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WO1999033961A1 - Nouvelle kinase - Google Patents

Nouvelle kinase Download PDF

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Publication number
WO1999033961A1
WO1999033961A1 PCT/JP1998/005974 JP9805974W WO9933961A1 WO 1999033961 A1 WO1999033961 A1 WO 1999033961A1 JP 9805974 W JP9805974 W JP 9805974W WO 9933961 A1 WO9933961 A1 WO 9933961A1
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WIPO (PCT)
Prior art keywords
dna
seq
sequence
activity
apoptosis
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PCT/JP1998/005974
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English (en)
Japanese (ja)
Inventor
Shizuo Akira
Taro Kawai
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Asahi Kasei Corp
Asahi Chemical Industry Co Ltd
Original Assignee
Asahi Chemical Industry Co Ltd
Asahi Kasei Kogyo KK
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Priority to AU16914/99A priority Critical patent/AU1691499A/en
Publication of WO1999033961A1 publication Critical patent/WO1999033961A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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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
    • 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
    • C07K14/4747—Apoptosis related proteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
    • 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 to a novel phosphorylase having an apoptosis-inducing activity and a DNA encoding the same. More specifically, the present invention relates to a phosphorylase having an apoptosis-inducing activity, which comprises the amino acid sequence of SEQ ID NO: 1, and a DNA encoding the same.
  • a drug useful for prevention and treatment of apoptosis-related diseases can be produced.
  • the phosphorylase of the present invention and the DNA encoding the same are useful in establishing a screening method for apoptotic regulators and a method for diagnosing apoptosis-related diseases.
  • the present invention also relates to a DNA or a derivative thereof comprising at least 12 bases in the base sequence of the above-mentioned DNA or its complementary strand; an expression vector capable of replicating the DNA or its complementary strand.
  • a replicable recombinant DNA comprising the same; a microorganism or a cell transformed with the above-mentioned replicable recombinant DNA; and at least one of the above-mentioned phosphorylases having at least one activity.
  • Conventional technology Conventional technology
  • Apoptosis or programmed cell death is one of the processes or modes of cell death proposed by Kerr, Wy11ie et al. [Kerr, J. F. R. et al., Bri and J. Cancer, 26: 239 (1972)].
  • Apoptosis is a phenomenon that occurs during physiological ontogenesis, disease, or the manifestation of a drug effect, and is thought to occur based on the activation of programs inherent in individual cells. Thus, apoptosis is distinguished in the form of cell death from necrosis, the process by which required cells are damaged and die.
  • apoptosis There are various stimuli that induce apoptosis, and their mechanisms are also diverse, but have common morphological characteristics.
  • the first observed morphological change is the formation of chromatin aggregates, which in most cases involve DNA fragmentation [Wyl 1 ie, AH, Nature, 284: 555 (1980)].
  • cytoplasmic condensation and the like occur, and the cells themselves form cell fragments called apoptotic bodies, and the formed apoptotic bodies are rapidly turned around.
  • Apoptosis is said to proceed by phagocytosis by cells and macrophages.
  • Apoptosis is deeply involved in the reduction of the somatic cells (lymphoid cells) that cause the disease in AIDS and other diseases.
  • Various anticancer drugs reduce cancer cell destruction by apoptosis.
  • apoptosis As a disease associated with apoptosis, symptoms may be manifested by attenuated induction of apoptosis, follicular lymphoma, carcinoma caused by mutation of P53, and hormone. Cancers caused by abnormalities such as breast, prostate and ovarian cancers; autoimmune diseases such as systemic erythematosus and immune-associated glomerulonephritis; herpes virus, adenowinores, box virus, etc. Virus infections. Conversely, AIDS, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, retinitis pigmentosa, and cerebellar mutations are considered to be symptoms that may be manifested by enhanced induction of apoptosis.
  • Myelodysplastic diseases such as aplastic anemia; ischemic diseases such as myocardial infarction and stroke; and toxic liver diseases caused by alcohol and the like [Thompson, B., Science, 267]. : 14 56 (1995)].
  • Other diseases involving apoptosis include chronic rheumatoid arthritis [Okamoto'K. Et al., Arthr it is Rheum., 40: 919 (1997)], ulcerative colitis [Iwamoto, M. et al., J. Patho 1.180: 152 (1996)], diabetes [Brown, DL et al., Surgery, 121: 372 (1997)], arteriosclerosis [Bochaton, PML et al., Am. J. Patho, 146]. : 1059 (1995)], myasthenia gravis [Shiono, H. et al., Eur. J. Immuno 1, 27: 805 (1997)].
  • Fas ligand activates interleukin-11 ⁇ converting enzyme (ICE) -like protease through Fas (CD95) and induces apoptosis [Nagata, S. et al. Science, 267: 1449 (1995); Enari, M. et al., Nature, 375: 78 (1995)].
  • ICE interleukin-11 ⁇ converting enzyme
  • CD95 Fas
  • MAPK 0 gen act ivated prote in k inase
  • This gene belongs to Jun kinases (JNKs) / st ress act ivat ed prote in kinases (SAPKs) and ext race Nat. 1. Atad. Sci. USA., 94: 3302 (1997)] and pp56 (1ck) were activated. [Gonz a 1 ez, GA et al., J. Immunol. 158: 4104 (1997)].
  • Tumor necrosis factor (TNF) associates with TNF receptor via TNF receptor Tumor necros isf actor receptor-assoc
  • apoptosis-inducing ceramide is used to generate mitogen ac ti vat ed prote in ki nas eki nase ki nas e (MAPKKK). 1) is involved in the activation of S APK / J NK and induces apoptosis [Shirakabe, K. et al., J. Bio and Chem. 272: 8141 (1997)]. Furthermore, apoptosis induced by interferon y (IFN ⁇ ) in HeLa cells and mouse fibroblasts, and dea th activated protein (DAP) kinas e [C hoen, 0.
  • IFN ⁇ interferon y
  • DAP dea th activated protein
  • DAP kinase involved in INF ⁇ -induced apoptosis is a phosphorylase that is not involved in the MAP kinase cascade [Choen, 0. et al. EMBO J., 16: 998 (1997)]. It is known that it has a structure similar to calmodulin-dependent phosphorylase (CaM kinase).
  • CaM kinase calmodulin-dependent phosphorylase
  • the present inventors have found that a Zip Interacting Protein kinase (Zi kinase) [Kawa T. et al. 18: 1642 (1998)].
  • an object of the present invention is to find a phosphorylase having a structure similar to CaM kinase and having a new apoptosis-inducing activity, and to provide a method for utilizing the same in the fields of medicine and medical treatment. It is in. Summary of the Invention
  • the present inventors have conducted intensive studies to solve the above-mentioned problems, and as a result, obtained a gene sequence having homology to the gene sequence of DAP kinase in a database (GenBank Release, 100, April, 1997). searched. As a result, three types of EST fragments were obtained, EST No. AA278574, EST No. AA419612, and EST No. R 19772. EST No. AA278574 and EST No. AA419612i were gene fragments showing homology of 55.6% (378 base) and 51.4% (389 base) with DAP kinase, respectively.
  • DRAK 1 and DRAK 2 have autophosphorylation activity, phosphatase activity for foreign substrates, and apoptosis-inducing activity.
  • the present inventors have found a novel phosphatase domain common to two phosphatases.
  • preparation of an expression system for DRAK1 and DRAK2 preparation of a drug screening system, and preparation of an antibody were performed, thereby completing the present invention.
  • one of the main objects of the present invention is to provide a substantially pure phosphorylase having an apoptosis-inducing activity, which comprises the amino acid sequence of SEQ ID NO: 1.
  • One other object of the present invention is to provide an isolated DNA encoding the above-mentioned phosphorylase.
  • Still another object of the present invention is to inhibit at least one activity of a oxidase selected from the group consisting of autophosphorylation activity, exogenous substrate phosphorylase activity, or apoptosis-inducing activity.
  • a oxidase selected from the group consisting of autophosphorylation activity, exogenous substrate phosphorylase activity, or apoptosis-inducing activity.
  • Still another object of the present invention is to provide an antibody capable of binding to a phosphatase.
  • sequence of SEQ ID NO: 1 is MLCK-1 e kinase doma in.
  • sequence of SEQ ID NO: 8 is a primer for subcloning of DRAK1 designed based on the complementary strand of GenBank entry AA 2785574.
  • SEQ ID NO: 9 is a primer for subcloning of DRAK1 designed based on the complementary strand of GenBank entry AA 2785574.
  • sequence of SEQ ID NO: 10 is a primer for subcloning at the 5 ′ end of DRAK2 designed based on the complementary strand of the AA419612 of GenBank.
  • sequence of SEQ ID NO: 11 is a primer for subcloning at the 5 'end of DRAK2 designed based on the complementary strand of the entry AA4196612 of GenBank.
  • sequence of SEQ ID NO: 12 is a primer for subcloning at the 3 ′ end of DRAK2 designed based on the sequence at the 5 ′ end of DRAK2.
  • sequence of SEQ ID NO: 13 is a primer for subcloning at the 3 'and 3' ends of DRAK 2 designed based on the sequence at the 5 'end of DRAK 2.
  • SEQ ID NO: 14 is a sense primer for adding a FLAG sequence to the N-terminus of DRAK1.
  • sequence of SEQ ID NO: 15 is an antisense primer for adding a FLAG sequence to the N-terminus of DRAK1.
  • SEQ ID NO: 16 applies the FLAG sequence to the N-terminus of DRAK 2. It is a sense primer to perform.
  • sequence of SEQ ID NO: 17 is an antisense primer for adding a FLAG sequence to the N-terminus of DRAK2.
  • sequence of SEQ ID NO: 18 is a primer for replacing Lys at the 90th position of DRAK1 with A1a.
  • SEQ ID NO: 19 is a primer for replacing Lys at position 62 of DRAK 2 with A1a.
  • SEQ ID NO: 20 is a sense primer for adding a Myc tag to the N-terminus of DRAK1.
