WO1997000690A1 - Proteine kinase associee au recepteur de l'interleukine-1 et dosages - Google Patents

Proteine kinase associee au recepteur de l'interleukine-1 et dosages Download PDF

Info

Publication number
WO1997000690A1
WO1997000690A1 PCT/US1996/009193 US9609193W WO9700690A1 WO 1997000690 A1 WO1997000690 A1 WO 1997000690A1 US 9609193 W US9609193 W US 9609193W WO 9700690 A1 WO9700690 A1 WO 9700690A1
Authority
WO
WIPO (PCT)
Prior art keywords
irak
sequence
pharmacological agent
receptor
ala
Prior art date
Application number
PCT/US1996/009193
Other languages
English (en)
Inventor
Zhaodan Cao
David V. Goeddel
Glenn E. Croston
Original Assignee
Tularik, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Tularik, Inc. filed Critical Tularik, Inc.
Priority to EP96919140A priority Critical patent/EP0839045A4/fr
Priority to JP9503857A priority patent/JPH11509085A/ja
Priority to AU61766/96A priority patent/AU702844B2/en
Priority to CA002225450A priority patent/CA2225450C/fr
Publication of WO1997000690A1 publication Critical patent/WO1997000690A1/fr

Links

Classifications

    • 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
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
    • C07K14/7155—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons for interleukins [IL]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • 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)
    • 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 field of this invention is a human interieukin receptor associated kinase and its use in drug screening.
  • IL-1 The cytokine interieukin-1 (IL-1) is a key mediator in the inflammatory response (for reviews, see Refs. 1-3) .
  • the importance of IL-1 in inflammation has been demonstrated by the ability of the highly specific IL-1 receptor antagonist protein to relieve inflammatory conditions (for review, see Refs. 1, 4) .
  • Many of the proinflammatory effects of IL-1 such as the upregulation of cell adhesion molecules on vascular endothelia, are exerted at the level of transcriptional regulation.
  • the transcriptional activation by IL-1 of cell adhesion molecules and other genes involved in the inflammatory response appears to be mediated largely by NF-KB (5-8) .
  • NF-cB inhibitory factor IKB In response to IL-1, the NF-cB inhibitory factor IKB is degraded and NF- ⁇ B is released from its inactive cytoplasmic state to localize within the nucleus where it binds DNA and activates transcription (9,10) . Elucidation of the IL-1 signal transduction pathway leading to NF-cB activation would provide valuable insight into mechanisms to alleviate inflammation. In particular, components of this pathway would provide valuable targets for automated, cost-effective, high throughput drug screening and hence would have immediate application in a broad range of domestic and international pharmaceutical and biotechnology drug development programs.
  • IL-1RI type I
  • IL-IRII type II
  • Both receptors have a single transmembrane domain, and an IgG-like extracellular domain.
  • the IL-IRII is found predominantly in B-cells, contains a cytoplasmic domain of only 29 amino acids, and may not play a direct role in intracellular signal transduction (for review, see Ref. 13) .
  • the human IL-1RI is found on most cell types and contains 552 amino acids in its mature form.
  • cytoplasmic domain of 212 amino acids is required for signaling activity (14-17) , but has no significant homology with protein kinases or any other mammalian factors involved in signal transduction.
  • the cytoplasmic domain of IL-1RI does share significant sequence homology with the Drosophila transmembrane protein Toll that is involved in dorsal- ventral patterning (18) . This homology may be functionally significant since other components of the Drosophila dorsal- ventral patterning pathway, Dorsal and Cactus, are homologous with NF- ⁇ B and I ⁇ B, respectively (19) .
  • mutation of the amino acids that are conserved between IL- 1RI and Toll inactivates IL-1RI signaling in T cells (15) .
  • the invention provides methods and compositions relating to a class of Interleukin-1 Receptor type I- Associated Protein Kinases (IRAK) .
  • Native full-length human IRAKs migrate in SDS polyacrylamide gel electrophoresis at an apparent molecular weight of approximately 100 kD.
  • the compositions include nucleic acids which encode IRAKs and hybridization probes and primers capable of hybridizing with the IRAK genes.
  • the invention includes methods for screening chemical libraries for lead compounds for a pharmacological agent useful in the diagnosis or treatment of disease associated an IRAK activity or an IRAK-dependent signal transduction.
  • the methods involve (1) forming a mixture comprising an IRAK, a natural intracellular IRAK substrate or binding target such as the Interieukin-1 receptor, and a candidate pharmacological agent; (2) incubating the mixture under conditions whereby, but for the presence of said candidate pharmacological agent, said IRAK selectively phosphorylates said substrate or binds said binding target; and (3) detecting the presence or absence of specific phosphorylation of said substrate by said IRAK or phosphorylation or binding of said IRAK to said binding target, wherein the absence of said selective binding indicates that said candidate pharmacological agent is a lead compound for a pharmacological agent capable of disrupting IRAK function.
