM DTT, lOμg/mL aprotinin, lμg/mL leupeptin and l μg/mL pepstatin). The peak IκBα kinase activity eluted at 40-48 mL, which corresponds to a molecular weight of 731 kD to 623 kD.
(3) HR 5/5 Mono Q (Pharmacia Biotech, Piscataway, NJ) - the column was run with a linear gradient starting with 0.0M NaCl Q Buffer and ending with
0.5M NaCl Q Buffer (without Brij detergent to prepare sample for Phenyl Superose column). The IκBα kinase activity eluted between 0.25 and 0.4 M NaCl.
(4) HR Phenyl Superose (Pharmacia Biotech, Piscataway, NJ) - the column was run with a linear gradient of 1.0M to 0.0M ammonium sulfate in Phenyl Superose Buffer (20mM Tris pH 8.0, 0.25mM EDTA, ImM EGTA, 20mM β- glycerophosphate, lOmM NaF, O. lmM Na2VO4, ImM benzamidine, ImM PMSF, ImM DTT, lOμg/mL aprotinin, lμg/mL leupeptin and lμg/mL pepstatin). The IκBα kinase activity eluted between 0.35 and 0.2 M ammonium sulfate.
(5) Gel Filtration Superdex 200 HR 10/30 (Pharmacia Biotech. Piscataway, NJ) - the column was run with Gel Filtration Buffer (see (2), above). The peak of activity eluted at 8-10 mL, which corresponds to a molecular weight of 720 kD to 600 kD.
(6) HR 5/5 Mono Q - the column was run as in (3) above except that the 0.05% Brij 35 was included in all Q buffers. IκBα kinase activity, with similar substrate specificity and molecular weight, was isolated from both the cytoplasmic and nuclear/membrane extracts.
At each step of the fractionation, IκB kinase activity was monitored using an in vitro assay. The assay was performed by combining 2 μg of the respective IKB substrate (GST-IκBα 1-54 (wildtype) or GST-IκBα (S32/36 to T), as described below) with 3-5 μL chromatographic fraction and 20 μL of Kinase Assay Buffer (20 mM HEPES pH 7.4, 10 mM MgCl2, 10 mM MnCl2, 20 mM NaCl, ImM DTT, 20mM PNPP, 20μM ATP, 20mM β-glycerophosphate, l OmM NaF, O. lmM Na2VO4, ImM benzamidine, ImM PMSF) containing γ32P-ATP, and incubating for 30 minutes at 30°C. The kinase reaction was terminated by adding 8 μL of 6x SDS-PAGE sample buffer. The entire sample was run on a 12% polyacrylamide gel, dried and subjected to autoradiography. IKB substrates for use in the above assay were prepared using standard techniques (see Haskill et al., Cell 65:1281-1289, 1991). The GST-IκBα 1-54 (wildtype) or GST-IκBα (S32/36 to T) substrates were prepared using standard techniques for bacterially expressed GST-protein. Bacterial cells were lysed, GST proteins were purified via binding to GST-agarose beads, washed several times, eluted from the beads with glutathione, dialyzed against 50 mM NaCl Kinase Assay Buffer and stored at -80°C. The TNFα-inducibility of IκB kinase activity was initially evaluated by Western blot analysis of the levels of IKB in HeLa S3 cytoplasmic extracts following gel filtration. IκBα was assayed by running 18 μL of the gel filtration fractions on 10% SDS PAGE, transferring to Nitrocellulose Membrane (Hybond-ECL, Amersham Life Sciences, Arlington Height, IL) using standard blotting techniques and probing with anti-IκBα antibodies (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). TNFα-inducibility was evaluated by comparing the level of IκBα in cells that were (Figure 3B) and were not (Figure 3A) exposed to TNFα (30 ng/mL for seven minutes, as described above). The IKB kinase activity of these cytoplasmic extracts was evaluated using the kinase assay described above. As shown in Figure 4B, the extract of TNFα-treated cells phosphorylated GST-IκBα 1-54 (wildtype), while the untreated cell extract showed significantly lower levels of IκBα kinase activity (Figure 4A).
Cytoplasmic extracts of TNFα-treated HeLa S3 cells (following Q Sepharose fractionation) were also subjected to in vitro kinase assays, using the N- terminal portion of IκBα (residues 1 to 54) as substrate. With the wild type substrate, phosphorylation of GST-IκBα 1-54 was readily apparent (Figure 5 A). In contrast, substrate containing threonine substitutions at positions 32 and 36 was not phosphorylated (Figure 5B). Following chromatographic fractionation by Q Sepharose. Superdex 200.
MonoQ Sepharose and Phenyl Superose, in vitro kinase assay showed substantial purification of the IKB kinase activity (Figure 6A). Further purification of the IKB kinase was achieved by passing the sample over, in series, an analytical Superdex 200 and Mono Q HR 5/5, resulting in 8 major protein bands as determined by silver staining. As before. the use of substrate containing threonine substitutions at positions 32 and 36 markedly reduced the phosphorylation (Figure 6B). These results demonstrate the purification of a stimulus-inducible IκBα kinase complex, which specifically phosphorylates serine residues at positions 32 and 36 of IκBα without the addition of exogenous factors.
Example 4 Immunoprecipitation of IKK Signalsome Using Anti MKP-1 Antibodies
This Example illustrates the immunoprecipitation of IκB kinase activity from cytoplasmic extracts prepared from stimulated cells.
A. Immunoprecipitation of IκB Kinase Complex from HeLa Cells
HeLa cells were TNF-α-treated (30 μg/mL, 7 minutes) and fractionated by gel filtration as described in Example 3. Twenty μL of gel filtration fraction #6 (corresponding to about 700 kD molecular weight) and lμg purified antibodies raised against MKP-1 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) were added to 400 μL of ice cold lx Pull Down Buffer (20mM Tris pH 8.0, 250 mM NaCl, 0.05% NP-40, 3mM EGTA, 5 mM EDTA, 10 mM β-glycerophosphate, 10 mM NaF, 10 mM PNPP, 300 μM Na3VO4, 1 mM benzamidine, 2 μM PMSF, 10 μg/ml aprotonin, 1 μg/ml leupeptin, 1 μg/ml pepstatin, 1 mM DTT). The sample was gently rotated for 1 hour at 4°C, at which time 40μL of protein A-agarose beads (50:50 slurry. Santa Cruz Biotechnology, Inc., Santa Cruz, CA) was added. The sample was then rotated for an additional 1.5 hours at 4°C. The protein A-agarose beads were pelleted at 3,000 φm for 2 minutes at 4°C and the pellet was washed three times with ice cold Pull Down Buffer (800 μL per wash).
The pellet was subjected to the standard in vitro IκBα kinase assay (as described above) using either 2 μg GST-IκB l-54 (wildtype) or 2 μg GST-IκBαl-54 (S32/36 to T) as the substrate.
The results, shown in Figure 7, demonstrate that antibodies directed against MKP-1 immunoprecipitate the stimulus-inducible IκBα kinase activity. The substrate specificity of this IκBα kinase activity corresponds to what has been described in vivo (strong phosphorylation of the GST-IκBαl-54 (wildtype) and no phosphorylation using GST-IκBαl-54 (S32/36 to T).
B. Characterization of Immunoprecipitated IKK Signalsome For these studies, small scale immunoprecipitation were performed using two 150 mm plates of HeLa cells (one stimulated and one unstimulated). Whole cell lysates were diluted 4-fold with 2x Pull-Down Buffer (40 mM Tris pH 8.0, 500 mM NaCl, 0.1% NP-40, 6 mM EDTA, 6 mM EGTA, 10 mM β-glycerophosphate, 10 mM NaF, 10 mM PNPP, 300 μM Na3VO4, 1 mM benzamidine, 2 μM PMSF, 10 μg/ml aprotonin, 1 μg/ml leupeptin, 1 μg/ml pepstatin, 1 mM DTT) and 2-4 μg of the indicated antibody was added. Lysates were incubated on ice for 1 -2 hours, 10 μl of Protein A or G beads were added, and lysates were left to incubate with gentle rotation for an additional 1 hour at 4°C. The immunoprecipitate was then washed 3 times with 2x Pull-Down Buffer, IX with kinase buffer without ATP and subjected to a kinase assay as described in Example 2. There was no noticeable loss in IκB kinase activity when the immunoprecipitate was subjected to more rigorous washing, such as in RIPA buffer (20 mM Tris, 250 mM NaCl, 1% NP-40, 1% DOC, 0.1% SDS, 3mM EDTA, 3mM EGTA, 10 mM β-glycerophosphate, 10 mM NaF, 10 mM PNPP, 300 μM Na3VO4, 1 mM benzamidine, 2 μM PMSF, 10 μg/ml aprotonin, 1 μg/ml leupeptin, 1 μg/ml pepstatin, 1 mM DTT) or washes up to 3.5 M urea.
Of a large panel of antibodies tested, one of three anti-MKP- 1 antibodies efficiently co-immunoprecipitated an inducible IκB kinase activity from HeLa cells as well as primary human umbilical vein endothelial cells (HUVEC). The co- immunoprecipitated kinase (IKK signalsome kinase) was inactive in unstimulated HeLa cells, but was rapidly activated within minutes of TNFα stimulation (Figure 8A, top panel). The IKK signalsome kinase did not phosphorylate a mutant GST-IκBα protein in which serine residues 32 and 36 had been mutated to threonine (Figure 8A top panel, even-numbered lanes). Activation of the signalsome kinase was maximal at 5 minutes and declined thereafter, a time course consistent with the time course of IκBα phosphorylation and degradation under the same conditions (Figure 8A, bottom panel). As expected, the signalsome IKB kinase was also activated by stimulation of cells with IL-1 or PMA (Figure 8B, lanes 1-4); moreover, its activity was inhibited in cells treated with TPCK, a known inhibitor of NFKB activation (Figure 8B, lane 7). Additionally, the IKK signalsome kinase specifically phosphorylated full-length wild-type IκBα, but not a mutant IκBα bearing the serine 32, 36 to alanine mutations, in the context of a physiological RelA-IκBα complex (Figure 8C, lanes 3, 4). Together these results indicate that the anti-MKP-1 antibody co-immunoprecipitated the IKK signalsome. The signalsome-associated IKB kinase met all the criteria expected of the authentic IκB kinase and had no detectable IκBα C-terminal kinase activity.