  • sequence of SEQ ID NO: 21 is an antisense primer for adding a Myc tag to the N-terminus of DRAK1.
  • sequence of SEQ ID NO: 22 is a sense primer for adding a Myc tag to the N-terminal of the amino acid sequence consisting of amino acids 1 to 345 of DRAK1.
  • sequence of SEQ ID NO: 23 is an antisense primer for adding a Myc tag to the N-terminal of the amino acid sequence consisting of amino acids 1 to 32 of DRAK1.
  • SEQ ID NO: 24 is a sense primer for adding a Myc tag to the N-terminus of DRAK 2.
  • sequence of SEQ ID NO: 25 is an antisense primer for adding a Myc tag to the N-terminus of DRAK 2.
  • SEQ ID NO: 26 adds a Myc tag to the N-terminal of the amino acid sequence consisting of amino acids 1 to 31 of DRAK 2. Is a sense primer.
  • sequence of SEQ ID NO: 27 is an antisense primer for adding a Myc tag to the N-terminal of the amino acid sequence consisting of amino acids 1 to 293 of DRAK2.
  • SEQ ID NO: 31 is a primer for subcloning of Trad designed based on the complement of the entry R 19772 of GenBank.
  • SEQ ID NO: 32 is a primer for subcloning of Trad designed based on the complement of the entry R 19772 of GenBank.
  • sequence of SEQ ID NO: 33 is a sense primer for adding a FLAG sequence to the N-terminal of Trad.
  • sequence of SEQ ID NO: 34 is an antisense primer for adding a FLAG sequence to the N-terminal of Trad.
  • SEQ ID NO: 35 is a primer for replacing Lys at position 1061 of Trad with Ala.
  • Figure 1 (a) shows the results of a northern blot t ing showing the tissue distribution of DRAK1.
  • FIG. 1 (b) shows the results of nor thern blot t ing showing the tissue distribution of DRAK2.
  • Fig. 2 (a) shows the wild-type and activity-deleted DRAK1
  • FLAG-DRAK1 in the figure indicates wild-type DRAK1
  • FLAG-DRAK1K9 OA indicates the activity-deficient DRAK1
  • Vector indicates the control.
  • Autophosphorylation ability and MLC indicate phosphorylation ability for MLC, respectively.
  • the lower row shows the results of detection of proteins obtained by the expression of each DNA in ⁇ FLAG B lot (western).
  • FIG. 2 (b) shows the measurement results of the autophosphorylation activity of wild-type and activity-deficient DRAK2 and the phosphorylation activity on foreign substrates.
  • FLAG-DRAK2 in the figure is wild-type DRAK2.
  • FLAG-DRAK2 K62A indicates activity-deficient DRAK2
  • Vector indicates a control
  • AUTO indicates self-oxidation ability
  • MLC indicates an oxidation ability for MLC.
  • the lower row shows the results of detection of proteins obtained by the expression of each DNA by ⁇ FLAG Blot (western).
  • FIG. 3 shows the intracellular localization of DRAK 1 and DRAK 2.
  • COS-7 cells transfected with DRAK 1 (FLAG-DRAK1) and DRAK 2 (FLAG-DRA K2) were stained with a FLAG antibody. The results show that DRAK 1 and DRAK 2 are each present throughout the cytoplasm. Further, the cell nuclei of the above cells were shown by DAPI staining.
  • FIG. 4 is a graph showing the apoptosis-inducing activity of wild-type DRAK1 and wild-type DRAK2 using NIH 3T3 cells, using cell morphology change as an index. Indicating the site of change, lack of activity DRAK 1 and lack of activity It shows that apoptosis is not induced in type DRAK2.
  • FIG. 5 shows DRAKs composed of an amino acid sequence common to the amino acid sequences of DRAK 1 and DRAK 2, and the inverted characters in the figure indicate the common amino acids.
  • Figure 6 shows the amino acid sequences of human ZIPkinase, human MLCK, mouse CaMkinase II a chain, human DAP kinase, human DRAK1 and human DRAK2.
  • the inverted characters in the figure indicate amino acids that are common.
  • Figure 7 (a) shows the autophosphorylation activity ("WT” or "1-345” in the figure) of DRAK 1 and DRAK 1 partially lacking the amino acid at the C-terminal. Changes in the phosphorylation activity of the foreign substrate ("MLC” in the figure) are shown.
  • Myc-DRAK1 in the figure is full-length DRAK1
  • Myc-DRAKl1-345 is the amino acid after the 346th amino acid.
  • DRAK1 and Myc-DRAKl 321 in which the sequence has been deleted are DRAK1 in which the amino acid sequence of the 32nd and subsequent amino acids have been deleted, and the vector indicates a control.
  • the lower part shows the results of detection of the proteins expressed by each DNA by a Myc Blot (western).
  • Figure 7 (b) shows the autophosphorylation activity of DRAK 2 and DRAK 2 partially lacking the amino acid at the C-terminus ("WT” or "1-293" in the figure and foreign
  • the figure shows the change in phosphorylation activity on the substrate (MLC).
  • Myc-DRAK2 is full-length DRAK2
  • yc-DRAK2 1-315 is DRAK2 lacking the amino acid sequence after the 31st position.
  • Myc-DRAK2 1-293 shows the amino acid sequence from the 294th position onwards.
  • DRAK 2 deleted, Vector indicates control.
  • the lower row shows the results of detection of proteins expressed by each DNA by ⁇ Myc Blot (western).
  • FIG. 8 shows the results of nor thern blotting, which shows the distribution of the filaments of Trad.
  • FIG. 9 shows the measurement results of the autophosphorylation activity of the wild-type and the activity-deficient T rad and the phosphorylation activity on the foreign substrate.
  • FLAG-Trad in the figure is the wild-type Trad, FLAG-Trad.
  • K1016A indicates activity-deficient T rad, Vector indicates a control, and the arrow indicates autophosphorylation possible.
  • the lower row shows the results obtained by detecting a protein obtained by the expression of each DNA using a FLAG Blot (western).
  • Figure 10 shows the results of comparison of the amino acid sequences of the phosphatase regions of Trad, Trio, DAPK, ZIPK, CaMKI I, and MLCK.
  • Figure 1 1 shows where Roh acid match (a) is, Trad, Tr io, the ka 1 ir in, 01) 1 11) 1 homologous (DH) result of comparison of a Mi acid sequence regions Show, Figure 11
  • (b) shows the results of comparison of the amino acid sequences in the plextrin-like (PH) region, and the inverted characters indicate the results where at least two molecules have the same amino acid sequence. Show.
  • Figure 12 shows that both T rad and cytoskeletal protein are equally localized in the cells.
  • the results of staining with antibodies [Fig. 4]
  • Fig. 4 shows the results of detection of intracellular skeletal proteins by Rhodamin to which Pha110Uin having affinity for intracellular skeletal proteins was bound.
  • a phosphorylase having an apoptosis-inducing activity which comprises the amino acid sequence of SEQ ID NO: 1;
  • An immobilizing enzyme is provided.
  • a substantially pure phosphorylase having an apoptosis-inducing activity which has the amino acid sequence of SEQ ID NO: 1.
  • a phosphorylase having apoptosis-inducing activity wherein one or several amino acids are deleted, substituted or added in the amino acid sequence of SEQ ID NO: 3 or 6.
  • a substantially pure phosphatase characterized by comprising an amino acid sequence.
  • 4. A peptide consisting of at least six amino acids whose amino acid sequence is selected from the group consisting of SEQ ID NOs: 1, 3, and 6.
  • DNA according to the above item 5 wherein the DNA is a nucleotide sequence of SEQ ID NO: 2 or 5, or a nucleotide sequence satisfying at least one of the following properties.
  • DNA or a derivative thereof comprising at least 12 bases in the base sequence of 5 or 6 above.
  • the DNA is a fragment of SEQ ID NO: 5 from 36 1st to 1 7.
  • a DNA comprising at least 12 bases in the base sequence of SEQ ID NO: 4 or 7, or a derivative thereof.
  • a replicable recombinant DNA obtained by incorporating the DNA according to any of the above items 5 to 10 into a replicable expression vector.
  • a phosphatase selected from the group consisting of autophosphorylation activity, exogenous substrate phosphatase activity, or apoptosis-inducing activity
  • the phosphorylase according to any one of the above items 1 to 3 or the peptide according to the above item 4 is brought into contact with a sample material, and A method comprising, using at least one of the activities as an indicator, a substance that suppresses or enhances the activity of a phosphorylase.
  • the left and right ends of the amino acid sequence described in the sequence listing are the amino terminal (hereinafter, N-terminal) and the carboxyl group terminal (hereinafter, C-terminal), respectively, and the left end of the base sequence. And the right end are the 5 'and 3' ends, respectively.
  • a in the DNA base sequence indicates adenine
  • C indicates cytosine
  • G indicates guanine
  • T indicates thymine
  • a la in the amino acid sequence is alanine, Arg is arginine, A sn is asno lagin, Asp is as no, laginic acid, Cys Is cystine, G1n is glutamine, G1u is glutamate, G1y is glycin, His is histidine, I1e is isoleucine, and Leu is leu.
  • Lys is lysine, Met is methionine, Phe is phenylalanine, Pro is proline, Ser is serine, Thr is threonine, and Trp is triptoline. Fan, Tyr is tyrosine, and Va1 is valine.
  • polypeptide includes those generally understood by those skilled in the art as peptides, oligopeptides, polypeptides, proteins and the like. Therefore, natural proteins and polypeptides obtained by chemical synthesis or recombinant techniques are also included, and polypeptides are subject to post-translational modifications such as sugar chain binding and phosphorylation. You may or may not have received it.
  • the phosphorylases of the present invention are homologous to the gene sequence of DAP kinase, and may be considered to have some relationship with apoptosis.
  • DRAKs as used in the present invention is a phosphorylase that includes a novel phosphatase domain represented by the amino acid sequence shown in SEQ ID NO: 1.