  • the nucleotide sequence of a natural cDNA encoding human IRAK-1 is shown as SEQUENCE ID N0:1 and the full conceptual translate is shown as SEQUENCE ID NO:2.
  • the IRAKs of the invention include natural derivatives of the IRAK gene and gene product.
  • IRAK-2 is encoded by a derivative of the IRAK-l cDNA where the coding region encompassing nucleotides 1514-1552 is deleted.
  • IRAK-3 is a derivative of IRAK-1 where the coding region encompassing nucleotides 1514-1558 is deleted.
  • the disclosed IRAKs include incomplete translates and deletion mutants of these cDNA sequences and deletion mutants, which translates or deletion mutants have IRAK- specific function such as the kinase activity described herein or IRAK self-association function.
  • the domain bound by residues 212 (Phe) through 523 (Ala) of SEQUENCE ID NO:2 defines an active kinase domain which may be used, independently or joined to other domains, in the subject methods.
  • the domain defined by the N- terminal 120 residues of SEQUENCE ID NO:2 defines an IRAK self-association domain. This domain finds use in methods involving higher order IRAK complexes which provide an important means of IRAK regulation. Hence, this domain may be used independently as a regulator or IRAK activity, as a reagent in an IRAK complex formation assay, etc.
  • the claimed IRAK proteins are isolated, partially pure or pure and are typically recombinantly produced.
  • An "isolated" protein for example, is unaccompanied by at least some of the material with which it is associated in its natural state and constitutes at least about 0.5%, preferably at least about 2%, and more preferably at least about 5% by weight of the total protein in a given sample; a partially pure protein constitutes at least about 10%, preferably at least about 30%, and more preferably at least about 60% by weight of the total protein in a given sample; and a pure protein constitutes at least about 70%, preferably at least about 90%, and more preferably at least about 95% by weight of the total protein in a given sample.
  • the invention provides IRAK-specific binding agents including substrates, natural intracellular binding targets, etc. and methods of identifying and making such agents, and their use in diagnosis, therapy and pharmaceutical development.
  • IRAK-specific agents are useful in a variety of diagnostic and therapeutic applications, especially where disease or disease prognosis is associated with improper utilization of a pathway involving an IRAK, e.g. IL-1 receptor activation.
  • Novel IRAK-specific binding agents include IRAK-specific antibodies and other natural intracellular binding agents identified with assays such as one- and two-hybrid screens, non-natural intracellular binding agents identified in screens of chemical libraries, etc.
  • Agents of particular interest modulate IRAK function, e.g. IRAK antagonists.
  • IRAK-specificity of the binding agent is shown by kinase activity (i.e. the agent demonstrates activity of an IRAK substrate, agonist, antagonist, etc.) or binding equilibrium constants (usually at least about IO 7 M " 1 , preferably at least about IO 8 M "1 , more preferably at least about IO 9 M _1) .
  • kinase activity i.e. the agent demonstrates activity of an IRAK substrate, agonist, antagonist, etc.
  • binding equilibrium constants usually at least about IO 7 M " 1 , preferably at least about IO 8 M "1 , more preferably at least about IO 9 M _1 .
  • a wide variety of cell-based and cell- free assays may be used to demonstrate IRAK-specific binding; preferred are rapid in vitro, cell-free assays such as mediating or inhibiting IRAK-protein (e.g. IRAK-IL-1 RI) binding, phosphorylation assays, immunoassays, etc.
  • the invention also provides nucleic acids encoding the subject proteins, which nucleic acids may be part of IRAK- expression vectors and may be incorporated into recombinant cells for expression and screening, transgenic animals for functional studies (e.g. the efficacy of candidate drugs for disease associated with expression of an IRAK) , etc., and nucleic acid hybridization probes and replication/amplification primers having an IRAK cDNA specific sequence contained in SEQUENCE ID NO:l.
  • Nucleic acids encoding IRAKs are isolated from eukaryotic cells, preferably human cells, by screening cDNA libraries with probes or PCR primers derived from the disclosed IRAK cDNAs.
  • the invention provides IRAK gene homologs sharing sufficient sequence similarity with that of the disclosed IRAK cDNAs to effect hybridization.
  • Such IRAK cDNA homologs are capable of hybridizing to the IRAK- encoding nucleic acid defined by SEQUENCE ID NO: 1 under low stringency conditions, e.g.
  • a hybridization buffer comprising 0% formamide in 0.9 M saline/0.09 M sodium citrate (SSC) buffer at a temperature of 37°C and remaining bound when subject to washing at 42°C with the SSC buffer at 37°C; or 30% formamide in 5 x SSPE (0.18 M NaCl, 0.01 M NaP0 4 , pH7.7, 0.001 M EDTA) buffer at a temperature of 42°C and remaining bound when subject to washing at 42°C with the 0.2 x SSPE.
  • SSC sodium citrate