The specificity of the IKK signalsome kinase was further established by kinetic analysis and by examining its activity on various peptides and recombinant protein substrates (Figure 9A). For these studies, synthetic peptides (Alpha Diagnostics International, San Antonio, TX) were prepared with the following sequences:
IκBα(21-41): CKKERLLDDRHDSGLDSMKDEE (SEQ ID NO: 1 1) IκBα(21 -41) S/T mutant: CKKERLLDDRHDJGLDJMKDEE (SEQ ID NO: 12) c-Fos(222-241 ): DLTGGPEVAT(PO3)PESEEAFLP (SEQ ID NO: 13) MKP-1 : CPTNSALNYLKSPITTSPS (SEQ ID NO: 14) cJun(56-70): CNSDLLTSPDVGLLK (SEQ ID NO: 15) cJun(65-79): CVGLLKLASPELERL (SEQ ID NO: 16) Phosphorylation of these peptides (100 μM) was performed using a kinase reaction as described above. Reactions were for one hour at room temperature and were terminated by the addition of SDS-PAGE loading buffer. SDS-PAGE with a 16% Tris/tricine gel (Novex, San Diego, CA) or a 4-20% Tris/glycine gel (Novex, San Diego. CA) was used to characterize the reaction products. Gels were washed, dried in vacuo. and exposed to autoradiographic film.
Inhibition of immunopurified IKK signalsome activity was measured by 32P incoφoration into GST-IκBα(l-54) in a discontinuous assay using the reaction conditions described above. The concentrations of GST-IκBα(l-54) and ATP used in the inhibition studies were 0.56 μM and 3 μM, respectively. Enzymatic reactions (32 μL) were carried out in wells of a 96 well assay plate for one hour at room temperature and terminated with the addition of trichloroacetic acid (TCA) (150 μL/well of 12.5% w/v). The subsequent 20 minute incubation with TCA precipitated the proteins but not peptides from solution. The TCA precipitate was collected on 96-well glass fiber plates (Packard) and washed 10 times with approximately 0.3 mL per well of Dulbecco's phosphate buffered saline pH 7.4 (Sigma) using a Packard Filtermate 196. Scintillation fluid (0.50 mL, Micro Scint, Packard) was added to each well and the plate was analyzed for 32P using a Packard TopCount scintillation counter. Less than 10% of ATP was turned over in the course of the assay reaction, ensuring that the kinetic data represented initial rate data. The inhibition constant of the P32, 36 peptide was determined by Dixon analysis (Dixon and Webb, In Enzymes (Academic Press: New York, ed. 3, 1979), pp. 350-51. The kinase displayed normal Michaelis-Menten kinetics, suggesting that it was not a mixture of diverse unrelated kinases. The kinase was capable of phosphorylating an lκBα (21-41) peptide (Figures 9A and 9B)) as well as two different IκBα (21-41) peptides, each bearing a free serine at either position 32 or 36 and phosphoserine at the other position (Figures 9A and 9B, lanes 2, 3). As expected, a peptide with phosphoserines at both positions was not phosphorylated at all (Figure 9B, top), indicating that there was no significant turnover of the phosphates under our reaction conditions. These experiments indicated that both serines 32 and 36 were phosphoacceptor sites for the IKK signalsome kinase, thus distinguishing it from other kinases such as pp90Rsk which phosphorylates IκBα only at serine 32 (Schouten, et al., EMBOJ 76:3133-44, 1997).
Although the IKK signalsome kinase efficiently phosphorylated IκB peptides, it did not phosphorylate the c-Fos phosphopeptide containing a free serine and a free threonine (Figure 9B, top), two c-Jun peptides containing serine 63 and 73, respectively, (Figure 9A, top panel, lanes 4, 5), or an MKP-1 peptide containing four serines and three threonines (Figure 9A, lane 6). The latter peptides were substrates for JNK2 (Figure 9A, bottom panel, lanes 4-6). An IκBα (21-41) peptide in which serines 32 and 36 were replaced by threonines was phosphorylated by the signalsome at least 10-fold less well than the wild-type serine-containing peptide, consistent with the slower phosphorylation and degradation kinetics of IκBα (S32/36 to T) in cells (DiDonato et al., Mol Cell. Biol. 76: 1295-1304. 1996), and the preference of the kinase for serine over threonine at positions 32, 36 in both full-length IκBα and GST-IκBα (1 -54) (Figures 8 A and C). In addition, the kinase phosphorylated GST-IκBβ (1-54), albeit with lower affinity. In most experiments, IκB kinase activity was also associated with strong RelA kinase activity (Figure 8C, lanes 3, 4), but no activity was observed towards several other substrates including myelin basic protein (MBP), GST-ATF2 (1 -1 12), GST-cJun (1-79), GST-ERK3, GST-Elk (307-428), GST-p38, and a GST fusion protein containing the C- terminal region of IκBα (242-314).
The specificity of the IKK signalsome kinase was further emphasized by its susceptibility to product inhibition (Figure 9B, bottom). The kinase was strongly inhibited by a doubly-phosphorylated IκBα peptide bearing phosphoserines at both positions 32 and 36, but not by the unrelated c-Fos phosphopeptide that contained a single phospho threonine. The singly-phosphorylated and the unphosphorylated IκBα peptides were less effective inhibitors.
Example 5 Absence of Free Ubiquitin in Purified IKK Signalsome
This example illustrates the absence of detectable free ubiquitin with a IKK signalsome prepared as in Example 3. Standard western blot procedures were performed (Amersham Life Science protocol, Arlington Heights, IL). 100 ng ubiquitin, 10 ng ubiquitin and 20 ul purified IkBα kinase complex was subjected to 16% Tricine SDS-PAGE (Novex, San Diego, CA), transferred to Hybond ECL Nitrocellulose membrane (Amersham Life Science, Arlington Heights, IL), and probed with antibodies directed against ubiquitin (MAB1510; Chemicon, Temecula, CA). The results are shown in Figure 10. Free ubiquitin could not be detected in the purified IkBα kinase preparation (even at very long exposures). The complexes described herein do not require addition of endogenous ubiquitin to detect IkBα kinase activity, nor is free ubiquitin a component in the purified IkBα kinase preparations of the present invention. Example 6 Purification of the NFKB Signalsome and Identification of IKK-1 and IKK-2
This Example illustrates a two-step affinity method for purification of the IKK signalsome, based on its recognition by the MKP-1 antibody (depicted in Figure 1 1 A) and the identification of IκB kinases.
For large scale IKK signalsome purification, HeLa S3 cells were stimulated for 7 minutes with 20 ng/ml TNFα (R&D Systems, Minneapolis, MN), harvested, whole cell lysates were prepared (1.2 g total protein) and approximately 5 mg of anti-MKP-1 antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) was added and incubated at 4°C for 2 hours with gentle rotation. Subsequently, 50 ml of Protein A agarose (Calbiochem, San Diego, CA) was added and the mixture was incubated for an additional 2 hours. The immunoprecipitate was then sequentially washed 4X Pull-Down Buffer, 2X RIPA buffer, 2X Pull-Down Buffer, IX 3.5 M urea-Pull-Down Buffer and 3X Pull -Down Buffer. The immunoprecipitate was then made into a thick slurry by the addition of 10 ml of Pull-Down Buffer, 25 mg of the specific MKP-1 peptide to which the antibody was generated (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) was added, and the mixture was incubated overnight at 4°C with gentle rotation. The eluted IKK signalsome was then desalted on PD 10 desalting columns (Pharmacia Biotech, Piscataway, NJ) equilibrated with 50 mM Q buffer and chromatographed on a Mono Q column (Pharmacia Biotech, Piscataway, NJ). Fractions containing peak IkB kinase activity were pooled, concentrated and subjected to preparative SDS-PAGE. The intensity of two prominent protein bands of -85 and -87 kDa (indicated by silver stain in Figure 1 1B as IKK-1 and IKK-2 respectively) correlated with the profile of IKB kinase activity. Coomassie stained -85 and -87 kDa bands were excised, in-gel digested with trypsin (Wil et al., Nature 57:466-69, 1 96) and a small aliquot of the supernatant was analyzed by high mass accuracy MALDI peptide mass mapping, as described by Shevchenko et al., Proc. Nail. Acad. Sci. USA 95:14440-45, 1996. The peptide mass map from the IKK-1 band was searched against a comprehensive protein sequence database using the program PeptideSearch developed in house at EMBL Heidelberg. Eight measured peptide masses matched calculated tryptic peptide masses from CHUK (conserved helix-loop-helix ubiquitous kinase; Connely and Marcu, Cell. Mol. Biol. Res. 47:537-49, 1995) within 30 ppm, unambiguously identifying the protein. The peptide mass map of the IKK-2 band did not result in a clear identification and therefore the sample was subjected to nanoelectrospray mass spectrometry (Wilm and Mann, Anal. Chem. 65:1-8, 1996). The peptide mixture obtained after extraction of the gel piece was micropurified on a capillary containing 50 nL of POROS R2 resin (PerSeptive Biosystems, Framingham, MA). After washing, the peptides were step-eluted with 0.5 μl of 50% MeOH in 5% formic acid into a nanoelectrospray needle. This needle was transferred to an APIII mass spectrometer (Perkin-Elmer, Sciex, Toronto, Canada) and the sample sprayed for approximately 20 minutes. During this time, peptide ions apparent from the mass spectrum were selected and isolated in turn and fragmented in the collision chamber of the mass spectrometer. From the tandem mass spectra, short stretches of sequence were assembled into peptide sequence tags (Mann and Wilm, Anal. Chem. 66:4390-99, 1994) and searched against a protein sequence database or against dbEST using PeptideSearch.