  • Representative examples of such a phosphatase are DRAK1 and DRAK2 of the present invention, each of which has an amino acid sequence as shown in SEQ ID NOS: 3 and 6, both of which are amino acids as shown in SEQ ID NO: 1. Phosphorylation involving an acid sequence.
  • Both DRAK1 and DRAK2 have autophosphorylase activity and phosphorylation activity on foreign substrates, and are enzymes having apoptosis-inducing activity.
  • a protein having high similarity to DRAK 1 of the present invention includes a novel human phosphorylase kinase gamma subunit (HPHK) (USP 5, 683, 910).
  • HPHK human phosphorylase kinase gamma subunit
  • DGENE Denssion ion Id. 19980920 uP
  • HPH KG was found to be almost identical to DRAK 1.
  • a comparison of DRAK1 and HPHKG reveals that the translation initiation position is different.
  • DRA ⁇ 1 is an enzyme found in the process of searching for a phosphorylating enzyme that regulates apoptosis activity
  • HPH KG is an enzyme that was found mainly for controlling glucose metabolism.
  • molecules having similarity to DRAK1 and DRAK2 include DAP kinase (International Publication WO95 / 106630) and ZIP kinase [Kawai, T. et al., Mol. Cell Enzyme: 18: 1642 (1998)], but all enzymes have amino acid sequences and gene sequences that are different from DRAK 1 and DRAK 2, and furthermore, localization in cells. Differences are observed. As shown in Example 9, DRAK1 and DRAK2 are localized throughout the cell, but DAP kinase is co-localized with cytoskeletal fibers in the cytoplasm and ZIP kinase is localized in the cell nucleus .
  • TRIO International Publication W097Z35997
  • TRIO is a molecule that has homology with the phosphorylase region common to DRAK1 and DRAK2. Both the amino acid sequence and the gene sequence of TRIO are different from those of DRAK1 and DRAK2.
  • TRIO is a molecule involved in the MAP kinase cascade, and is associated with cell morphology and cell motility [Bell langer, JMeta 1., Oncogene, 16: 147]. (1998)]. None of the phosphorylases of the present invention are limited to the amino acid sequences shown in the sequence listing.
  • the phosphatase of the present invention also includes "an amino acid sequence in which one or several amino acids are deleted, substituted or added".
  • an amino acid sequence refers to the amino acid sequence of an allelic or spontaneous mutation found in nature, or a mutant obtained by artificial mutation or genetic recombination technology.
  • modification and substitution of amino acid reference can be made to, for example, the patent application of Bennett et al. (International Publication WO96 / 26445).
  • the amino acid sequence encompassed by the present invention is all a polypeptide having the activity of the novel phosphatase of the present invention, and is a modification of one amino acid residue.
  • an amino acid sequence containing a change that causes loss of its activity is not included in the present invention.
  • DRAK1 and DRAK2 only polypeptides having their autophosphorylation activity, phosphorylation activity on foreign substrates, and apoptosis-inducing activity are included in the present invention.
  • a peptide comprising at least six amino acids of a phosphorylase.
  • the full-length protein and its partial peptides of the present invention can be used to prepare a system for measuring enzyme activity for diagnosis or therapy, to prepare antibodies, and to elucidate new regulatory proteins that further regulate the enzyme activity.
  • a peptide consisting of amino acid 3 at position 23 of SEQ ID NO: 3 and amino acids 3 to 4 at position 29 of SEQ ID NO: 6 The peptide consisting of the amino acid of the eye is a region corresponding to the phosphatase active domain shown in SEQ ID NO: 1, and as shown in FIG. 6, it is unique to the phosphatase of the present invention. Is an array. These sequences are useful when creating a system for measuring enzyme activity.
  • the peptide consisting of the amino acid at positions 35 and 36 of SEQ ID NO: 3 and the peptide consisting of the amino acids at positions 319 to 31 of SEQ ID NO: 6 are As shown in Examples 16 and 17, it is useful for producing antibodies. Furthermore, as performed in Example 20 and Example 21, the peptide consisting of the amino acid at the 3rd and 4th positions of SEQ ID NO: 3 and the 293rd position of the amino acid at the 4th position of SEQ ID NO: 6 The peptide consisting of the amino acid at position 37-2 has the ability to regulate enzyme activity, and is useful for elucidating a novel activity regulating protein that binds to this region.
  • a DNA encoding the above-described phosphorylase of the present invention there is provided a DNA encoding the above-described phosphorylase of the present invention.
  • the DNAs of the phosphatase of the present invention, DRAK1 and DRAK2 are shown in SEQ ID NO: 2 and SEQ ID NO: 5 in the sequence listing.
  • the DNA encoding the phosphorylase of the present invention is not limited to these sequences, and DNA obtained by degeneracy of the gene code is also included in the present invention.
  • Is composed of (1) a DNA comprising the nucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 5 and a DNA hybridizing under stringent conditions; and (2) a DNA comprising the nucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 5 Over 70% homology with DNA DNA is included.
  • DNA that hybridizes under stringent conditions refers to washing conditions after hybridization, for example, by appropriately changing the temperature and the salt concentration to obtain nonspecific high-molecular-weight DNA.
  • a highly complementary sequence, identified under conditions of reduced hybridization Specifically, the specificity between the hybridizing polynucleotides, such as 2 XSSC, 0.1% SDS, 65 ° C, etc. in Examples 3 and 4 is guaranteed. DNA that hybridizes under the conditions.
  • a DNA comprising at least 12 bases selected from the base sequence of the DNA of the present invention or a derivative thereof.
  • Such DNAs include, for example, at least 12 or more, preferably 16 or more, and more preferably 18 or more selected from the nucleotide sequences of SEQ ID NO: 2 and SEQ ID NO: 6.
  • a DNA consisting of the above bases is exemplified.
  • at least 12 or more, preferably 16 or more, selected from the nucleotide sequences of SEQ ID NO: 3 and SEQ ID NO: 6, which are complementary to the nucleotide sequences of SEQ ID NO: 2 and SEQ ID NO: 5, and DNA consisting of preferably 18 or more bases is also very useful.
  • DNA makes it possible to detect cDNA clones of phosphatase, cDNA, genomic DNA, and genomic gene clones.
  • the required length of DNA depends on the specificity of the sequence, the stability of binding to the DNA to be detected, etc. 1
  • PCR polymerase chain reaction
  • Tm double-stranded dissociation temperature
  • a nucleic acid of 12 bases or more is required when the GC content is high, and a nucleic acid of 16 bases or more is required for a general region where the GC content is about 50%.
  • a nucleic acid derivative having stable binding to DNA is used, the target DNA can be detected even with a shorter nucleic acid.
  • a derivative of the DNA a derivative in which the above-mentioned DNA is, for example, methylated, methylphosphated, deaminated, or thiophosphated can be used. .
  • Examples of a method for genetic diagnosis using such DNA or a derivative thereof include techniques such as hybridization and PCR.
  • the cloning of genes on the genome including humans is also possible, and the cloned genes can be used, for example, transgenic mice, gene targeting mice, and the present invention.
  • the use of recent gene manipulation techniques such as double knockout mice in which both genes and related genes have been inactivated, more detailed functions of the phosphorylase of the present invention can be obtained. It can be clarified.
  • the DNA and the derivative thereof of the present invention include the nucleotide sequence of SEQ ID NO: 2, which corresponds to the sequence unique to the phosphatase of the present invention (that is, the region corresponding to the sequence of SEQ ID NO: 1). It is preferable to select from DNA consisting of bases or DNA consisting of bases 361 to 1146 described in SEQ ID NO: 5.
  • DNA By using such DNA to enhance or delete the enzyme activity specific to DRAK 1 or DRAK 2 in mice, etc., it is possible to analyze the functions of DRAK 1 or DRAK 2 in more detail. is there.
  • a two-hybrid evaluation method in which gene analysis is performed by applying protein-protein synthesis in cells [Hsu, H. et al., Proc. Natl. Acad. Sc. USA., 91: 3181 (1994) ] Can be used to analyze a group of signaling molecules.
  • DNA consisting of nucleotides 108 to 1359 of SEQ ID NO: 2 and 1 1 4 1st primer of SEQ ID NO: 5 13 77 The DNA consisting of the 7th base is an important region for regulating the phosphatase activity of DRAK1 and DRAK2.
  • the above-mentioned sequence is useful for analysis of a group of signaling molecules specific to DRAK1 and DRAK2. Further, if an abnormality of the gene of the present invention is found on the genome, it can be applied to gene diagnosis and gene therapy.
  • the present invention is selected from the nucleotide sequences of SEQ ID NO: 4 and SEQ ID NO: 7, which are complementary strands of the cDNAs of DRAK 1 and DRAK 2.
  • DNA comprising at least 12 bases, preferably at least 16 bases, more preferably at least 18 bases, and derivatives thereof. By using such a DNA, it is possible to detect the mRNA of the phosphorylase of the present invention.
  • antisense nucleic acids for example, DNA or a derivative thereof, which is a fragment of the nucleotide sequence of SEQ ID NO: 4 or SEQ ID NO: 7, or DNA
  • antisense RNA designed based on the sequence, it is possible to perform techniques such as hybridization, primer extension, nuclease, protection, and atsushi. You.
  • administration of an antisense nucleic acid to a cell or a living body may be considered.
  • the excessive reaction of the phosphorylase of the present invention is a pathological condition
  • suitable vector for examples of preparation and use of these antisense nucleic acids, reference can be made to Murray, J. AH, edited by ANTISENSE RNA AND DNA, Wiley-Liss, Inc., 1992.
  • nucleotide sequence from the 149th to the 154th nucleotide of SEQ ID NO: 4 and the nucleotide sequence from the 345th to the 37th nucleotide of SEQ ID NO: 7 described in the sequence listing and 5 95 The nucleotide sequences from the 5th to the 62nd nucleotide were all the same as in Examples 1 and 2. It can be used for detecting DRAK 1 and DRAK 2, and is effective for detecting the phosphorylase of the present invention for diagnostic purposes.