  • hybridization buffer comprising 20% formamide in 0.9 M saline/0.09 M sodium citrate (SSC) buffer at a temperature of 42°C and remaining bound when subject to washing at 42°C with 2 X SSC buffer at 42°C; or a hybridization buffer comprising 50% formamide in 5 x SSPE buffer at a temperature of 42°C and remain bound when subject to washing at 42°C with 0.2 x SSPE buffer at 42°C.
  • More preferred nucleic acids encode kinases comprising kinase domains with at least about 25%, preferably at least about 50% pair-wise identity to a disclosed IRAK kinase domain.
  • the subject nucleic acids are recombinant, meaning they comprise a sequence joined to a nucleotide other than that which it is joined to on a natural chromosome and are often isolated, i.e. constitute at least about 0.5%, preferably at least about 5% by weight of total nucleic acid present in a given fraction.
  • the recombinant nucleic acids may be contained within vectors, cells or organisms.
  • the subject nucleic acids find a wide variety of applications including use as translatable transcripts, hybridization probes, PCR primers, therapeutic nucleic acids, etc.; use in detecting the presence of IRAK genes and gene transcripts, in detecting or amplifying nucleic acids encoding additional IRAK homologs and structural analogs, and in gene therapy applications.
  • the invention provides efficient methods of identifying pharmacological agents or lead compounds for agents active at the level of an IRAK modulatable cellular function, particularly IRAK mediated IL-1 signal transduction, especially in inflammation.
  • these screening methods involve assaying for compounds which interfere with an IRAK activity such as kinase activity or IL-1 receptor I binding.
  • the methods are amenable to automated, cost- effective high throughput screening of chemical libraries for lead compounds.
  • Identified reagents find use in the pharmaceutical industries for animal and human trials; for example, the reagents may be derivatized and rescreened in in vitro and in vivo assays to optimize activity and minimize toxicity for pharmaceutical development.
  • Target therapeutic indications are limited only in that the target cellular function be subject to modulation, usually inhibition, by disruption of the formation of a complex comprising an IRAK and one or more natural IRAK intracellular binding targets including substrates.
  • Target indications may include infection, genetic disease, cell growth and regulatory disfunction, such as neoplasia, inflammation, hypersensitivity, etc.
  • assays for binding agents are provided including labeled in vitro kinase assays, protein- protein binding assays, immunoassays, cell based assays, etc.
  • the IRAK compositions used the methods are usually added in an isolated, partially pure or pure form and are typically recombinantly produced.
  • the IRAK may be part of a fusion product with another peptide or polypeptide, e.g. a polypeptide that is capable of providing or enhancing protein-protein binding, stability under assay conditions
  • the assay mixtures comprise a natural intracellular IRAK binding target including substrates, such as the C-terminus IL-1 RI or, in the case of an autophosphorylation assay, the IRAK itself can function as the binding target.
  • An IRAK derived pseudosubstrate may be used or modified (e.g. A to S/T substitutions) to generate effective substrates for use in the subject kinase assays.
  • the use of serine/threonine kinase pseudosubstrate peptides and the generation of substrate peptides therefrom are well known in the art . While native binding targets may be used, it is frequently preferred to use portions (e.g.
  • the assay mixture also comprises a candidate pharmacological agent.
  • Candidate agents encompass numerous chemical classes, though typically they are organic compounds; preferably small organic compounds and are obtained from a wide variety of sources including libraries of synthetic or natural compounds.
  • a variety of other reagents may also be included in the mixture. These include reagents like salts, buffers, neutral proteins, e.g. albumin, detergents, etc. which may be used to facilitate optimal binding and/or reduce non-specific or background interactions, etc.
  • reagents that otherwise improve the efficiency of the assay such as protease inhibitors, nuclease inhibitors, antimicrobial agents, etc. may be used.
  • the resultant mixture is incubated under conditions whereby, but for the presence of the candidate pharmacological agent, the IRAK specifically binds the cellular binding target, portion or analog.
  • the mixture components can be added in any order that provides for the requisite bindings. Incubations may be performed at any temperature which facilitates optimal binding, typically between 4 and 40°C, more commonly between 15° and 40°C. Incubation periods are likewise selected for optimal binding but also minimized to facilitate rapid, high-throughput screening, and are typically between .1 and 10 hours, preferably less than 5 hours, more preferably less than 2 hours.
  • a separation step is often used to separate bound from unbound components. Separation may be effected by precipitation (e.g. TCA precipitation, immunoprecipitation, etc.), immobilization (e.g on a solid substrate) , etc., followed by washing by, for examples, membrane filtration (e.g. Whatman's P-81 ion exchange paper, Polyfiltronic' s hydrophobic GFC membrane, etc.), gel chromatography (e.g. gel filtration, affinity, etc.) .