Three peptides matched the IKK-1 sequence. Al : IIDLGYAK (SEQ ID NO: 17); A2: VEVALSNIK (SEQ ID NO: 18); A3 SIQLDLER (SEQ ID NO: 19). Three other peptides matched human EST sequences in dbEST: Bl : ALELLPK (SEQ ID NO:20), B2: VIYTQLSK (SEQ ID NO:21), B6: LLLQAIQSFEK (SEQ ID NO:22) all match EST clone AA326115. The peptide B4 with the sequence LGTGGFGNVIR (SEQ ID NO:23) was found in clone R06591. After the full-length IKK-2 sequence was obtained (as described below) two more peptides B3: ALDDILNLK (SEQ ID NO:24) and B5: DLKPENIVLQQGEQR (SEQ ID NO:25) were found in the sequence. Peptide Al is present in both IKK-1 and IKK-2 sequences. All the EST clones identified were clearly homologous to human and mouse CHUK, a serine/threonine kinase of hitherto unknown function. Once the complete coding sequence of IKK-2 was obtained (as described below), all sequenced peptides (apart from two peptides derived from IKK-1 ) could be assigned to this protein.
Representative mass spectra are shown in Figures 12A and 12B. In Figure 12A. peaks labeled A were matched to the tryptic peptides of IKK-1 upon fragmentation followed by database searching with peptide sequence tags. Peaks labeled B2. B4, B6 were not found in protein databases but instead matched human EST sequences. One more peptide (Bl) matching a human EST clone was observed at m/z 392.2 and is not shown in panel A. In Figure 12B, a continuous series of C-terminal-containing fragments (Y"- ions) was used to construct a peptide sequence tag as shown by boxed letters. Search of this tag resulted in a match to the peptide LLLQALQSFEK (SEQ ID NO:22) in human EST clone AA3261 15. Two more peptides, Bl (ALELLPK; SEQ ID NO:20) and B2 (VIYTQLSK; SEQ ID NO:21) were found in the sequence of the same EST clone.
Full-length human IKK-1 and IKK-2 cDNAs were cloned based on the. human EST clones, which were obtained from Genome Systems, Inc. (St. Louis, MO). The precise nucleotide sequences were determined and used to design primers to PCR clone human IKK-2 from a human HeLa cell cDNA library (Clontech, Inc., Palo Alto, CA). Several IKK-2 cDNA clones were isolated and sequenced. Full-length mouse IKK- 1 and a partial human IKK-1 nucleotide sequence was available in the comprehensive database, primers were designed to PCR clone the respective human and mouse IKK-1 cDNAs. The partial human IKK-1 coding region was used to probe a HeLa cDNA phage library (Stratagene, Inc., La Jolla, CA) to obtain the full-length human IKK-1 cDNA clone using standard procedures.
Sequence analysis of these clones revealed that IKK-1 and IKK-2 were related protein serine kinases (51% identity) containing protein interaction motifs (Figure 13 A). Both IKK-1 and IKK-2 contain the kinase domain at the N-terminus. and a leucine zipper motif and a helix-loop-helix motif in their C-terminal regions (Figure 13 A). Northern analysis indicated that mRNAs encoding IKK-2 were widely distributed in human tissues, with transcript sizes of -4.5 kb and 6 kb (Figure 13B). The distribution of IKK-1 (CHUK) transcripts has been reported previously (Connely et al., Cell. Mol. Biol. Res. 47:537-49, 1995). IKK-1 and IKK-2 mRNAs are constitutively expressed in Jurkat. HeLa and HUVEC cell lines, and their levels are not altered for up to 8 hours following stimulation with NFKB inducers such as TNFα (HeLa, HUVEC) or anti-CD28 plus PMA (Jurkat).
To further characterize the properties of IKK-1 and IKK-2, recombinant HA-tagged IKK-1 and Flag-tagged IKK-2, either separately or alone, were in vitro transcribed and translated in wheat germ or rabbit reticulocyte lysate (Promega. Madison. WI). The reactions were performed exactly as described in the manufacturer's protocol. Epitope-tagged IKK-1 and IKK-2 then immunoprecipitated with the appropriate anti-tag antibody. Immunoprecipitates containing these proteins phosphorylated IκBα and IκBβ with the correct substrate specificity (i.e., immunoprecipitates of IKK-1 and IKK-2 phosphorylated both GST-IκBα (Figure 14A, panel 3) and GST-IκBβ (panel 4), but did not phosphorylate the corresponding S32/36 to T mutant protein). IKK-1 expressed in rabbit reticulocyte lysates was also capable of autophosphorylation (Figure 14A, panel 2, lane 1), whereas a kinase-inactive version of IKK-1 , in which the conserved lysine 44 had been mutated to methionine, showed no autophosphorylation. In contrast IKK-2, although expressed at equivalent levels in the lysates (panel 1), showed very weak autophosphorylation (panel 2, lane 2).
Expression of the kinase inactive mutants (K to M) of IKK-1 and IKK-2 indicate that both play critical roles in NFKB activation as demonstrated by immunofluorescent studies (Figures 14B and 14C). For these studies, HeLa cells were transiently transfected with either HA-tagged IKK-1 or Flag-tagged IKK-2. Cells were fixed for 30 minutes with methanol. For immunofluorescence staining, the cells were incubated sequentially with primary antibody in PBS containing 10% donkey serum and 0.25% Triton X-100 for 2 hours followed by fluorescein-conjugated or Texas red- conjugated secondary antibody (Jackson Immunoresearch Laboratories, Inc., West Grove, PA; used at 1 :500 dilution) for 1 hour at room temperature. The coverslips were rinsed and coverslipped with Vectashield (Vector Laboratories, Burlingame, CA) before scoring and photographing representative fields. Primary antibodies used for immunofluorescence staining included antibodies against Rel A (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), HA tag (Babco, Berkeley, CA) and Flag tag (IBI- Kodak, New Haven, CT).
Kinase-inactive versions (K44 to M) of IKK-1 and IKK-2 had no effect on the subcellular localization of RelA in unstimulated HeLa cells, since RelA remained cytoplasmic both in cells expressing the epitope-tagged proteins and in the adjacent untransfected cells (Figures 14B and 14C. top panels). In contrast, both mutant proteins inhibited RelA nuclear translocation in TNFα-stimulated cells (Figures 14B and 14C, bottom panels). The inhibition mediated by the IKK-2 mutant was striking and complete (Figure 14C: compare mutant IKK-2-expressing cells with untransfected cells in the same field), whereas that mediated by the mutant IKK-1 protein, expressed at apparently equivalent levels, was significant but incomplete (Figure 14B). This difference in the functional activities of the two mutant kinases may point to distinct roles for these two kinases in NFKB activation.
The presence of the leucine zipper and helix-loop-helix motif in IKK-1 and IKK-2 suggested that they interacted functionally with other proteins in the signalsome. An obvious possibility was that the proteins formed hetero- or homodimers with one another. HA-tagged IKK-1 and FLAG-tagged IKK-2 were translated in rabbit reticulocyte lysates, either alone or together, and then immunoprecipitated with antibodies to the appropriate epitope tags. This experiment demonstrated clearly that IKK-2 was present in IKK-1 immunoprecipitates (Figure 15A, lane 3) and vice versa (lane 4), suggesting that these proteins either associated directly or via adapter proteins/IKK signalsome components present in the rabbit reticulocyte lysates. In contrast, however, there was no evidence for association of IKK-1 and IKK-2 that had been cotranslated in wheat germ lysates (Figure 15B), suggesting that the proteins did not heterodimerize directly. When full-length IKK-1 was translated together in wheat germ extracts with a truncated version of IKK-1 that still possessed the protein interaction motifs, there was also no evidence of association, suggesting that IKK-1 was also not capable of forming homodimers under these conditions.
Both IKK-1 and IKK-2 kinases were active when expressed in wheat germ extracts, since they were capable of autophosphorylation, but they were inactive with respect to phosphorylation of IκB substrates. Since both autophosphorylation and substrate phosphorylation were intact in rabbit reticulocyte lysates, there appeared to be a direct correlation between the association of IKK-1 and IKK-2 into a higher order protein complex and the presence of specific IκB kinase activity in IKK-1 and IKK-2 immunoprecipitates. This higher order complex is most likely the IKK signalsome itself. Indeed, immunoprecipitation of rabbit reticulocyte lysates with anti-MKP-1 antibody pulls down a low level of active IKB kinase activity characteristic of the IKK signalsome. It is clear that the IKK signalsome contains multiple protein components in addition to IKK-1 and IKK-2 (Figure 1 IB). Some of these may be upstream kinases such as MEKK-1 (Chen et al., Cell 54:853-62, 1996) or NIK (Malinin, et al., Nature 555:540-44, 1997); others may be adapter proteins that mediate the IKK-1 :IKK-2 interaction. Indeed MEKK-1 copurifies with IKK signalsome activity (Figure IC), and two other signalsome proteins have been functionally identified. The protein crossreactive with anti-MKP-1 is an intrinsic component of the IKK signalsome kinases, since the IκB kinase activity coprecipitated with this antibody is stable to washes with 2-4 M urea. Moreover, both IKK-1 immunoprecipitates and MKP-1 immunoprecipitates containing the IKK signalsome (Figure 8C) contain an inducible RelA kinase whose kinetics of activation parallel those of the IKB kinase in the same immunoprecipitates. Another strong candidate for a protein in the signalsome complex is the E3 ubiquitin ligase that transfers multiubiquitin chains to phosphorylated IKB (Hershko et al., Annu. Rev. Biochem. 67:761-807, 1992).
These results indicate that IKK- 1 and IKK-2 are functional kinases within the IKK signalsome, which mediate IKB phosphorylation and NFKB activation. Appropriate regulation of IKK-1 and IKK-2 may require their assembly into a higher order protein complex, which may be a heterodimer facilitated by adapter proteins, the complete IKK signalsome, or some intermediate subcomplex that contains both IKK-1 and IKK-2.