  • a base sequence consisting of 20 to 40 bases near the 2541st to 254th positions of SEQ ID NO: 4 and the 1 Base sequences of 20 to 40 bases near the 396th to 1398th positions are useful.
  • a recombinant DNA comprising any one of the above-mentioned DNAs of the present invention.
  • the vector used for preparing the recombinant DNA of the present invention is not particularly limited, and any commonly used vector can be used.
  • the vector pEFBOS [Misushima, S.
  • the recombinant DNA of the present invention into a known host. That is, according to the present invention, there is provided a microorganism or cell transformed with the recombinant DNA.
  • the host into which the recombinant DNA of the present invention is introduced is not particularly limited, but is a microorganism or a cell capable of expressing the recombinant DNA of the present invention.
  • prokaryotic cells such as Escherichia sp. (Escherichia coli) and Bacillus sp. (Bacillus subtilis) are recombinantly prepared using the calcium chloride method or the like. DNA can be introduced.
  • Examples of the genus Escherichia include Escherichia coli K12, HB101, MC1061, L ⁇ 392, JM109, and INVaF '.
  • Bacillus genus include Bacillus subtilis Ml 114.
  • Phage vectors can be introduced into, for example, grown Escherichia coli using the in vitro packaging method (Pr0c. Nat 1. Acid Sci. 74: 3259-3263, 1977). It can be. Eukaryotic cells such as animal cells and insect cells can also be used as hosts.
  • a transformed cell E.co1i DH5 in which Escherichia coli DT5 ⁇ was transfected with plasmid hDRAK2-pT7 containing cDNA encoding the entire amino acid sequence of DRAK2 of the present invention.
  • DRAK1 and DRAK2 can be easily produced.
  • phosphorylase expression system using transformants see Kriegler, Gene Transfer and Expression-A Laoratory Manual, Stockton Press, (1990) and Yokota et al., Biomanual Series 4, Gene transfer and expression. ⁇ It can be constructed with reference to analysis method, Yodosha, 1996 (Japan), etc.
  • the recombinant DNA used to produce the phosphatase is the translation initiation codon at the 5 'and 5' ends of the DNA encoding the phosphatase inserted in the vector, and the 3 'end It may have a stop codon.
  • a translation initiation codon and a translation termination codon can also be added using an appropriate synthetic nucleic acid adapter.
  • a promoter is placed upstream of the DNA. — It is preferable to connect
  • the promoter used in the present invention is not particularly limited as long as it is a promoter corresponding to a host used for gene expression. If the host is a bacterium belonging to the genus Escherichia, a tac promoter, a trp promoter, a 1ac promoter, etc. are preferred. If the host is a bacterium belonging to the genus Bacillus, an SPO1 promoter, an SPO2 promoter, etc. Is preferred.
  • the recombinant DNA to be introduced preferably has a ribosome binding site together with a promoter. When the host is yeast, a PGK promoter, a GAP promoter, an ADH promoter, etc. are preferred.
  • a SV40-derived promoter, a retroviral promoter, Metabolic promoters and heat shock promoters can be used.
  • the DNA used for producing the phosphatase of the present invention is not particularly limited as long as it encodes a phosphatase including the amino acid sequence of SEQ ID NO: 1.
  • the DNA of SEQ ID NO: 2 encoding DRAK1 and the DNA of SEQ ID NO: 5 encoding DRAK2 can be used.
  • a known nucleotide sequence can be bound to DNA encoding the phosphatase.
  • nucleic acids encoding antigen epitopes can be added to facilitate detection of the produced protein. This For an example of such a technique, see Choe, H. et al., Cell, 85, 1135-1148, 1996.
  • a transformant for producing a phosphatase is obtained by introducing the recombinant DNA constructed as described above into a host cell capable of expressing DNA.
  • a host cell capable of expressing DNA.
  • the above-mentioned Escherichia genus, Bacillus genus, yeast, animal cells and the like can be used.
  • animal cells are preferred, and monkey cells such as COS-7, Vero cells, Chinese hamster cells CHO: silkworm cells SF9, and the like can be mentioned.
  • a transformant can be produced by introducing the above-described expression vector into a COS-7 cell.
  • DRAK1 or DRAK2 as the phosphorylating enzyme of the present invention can be produced.
  • the production of the phosphorylase from the cultured transformant can be confirmed by the Western bloUing method used in Examples 7 and 8.
  • the phosphorylases DRAK 1 and DRAK 2 of the present invention are enzymes obtained based on the genetic information of DAP kinase, which is known to induce apoptosis in HeLa cells.
  • the experiments in Examples 10 and 11 confirmed that DRAK1 and DRAK2 had apoptosis-inducing activity. An experiment using DRAK 1 is described below.
  • FLAG-DRAKl (p EFBOS-FLAG-DRAK 1), wild-type DRAK 1, FLAG-DRAK1K90A (pE FBOS-FLAG-DRAK1K90A), DRAK 1 with phosphatase activity, and Vector as control (pEFBOSmock) was transiently introduced into NIH 3T3 cells together with the LacZ expression vector (pEFBOS-LacZ) using the lipofection method. X-ga1 staining was performed 36 hours after transfection. As a result, wild-type DRAK1-transfected cells showed typical apoptotic morphology with nuclear aggregation.
  • the percentages of cells showing apoptotic morphology of wild-type DRAK1-introduced cells and deletion-type DRAK1-introduced cells were 25.3% and 2.1%, respectively. Similar results have been obtained for DRAK 2.
  • the transformed DRAK1 or DRAK2 cells of the present invention show an apoptosis morphology, which is effective for screening compounds having apoptosis inhibitory activity. .
  • the present inventors examined the relationship between the apoptosis-inducing activity of DRAK1 and DRAK2 and the activity of its phosphatase by conducting the experiments shown in Examples 10 to 15. It has been confirmed that elementary activity is essential for induction of apoptosis. Therefore, the phosphatase activity of the phosphatase of the present invention is also effective for screening substances that induce or suppress apoptosis. More specifically, according to the present invention, the phosphatase of the present invention or its peptide is brought into contact with a sample material, and the activity of the phosphatase (autophosphorylation activity, exogenous substrate phosphorylase activity) is obtained. Or apoptosis-inducing activity) as an indicator, and detecting a substance that suppresses or enhances the activity of the phosphorylase.
  • Antibodies that specifically recognize the phosphorylase DRAK1 or DRAK2 of the present invention can be prepared as described in Examples 16 and 17.
  • the length of the peptide used for preparing the antibody is not particularly limited, but may be any length that can characterize the phosphorylase protein of the present invention, and is preferably 6 amino acid residues or more. Can use a peptide having 8 amino acid residues or more.
  • the peptide is adjuvanted as it is, or after cross-linking with KLH (keyho 1 e-1 imp et hemocyanin), ⁇ -3 ⁇ 4, Dv i nse rum al bumin) and the carrier protein.
  • the antibody is inoculated into an animal, and the serum is collected to obtain an antiserum containing an antibody (polyclonal antibody) that recognizes a phosphorylase (DRAK1 or DRAK2). It is also possible to purify the antibody from the obtained antiserum and use it.
  • the animals to be inoculated with the antigen sheep, rabbits, goats, rabbits, mice, rats, etc. are used, but sheep and rabbits are used for the production of polyclonal antibodies. I like it.
  • amino acid sequence shown in SEQ ID NOs: 1, 3 and 6, or more than 6 amino acid residues selected from the amino acid sequence, preferably 8 amino acid residues can be fused with GST (daltathione S-transferase) or the like, and the resulting peptides can be used as antigens, either as purified or unpurified.
  • the antibody of the present invention can be prepared by various methods and gene cloning methods shown in a companion document (Antibodi es aboratory manual, E. Harlow et al., Old Spring Harbor Laboratory). Using the isolated immunoglobulin gene, it can also be produced as a recombinant antibody expressed in cells. Antibodies produced by such a method can also be used for purification of the phosphorylase of the present invention.
  • phosphatase having homology to DAP kinase.
  • the cDNA for the entire coding region of this phosphatase is a GenBank library.
  • EST fragment No. R 19772 [homology with DAP kinase is 56.3% (439 base)] extracted from 1997, Hybrid iz at shown in Reference Example 1. Obtained using the ion method.
  • the present inventors have designated the obtained protein encoded by cDNA as Trad. Search for the amino acid sequence of Trad, the DNA sequence encoding it, and the sequence of cDNA complementary to Trad in the database (GenBank Release, 100, April, 1997), respectively. All of them were new sequences.
  • Trad a novel protein, has a Dbl homology (DH) region, a prestrin-like region, and a phosphorylase region in a single polypeptide chain. It is a phosphatase having a domain structure similar to TRIO as shown in FIG. 0 and having autophosphorylation activity as shown in Reference Example 5. Trad is a phosphorylase that is different from DRAKI and DRAK2 of the present invention and does not show apoptosis-inducing activity in transformed cells.
  • DH Dbl homology
  • T rad is activated as a result of induction of apoptosis, and as a result of activation of T rad, cytoplasmic aggregation observed in apoptosis-inducing cells is caused by the action of plasma membrane skeletal proteins. It is estimated to be caused by
  • Tr The physiological function of ad was obtained from the Rho subfamily. That is, Ras, Rab, Rho, Ran, and the like are well known as low molecular weight GTP-binding proteins (G proteins) [Barbacid, M., Annu. Rev. Biochem. 56: 779 (1987); Salminen, A. et al., Cell 1, 49: 527 (1987); Bischof f, FR et al., Nature, 354: 80 (1991)].
  • G proteins G proteins
  • the G proteins exist inactivating the activated and GDP and binding that binds GTP, guanine nuc leot to activation of G protein ide exchange f actors GF s) force s fe Watte Rereru.
  • a typical example of 0 £ 3 is 0 13 1 [13, ⁇ 1. Et al., Nature, 4: 311 (1991)], but proteins that show GEF s activity against the Rho subfamily include proteins. Many have a Db1 homology (DH) region [Quilliam, LA et al., BioEssays, 17: 395 (1995)].