  • precipitation e.g. TCA precipitation, immunoprecipitation, etc.
  • immobilization e.g on a solid substrate
  • washing for examples, membrane filtration (e.g. Whatman's P-81 ion exchange paper, Polyfiltronic' s hydrophobic GFC membrane, etc.), gel chromatography (e.g. gel filtration, affinity, etc.) .
  • binding is detected by a change in the kinase activity of the IRAK. Detection may be effected in any convenient way.
  • one of the components usually comprises or is coupled to a label.
  • labels may be employed - essentially any label that provides for detection of bound protein.
  • the label may provide for direct detection as radioactivity, luminescence, optical or electron density, etc. or indirect detection such as an epitope tag, an enzyme, etc.
  • a variety of methods may be used to detect the label depending on the nature of the label and other assay components. For example, the label may be detected bound to the solid substrate or a portion of the bound complex containing the label may be separated from the solid substrate, and thereafter the label detected.
  • Labels may be directly detected through optical or electron density, radiative emissions, nonradiative energy transfers, etc. or indirectly detected with antibody conjugates, etc.
  • emissions may be detected directly, e.g. with particle counters or indirectly, e.g. with scintillation cocktails and counters.
  • IL-1RI intracellular region of IL-1RI interacts with other factors to transduce IL-l signals.
  • To measure NF- ⁇ B activation we utilized an assay in which expression vectors for IL-1RI mutants were cotransfected with an E-selectin promoter- luciferase reporter plasmid into the human 293 cell line. Stimulation of E-selectin transcription by IL-1 is known to occur primarily through the activation of NF-KB (24, 25) .
  • Luciferase activity in transiently transfected 293 cells was determined in the presence or absence of IL-1 stimulation.
  • IL-1 (1 ng/ml) induced a low level of transcriptional activation through endogenous IL-1RI.
  • IL-1 dependent transcriptional activation was observed in cells transiently transfected with wild type IL-1RI. This result demonstrates that the majority of reporter activity in transiently transfected cells is signaled by transfected IL- 1RI, and validates the use of this system for the analysis of IL-1RI mutants.
  • IL-1RI Five different C-terminal truncation mutants of IL-1RI were examined for their ability to activate the E-selectin reporter in response to IL-1. Removal of 20, 25 or 31 amino acids from the C-terminus did not appreciably affect the ability of IL-1RI to activate NF- ⁇ B. Deletion of 45 or 75 C-terminal amino acids eliminated the ability of IL-1RI to activate NF-KB. Therefore, the region defined by the -31 and -45 deletions (residues 508-521) includes sequences required for the activation of NF-KB by IL-1. Furthermore, the -45 and -75 deletion mutants behaved as dominant negative mutations and blocked the ability of the endogenous IL-1RI to activate NF- ⁇ B.
  • amino acids 508 to 521 of IL-1RI appear necessary for signal transduction, this region was examined more closely by constructing receptors with sets of three amino acids mutated to alanine. These mutants, which include 510- 512A, 513-515A, and 518-520A, were analyzed in the NF-KB reporter assay for their ability to activate NF-/cB. By this analysis the 510-512A mutant is active, while the 513-515A and 518-520A mutants are inactive. Amino acids 510, 511, and 512 of the IL-1RI are not conserved in Toll, while conserved amino acids are present in both the 513-515 and 518-520 regions. The requirement of these conserved residues for IL-1RI function may indicate that these amino acids directly contact signaling molecules or are critical to overall receptor structure.
  • IRAK I.L-1RI Associated-Kinase
  • the major target of the IRAK in this reaction is an endogenous substrate of approximately 100 kDa.
  • the specificity of the receptor-kinase interaction is supported by the absence of activity in the preimmune precipitate, and by the ability of an IL-IRI-IgG fusion protein to compete away the kinase activity when added to the immunoprecipitation.
  • Kinase activation occurred rapidly, reaching an optimum within two minutes of exposure of cells to IL-1, suggesting that activation of the kinase occurs proximally to the IL-1 receptor.
  • IRAK is involved in NF- ⁇ B activation
  • the activity of the kinase in immunoprecipitates of mutated receptor should correlate with in vivo activation of the E- selectin reporter by mutated receptors.
  • the C-terminal deletions mutants of IL-1RI were transiently expressed in 293 cells, receptor was immunoprecipitated, and examined for associated IL-1 inducible kinase activity. In the absence of transfected receptor, 293 cells display low but detectable levels of IRAK activity. All three C-terminal deletion mutants (-20, -25, -31) that can activate NF-zcB display associated kinase activity that is indistinguishable from that associated with intact IL-IRI.