From the foregoing, it will be appreciated that, although specific embodiments of the invention have been described herein for the puφose of illustration, various modifications may be made without deviating from the spirit and scope of the invention.
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(l) APPLICANTS: Mercurio, Frank Zhu, Hengyi Barbosa, Miguel
Figure imgf000045_0001
Murray, Brion W.
(n) TITLE OF INVENTION: STIMULUS-INDUCIBLE PROTEIN KINASE COMPLEX AND METHODS OF USF THEREFOR
(m) NUMBER OF SEQUENCES: 25
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(D) SOFTWARE: Patentln Release #1.0, Version #1.30
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: US
(B) FILING DATE: 12-AUG-1997
(C) CLASSIFICATION:
(vm) ATTORNEY/AGENT INFORMATION: (A) NAME: Maki, David J. (B) REGISTRATION NUMBER: 31,392
(C) REFERENCE/DOCKET NUMBER: 860098.413C1
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (206) 622-4900
(B) TELEFAX: (206) 682-6031
(2) INFORMATION FOR SEQ ID NO : 1 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 317 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 1 :
Met Phe Gin Ala Ala Glu Arq Pro Gin Glu Trp Ala Met Glu Gly Pro 1 5 10 15
Arg Asp Gly Leu Lys Lys Glu Arg Leu Leu Asp Asp Arg His Asp Ser 20 25 30
Gly Leu Asp Ser Met Lys Asp Glu Glu Tyr Glu Gin Met Val Lys Glu 35 40 45
Leu Gin Glu He Arg Leu Glu Pro Gin Glu Val Pro Arg Gly Ser Glu 50 55 60
Pro Trp Lys Gin Gin Leu Thr Glu Asp Gly Asp Ser Phe Leu His Leu 65 70 75 80 Ala He He His Glu Glu Lys Ala Leu Thr Met Glu Val He Arg Gin 85 90 95
Val Lys Gly Asp Leu Ala Phe Leu Asn Phe Gin Asn Aεn Leu Gin Gin 100 105 110
Thr Pro Leu His Leu Ala Val He Thr Asn Gin Pro Glu He Ala Glu 115 120 125
Ala Leu Leu Gly Ala Gly Cys Asp Pro Glu Leu Arg Asp Phe Arq Gly 130 135 140
Asn Thr Pro Leu His Leu Ala Cys Glu Gin Gly Cys Leu Ala Ser Val 145 150 155 160
Gly Val Leu Thr Gin Ser Cys Thr Thr Pro His Leu His Ser He Leu 165 170 175
Lys Ala Thr Asn Tyr Asn Gly His Thr Cys Leu His Leu Ala Ser He 180 185 190
His Gly Tyr Leu Gly He Val Glu Leu Leu Val Ser Leu GJ y Ala Asp 195 200 205
Val Asn Ala Gin Glu Pro Cys Asn Gly Arg Thr Ala Leu His Leu Ala 210 215 220
Val Asp Leu Gin Asn Pro Asp Leu Val Ser Leu Leu Leu Lys Cys G.1 y 225 230 235 240
Ala Asp Val Asn Arg Val Thr Tyr Gin Gly Tyr Ser Pro Tyr Gin Leu 245 250 255
Thr Trp Gly Arg Pro Ser Thr Arg He Gin Gin Gin Leu Gly Gin Leu 260 265 270
Thr Leu Glu Asn Leu Gin Met Leu Pro Glu Ser Glu Asp Glu Glu Ser 275 280 285
Tyr Asp Thr Glu Ser Glu Phe Thr Glu Phe Thr Glu Asp Glu Leu Pro 290 295 300
Tyr Asp Asp Cys Val Phe Gly Gly Gin Arq Leu Thr Leu 305 310 315
(2) INFORMATION FOR SEQ ID NO: 2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 359 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : :
Met Ala Gly Val Ala Cys Leu Gly Lys Thr Ala Asp Ala Asp Glu Trp 1 5 10 15
Cys Asp Ser Gly Leu Gly Ser Leu Gly Pro Asp Ala Ala Ala Pro Gly 20 25 30
Gly Pro Gly Leu Gly Ala Glu Leu Gly Pro Glu Leu Ser Trp Ala Pro 35 40 45
Leu Val Phe Gly Tyr Val Thr Glu Asp Gly Asp Thr Ala Leu His Leu 50 55 60
Ala Vdl He His Gin His Glu Pro Phe Leu Asp Phe Leu Leu Gly Phe
65 70 75 80 Ser Ala Gly His Glu Tyr Leu Asp Leu Gin Asn Asp Leu Gly Gin Thr 85 90 95
Ala Leu His Leu Ala Ala He Leu Gly Glu Ala Ser Thr Val Glu Lys 100 105 110
Leu Tyr Ala Ala Gly Ala Gly Val Leu Val Ala Glu Arg Gly Gly His 115 120 125
Thr Ala Leu His Leu Ala Cys Arg Val Arg Ala His Thr Cys Ala Cys 130 135 140
Val Leu Leu Gin Pro Arg Pro Ser His Pro Arq Asp Ala Ser Asp Thr 145 150 155 160
Tyr Leu Thr Gin Ser Gin Asp Cys Thr Pro Asp Thr Ser His Ala Pro 165 170 175
Ala Ala Val Asp Ser Gin Pro Asn Pro Glu Asn Glu Glu Glu Pro Arg 180 185 190
Asp Glu Asp Trp Arg Leu' Gin Leu Glu Ala Glu Asn Tyr Asp Gly His 195 200 205
Thr Pro Leu His Val Ala Val He His Lys Asp Ala Glu Met VaJ Arg 210 215 220
Leu Leu Arg Asp Ala Gly Ala Asp Leu Asn Lys Pro Glu Pro Thr Cys 225 230 235 240
Gly Arg Thr Pro Leu His Leu Ala Val Glu Ala Gin Ala Ala Ser Val 245 250 255
Leu Glu Leu Leu Leu Lys Ala Gly Ala Asp Pro Thr Ala Arg Met Tyr 260 265 270
Gly Gly Arg Tnr Pro Leu Gly Ser Ala Leu Leu Arg Pro Asn Pro He 275 280 285
Leu Ala Arq Leu Leu Arg Ala His Gly Ala Pro Glu Pro Glu Asp Glu 290 295 300
Asp Asp Lys Leu Ser Pro Cys Ser Ser Ser Gly Ser Asp Ser Asp Ser 305 310 315 320
Asp Asn Arq Asp Glu Gly Asp Glu Tyr Asp Asp lie Val Val His Ser 325 330 335
Gly Arg Ser Gin Asn Arg Gin Pro Pro Ser Pro Ala Ser Lys Pro Leu 340 345 350
Pro Asp Asp Pro Asn Pro Ala 355
(2) INFORMATION FOR SEQ ID NO: 3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 282 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 3 :
Met Ser Pro He Leu Gly Tyr Trp Lys He Lys Gly Leu Val Gin Pro 1 5 10 15
Thr Arg Leu Leu Leu Glu Tyr Leu Glu Glu Lys Tyr Glu Glu His Leu 20 25 30 Tyr Glu Arg Asp Glu Gly Asp Lys Trp Arg Asn Lys Lys Phe Glu Leu
35 40 45
Gly Leu Glu Phe Pro Asn Leu Pro Tyr Tyr He Asp Gly Asp Val Lys 50 55 60
Leu Thr Gin Ser Met Ala He He Arg Tyr He Ala Asp Lys His Asn 65 70 75 80
Met Leu Gly Gly Cys Pro Lys Glu Arg Ala Glu He Ser Met Leu Glu 85 90 95
Gly Ala Val Leu Asp He Arg Tyr Gly Val Ser Arg He Ala Tyr Ser 100 105 110
Lys Asp Phe Glu Thr Leu Lys Val Asp Phe Leu Ser Lys Leu Pro Glu 115 120 125
Met Leu Lys Met Phe Glu Asp Arg Leu Cys His Lys Tnr Tyr Leu Asn 130 135 140
Gly Asp His Val Thr His Pro Asp Phe Met Leu Tyr Asp Ala Leu Asp 145 150 155 160
Val Val Leu Tyr Met Asp Pro Met Cys Leu Asp Ala Phe Pro Lys Leu 165 170 175
Val Cys Phe Lys Lys Arg He Glu Ala He Pro Gin He Asp Lys Tyr 180 185 190
Leu Lys Ser Ser Lys Tyr He Ala Trp Pro Leu Gin GJ y Trp Gin Ala 195 200 205
Thr Phe Gly Gly Gly Asp His Pro Pro Lys Ser Asp Pro Arg Glu Phe 210 215 220
He Val Thr Asp Met Phe Gin Ala Ala Glu Arg Pro Gin Glu Trp Ala 225 230 235 240
Met Glu Gly Pro Arg Asp Gly Leu Lys Lys Glu Arg Leu Leu Asp Asp 245 250 255
Arg His Asp Ser Gly Leu Asp Ser Met Lys Asp Glu Glu Tyr Glu Gin 260 265 270
Met Val Lys Glu Leu Gin Glu He Arg Leu 275 280
(2) INFORMATION FOR SEQ ID NO: :
(J) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 272 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: :
Met Ser Pro He Leu Gly Tyr Trp Lys He Lys Gly Leu Val Gin Pro
1 5 10 15
Thr Arg Leu Leu Leu Glu Tyr Leu Glu Glu Lys Tyr Glu Glu His Leu 20 25 30
Tyr Glu Arg Asp Glu Gly Asp Lys Trp Arq Asn Lys Lys Phe Glu Leu 35 40 45
Gly Leu Glu Phe Pro Asn Leu Pro Tyr Tyr He Asp Gly Asp Val Lys 50 55 60 Leu Thr Gin Ser Met Ala He He Arg Tyr He Ala Asp Lys His Asn 65 70 75 80
Met Leu Gly Gly Cys Pro Lys Glu Arg Ala Glu He Ser Met Leu Glu 85 90 95
Gly Ala Val Leu Asp He Arg Tyr Gly Val Ser Arg He Ala Tyr Ser 100 105 110
Lys Asp Phe Glu Thr Leu Lys Val Asp Phe Leu Ser Lys Leu Pro Glu 115 120 125
Met Leu Lys Met Phe Glu Asp Arq Leu Cys His Lys Thr Tyr Leu Asn 130 135 140