  • Rho-kinase LMa-tsui, T. et al., EMBO J. 15: 2208 (1996), which has Rho-binding activity, is a molecule involved in Rho signaling.
  • PDN Protein kinase N
  • Rho-kinase, MACKs, and others characterize the phosphorylase region of such molecules as Myotonic dystrophy kinase (DMK) [Vonder Ven, PF, and others, involved in muscular dystrophy. H leak 1. Gene 2: 1889 (1993)], and Trio is a DAP kinase that has apoptosis-inducing activity [Deiss, LP et al., Genes Dev. , 9:15 (1995)] and Zip kinase [Kawai, T. et al., Mo. Ce 11. Bio, 18: 1642 (1998)].
  • DMK Myotonic dystrophy kinase
  • T rad shows localization only in skeletal muscle, and also shows the same localization as intracellular skeletal protein, it is considered that T rad is useful for screening drugs that control skeletal muscle function.
  • T rad The amino acid sequence of T rad is described in SEQ ID NO: 29, but T ra d is not limited to this amino acid sequence. Therefore, Trad also includes “an amino acid sequence in which one or several amino acids have been deleted, replaced or added”. Such a sequence refers to an allelic or spontaneous mutation found in nature, as well as an amino acid sequence of a mutant obtained by using an artificial mutation or a genetic recombination technique. For such modification or substitution of amino acid, reference can be made to, for example, a patent application (International Publication WO96 / 26445) by Bennett et al.
  • T rad The amino acid sequence included as T rad is a polypeptide having phosphorylation activity possessed by T rad, and even if one amino acid residue is modified even if it is a modification of one amino acid residue. Amino acid sequences containing changes that result in loss of activity are not included.
  • peptide comprising at least 6 amino acids obtained from the full-length amino acid sequence of Trad described in SEQ ID NO: 29 is also useful.
  • Trad full-length protein and its partial peptides can be used for the preparation of a system for measuring enzyme activity for the purpose of diagnosis or therapy, the production of antibodies, Furthermore, it is useful for elucidation of a novel regulatory protein that regulates the enzyme activity.
  • the sequence consisting of the amino acids 987 to 1241 of SEQ ID NO: 29 is a sequence specific to a phosphatase, as shown in FIG. A peptide having this sequence is useful for preparing a system for measuring enzyme activity.
  • the sequence consisting of the amino acid at position 406 and the amino acid at position 406 in SEQ ID NO: 29 is a sequence unique to Db1-like protein, and the function of the Rho subfamily is examined.
  • sequence consisting of amino acids at positions 4334 to 5337 of SEQ ID NO: 29 is a sequence specific to a referencestatin-like protein. This sequence is known to be involved in protein binding [Musacchio, A et al., Trends
  • the peptide represented by SEQ ID NO: 29 represented by the first force, the first force, the first force, the second force, the second position, the second position, and the second position, Trad As shown in Example 28, this is a region presumed to be an epitope, and is useful for producing an antibody.
  • the present inventors described the nucleotide sequence of the DNA encoding T rad in SEQ ID NO: 28 of the sequence listing.
  • the DNA coding for Trad is not limited to these sequences, and DNA obtained by degeneracy of gene code and the like is also included in DNA for Trad. Therefore, the DNA of T rad includes (1) a DNA consisting of the nucleotide sequence of SEQ ID NO: 28 and a stringent And DNA having a homology of 70% or more with the nucleotide sequence of SEQ ID NO: 28.
  • DNA that hybridizes under stringent conditions refers to washing conditions after hybridization, for example, by appropriately changing the temperature and the salt concentration to obtain nonspecific high-molecular-weight DNA.
  • a DNA consisting of at least 12 bases or a derivative thereof selected from the base sequence of the Trad DNA is also very useful.
  • Such DNAs include, for example, at least 12 or more, preferably 16 or more, and more preferably 18 or more bases selected from the base sequence of SEQ ID NO: 28.
  • at least 12 or more, preferably 16 or more, and more preferably 18 or more selected from the nucleotide sequence of SEQ ID NO: 30 which is a complementary strand of the nucleotide sequence of SEQ ID NO: 28 DNA consisting of the above bases is also very useful.
  • DNA When target DNA is detected by polymerase chain reaction (PCR), the T m (double-stranded dissociation temperature) It is desirable to use a DNA fragment with a temperature of 45 ° C or higher.
  • PCR polymerase chain reaction
  • the Tm can be estimated by summing one GC bond at 4 ° C and one AT bond at 2 ° C. Therefore, when the GC content is high, 12 bases of DNA are required, and when the GC content is about 50% in a general region, about 16 bases of DNA are required.
  • the target DNA can be detected even with a shorter nucleic acid.
  • the derivative of DNA the above-mentioned DNA is, for example, a derivative obtained by methylation, methylphosphate formation, deamination, or thiophosphate formation.
  • Examples of a method for genetic diagnosis using such DNA or its derivative include techniques such as hybridization and PCR.
  • the cloning of genes on the genome including humans is also possible, and the cloned genes can be used, for example, transgenic mice, gene-getting mice, and the present invention.
  • Recent genetic manipulations such as double knockout mice in which both genes and related genes are inactivated Using the technique, the more detailed function of the phosphorylase of the present invention can be elucidated.
  • DNA is the DNA consisting of the 3840th base from the 3706th base of SEQ ID NO: 28 or the 23rd base to the 33rd base of the Db1 region of SEQ ID NO: 28
  • a two-hybrid evaluation method for performing gene analysis utilizing the integrated action of protein and protein in cells [Hsu, H. et al., Proc. Natl. Acad. ScI. USA.
  • the sequence consisting of the 1417th to 1728th bases of SEQ ID NO: 28 is a sequence specific to the prestrin-like protein, and this sequence is represented by This is useful for analyzing unique signaling molecules.
  • the base of SEQ ID NO: 30 which is the complementary strand of the cDNA of Trad is also available.
  • the use of 12 or more, preferably 16 or more, more preferably 18 or more DNAs in the sequence and its derivatives enables detection of Trad mRNA.
  • antisense nucleic acids for example, DNA or a derivative thereof, which is a fragment of the nucleotide sequence of SEQ ID NO: 30, or a design based on the DNA sequence
  • it is possible to carry out techniques such as hybridization, primer extension, nuclease protection and the like.
  • administration of antisense nucleic acids to cells or living organisms may be considered for the purpose of clarifying more detailed functions of Trad.
  • T rad is a pathological condition, it is possible to treat the disease by suppressing gene expression using the above-mentioned antisense nucleic acid.
  • antisense nucleic acid into a suitable vector and use that vector for therapy.
  • suitable vector for example, a suitable vector and use that vector for therapy.
  • antisense nucleic acids See Murray: J. AH, edited by ANTISENSE RNA AND DNA, Wiley-Liss, Inc., 1992.
  • the sequence consisting of the 179th base to 209th base of SEQ ID NO: 30 described in the sequence listing is used for detection of Trad. It is possible to read it and it is effective for detecting T rad for diagnostic purposes.
  • a base sequence consisting of 20 to 40 bases in the vicinity of the 52nd and 36th positions of SEQ ID NO: 30 is effective. .
  • a recombinant DNA containing any of the above-mentioned DNAs can be prepared.
  • the vector used for preparing the recombinant DNA is not particularly limited, but a commonly used vector can be used. Specifically, PBR32, PUC8, pUC19, PUC18, and pUC119 derived from Escherichia coli (all manufactured by Takara Shuzo Co., Ltd. in Japan), Bacillus subtilis-derived plasmid, and yeast-derived Plasmid and other positive vector vectors, and bacteriophage vectors such as gt10 and ⁇ gt11 (both manufactured by Stratagene, USA); However, any other substances can be used as long as they can be propagated in the host.
  • a vector of T rad is described in the vector p EFBOS LMisushima, S, et al., Nucleic Acids Res., 18: 5322 (1990)].
  • the plasmid pEFBOS-FLAG-hTrad obtained by insertion is listed.
  • the recombinant DNA is introduced into a known host. That is, it is a microorganism or cell transformed with the recombinant DNA.
  • the host into which the recombinant DNA is introduced is not particularly limited, but is a microorganism or a cell capable of expressing the recombinant DNA.
  • prokaryotic cells such as Escherichia sp. (Escherichia coli) and Bacillus sp. Vectors can be introduced.
  • Escherichia bacteria include Escherichia coli K12, ⁇ 101, MC1061, LE392, JM109, and INVaF '. . Bacillus spp.
  • Bacillus spp. An example of this is Bacillus subtilis Ml114.
  • the phage vector can be introduced, for example, into the grown E. coli using the in vitro packaging method (Proc. Nat 1. Acad. Sc. 74: 3259-3263, 1977). it can.
  • Eukaryotic cells such as animal cells and insect cells can also be used as hosts.
  • Transformed cells obtained by transfecting Escherichia coli DH5a with a plasmid pEFBOS-FLAG-hTrad containing cDNA encoding the entire amino acid sequence of T rad E.co1i: DH5 Hiichi pEFBOS— FLAG—hTrad was established in the Institute of Biotechnology and Industrial Technology at the Ministry of International Trade and Industry of Japan (U.S.A. 3105-1006, 1-3-1-3 Higashi, Tsukuba, Ibaraki, Japan). The deposit number was FERMBP — 6301 on March 19, 1990 on March 22nd, 1999, and was received.
  • Trad can be easily produced.
  • the expression system of T rad by the transformant see Kriegler, Gene Transfer nd Expres sion-A La or at ory Manual, Stockt on Pres s, (1990); and Yokota It can be constructed with reference to Biomanual Series 4, Gene transfer and expression / analysis methods, Yodosha, 1996 (Japan).
  • the recombinant DNA used to produce T rad has a translation initiation codon at the 5 'end of the T rad coding DNA inserted in the vector and a translation stop codon at its 3' end. It may be.