  • IRAK activity does not coprecipitate with the -45 deletion mutant that was unable to activate NF- ⁇ B.
  • IRAK activity does not coprecipitate with the -45 deletion mutant that was unable to activate NF- ⁇ B.
  • the triple alanine scan mutants of IL-IRI were examined by the coimmunoprecipitation assay following transfection into 293 cells. IRAK activity was observed with the 510-512A mutant, but not with the 513- 515 Ala or 518-520 Ala mutants. Once again there was a direct correlation between the ability of an IL-IRI mutant to interact with IRAK and to induce NF- ⁇ B activation.
  • pplOO In order to purify pplOO, we stably transfected 293 cells with IL-IRI expression plasmid.
  • the 293/IL-1RI cells express IL-IRI at a level at least two orders of magnitude greater than that of parental 293 cells as shown by FACS analyses.
  • the cells were grown in suspension and treated briefly with IL-1 before harvest and extract preparation.
  • pplOO was purified from extracts prepared from 100 liters of cells by a large scale immunoprecipitation using rabbit antibodies to the extracellular domain of IL-IRI. To follow pplOO, immunoprecipitants were subjected to an in vitro kinase reaction in the presence of ⁇ 32 P-ATP.
  • pplOO eluted from the IL-IRI immunocomplex was further purified by Q sepharose column chromatography. Protein fractions containing radiolabeled pplOO were subjected to two-dimensional gel electrophoresis and blotted to polyvinylidene difluoride (PVDF) membrane. pplOO (about 0.4 ⁇ g) was identified by autoradiography and digested with lysine-C and trypsin. The resulting peptides were fractionated by capillary high-performance liquid chromatography. Amino acid sequences of 10 polypeptides were obtained, which were used to design degenerate oligonucleotides as primers for polymerase chain reaction (PCR) .
  • PCR polymerase chain reaction
  • a DNA fragment of 356 nucleotides was amplified from cDNA prepared using mRNA from 293 cells. This DNA fragment encodes the peptide used to design the PCR primers as well as three other sequenced peptides. Using this DNA fragment as a probe, we isolated corresponding cDNA clones from a human teratocarcinoma cDNA library. The longest clone obtained is 3.5 kilobase pair in length (SEQUENCE ID NO:l) and encodes a protein of 699 amino acids (SEQUENCE ID NO:2) . An in-frame stop codon was located 36 nucleotides upstream from the first methionine, indicating that the clone encodes a full length protein.
  • Plasmid Construction and Antiserum Preparation The human IL-IRI cDNA was cloned into pRK5 (20) to give the plasmid pRK-IL-IRI in which expression is under the control of the cytomegalovirus immediate early promoter-enhancer.
  • Expression plasmids for the C-terminal deletion mutants of IL-1 receptor were generated from pRK-IL-IRI by introducing stop codons into the IL-IRI coding region by polymerase chain reaction (PCR) .
  • the internal triple mutants were made by a procedure involving two rounds of PCR. The first round of PCR generated overlapping fragments with the corresponding mutations in the center of the overlapped region.
  • IL-IRI a fusion protein consisting of the mature IL-IRI extracellular domain fused to human IgG as described (22), was expressed transiently in 293 cells.
  • Cell culture medium containing the chimeric protein was harvested on 3 and 7 days after transfection.
  • the IL-1RI- IgG fusion protein was purified by protein A-agarose chromatography and used to immunize rabbits by BAbCo (Richmond, CA) .
  • Extracts for immunoprecipitations and in vi tro phosphorylation assays were prepared as follows: 293 cells were seeded at 50% density in 100 mm plates and transfected with IL-IRI expression plasmids on the following day. 40 to 48 hours later, IL-1 (20 ng/ml) was added to the media. After incubation at 37°C for the indicated times, media was removed and the plates were chilled on ice immediately.
  • the cells were washed twice with 5 ml of ice-cold phosphate buffered saline (PBS) and scraped off the plates in 5 ml of PBS containing 1 mM EDTA. Cells were pelleted by 1200 x g centrifugation for 3 minutes and suspended in 1 ml of lysis buffer (50 mM HEPES pH 7.6, 250 mM NaCl, 1 mM dithiothreitol
  • PBS ice-cold phosphate buffered saline
  • the reactions were mixed with 20 ul of the protein A-agarose slurry and incubated for an additional 1 hour.
  • Protein A beads were collected by centrifugation in a microcentrifuge for 10 seconds, and washed 5 times with 1 ml of lysis buffer.
  • the beads were then suspended in 20 ul of kinase buffer containing 20 mM Tris-HCl pH 7.6, 20 mM MgCl 2 20 mM ⁇ -glycerophosphate, 20 mM p-nitrophenyl phosphate, 1 mM Na orthovanadate, ImM benzamidine, 0.4 mM PMSF, 1 M Na metabisulfite, 2 uM cold ATP and 10 uCi [ 32 P] ⁇ -ATP.
  • the kinase reactions were allowed to proceed at 30°C for 30 minutes and terminated with 20 ml of SDS sample buffer. After boiling for 3-5 minutes, 20 ml reaction aliquots were separated by 8% SDS-PAGE. Radiolabeled proteins were visualized by autoradiography.
  • 293 cells were cultured in Dulbeco's Modification of Eagle's Medium with 4.5 gram/ml glucose and L-glutamine (Mediatech) supplemented with 10% fetal bovine serum, 100 ug/ml streptomycin and 100 ug/ml penicillin.