Gly Asp His Val Thr His Pro Asp Phe Met Leu Tyr Asp Ala Leu Asp 145 150 155 160
Val Val Leu Tyr Met Asp Pro Met Cys Leu Asp Ala Phe Pro Lys Leu 165 170 175
Val Cys Phe Lys Lys Arq He Glu Ala He Pro Gin He Asp Lys Tyr 180 185 190
Leu Lys Ser Ser Lys Tyr He Ala Trp Pro Leu Gin Gly Trp Gin Ala 195 200 205
Thr Phe Gly Gly Gly Asp His Pro Pro Lys Ser Asp Pro Arg Glu Phe 210 215 220
He Val Thr Asp Met Ala Gly Val Ala Cys Leu Gly Lys Thr Ala Asp 225 230 235 240
Ala Asp Glu Trp Cys Asp Ser Gly Leu Gly Ser Leu Gly Pro Asp Aid 245 ?50 255
AJa Ala Pro Gly Gly Pro Gly Leu Gly Ala Glu Leu Gly Pro Glu Leu 260 265 270
(2) INFORMATION FOR SEQ ID NO: 5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 282 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 5 :
Met Ser Pro He Leu Gly Tyr Trp Lys He Lys Gly Leu Val Gin Pro
1 5 10 15
Thr Arg Leu Leu Leu Glu Tyr Leu Glu Glu Lys Tyr Glu Glu His Leu 20 25 30
Tyr Glu Arg Asp Glu Gly Asp Lys Trp Arg Asn Lys Lys Phe Glu Leu 35 40 45
Gly Leu Glu Phe Pro Asn Leu Pro Tyr Tyr He Asp Gly Asp Val Lys
50 55 60
Leu Thr Gin Ser Met Ala He He Arg Tyr He Ala Asp Lys His Asn 65 70 75 80
Met Leu Gly Gly Cys Pro Lys Glu Arg Ala Glu He Ser Met Leu Glu
85 90 95
Gly Ala Val Leu Asp He Arg Tyr Gly Val Ser Arg He Ala Tyr Ser 100 105 110 Lys Asp Phe Glu Thr Leu Lys Val Asp Phe Leu Ser Lys Leu Pro Glu 115 120 125
Met Leu Lys Met Phe Glu Asp Arg Leu Cys His Lys Thr Tyr Leu Asn 130 135 140
Gly Asp His Val Thr His Pro Asp Phe Met Leu Tyr Asp Ala Leu Asp 145 150 155 160
Val Val Leu Tyr Met Asp Pro Met Cys Leu Asp Ala Phe Pro Lys Leu 165 170 175
Val Cys Phe Lys Lys Arg He Glu Ala He Pro Gin He Asp Lys Tyr 180 185 190
Leu Lys Ser Ser Lys Tyr He Ala Trp Pro Leu Gin Gly Trp Gin Ala 195 200 205
Thr Phe Gly Gly Gly Asp His Pro Pro Lys Ser Asp Pro Arg Glu Phe 210 215 220
He Val Thr Asp Met Phe Gin Ala Ala Glu Arg Pro Gin Glu Trp Ala 225 230 235 240
Met Glu Gly Pro Arg Asp Gly Leu Lys Lys Glu Arg Leu Leu Asp Asp 245 250 255
Arg His Asp Thr Gly Leu Asp Thr Met Lys Asp Glu Glu Tyr Glu Gin 260 265 270
Met Val Lys Glu Leu Gin Glu He Arg Leu 275 280
(2) INFORMATION FOR SEQ ID NO: 6:
(l) SEQUENCE CHARACTERISTICS: (A) LENGTH: 272 ammo acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
( xi ) SEQUENCE DESCRI PTION : SEQ I D NO : 6 :
Me t Ser Pro He Leu Gl y Tyr Trp Lys He Lys Gl y Leu Va l Gin Pro
1 5 1 0 15
Thr Arg Leu Leu Leu Glu Tyr Leu Glu Glu Lys Tyr Glu Glu His Leu 20 25 30
Tyr Glu Arg Asp Glu Gly Asp Lys Trp Arg Asn Lys Lys Phe Glu Leu 35 40 45
Gly Leu Glu Phe Pro Asn Leu Pro Tyr Tyr He Asp Gly Asp Val Lys 50 55 60
Leu Thr Gin Ser Met Ala He He Arg Tyr He Ala Asp Lys His Asn 65 70 75 80
Met Leu Gly Gly Cys Pro Lys Glu Arg Ala Glu He Ser Met Leu Glu 85 90 95
Gly Ala Val Leu Asp He Arg Tyr Gly Val Ser Arg He Ala Tyr Ser 100 105 110
Lys Asp Phe Glu Thr Leu Lys Val Asp Phe Leu Ser Lys Leu Pro Glu 115 120 125
Met Leu Lys Met Phe Glu Asp Arg Leu Cys His Lys Thr Tyr Leu Asn 130 135 140 Gly Asp His Val Thr His Pro Asp Phe Met Leu Tyr Asp Ala Leu Asp 145 150 155 160
Val Val Leu Tyr Met Asp Pro Met Cys Leu Asp Ala Phe Pro Lys Leu 165 170 175
Val Cys Phe Lys Lys Arg He Glu Ala He Pro Gin He Asp Lys Tyr 180 185 190
Leu Lys Ser Ser Lys Tyr He Ala Trp Pro Leu Gin Gly Trp Gin Ala 195 200 205
Thr Phe Gly Gly Gly Asp His Pro Pro Lys Ser Asp Pro Arq Glu Phe 210 215 220
He Val Thr Asp Met Ala Gly Val Ala Cys Leu Gly Lys Thr Ala Asp 225 230 235 240
Ala Asp Glu Trp Cys Asp Ala Gly Leu Gly Ala Leu Gly Pro Asp Ala 245 250 255
Ala Ala Pro Gly Gly Pro Gly Leu Gly Ala Glu Leu Gly Pro Glu Leu 260 265 270
(2) INFORMATION FOR SEQ ID NO: 7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2251 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 7 :
GGCACGAGGC CCCATGGAGC GGCCCCCGGG GCTGCGGCCG GGCGCGGGCG GGCCCTGGGA 60
GATGCGGGAG CGGCTGGGCA CCGGCGGCTT CGGGAACGTC TGTCTGTACC AGCATCGGGA 120
ACTTGATCTC AAAATAGCAA TTAAGTCTTG TCGCCTAGAG CTAAGTACCA AAAACAGAGA 180
ACGATGGTGC CATGAAATCC AGATTATGAA GAAGTTGAAC CATGCCAATG TTGTAAAGGC 240
CTGTGATGTT CCTGAAGAAT TGAATATTTT GATTCATGΛT GTGCCTCTTC TAGCAATGGA 300
ATACTGTTCT GGAGGAGATC TCCGAAAGCT GCTCAACAAA CCAGAAAATT GTTGTGGACT 360
TAAAGAAAGC CAGATACTTT CTTTACTAAG TGATATAGGG TCTGGGATTC GATATTTGCA 420
TGAAAACAAA ATTATACATC GAGATCTAAA ACCTGAAAAC ATAGTTCTTC AGGATGTTGG 480
TGGAAAGATA ATACATAAAA TAATTGATCT GGGATATGCC AAAGATGTTG ATCAAGGAAG 540
TCTGTGTACA TCTTTTGTGG GAACACTGCA GTATCTGGCC CCAGAGCTCT TTGAGAATAA 600
GCCTTACACA GCCACTGTTG ATTATTGGAG CTTTGGGACC ATGGTATTTG AATGTATTGC 660
TGGATATAGG CCTTTTTTGC ATCATCTGCA GCCATTTACC TGGCATGAGA AGATTAAGAA 720
GAAGGATCCA AAGTGTATAT TTGCATGTGA AGAGATGTCA GGAGAAGTTC GGTTTAGTAG 780
CCATTTACCT CAACCAAATA GCCTTTGTAG TTTAATAGTA GAACCCATGG AAAACTGGCT 840
ACACTTGATG TTGAATTGGG ACCCTCAGCA GAGAGGAGGA CCTGTTGACC TTACTTTGAA 900
GCAGCCAAGA TGTTTTGTAT TAATGGΛTCA CATTTTGAAT TTGAAGATAG TACACATCCT 960
AAATATGACT TCTGCAAAGA TAATTTCTTT TCTGTTACCA CCTGATGAAA GTCTTCATTC 1020 ACTACAGTCT CGTATTGAGC GTGAAACTGG AATAAATACT GGTTCTCAAG AACTTCTTTC 1080
AGAGACAGGA ATTTCTCTGG ATCCTCGGAA ACCAGCCTCT CAATGTGTTC TAGATGGAGT 1140
TAGAGGCTGT GATAGCTATA TGGTTTATTT GTTTGATAAA AGTAAAACTG TATATGAAGG 1200
GCCATTTGCT TCCAGAAGTT TATCTGATTG TGTAAATTAT ATTGTACAGG ACAGCAAAAT 1260
ACAGCTTCCA ATTATACAGC TGCGTAAAGT GTGGGCTGAA GCAGTGCACT ATGTGTCTGG 1320
ACTAAAAGAA GACTATAGCA GGCTCTTTCA GGGACAAAGG GCAGCAATGT TAAGTCTTCT 1380
TAGATATAAT GCTAACTTAA CAAAAATGAA GAACACTTTG ATCTCAGCAT CACAACAACT 1440
GAAAGCTAAA TTGGAGTTTT TTCACAAAAG CATTCAGCTT GACTTGGAGA GATACAGCGA 1500
GCAGATGACG TATGGGATAT CTTCAGAAAA AATGCTAAAA GCATGGAAAG AAATGGAAGA 1560
AAAGGCCATC CACTATGCTG AGGTTGGTGT CATTGGA AC CTGGAGGATC AGATTATGTC 1620
TTTGCATGCT GAAATCATGG AGCTACAGAA GAGCCCCTAT GGAAGACGTC AGGGAGACTT 1680
GATGGAATCT CTGGAACAGC GTGCCATTGA TCTATATAAG CAGTTAAAAC ACAGACCTTC 1740
AGATCACTCC TACAGTGACA GCACAGAGAT GGTGAAAATC ATTGTGCACA CTGTGCAGAG 1800
TCAGGACCGT GTGCTCAAGG AGCGTTTTGG TCATTTGAGC AAGTTGTTGG GCTGTAAGCA 1860
GAAGATTATT GATCTACTCC CTAAGGTGGA AGTGGCCCTC AGTAΛTATCA AAGAAGCTGA 1920
CAATACTGTC ATGTTCATGC AGGGAAAAAG GCAGAAAGAA ATATGGCATC TCCTTAAAAT 1980
TGCCTGTACA CAGAGTTCTG CCCGCTCTCT TGTAGGATCC AGTCTAGAAG GTGCAGTAAC 2040
CCCTCAAGCA TACGCATGGC TGGCCCCCGA CTTAGCAGAA CATGATCATT CTCTGTCATG 2100
TGTGGTAACT CCTCAAGATG GGGAGACTTC AGCACAAATG ATAGAAGAAA ATTTGAACTG 2160 CCTTGGCCAT TTAAGCACTA TTATTCATGA GGCAAATGAG GAACAGGGCA ATAGTATGAT 2220
GAATCTTGAT TGGAGTTGGT TAACAGAATG A 2251
(2) INFORMATION FOR SEQ ID NO: 8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2271 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:
ATGAGCTGGT CACCTTCCCT GACAACGCAG ACATGTGGGG CCTGGGAAAT GAAAGAGCGC 60
CTTGGGACAG GGGGATTTGG AAATGTCATC CGATGGCACA ATCAGGAAAC AGGTGAGCAG 120
ATTGCCATCA AGCAGTGCCG GCAGGAGCTC AGCCCCCGGA ACCGAGAGCG GTGGTGCCTG 180
GAGATCCAGA TCATGAGAAG GCTGACCCAC CCCAATGTGG TGGCTGCCCG AGATGTCCCT 240