  • the translation start codon and translation stop codon should be It can also be added using an acid adapter. Further, it is preferable to connect a promoter upstream of the DNA in order to express the desired DNA.
  • the promoter used in the present invention is not particularly limited as long as it is a promoter corresponding to a host used for gene expression. If the host is a bacterium belonging to the genus Escherichia, a tac promoter, a trp promoter, a 1 ac promoter are preferred, and if the host is a bacterium belonging to the genus Bacillus, an SPO1 promoter, an SPO2 promoter, etc. are preferred. .
  • the recombinant DNA to be introduced preferably has a ribosome binding site together with a promoter.
  • yeast PGK promoter, GAP promoter, ADH promoter and the like are preferable.
  • the host is animal cells, SV40-derived promoter and retrovirus promoter are preferable. And promoters such as metal, methanolic promoter, and heat shock promoter.
  • DNA used for the production of Trad there is no particular limitation on the DNA used for the production of Trad, as long as it encodes the amino acid sequence of SEQ ID NO: 29.
  • the DNA of SEQ ID NO: 28 can be used.
  • a known nucleotide sequence can be bound to the DNA encoding Trad in order to produce a protein having a special function. For example, it may be necessary to add nucleic acids encoding antigen epitopes to facilitate detection of the produced protein. it can.
  • a transformant for producing Trad is obtained by introducing the recombinant DNA constructed as described above into a host cell capable of expressing DNA.
  • a host cell capable of expressing DNA.
  • the above-mentioned Escherichia genus, Bacillus genus, yeast, animal cells and the like can be used as the host.
  • animal cells are preferred, and examples include monkey cells such as COS—7, Vero cells, Chinese hamster cells CH ⁇ , and silkworm cells SF9.
  • a transformant can be produced by introducing the recombinant DNA into COS-7 cells. By culturing the transformant, Trad can be produced. The production of Trad by the cultured transformant can be confirmed by the Westernb1ottint method used in Reference Example 4.
  • Trad is a phosphorylase obtained from the genetic information of DAP kinase, which is known to induce apoptosis in HeLa cells, as shown in Fig. 10.
  • the phosphorylation enzyme region of T rad has 58, 41, 41, 31 and 30% homology with the phosphorylation enzyme regions of Trio, DAP kinase, Zip kinase, CaM kinase II and MLCK, respectively. Has the property.
  • the experiment in Reference Example 5 confirmed that Trad had phosphorylation activity.
  • wild-type Trad pEFBOS-FLAG-hTrad
  • TradK1016A pEFBOS-FLAG-hTradK1016A
  • Vector as a control pEFBOS-FLAG-hTradK1016A
  • p EFBOSmock was transiently transfected into COS 7 cells using the Lipoff extraction method. 36 hours after gene transfer, immunoprecipitates were prepared using a FLAG tag, and the autophosphorylation activity was evaluated. As a result, as shown in Reference Example 5, it was revealed that only wild-type Trad had autophosphorylation activity.
  • the DH region of T rad is composed of T rio Ka 1 ir in [Ra shi du 1, MA et al., J. Bio 1. Chem., 272: 12667 (1997) ] And 0 1) have a homology of 67, 38, and 42% with the respective 011 regions.
  • many proteins exhibiting GEFs activity against the Rho subfamily have a Db1 homology (DH) region.
  • DH Db1 homology
  • Trad is a protein showing selective tissue localization in skeletal muscle. Based on the above, it is considered that it is possible to screen a substance having an activity of regulating the function of skeletal muscle using the phosphorylation activity of Trad for itself as an index, and as shown in Reference Example 8. Screening Created a method.
  • An antibody that specifically recognizes T rad can be prepared as shown in Reference Example 7.
  • the length of the peptide used for preparing the antibody is not particularly limited, but may be any length that is characteristic of the Trad protein, and may be at least 6 amino acid residues, preferably 8 amino acids.
  • a peptide having an acid residue or more can be used. This peptide is used as is, or after cross-linking with KLH (keyhole-l-impet hemocyanin) or BSA (bovine serum alumin and a recognizable carrier protein), and then inoculated into an animal with an adjuvant, if necessary, and the serum is recovered.
  • KLH keyhole-l-impet hemocyanin
  • BSA bovine serum alumin and a recognizable carrier protein
  • an antiserum containing an antibody (polyclonal antibody) that recognizes Trad can be obtained, and the antibody can be purified and used from the obtained antiserum.
  • the animals to be inoculated with oocytes include sheep, rabbits, goats, rabbits, mice, rats, etc., but sheep and rabbits are used for the production of polyclonal antibodies. It is also possible to produce a monoclonal antibody by a known method for producing hybridoma cells, in which case a mouse is preferred, and the mouse shown in SEQ ID NO: 29 is preferable.
  • the full length of the amino acid sequence or 6 ⁇ Mi acid residue or more selected Li by ⁇ Mi acid sequence, the preferred municipal district is 8 A Mi acid residue or more of the peptide GST (Darutachion S).
  • the resulting peptide can also be used as an antigen, purified or unpurified, by fusing it with the enzyme (transferase).
  • the antibody of the present invention can be used for various methods and gene cloning methods shown in a compendium (Antiboeies al aboratory manua l, E. Harlow et al. 1, Cold Spring Har or Laboratory). Using the immunoglobulin gene thus isolated, it can also be produced as a recombinant antibody expressed in cells. Antibodies produced by such a method can also be used for purification of Trad.
  • pT7Blue manufactured by Novagen, USA; hereinafter, referred to as Tiector
  • Tiector The mixture was mixed so that the molar ratio of the vector to the DNA was 3: 3.
  • DNA was incorporated into the vector using t (TaKaRa, Japan).
  • the DNA-integrated vector T-vector was transfected into E.
  • coli DH5a (manufactured by T0Y0B0, Japan), and ampicillin (manufactured by Sigma, USA) 50; ug Zml and X-gal L-Broth (manufactured by Nacalai, Japan) containing 200 gml was seeded on a plate of semi-solid medium and left at 37 ° C for about 12 hours. The white colonies that appeared were randomly selected, inoculated in 2 ml of L-Broth liquid medium containing the same concentration of ampicillin, and cultured with shaking at 37 ° C for about 8 hours.
  • the cells were collected, the plasmid was separated using a wizard domiprep (Promega, USA) according to the attached instructions, and the plasmid was separated with the restriction enzyme EcoRI (Japan). (TOYOB0, Japan) and restriction enzyme Sa1I (T0Y0B0, Japan). It was confirmed that the above PCR product was incorporated into the vector by cutting out about 400 bp of DNA. Determine the nucleotide sequence of the incorporated cDNA for the clones confirmed to retain the PCR product did.
  • the nucleotide sequence of the imported cDNA fragment was determined using a fluorescent sequencer manufactured by Applied Biosystems, USA. Sequence samples were prepared using PRISM, Ready Reaction Dye Terminator Cycle Sequencing Kit (Applied B systems, USA). In a 0.2 ml micro tube, add 10 .1 ⁇ of the reaction stock solution and 2 .10 ⁇ l of 1.6 pmo 1 / ⁇ 1 ⁇ 7 promoter primer (GIBC0 BRL, USA) ) And 8.01 of 0.10 / g Zl DNA for sequencing are added and mixed, and placed at 96 ° C for 10 seconds, 50 ° C.
  • a PCR amplification reaction was performed for 25 cycles, with C being 5 seconds and 60 ° C for 4 minutes as one cycle, and incubated at 4 ° C for 5 minutes.
  • 2.0 ⁇ l of sodium triacetate ( ⁇ 5.2) and 501 of ethanol were added, and the mixture was stirred and left at room temperature for 15 minutes.
  • the precipitate was collected by centrifugation at rpm for 15 minutes.
  • After the precipitate was washed with 70% ethanol, it was allowed to stand under vacuum for 2 minutes and dried to obtain a sample for sequencing.
  • the sequence sample is dissolved in 6. ⁇ ⁇ of formamide containing 10 mM EDTA, denatured at 90 ° C for 2 minutes, cooled on ice, and the denatured sample 2.
  • the 2P-labeled probe was prepared as follows. The base sequence from the 4th to 394th bases of AA2 7 8 7 7 4 is inserted into the vector T-Vect0r into which this sequence is incorporated, and the restriction enzyme Ec0RI (Japan, T0Y0B0 ) And restriction enzyme Sa1I (T0Y0B0, Japan), and cut out with 1.0% agarose. Electrophoresis was performed in the gel. After staining with ethidium mouth mouth (manufactured by Nippon Gene Co., Ltd., Japan), the band was observed under ultraviolet light, a band of about 400 bp was cut out from the gel, and the band was cut out with Wizard (Promega, USA, USA).
  • DNA fragment was labeled with a DNA labeling kit (Me gap lime DNA labeling system: Amersham, UK).
  • DNA 1 OSOng Z 1 was added to 5 ⁇ l of primer solution and deionized water to bring the total volume to 33 ⁇ l, followed by a boiling water bath for 5 minutes, and then 5X reaction solution 10 / ⁇ 1,- 3 2 P] dCTP (Amersham, UK) 5 ⁇ 1 and Klenow enzyme solution (T0Y0B0, Japan) 2 ⁇ 1 are added, and the mixture is water-bathed at 37 ° C for 10 minutes and radiolabeled.
  • the DNA fragment of AA27857574 was synthesized. After that, a Sephadex column (Probe eQuant G-50
  • the DNA fragment was purified by Microcosm 1 umns (Pharmacia, Sweden), boiled in a water bath for 5 minutes, and cooled with ice for 2 minutes to obtain a probe.
  • the filter prepared by the above method was applied to a final concentration of each component of SSC solution with 6 times concentration, Denhardt's solution with 5 times concentration (Wako Pure Chemical Industries, Japan), 1% SDS (dodecyl sulfate sodium).