  • 293 cells were seeded on 100 mm plates at 30% density and were transfected on the following day with 10 mg IL-IRI expression plasmid (supra) and 1 mg pNeo by calcium phosphate precipitation.
  • Stably transfected cells were selected with culture medium containing 500 ⁇ g/ml of G418 (Gibco) .
  • Extract Preparation Cells from suspension culture (100 liters) were pelleted in a Sorvall GS-3 rotor at 2500 RPM for 5 minutes and re-suspended in 5 liters of pre-warmed serum-free MEM medium. The cells were incubated with 200 ng/ml recombinant human IL-13 (Genentech) at 37°C for 3 minutes and pelleted by centrifugation at 4°C. All of the following steps were performed at 4°C.
  • the cells were suspended in 5 pelleted-cell-volumes of buffer containing 50 mM Hepes pH 7.9, 250 mM NaCl, 5 mM dithiothreitol (DTT), 1 mM EDTA, 0.1% NP-40, 10% (v/v)glycerol, 20 mM b glycerophosphate, 5 mM p-nitrophenyl phosphate, 1 mM Na orthovanata e, 1 mM benzamidine, 0.4 mM phenylmethylsulfonyl fluoride (PMSF) , 1 mM Na metabisulfite, 10 ug/ml leupeptin and 10 ug/ml aprotinin.
  • buffer containing 50 mM Hepes pH 7.9, 250 mM NaCl, 5 mM dithiothreitol (DTT), 1 mM EDTA, 0.1% NP-40, 10% (v/v)glycerol
  • the cell lysate was centrifuged in a Sorvall H6000A rotor at 4000 RMP for 10 minutes. The supernatants were collected and centrifuged in a Beckman 45 TI rotor at 40,000 RPM for 2 hours. The supernatants were aliquoted and stored at -70°C.
  • ppl OO Purification of ppl OO : the extracts were thawed and spun in a Beckman 45 TI at 40,000 RPM for 2 hours. The supernatants were incubated with 40 mg of rabbit IgG against the extracellular domain of the IL-1R at 4°C for 2 hours with rocking. 25 ml of protein A sepharose CL4B (Pharmacia) were mixed with the extracts and the incubation continued for another 2 hours .
  • the protein A beads were collected in a column and washed with 250 ml of washing buffer #1 containing 50 mM Hepes pH 7.9, 250 mM NaCl, 5 mM dithiothreitol (DTT), 1 mM EDTA, 0.1% NP-40, 20 M ⁇ glycerophosphate, 1 mM Na orthovanatate, 1 mM benzamidine, 0.4 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM Na metabisulfite.
  • the beads were then suspended in 50 ml kinase buffer containing 20 mM Tris-HCl pH 7.6, 20 mM MgCl 2 , 20 mM ⁇ glycerophosphate, 20 mM p-nitrophenylphosphate, 1 mM EDTA, 1 mM Na orthovanadate, 1 mM benzamidine, 0.4 mM PMSF, 1 mM Na metabisulfite, 5 mM cold ATP and 100 mCi [ ⁇ 2 P]g -ATP and incubated at 30°C for 15 minutes. The kinase reaction was chased with 100 mM of unlabeled ATP for an additional 15 minutes.
  • Protein A beads were collected in an empty column and washed with 150 ml of washing buffer #2 containing 150 ml of buffer consisted of 50 mM Hepes, pH 7.9, 1 M NaCl, 5 mM DTT, 1 mM EDTA and 0.1% NP40, then 150 ml of washing buffer #3 consisting of 50 mM Hepes, pH 7.9, 100 mM NaCl, 2 M urea, 5 mM DTT, 1 mM EDTA and 0.1% NP40.
  • the proteins were then eluted with 50 ml of elution buffer containing 50mM Hepes, pH 7.9, 100 mM NaCl, 5 mM DTT, 1 mM EDTA, 0.1% NP-40 and 7 M urea at 4°C overnight with rocking.
  • the eluted materials were loaded on a 0.5 ml Q Sepharose column equilibrated in the elution buffer. The column was washed extensively with the elution buffer before bound proteins were eluted with buffer containing 0.5 M NaCl.
  • the high salt eluate was concentrated in a Centricon 50 (Microcon) to 50 ⁇ l , diluted with 1 ml isoelectric focusing sample buffer (O'Farrell (1975) J. Biol Chem), concentrated down again to 50 ⁇ l .
  • the sample was then subjected to two-dimensional gel electrophoresis.
  • Two-dimensional gel electrophoresis and micro peptide sequencing Isoelectric focusing was used as the first dimensional separation. The preparation and running conditions were described previously. The pH gradient was created with ampholines pH 5.0-7.0 and pH 3.5-9.5 blended at a radio of 1 :1. 7% acrylamide SDS gel electrophoresis was used as second dimension separation. After the electrophoresis, the proteins were transferred to a polyvinylidenedifluoride membrane (Milipore) and stained with Coomassie blue R-250 in 40% methanoland 10% acetic acid for 30 seconds, followed by a 5 minute de-staining in 40% methanol and 10% acetic acid.
  • - Neutralite Avidin 20 ⁇ g/ml in PBS.
  • - IRAK IO "8 - IO "5 M biotinylated IRAK-1 at 20 ⁇ g/ml in PBS.
  • Blocking buffer 5% BSA, 0.5% Tween 20 in PBS; 1 hour at room temperature.
  • - Protease inhibitor cocktail 1000X: 10 mg Trypsin Inhibitor (BMB # 109894) , 10 mg Aprotinin (BMB # 236624) , 25 mg Benzamidine (Sigma # B-6506) , 25 mg Leupeptin (BMB # 1017128), 10 mg APMSF (BMB # 917575), and 2mM NaVo 3 (Sigma # S-6508) in 10 ml of PBS.
  • Blocking buffer 5% BSA, 0.5% Tween 20 in PBS; 1 hour at room temperatur .
  • - Protease inhibitor cocktail 1000X: 10 mg Trypsin Inhibitor (BMB # 109894) , 10 mg Aprotinin (BMB # 236624) , 25 mg Benzamidine (Sigma # B-6506) , 25 mg Leupeptin (BMB # 1017128) , 10 mg APMSF (BMB # 917575) , and 2mM NaVo 3 (Sigma # S-6508) in 10 ml of PBS.
  • IL-IRI IO "8 - IO "5 M biotinylated IL-IRI intracellular domain (residues 327-527) in PBS.
  • ADDRESSEE FLEHR, HOHBACH, TEST, ALBRITTON & HERBERT
  • GTTGCCATCC TCAGCCTCCA CCTTCCTCTC CCCAGCTTTT CCAGGCTCCC AGACCCATTC 540
  • CACACTGCAA GCAGGTCTGG CTGCAGATGC CTGGGCTGCT CCCATCGCCA TGCAGATCTA 1500
  • CAAGAAGCAC CTGGACCCCA GGCCCGGGCC CTGCCCACCT GAGCTGGGCC TGGGCCTGGG 1560