GAGGGGATGC AGAACTTGGC GCCCAATGAC CTGCCCCTCC TGGCCATGGA GTACTGCCAA 300
GGAGGAGATC TCCGGAAGTA CCTGAACCAG TTTGAGAACT GCTGTGGTCT GCGGGAAGGT 360
GCCATCCTCA CCTTGCTGAG TGACATTGCC TCTGCGCTTA GATACCTTCA TGAAAACAGA 420
ATCATCCΛTC GGGATCTAAA GCCAGAAAAC ATCGTCCTGC AGCAAGGAGA ACAGAGGTTA 480
ATACACAAAA TTATTGACCT AGGATATGCC AAGGAGCTGG ATCAGGGCAG TCTTTGCACA 540 TCATTCGTGG GGACCCTGCA GTACCTGGCC CCAGAGCTAC TGGAGCAGCA GAAGTACACA 600
GTGACCGTCG ACTACTGGAG CTTCGGCACC CTGGCCTTTG AGTGCATCAC GGGCTTCCGG 660
CCCTTCCTCC CCAACTGGCA GCCCGTGCAG TGGCATTCAA AAGTGCGGCA GAAGAGTGAG 720
GTGGACATTG TTGTTAGCGA AGACTTGAAT GGAACGGTGA AGTTTTCAAG CTCTTTACCC 780
TACCCCAATA ATCTTAACAG TGTCCTGGCT GAGCGACTGG AGAAGTGGCT GCAACTGATG 840
CTGATGTGGC ACCCCCGACA GAGGGGCACG GATCCCACGT ATGGGCCCAA TGGCTGCTTC 900
AAGGCCCTGG ATGACATCTT AAACTTAAAG TTGGTTCATA TCTTGAACAT GGTCACGGGC 960
ACCATCCACA CCTACCCTGT GACAGAGGAT GAGAGTCTGC AGAGCTTGAA GGCCAGAATC 1020
CAACAGGACA CGGGCATCCC AGAGGAGGAC CAGGAGCTGC TGCAGGAAGC GGGCCTGGCG 1080
TTGATCCCCG ATAAGCCTGC CACTCΛGTGT ATTTCAGACG GCAAGTTAAA TGAGGGCCAC 11 0
ACATTGGACΛ TGGATCTTGT TTTTCTCTTT GACAACAGTA AAATCACCTA TGAGACTCAG 1200
ATCTCCCCAC GGCCCCAACC TGAAAGTGTC AGCTGTATCC TTCAAGAGCC CAAGAGGAAT 1260
CTCGCCTTCT TCCACCTGAG GAAGGTGTGG GGCCAGGTCT GGCACAGCAT CCAGACCCTG 1320
AAGGAAGATT GCAACCGGCT GCAGCAGGGA CAGCGAGCCG CCATGATGAA TCTCCTCCGA 1380
AACAACAGCT GCCTCTCCAA AATGAAGAAT TCCATGGCTT CCATGTCTCA GCAGCTCAAG 1440
GCCAAGTTGG ATTTCTTCAA AACCAGCATC CAGATTGACC TGGAGAAGTA CAGCGAGCAA 1500
ACCGAGTTTG GGATCACATC AGATAAACTG CTGCTGGCCT GGAGGGAAAT GGAGCAGGCT 1560
GTGGAGCTCT GTGGGCGGGA GAACGAAGTG AAACTCCTGG TAGAACGGAT GATGGCTCTG 1620
CAGACCGACA TTGTGGACTT ACAGAGGAGC CCCATGGGCC GGAAGCAGGG GGGAACGCTG 1680 GACGACCTAG AGGAGCAAGC AAGGGAGCTG TACAGGAGAC TAAGGGAAAA ACCTCGAGAC 1740
CAGCGAACTG AGGGTGACAG TCAGGAAATG GTACGGCTGC TGCTTCAGGC AATTCAGAGC 1800
TTCGAGAAGA AAGTGCGAGT GATCTATACG CAGCTCAGTA AAACTGTGGT TTGCAAGCAG 1860
AAGGCCCTGG AACTGTTGCC CAAGGTGGAA GAGGTGGTGA GCTTAATGΛA TGAGGATGAG 1920
AAGACTGTTG TCCGGCTGCA GGAGAAGCGG CAGAAGGAGC TCTGGAATCT CCTGAAGATT 1980
GCTTGTAGCA AGGTCCGTGG TCCTGTCAGT GGAAGCCCGG ΛTΛGCATGAA TGCCTCTCGA 2040
CTTAGCCAGC CTGGGCAGCT GATGTCTCAG CCCTCCACGG CCTCCAACAG CTTACCTGAG 2100
CCAGCCAAGA AGAGTGAAGA ACTGGTGGCT GAAGCACATA ACCTCTGCAC CCTGCTAGAA 2160
AATGCCATΛC AGGACACTGT GAGGGAACAΛ GACCAGAGTT TCACGGCCCT AGACTGGAGC 2220
TGGTTACAGA CGGΛAGAAGA AGAGCACAGC TGCCTGGAGC AGGCCTCATG A 2271
(2) INFORMATION FOR SEQ ID NO: 9:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 756 ammo acids
(B) TYPE: ammo acid
( C ) STRANDEDNESS :
( D ) TOPOLOGY : linear
( xi ) SEQUENCE DESCRI PT ION : SEQ I D NO : 9 :
Met Ser Trp Ser Pro Ser Leu Thr Thr Gin Thr Cys Gl y Al a Trp Gl u 1 1 0 15 Met Lys Glu Arg Leu Gly Thr Gly Gly Phe Gly Asn Val He Arg Trp 20 25 30
His Asn Gin Glu Thr Gly Glu Gin He Ala He Lys Gin Cys Arg Gin 35 40 45
Glu Leu Ser Pro Arg Asn Arg Glu Arg Trp Cys Leu Glu He Gin He 50 55 60
Met Arg Arg Leu Thr His Pro Asn Val Val Ala Ala Arg Asp Val Pro 65 70 75 80
Glu Gly Met Gin Asn Leu Ala Pro Asn Asp Leu Pro Leu Leu Ala Met 85 90 95
Glu Tyr Cys Gin Gly Gly Asp Leu Arg Lys Tyr Leu Asn Gin Phe Glu 100 105 110
Asn Cys Cys Gly Leu Arg Glu Gly Ala He Leu Thr Leu Leu Ser Asp 115 120 125
He Ala Ser Ala Leu Arg Tyr Leu His Glu Asn Arg He He His Arg 130 135 140
Asp Leu Lys Pro Glu Asn He Val Leu Gin Gin Gly Glu Gin Arg Leu 145 150 155 160
He His Lys He He Asp Leu Gly Tyr Ala Lys Glu Leu Asp Gin Gly 165 170 175
Ser Leu Cys Thr Ser Phe Val Gly Thr Leu Gin Tyr Leu Ala Pro Glu 180 185 190
Leu Leu Glu Gin Gin Lys Tyr Thr Val Thr Val Asp Tyr Trp Ser Phe 195 200 205 Gly Thr Leu Ala Phe Glu Cys He Thr Gly Phe Arq Pro Phe Leu Pro 210 215 220
Asn Trp Gin Pro Val Gin Trp His Ser Lys Val Arq Gin Lys Ser Glu 225 230 235 240
Val Asp He Val Val Ser Glu Asp Leu Asn Gly Thr Val Lys Phe Ser 245 250 255
Ser Ser Leu Pro Tyr Pro Asn Asn Leu Asn Ser Val Leu Ala Glu Arg 260 265 270
Leu Glu Lys Trp Leu Gin Leu Met Leu Met Trp His Pro Arg Gin Arg 275 280 285
Gly Thr Asp Pro Thr Tyr Gly Pro Asn Gly Cys Phe Lys Ala Leu Asp 290 295 300
Asp He Leu Asn Leu Lys Leu Val His He Leu Asn Met Val Thr Gly 305 310 315 320
Thr He His Thr Tyr Pro' Val Thr Glu Asp Glu Ser Leu Gin Ser Leu 325 330 335
Lys Ala Arg He Gin Gin Asp Thr Gly He Pro Glu Glu Asp Gin Glu 340 345 350
Leu Leu Gin Glu Ala Gly Leu Ala Leu He Pro Asp Lys Pro Ala Thr 355 360 365
Glr. Cys He Ser Asp Gly Lys Leu Asn Glu Gly His Thr Leu Asp Met 370 375 380
Asp Leu Val Phe Leu Phe Asp Asn Ser Lys He Thr Tyr Glu Thr Gin 385 390 395 400
He Ser Pro Arg Pro Gin Pro Glu Ser Val Ser Cys He Leu Gin Glu 405 41 0 4 15
Pro Lys Arg Asn Leu Ala Phe Phe His Leu Arg Lys Val Trp Gly Gin 420 425 430
Val Trp His Ser He Gin Thr Leu Lys Glu Asp Cys Asn Arg Leu Gin 435 440 445
Gin Gly Gin Arg Ala Ala Met Met Asn Leu Leu Arg Asn Asn Ser Cys 450 455 460
Leu Ser Lys Met Lys Asn Ser Met Ala Ser Met Ser Gin Gin Leu Lys 465 470 475 480
Ala Lys Leu Asp Phe Phe Lys Thr Ser He Gin He Asp Leu Glu Lys 485 490 495
Tyr Ser Glu Gin Thr Glu Phe Gly He Thr Ser Asp Lys Leu Leu Leu 500 505 510
Ala Trp Arg Glu Met Glu Gin Ala Val Glu Leu Cys Gly Arg Glu Asn 515 520 525
Glu Val Lys Leu Leu Val Glu Arg Met Met Ala Leu Gin Thr Asp He 530 535 540
Val Asp Leu Gin Arg Ser Pro Met Gly Arg Lys Gin Gly Gly Thr Leu 545 550 555 560
Asp Asp Leu Glu Glu Gin Ala Arg Glu Leu Tyr Arg Arg Leu Arg Glu 565 570 575
Lys Pro Arg Asp Gin Arg Thr Glu Gly Asp Ser Gin Glu Met Val Arg 580 585 590
Leu Leu Leu Gin Ala He Gin Ser Phe Glu Lys Lys Val Arg Val He 595 600 605 Tyr Thr Gin Leu Ser Lys Thr Val Val Cys Lys Gin Lys Ala Leu Glu 610 615 620
Leu Leu Pro Lys Val Glu Glu Val Val Ser Leu Met Asn Glu Asp Glu 625 630 635 640
Lys Thr Val Val Arg Leu Gin Glu Lys Arg Gin Lys Glu Leu Trp Asn 645 650 655
Leu Leu Lys He Ala Cys Ser Lys Val Arg Gly Pro Val Ser Gly Ser 660 665 670
Pro Asp Ser Met Asn Ala Ser Arg Leu Ser Gin Pro Gly Gin Leu Met 675 680 685
Ser Gin Pro Ser Thr Ala Ser Asn Ser Leu Pro Glu Pro Ala Lys Lys 690 695 700
Ser Glu Glu Leu Val Ala Glu Ala His Asn Leu Cys Thr Leu Leu Glu 705 710 715 720
Asn Ala He Gin Asp Thr Val Arg Glu Gin Asp Gin Ser Phe Thr Ala 725 730 735
Leu Asp Trp Ser Trp Leu Gin Thr Glu Glu Glu Glu His Ser Cys Leu 740 745 750
Glu Gin Ala Ser 755
(2) INFORMATION FOR SEQ ID NO: 10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 745 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: (D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:
Met Glu Arg Pro Pro Gly Leu Arg Pro Gly Ala Gly Gly Pro Trp Glu
1 5 10 15
Met Arg Glu Arg Leu Gly Thr Gly Gly Phe Gly Asn Val Cys Leu Tyr
20 25 30
Gin His Arg Glu Leu Asp Leu Lys He Ala He Lys Ser Cys Arg Leu 35 40 45
Glu Leu Ser Thr Lys Asn Arg Glu Arg Trp Cys His Glu He Gin He 50 55 60
Met Lys Lys Leu Asn His Ala Asn Val Val Lys Ala Cys Asp Val Pro 65 70 75 80