  • SSC solution Denhardt's solution with 5 times concentration
  • SDS dodecyl sulfate sodium
  • a hybridized solution containing denatured salmon sperm DNA (Sigma, USA) in a boiling water bath of 100,000 g / ml lime, Japan (Wako Pure Chemical Industries, Ltd.) And shaken at 65 ° C from 0.5 force for 1 hour. Then added 32 P-labeled probe in the hive Li Dizeshiyo down liquid, to try O by shaking 1 6 h at 6 5 ° C, Neubridization was performed.
  • the filter is immersed in a SSC solution containing 0.1% SDS containing 2% final concentration of each component, washed once at 65 ° C, and further added with 0.2% SSC.
  • the plate was washed twice with a washing solution containing 0.1% SDS at 65 ° C for 30 minutes.
  • the washed finoletter was subjected to autoradiography at 185 ° C using a sensitizing screen. As a result, he picked up the strongly exposed portion of the clone, re-plated the plaque again, performed screening twice as described above, and completely isolated the single clone.
  • the purified DNA was digested with the restriction enzyme EcoRI, and incorporated into a plasmid pBluescript II KS (+) (Stratagene, USA) similarly digested with the restriction enzyme EcoRI.
  • the DNA sequence of these clones was analyzed using a DNA sequencer to determine the sequence of DRAK1 of SEQ ID NO: 3.
  • a transformed cell E.co1i DH5 ⁇ - transformed with a plasmid hDRAKl-pBS containing cDNA encoding the entire amino acid sequence of DRAK1 of the present invention into Escherichia coli DH5a hDRAKl-pBS is Deposited with the National Institute of Advanced Industrial Science and Technology, the Ministry of International Trade and Industry of Japan on January 21, 1997 under the accession number: FE RM BP — 6180.
  • the 5 'end was closed by the following method.
  • the synthetic oligonucleotide described in SEQ ID NO: 10 prepared based on the nucleotide sequence information of AA4196612 and the adapter primer (AP-1) attached to the kit )
  • the PCR was performed for 30 cycles.
  • the synthetic oligonucleotide described in SEQ ID NO: 11 prepared from the nucleotide sequence information of AA4196612 and the adapter primer attached to the kit ( 30 cycle PCR was performed using AP-2), and the clone having the 5 'end of DRAK 2 was cloned.
  • the 3 'end was cloned.
  • the synthetic oligonucleotide described in SEQ ID NO: 12 and the AP attached to the kit were prepared based on the nucleotide sequence information of the 5 'end of DRAK2 determined previously. — 30 cyclnore PCR reactions were performed using 1 primer. This PCR product was subjected to 30-cycle PCR using ⁇ , the synthetic oligonucleotide shown in SEQ ID NO: 13 and ⁇ II-2 primer, and a clone having a DRAK2 3'-end. Was cloned.
  • DRAK2 was amplified by PCR, it was purified by Wizard (Promega, USA) and then cloned using a TA cloning kit (Novagen, USA).
  • T-vector pT7Blue (available from Novagen, USA; hereinafter referred to as T-vector) as a vector
  • mixing was performed so that the molar specific force between the vector and the DNA was S1: 3.
  • the DNA was incorporated into the vector using the Ligation kit (TaKaRa, Japan).
  • DNA is integrated Tabeku terpolymers T-vec tor E. coli DH 5 a (Japan, Ltd.
  • T0Y0B0 companies were transfected into, A down Pishi Li emissions (US, manufactured 318111 3 companies) 5 0 ⁇ Seed on a plate of L-Broth (TaKaRa, Japan) semi-solid medium containing 200 g / ml of Zml and X-gal (manufactured by Nakarai, Japan) for about 12 hours 3 7 Left at ° C.
  • the white colonies that appeared were randomly selected, inoculated in 2 ml of L-Broth liquid medium containing the same concentration of ampicillin, and cultured with shaking at 37 ° C for about 8 hours. Thereafter, the cells were collected, and after collecting the plasmid, the sequence of the integrated full-length DNA was determined.
  • the total length of human DRAK2 was finally determined, and the DNA sequence of DRAK2 of SEQ ID NO: 5 was determined.
  • a plasmid h containing a cDNA encoding the entire amino acid sequence of DRAK 2 of the present invention h DRAK 2 — pT7 is transformed into E. coli DH5a.
  • a transformed cell E.co1i: DH5 ⁇ -hDRAK2- ⁇ 7 was deposited internationally with the Ministry of International Trade and Industry of the Ministry of International Trade and Industry of Japan on January 21, 1997 under the accession number: FERMBP — 6181.
  • Northern hybridization was performed using the translation region of human DRAK1 as a probe, using Human Mul tip 1 e Tissue Northern Blot (manufactured by CLONTECH, USA). .
  • the radiolabel of the probe is Megaprim e DNA l abe l ing sys t em ( UK, Ame rsh am Inc.) and [c - 32 P] d CT P ( UK, Amersham Corp.) was used.
  • DRAK2 expression was observed in almost all tissues.
  • DRAK1 with FLAG epitope added to the N-terminal side was amplified by PCR. Amplification was performed using Amp1 iTaq (Norkin Kinmera, USA) using the synthetic oligonucleotide described in SEQ ID NO: 14 as the sense primer and the antisense primer as the antisense primer. The synthetic oligonucleotide described in No. 15 was used. PCR is performed at 94 ° C for 1 minute, followed by two cycles of 94 ° C for 30 seconds, 56 ° C for 30 seconds, and 72 ° C for 1 minute, followed by 72 ° C for 10 minutes. Was.
  • the amplified PCR product was digested with restriction enzyme Sa1I (manufactured by TOY0B0, Japan).
  • the expression vector (pEF-BOS) [Mi zush ima, S. et al., Nucl eic Acids Res., 18: 5322 (1990)], digested with restriction enzyme XbaI (TaKaRa, Japan), and then Blunting kit (TaKaRa, Japan).
  • the ends were blunted, and Sai 1 inker was connected with 1 igation kit (TaKaRa, Japan).
  • the obtained plasmid was cut with a restriction enzyme Sa1I (manufactured by T0Y0B0, Japan), and the PCR products prepared above were connected to obtain pEFBOS-FLAG-DRAK1.
  • a phosphatase activity-deficient mutant in which the 90th lysine was substituted with alanine was synthesized with the synthetic oligonucleotide shown in SEQ ID NO: 18 and a Trn product manufactured by CL0NTECH, USA.
  • An ans former Site-Directed Mutagenesis Kit was prepared according to the attached protocol to obtain pEFBOS-FLAG-DRAK1K90A.
  • the expression vector (pEF-BOS) [Mizushima, S, et al., Nuc eic Acids Res., 18: 5322 (1990)] was cleaved with restriction enzyme XbaI (TaKaRa, Japan). Thereafter, the ends were blunted with Blunt ing kit (TaKaRa, Japan), and Sail 1 inker was connected with ligat ion kit (TaKaRa, Japan).
  • the obtained plasmid is cut with restriction enzyme Sa11 (manufactured by T0Y0B0, Japan), the PCR products prepared above are connected to vectors, respectively, and pEFBOS-Myc-DRAK1, pEFBOS- My c-DRAKl 345 and pEFB0S-My c-DRAK1 321 were obtained.
  • DRAK 2 with FLAG epitope added to the N-terminal side was amplified by PCR. Amplification was performed using Amp 1 i Taq (produced by Nokinkin Elmer Inc., USA). The synthetic oligonucleotide described in SEQ ID NO: 16 was used as a sense primer, and the SEQ ID NO was used as an antisense primer. The synthetic oligonucleotide described in 17 was used. After 1 minute at 94 ° C, the PCR was performed for 2 cycles at 30 ° C for 30 seconds, 30 seconds for 56 ° C, and 1 minute for 72 ° C, followed by 10 minutes at 72 ° C. I got it.
  • the amplified PCR product was digested with restriction enzyme Sa1I (manufactured by T0Y0B0, Japan).
  • restriction enzyme Sa1I manufactured by T0Y0B0, Japan
  • pEF-BOS restriction enzyme
  • XbaI restriction enzyme
  • kit kit
  • the sail inker was connected with liga ion kit (Japan, TaKaRa).
  • the obtained plasmid was digested with restriction enzyme Sa1I (manufactured by TOYOB0, Japan), and the PCR products prepared above were connected to obtain PEFBOS-FLAG-DRAK2.
  • a phosphatase activity-deficient mutant (DRAK2K62A) in which the second lysine was substituted with alanine was synthesized with the synthetic oligonucleotide described in SEQ ID NO: 19 and a Transformer manufactured by CL0NTECH, USA.
  • the Site-Directed Mutagenes is Kit, the attached protocol (prepared in accordance therewith-pEFBOS-FLAG-DRAK2K62A was obtained.
  • Amplification of Myc—DRAK2, Myc—DRAK2 1-315, Myc—DRAK2 1—293 using Amp 1 i Taq (manufactured by Pakinkin Elma Inc., USA)
  • the synthetic oligonucleotide described in SEQ ID NO: 24 was used as a sense primer for amplification, and the synthetic oligonucleotide described in SEQ ID NO: 25 was used as an antisense primer for amplification of Myc-DRAK2.
  • Nucleotide was used as the antisense primer for amplification of Myc-DRAK21-315, and the synthetic oligonucleotide described in SEQ ID NO: 26 was used as Myc-DRAK21--2.
  • the synthetic oligonucleotide described in SEQ ID NO: 27 was used as an antisense primer for amplification of 93. PCR is performed at 94 ° C for 1 minute, followed by two cycles of 94 ° C for 30 seconds, 56 ° C for 30 seconds, and 72 ° C for 1 minute, and then for 72 ° C for 10 minutes. Was.
  • the amplified PCR product was digested with restriction enzyme Sa1I (manufactured by T0Y0B0, Japan).
  • the expression vector-[(pEF-BOS)] iz ush ima, S.
  • Wild-type DRAK 1 (pEFBOS-FLAG-DRAK1), phosphatase-deficient DRAKlK90A (pEFBOS-FLAG-DRAK1K90A), and a control (pEFBO Smock) were separately transferred to COS-17 cells (ATCC No. CR-1651). In accordance with the Huxion Law, it was introduced on a temporary basis. 36 hours later, 0.5% NP — 40, 10 mM Tris — HCl (pH 7.5), preparation of 500 jl cell extract of 150 mM NaCl The cells were solubilized in a buffer to prepare a cell extract.