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Medicinal Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Biochemistry (AREA)
  • Immunology (AREA)
  • Wood Science & Technology (AREA)
  • Molecular Biology (AREA)
  • Public Health (AREA)
  • Biomedical Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Cell Biology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Biophysics (AREA)
  • Veterinary Medicine (AREA)
  • Toxicology (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • General Engineering & Computer Science (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Peptides Or Proteins (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

Cette invention concerne des protéines kinases humaines associées au récepteur de l'Interleukine-1 (IRAK), les acides nucléiques qui codent les IRAK et les sondes et les armorces d'hybridation moléculaire capables de s'hybrider avec les gènes IRAK et les procédés d'utilisation des compositions en question; en particulier des procédés tels que des dosages de liaison in vitro à base d'IRAK et des dosages de phosphorylation destinés au criblage de banques chimiques pour détecter des composés principaux pour des agents pharmaceutiques.
PCT/US1996/009193 1995-06-23 1996-06-05 Proteine kinase associee au recepteur de l'interleukine-1 et dosages WO1997000690A1 (fr)

Priority Applications (4)

Application Number Priority Date Filing Date Title
EP96919140A EP0839045A4 (fr) 1995-06-23 1996-06-05 Proteine kinase associee au recepteur de l'interleukine-1 et dosages
JP9503857A JPH11509085A (ja) 1995-06-23 1996-06-05 インターロイキン−１レセプター関連プロテインキナーゼ及び検定
AU61766/96A AU702844B2 (en) 1995-06-23 1996-06-05 Interleukin-1 receptor-associated protein kinase and assays
CA002225450A CA2225450C (fr) 1995-06-23 1996-06-05 Proteine kinase associee au recepteur de l'interleukine-1 et dosages

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US49400695A 1995-06-23 1995-06-23
US08/494,006 1995-06-23

Publications (1)

Publication Number Publication Date
WO1997000690A1 true WO1997000690A1 (fr) 1997-01-09

Family

ID=23962617

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1996/009193 WO1997000690A1 (fr) 1995-06-23 1996-06-05 Proteine kinase associee au recepteur de l'interleukine-1 et dosages

Country Status (5)

Country Link
EP (1) EP0839045A4 (fr)
JP (1) JPH11509085A (fr)
AU (1) AU702844B2 (fr)
CA (1) CA2225450C (fr)
WO (1) WO1997000690A1 (fr)

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999027112A1 (fr) * 1997-11-26 1999-06-03 Human Genome Sciences, Inc. Irak-2 humaine, kinase-2 humaine associee au recepteur de l'interleukine-1
WO2000075310A1 (fr) * 1999-06-08 2000-12-14 Glaxo Group Limited Kinases associees au recepteur de l'interleukine-1
US6262228B1 (en) * 1998-08-17 2001-07-17 Tularik Inc. IRAK3 polypeptides and methods
JP2001512319A (ja) * 1997-02-18 2001-08-21 シグナル ファーマシューティカルズ，インコーポレイテッド 細胞のＮＦ−κＢ活性化を調節するための組成物および方法
WO2002040680A2 (fr) * 2000-11-17 2002-05-23 The Burnham Institute Nouvelles proteines du domaine de mort cellulaire
WO2005051314A2 (fr) * 2003-11-24 2005-06-09 Exelixis, Inc. Genes irak utilises en tant que modificateurs de morphogenese de ramification et procedes d'utilisation associes
US6965023B2 (en) 2000-11-17 2005-11-15 The Burnham Institute Death domain proteins

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
EUROPEAN JOURNAL OF IMMUNOLOGY, July 1994, Vol. 24, MARTIN et al., "Interleukin-1-Induced Activation of a Protein Kinase Co-Precipitating with the Type I Interleukin-1 Receptor in T Cells", pages 1566-1571. *
SCIENCE, 23 February 1996, Vol. 271, CAO et al., IRAK: "A Kinase Associated With the Interleukin-1 Receptor", pages 1128-1131. *
See also references of EP0839045A4 *
THE JOURNAL OF BIOLOGICAL CHEMISTRY, 14 July 1995, Vol. 270, No. 28, CROSTON et al., "NF-kB Activation by Interleukin-1 (IL-1) Requires an IL-1 Receptor-Associated Protein Kinase Activity", pages 16514-16517. *
THE JOURNAL OF BIOLOGICAL CHEMISTRY, 28 January 1994, Vol. 269, No. 4, LIU et al., "Renaturation and Tumor Necrosis Factor-alpha Stimulation of a 97kDa Ceramide-Activated Protein Kinase", pages 3047-3052. *
THE NEW ENGLAND BIOLABS CATALOG, 1993/1994 Edition, page 97. *