Glu Glu Leu Asn He Leu He His Asp Val Pro Leu Leu Ala Met Glu 85 90 95
Tyr Cys Ser Gly Gly Asp Leu Arg Lys Leu Leu Asn Lys Pro Glu Asn 100 105 110
Cys Cys Gly Leu Lys Glu Ser Gin He Leu Ser Leu Leu Ser Asp He 115 120 125
Gly Ser Gly He Arg Tyr Leu His Glu Asn Lys He He His Arg Asp 130 135 140
Leu Lys Pro Glu Asn He Val Leu Gin Asp Val Gly Gly Lys He He 145 150 155 160 His Lys He He Asp Leu Gly Tyr Ala Lys Asp Val Asp Gin Gly Ser 165 170 175
Leu Cys Thr Ser Phe Val Gly Thr Leu Gin Tyr Leu Ala Pro Glu Leu 180 185 190
Phe Glu Asn Lys Pro Tyr Thr Ala Thr Val Asp Tyr Trp Ser Phe Gly 195 200 205
Thr Met Val Phe Glu Cys He Ala Gly Tyr Arg Pro Phe Leu His His 210 215 220
Leu Gin Pro Phe Thr Trp His Glu Lys lie Lys Lys Lys Λsp Pro Lys 225 230 235 240
Cys He Phe Ala Cys Glu Glu Met Ser Gly Glu Val Arg Phe Ser Ser 245 250 255
Has Leu Pro Gin Pro Asn Ser Leu Cys Ser Leu He Val Glu Pro Met 260 265 270
Glu Asn Trp Leu Gin Leu Met Leu Asn Trp Asp Pro Gin Gin Arg Gly 275 280 285
Gly Pro Val Asp Leu Thr Leu Lys Gin Pro Arg Cys Phe Val Leu Met 290 295 300
Asp Has He Leu Asn Leu Lys He Vdl Has He Leu Λsn Met Thr Ser 305 310 315 320
Ala Lys lie He Ser Phe Leu Leu Pro Pro Asp Glu Ser Leu His Ser 325 330 335
Leu Gin Ser Arg He Glu Arg Glu Thr Gly He Asn Thr Gly Ser Gin 340 345 350 Glu Leu Leu Ser Glu Thr Gly He Ser Leu Asp Pro Arg Lys Pro Ala 355 360 365
Ser Gin Cys Val Leu Asp Gly Val Arg Gly Cys Asp Ser Tyr Met Val 370 375 380
Tyr Leu Phe Asp Lys Ser Lys Thr Val Tyr Glu Gly Pro Phe Ala Ser 385 390 395 400
Arg Ser Leu Ser Asp Cys Val Asn Tyr He Val Gin Asp Ser Lys He 405 410 415
Gin Leu Pro He He Gin Leu Arg Lys Val Trp Ala Glu Ala Val His 420 425 430
Tyr Val Ser Gly Leu Lys Glu Asp Tyr Ser Arg Leu Phe Gin Gly Gin 435 440 445
Arg Ala Ala Met Leu Ser Leu Leu Arg Tyr Asn Ala Asn Leu Thr Lys 450 455 460
Met Lys Asn Thr Leu He Ser Ala Ser Gin Gin Leu Lys Ala Lys Leu 465 470 475 480
Glu Phe Phe His Lys Ser He Gin Leu Asp Leu Glu Arq Tyr Ser Glu 485 490 495
Gin Met Thr Tyr Gly He Ser Ser Glu Lys Met Leu Lys Ala Trp Lys 500 505 510
Glu Met Glu Glu Lys Ala He His Tyr Ala Glu Val Gly Val He Gly 515 520 525
Tyr Leu Glu Asp Gin He Met Ser Leu His Ala Glu He Met Glu Leu 530 535 540
Gin Lys Ser Pro Tyr Gly Arg Arg Gin Gly Asp Leu Met Glu Ser Leu 545 550 555 560
Glu Gin Arg Ala He Asp Leu Tyr Lys Gin Leu Lys His Arg Pro Ser 565 570 575
Asp His Ser Tyr Ser Asp Ser Thr Glu Met Val Lys He He Val His 580 585 590
Thr Val Gin Ser Gin Asp Arg Val Leu Lys Glu Arg Phe Gly His Leu 595 600 605
Ser Lys Leu Leu Gly Cys Lys Gin Lys He He Asp Leu Leu Pro Lys
610 615 620
Val Glu Val Ala Leu Ser Asn He Lys Glu Ala Asp Asn Thr Val Met 625 630 635 640
Phe Met Gin Gly Lys Arg Gin Lys Glu He Trp His Leu Leu Lys He 645 650 655
Ala Cys Thr Gin Ser Ser Ala Arg Ser Leu Val Gly Ser Ser Leu Glu 660 665 670
Gly Ala Val Tnr Pro Gin Ala Tyr Ala Trp Leu Ala Pro Asp Leu Ala 675 680 685
Glu His Asp His Ser Leu Ser Cys Val Val Thr Pro Gin Asp Gly Glu 690 695 700
Thr Ser Ala Gin Met He Glu Glu Asn Leu Asn Cys Leu Gly His Leu 705 710 715 720
Ser Thr He He His Glu ALa Asn Glu Glu Gin Gly Asn Ser Met Met 725 730 735
Asn Leu Asp Trp Ser Trp Leu Thr Glu 740 745 (2) INFORMATION FOR SEQ ID NO: 11:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 amino acids
(B) TYPE: ammo acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:
Cys Lys Lys Glu Arg Leu Leu Asp Asp Arg His Asp Ser Gly Leu Asp 1 5 10 15
Ser Met Lys Asp Glu Glu 20
(2) INFORMATION FOR SEQ ID NO: 12:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:
Cys Lys Lys Glu Arg Leu Leu Asp Asp Arg His Asp Thr Gly Leu Asp 1 5 10 15 Thr Met Lys Asp Glu Glu 20
(2) INFORMATION FOR SEQ ID NO: 13:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 amino acids
(B) TYPE: ammo acαd
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(lx) FEATURE:
(A) NAME/KEY: Modifled-site
(B) LOCATION: 10
(D) OTHER INFORMATION: /note- "Where Xaa s a Phosphate Ester of Threonine"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : ] 3 :
ASD Leu Thr Gly Gly Pro Glu Val Ala Xaa Pro Glu Ser Glu Glu Ala 1 5 10 15
Phe Leu Pro
(2) INFORMATION FOR SEQ ID NO: 14:
(l, SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 ammo acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear (xi ) SEQUENCE DESCRIPTION : SEQ ID NO : 14 :
Cys Pro Thr Asn Ser Ala Leu Asn Tyr Leu Lys Ser Pro He Thr Thr 1 5 10 15
Ser Pro Ser
(2) INFORMATION FOR SEQ ID NO: 15:
(α) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 ammo acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15:
Cys Asn Ser Asp Leu Leu Thr Ser Pro Asp Val Gly Leu Leu Lys
1 5 10 15
(2) INFORMATION FOR SEQ ID NO: 16:
(1) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 ammo acids
Figure imgf000073_0001
(C) STRANDEDNESS:
(D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:
Cys Val Gly Leu Leu Lys Leu Ala Ser Pro Glu Leu Glu Arg Leu
1 5 10 15
(2) INFORMATION FOR SEQ ID NO: 17:
(α) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(κi) SEQUENCE DESCRIPTION: SEQ ID NO:!/:
He He Asp Leu Gly Tyr Ala Lys 1 5
(2) INFORMATION FOR SEQ ID NO: 18:
( ) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: ammo acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: If
Val Glu Val Ala Leu Ser Asn He Lys
1 5
(2) INFORMATION FOR SEQ ID NO: 19:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 ammo acids
(B) TYPE: ammo acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(xα) SEQUENCE DESCRIPTION: SEQ ID NO: 19:
Ser He Gin Leu Asp Leu Glu Arg 1 5
(2) INFORMATION FOR SEQ ID NO:20:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 ammo acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20: Ala Leu Glu Leu Leu Pro Lys 1 5
(2) INFORMATION FOR SEQ ID NO: 21:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:21
Val He Tyr Thr Gin Leu Ser Lys 1 5
(2) INFORMATION FOR SEQ ID NO: 22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 11 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(xi ) SEQUENCE DESCRIPTION: SEQ ID NO: 22:
Leu Leu Leu Gin Ala He Gin Ser Phe Glu Lys 1 5 10 (2) INFORMATION FOR SEQ ID NO: 23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 11 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23:
Leu Gly Thr Gly Gly Phe Gly Asn Val He Arq 1 5 10
(2) INFORMATION FOR SEQ ID NO:24:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24:
Ala Leu Asp Asp He Leu Asn Leu Lys 1 5
(2) INFORMATION FOR SEQ ID NO: 25:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 25:
Asp Leu Lys Pro Glu Asn He Val Leu Gin Gin Gly Glu Gin Arg 1 5 10 15

Claims

Claims
1. An IKK signalsome capable of specifically phosphorylating IκBα at residues S32 and S36, and IκBβ at residues 19 and 23, without the addition of exogenous cofactors.
2. An IKK signalsome according to claim 1 wherein the signalsome is derived from a human tissue or cell line.