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2001512319A (ja) * 1997-02-18 2001-08-21 シグナル ファーマシューティカルズ，インコーポレイテッド 細胞のＮＦ−κＢ活性化を調節するための組成物および方法
JP2010063461A (ja) * 1997-02-18 2010-03-25 Celgene Corp 細胞のＮＦ−κＢ活性化を調節するための組成物および方法
WO1999027112A1 (fr) * 1997-11-26 1999-06-03 Human Genome Sciences, Inc. Irak-2 humaine, kinase-2 humaine associee au recepteur de l'interleukine-1
US6222019B1 (en) 1997-11-26 2001-04-24 Human Genome Sciences, Inc. Human IRAK-2 antibodies
US6653452B2 (en) 1997-11-26 2003-11-25 Human Genome Sciences, Inc. Human IRAK-2
US6262228B1 (en) * 1998-08-17 2001-07-17 Tularik Inc. IRAK3 polypeptides and methods
WO2000075310A1 (fr) * 1999-06-08 2000-12-14 Glaxo Group Limited Kinases associees au recepteur de l'interleukine-1
WO2002040680A2 (fr) * 2000-11-17 2002-05-23 The Burnham Institute Nouvelles proteines du domaine de mort cellulaire
WO2002040680A3 (fr) * 2000-11-17 2003-03-13 Burnham Inst Nouvelles proteines du domaine de mort cellulaire
US6965023B2 (en) 2000-11-17 2005-11-15 The Burnham Institute Death domain proteins
WO2005051314A2 (fr) * 2003-11-24 2005-06-09 Exelixis, Inc. Genes irak utilises en tant que modificateurs de morphogenese de ramification et procedes d'utilisation associes
WO2005051314A3 (fr) * 2003-11-24 2007-04-26 Exelixis Inc Genes irak utilises en tant que modificateurs de morphogenese de ramification et procedes d'utilisation associes

Also Published As

Publication number Publication date
JPH11509085A (ja) 1999-08-17
EP0839045A1 (fr) 1998-05-06
CA2225450C (fr) 2001-05-29
CA2225450A1 (fr) 1997-01-09
AU6176696A (en) 1997-01-22
AU702844B2 (en) 1999-03-04
EP0839045A4 (fr) 1999-06-09

Similar Documents

Publication Publication Date Title
US5563039A (en) TNF receptor-associated intracellular signaling proteins and methods of use
JP3472584B2 (ja) ＩＫＫ−βタンパク質、核酸及び方法
AU724178B2 (en) NIK proteins, nucleic acids and methods
KR100536394B1 (ko) Fas/ap01수용체들의기능조절인자
WO1998055621A1 (fr) Membre ntn-2 de la famille des ligands du facteur de necrose tumorale (tnf)
WO1998039410A1 (fr) IλB $i(KINASES)
JP4435574B2 (ja) ＩＬ−２レセプターγ鎖の誘導体、その製法および使用
US6030788A (en) Cyclin-dependent protein kinase
US6280955B1 (en) Interleukin-1 receptor accessory proteins, nucleic acids and methods
US6222019B1 (en) Human IRAK-2 antibodies
AU702844B2 (en) Interleukin-1 receptor-associated protein kinase and assays
US5654397A (en) Interleukin-1 receptor-associated protein kinase and assays
US5871960A (en) Nucleic acids encoding CR5 polypeptide, vector and transformed cell thereof, and expression thereof
WO1996026292A1 (fr) Transducteurs du signal de l'interleukine-2 et dosages de liaison
AU706887B2 (en) Novel protein - TRAF6
WO1998001541A1 (fr) Proteine kinase associee a traf-2 et dosages
US5871961A (en) Nucleic acids encoding CR2 polypeptides, vector and transformed cell thereof, and expression thereof
US6458561B1 (en) Human NIM1 kinase
US6172195B1 (en) IKAP proteins and methods
WO2001016314A1 (fr) PROTEINE IREN (IλB REGULATOR), PRODUCTION ET UTILISATION
US6803204B2 (en) Biologically active alternative form of the IKKα IκB kinase
US20040109854A1 (en) Novel traf6 inhibiting protein
AU767967B2 (en) Modulators of TNF receptor associated factor (TRAF), their preparation and use
WO2000005370A1 (fr) Recepteur de cytokine orphelin
IL126428A (en) Modulators of tnf receptor associated factor (traf), their preparation and use

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AU CA JP

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FI FR GB GR IE IT LU MC NL PT SE

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
121 Ep: the epo has been informed by wipo that ep was designated in this application
ENP Entry into the national phase

Ref document number: 2225450

Country of ref document: CA

Ref country code: CA

Ref document number: 2225450

Kind code of ref document: A

Format of ref document f/p: F

ENP Entry into the national phase

Ref country code: JP

Ref document number: 1997 503857

Kind code of ref document: A

Format of ref document f/p: F

WWE Wipo information: entry into national phase

Ref document number: 1996919140

Country of ref document: EP

WWP Wipo information: published in national office

Ref document number: 1996919140

Country of ref document: EP

WWW Wipo information: withdrawn in national office

Ref document number: 1996919140

Country of ref document: EP