3. A polypeptide comprising a component of an IKK signalsome according to claim 1 , or a variant of such a component, wherein the component has a sequence recited in SEQ ID NO:9.
4. An isolated DNA molecule encoding a polypeptide according to claim 3.
5. A recombinant expression vector comprising a DNA molecule according to claim 4.
6. A host cell transformed or transfected with an expression vector according to claim 5.
7. A host cell according to claim 6, wherein the host cell is selected from the group consisting of bacteria, yeast, baculovirus infected insect cells and mammalian cells.
8. A method for preparing an IKK signalsome, comprising combining components of an IKK signalsome in a suitable buffer.
9. A method for phosphorylating a substrate of an IKK signalsome, comprising contacting a substrate with a signalsome according to claim 1 and thereby phosphorylating the substrate.
10. A method for phosphorylating a substrate of an IKK signalsome, comprising contacting a substrate with a polypeptide comprising a component of an IKK signalsome having IKB kinase activity, and thereby phosphorylating the substrate.
1 1. A method according to claim 10, wherein the polypeptide comprises IKK-1 (SEQ ID NO: 10).
12. A method according to claim 10, wherein the polypeptide comprises IKK-2 (SEQ ID NO:9).
13. The method of either of claims 9 or 10, wherein the substrate is IκBα or a variant thereof.
14. A method for screening for an agent that modulates IKK signalsome activity, comprising:
(a) contacting a candidate agent with an IKK signalsome according to claim 1 , wherein the step of contacting is carried out under conditions and for a time sufficient to allow the candidate agent and the IKK signalsome to interact; and
(b) subsequently measuring the ability of the candidate agent to modulate a IKK signalsome activity.
15. A method according to claim 14, wherein the IKK signalsome activity modulated is selected from the group consisting of IκB kinase activity, p65 kinase activity and IKK phosphatase activity.
16. A method for screening for an agent that modulates IKK signalsome activity, comprising: (a) contacting a candidate agent with a polypeptide comprising a component of an IKK signalsome according to claim 1, wherein the step of contacting is carried out under conditions and for a time sufficient to allow the candidate agent and the polypeptide to interact; and
(b) subsequently measuring the ability of the candidate agent to modulate the ability of the polypeptide to phosphorylate an I B protein.
17. A method according to claim 16, wherein the polypeptide comprises IKK-1 (SEQ ID NO: 10).
18. A method according to claim 16, wherein the polypeptide comprises IKK-2 (SEQ ID NO:9).
19. An antibody that binds to IKK-1 (SEQ ID NO: 10) and/or IKK-2 (SEQ ID NO:9).
20. An antibody according to claim 19, wherein the antibody inhibits the phosphorylation of an IκB protein by an IKK signalsome.
21. A composition comprising an agent that modulates IKK signalsome activity in combination with a pharmaceutically acceptable carrier, for use in the manufacture of a medicament for modulating NF-κB activity in a patient.
22. The composition of claim 21, wherein the agent inhibits activation of an IKK signalsome.
23. The composition of claim 21, wherein the agent inhibits kinase activity of an activated IKK signalsome.
24. A composition comprising an agent that modulates IKK signalsome activity in combination with a pharmaceutically acceptable carrier, for use in the manufacture of a medicament for treating a patient afflicted with a disorder associated with the activation of an IKK signalsome.
25. The composition of any one of claims 21 -24, wherein the agent is a monoclonal antibody.
26. The composition of any one of claims 21-24, wherein the agent comprises a polynucleotide.
27. A method for detecting IKK signalsome activity in a sample, comprising:
(a) contacting a sample with an antibody that binds to an IKK signalsome under conditions and for a time sufficient to allow the antibody to immunoprecipitate an IKK signalsome;
(b) separating immunoprecipitated material from the sample; and
(c) determining the ability of the immunoprecipitated material to phosphorylate an IKB protein with in vivo specificity.
28. A method according to claim 27, wherein the immunoprecipitated material phosphorylates IκBα at residues S32 and S36.
29. A kit for detecting IKK signalsome activity in a sample, comprising an antibody that binds to a IKK signalsome in combination with a suitable buffer.
30. A method for identifying an upstream kinase in the NF- B signal transduction cascade, comprising evaluating the ability of a candidate upstream kinase to phosphorylate and induce enzymatic activity of an IKK signalsome or a component or variant thereof, and thereby identifying an upstream kinase in the NF-κB signal transduction cascade.
31. A method for identifying a component of an IKK signalsome, comprising:
(a) isolating an IKK signalsome;
(b) separating the signalsome into components; and
(c) obtaining a partial sequence of a component, and thereby identifying a component of an IKK signalsome.
32. A method for preparing an IKK signalsome from a biological sample, comprising:
(a) separating a biological sample into two or more fractions; and
(b) monitoring IκB kinase activity in the fractions.
PCT/US1997/015003 1996-08-26 1997-08-26 Stimulus-inducible i (kappa)b kinase [ikk] signalsome WO1998008955A1 (en)

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JP51184098A JP4695735B2 (en) 1996-08-26 1997-08-26 Stimulation-inducible I (κ) B kinase [IKK] signalsome
EP97938616A EP0920518A1 (en) 1996-08-26 1997-08-26 Stimulus-inducible i (kappa)b kinase ikk] signalsome
CA2263835A CA2263835C (en) 1996-08-26 1997-08-26 Stimulus-inducible i (kappa)b kinase [ikk] signalsome
AU40904/97A AU726383B2 (en) 1996-08-26 1997-08-26 Stimulus-inducible I (KAPPA)B kinase (IKK) signalsome

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US08/697,393 US5972674A (en) 1996-08-26 1996-08-26 Stimulus-inducible protein kinase complex and methods of use therefor
US08/697,393 1996-08-26
US08/910,820 US6258579B1 (en) 1996-08-26 1997-08-13 Stimulus-inducible protein kinase complex and methods of use therefor
US08/910,820 1997-08-13

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US20090169564A1 (en) 2009-07-02
JP2001502892A (en) 2001-03-06
EP0920518A1 (en) 1999-06-09
US6576437B2 (en) 2003-06-10
US20030100026A1 (en) 2003-05-29
AU726383B2 (en) 2000-11-02
CA2263835C (en) 2012-02-21
AU4090497A (en) 1998-03-19
JP4695735B2 (en) 2011-06-08
US20020151021A1 (en) 2002-10-17
US7285654B2 (en) 2007-10-23
CA2263835A1 (en) 1998-03-05

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