  • the cells were exposed to an X-ray film using Renaissance (manufactured by DuPont, USA) to confirm the molecular weight of the expressed protein.
  • Fig. 2 (a) the FLAG sequences of the expressed wild-type DRAK1 (FLAG-DRAK1), the activity-deficient DRAK1 (FLAG-DRAK1 K90A), and the control (Vector) were used.
  • FLAG Blot western
  • identified that the molecular weight of DRAK 1 is 54 kDa.
  • Myc — DRAK 1 (p EFBOS-My c-DRAKl), Myc-DRAK l 1 — 3 4 5 (pEFBOS-y c-DRAKl 1 -345), Myc-DRAK l 1 — 3 2 1 (p EFBOS-My c-DRAKl 1-321) and a control (Vector) were each used for COS-17 cells (ATCC No. CRL-1).
  • the cells were solubilized in a 500 / xl cell extract preparation buffer consisting of (pH 7.5) and 150 mM NaCl, and a cell extract was prepared.
  • a 500 / xl cell extract preparation buffer consisting of (pH 7.5) and 150 mM NaCl
  • a cell extract was prepared.
  • Add 20 ⁇ l of Laemml is amp 1 eb uf fer (manufactured by TEFCO, Japan) to 20 ⁇ l of this cell extract, and add it at 94 ° C for 5 minutes.
  • Wild-type DRAK 2 (PEFB0S-FLAG-DRAK2), phosphatase-deficient DRAK2K62A (pEFBOS-FLAG-DRAK2K62A), and control (pEFBO Smock) were each separately transferred to COS-7 cells (ATCC No. CRL-1651). In accordance with the Huxion Law, it was introduced on a temporary basis. After 36 hours, 0.5% NP — 40, 10 mM Tris — HCl (pH 7.5), 500 mM NaCl, extraction of 501 cells The cells were solubilized in a liquid preparation buffer to prepare a cell extract.
  • the filter was reacted with an HRP-labeled anti-Myc antibody (manufactured by Invitrogen, The Netherlands) diluted 100-fold, washed with TBS-0.5% Tween20, and renaisance (US, DuPont) was used to expose the X-ray film, and the molecular weight of the expressed protein was determined.
  • HRP-labeled anti-Myc antibody manufactured by Invitrogen, The Netherlands
  • TBS-0.5% Tween20 washed with TBS-0.5% Tween20
  • renaisance US, DuPont
  • Blocking was performed for 60 minutes at room temperature using (1). Next, perform primary staining for 60 minutes with the anti-01 ⁇ 2 antibody (manufactured by Kodak Company, USA) diluted 300 times, and wash three times with PBS (-).
  • FITC-conjugated anti-mouse antibody U.S.A., manufactured by Bio Source International Inc.-Tago Products
  • DAPI 6-diamidino-2-phenyl indole
  • AX80 (Olympus, Japan) used for microscopic observation For detection of DRAK1 and DRAK2, excitation wavelength of 470 to 490 nm for detecting FITC, excitation wavelength of 515 to 550 nm, and excitation of DAPI for staining nuclei are detected. Wavelengths of 345 to 364 nm and detection wavelengths of 455 to 461 nm were used.
  • DRAK1 is a phosphorylase obtained based on the genetic information of DAP kinase. Since DAP kinase induces apoptosis in HeLa cells, it was confirmed whether DRAK1 has apoptosis-inducing activity.
  • Wild-type DRAK 1 (pEFBOS-FLAG-DRAK 1), phosphatase-deficient DRAK1K90A (pEFBOS-FLAG-DRAK1K90A), and control (pEFBO Smock) were each used to express the LacZ expression vector (pEFBOS-LacZ).
  • pEFBOS-LacZ phosphatase-deficient DRAK1K90A
  • pEFBO Smock pEFBO Smock
  • Fig. 4 (a) The ratio of cells that produced wild-type DRAK 1 or DRAK 1K90A lacking phosphatase activity (that is, cells that developed color) was compared to the control cells, respectively. Compared. The results are shown in Fig. 4 (a). As shown in Fig. 4 (a), wild-type DRAK 1 exhibited a typical apoptosis morphology with nuclear aggregation. The percentage of cells showing apoptosis morphology due to the introduction of wild-type DRAK1 and deletion-type DRAK1 was 25.3% and 2.1%, respectively.
  • DRAK2 is a phosphorylase obtained based on the genetic information of DAPkinase. Since DAPnase induces apoptosis in HeLa cells, it was confirmed whether DRAK2 has apoptosis-inducing activity.
  • Wild-type DRAK 2 (pEFB0S-FLAG-DRAK2), phosphatase-deficient DRAK2K62A (pEFBOS-FLAG-DRAK2K62A), and control (pEFBO Smock) were each used for the LacZ expression vector (pEFBOS-LacZ).
  • transiently transfected into NIH 3T3 cells (ATCC No. CRL-1658) using Trans IT LT-1 (TaKaRa, Japan) according to the lipofusion method did.
  • X-ga 1 staining was performed 36 hours after the introduction.
  • the DRAK 1 phosphorylase domain (SEQ ID NO: 3 from positions 61 to 32 3) has high homology to the calmodulin-dependent phosphorylase family (CaM kinase fami i y). In the comparison of the amino acid sequences of DAP kinase and DRAK1 as described above, it was found that the phosphorylase domain was conserved in DRA ⁇ 1. Figure 6 shows a comparison of the amino acid sequences. Therefore, the phosphorylase activity of DRAK1 was directly confirmed.
  • Wild-type DRAK 1 (pEFBOS-FLAG-DRAK 1), phosphatase-deficient DRAKlK90A (pEFBOS-FLAG-DRAK1K90A), and control (pEFBO Smock) were each transferred to COS-7 cells (ATCC No. CRL-1651). Introduced in accordance with the Poffex Law. After 36 hours, 0.5% NP—40, 10 mM Tris—HC 1 (pH 7.5), 500 mM NaCl, 500 / l cell extract The cells were solubilized with the preparation buffer to prepare a cell extract.
  • the non-specifically adsorbed protein is removed twice from the cell extract using 50 ⁇ l of Protein G agarose beads (Pharmacia, Sweden) on the cell extract of 100 ju1 according to a standard method.
  • Lg anti-FLAG antibody Kodak, USA
  • 50 ⁇ l of Protein G agarose beads Pharmacia, Sweden
  • Beads 0 ⁇ l were reacted overnight at 4 ° C.
  • the beads after the reaction were washed three times with a cell extract preparation buffer 1003 to prepare an immunoprecipitate.
  • Wild-type FLAG-DRAK2 (p EFB0S-FLAG-DRAK2), FLAG-DRAK2K62A (pEFBOS_FLAG-DRAK2K62A) lacking phosphorylation enzyme activity, and a control vector (pEFBOSmock) were each treated with COS — 7 cells (ATCC number CR / 1651). ) was temporarily introduced in accordance with the Lipofyxion Law. After 36 hours, 0.5% NP — 40, 10 mM Tris — HCl (pH 7.5), 500 mM NaCl, 500 ⁇ l cell extract The cells were solubilized with the preparation buffer to prepare a cell extract.
  • Peptides from Asn at position 353 to Ser at position 364 of SEQ ID NO: 3 were synthesized, and immunized to rabbits as an immunogen, and the antibody titer was measured. Thereafter, whole blood is collected, serum is collected, and an anti-human DRAK1 protein is used in an Econopack serum IgG purification kit (Bio-Rad, USA) according to the attached instruction manual. The local antibody was purified.
  • Peptides from Arg at position 319 to Asn at position 331 in SEQ ID NO: 6 were synthesized, and immunized to rabbits as an immunogen, and the antibody titer was measured. Thereafter, whole blood is collected, serum is collected, and an anti-human DRAK2 protein and a heron polyclonal antibody are purified using an Econopack serum IgG purification kit manufactured by Bio-Rad, USA, according to the attached instruction manual. Was purified and produced.
  • Wild-type DRAK1 (pEFBOS-FLAG-DRAK1) was introduced into COS-7 cells (ATCC No. CR-1651) in a reproducible manner according to the lipofiction method. After 36 hours, 0.5% NP_40, 50 mM MTris-HC1 (pH 7.5), 500 mM cell extract consisting of 150 mM MNaCl The cells were solubilized in a preparation buffer to prepare a cell extract. Next, an immunoprecipitate was prepared from the cell extract using an anti-FLAG antibody (manufactured by Kodak, USA) and Protein Gagarose beads (manufactured by Pharmacia, Sweden) according to a standard method.
  • an anti-FLAG antibody manufactured by Kodak, USA
  • Protein Gagarose beads manufactured by Pharmacia, Sweden
  • Chelerythrin Chloride (manufactured by CALB I OCHEM, USA), which is commercially available as a phosphatase inhibitor, was used as an evaluation compound, and the concentration of DMSO (manufactured by GIBO BRL, USA) was 10 mM. Dissolved. This was used as a stock solution and diluted with distilled water to prepare a solution of 400 mg. For comparison, a solution obtained by diluting DMSO with distilled water so as to have the same concentration as the evaluation concentration was used.
  • the degree of phosphorylation of the protein was determined by measuring the degree of blackening of the film, specifically BAS-200 Bio. Imaging Analyzer (Fuji Photo Film, Japan) The amount of radioactivity when a DMSO solution containing no evaluation compound was used was used as a control, and the inhibitory activity was determined based on the decrease rate. Concentration) does not show inhibitory activity Was.
  • Wild-type DRAK2 (PEFB0S-FLAG-DRAK2) was transiently introduced into COS-7 cells (ATCC No. CRL-1651) according to the lipofection method. After 36 hours, 0.5% NP-40, 10 mM MTris-HC 1 (pH 7.5), 150 mM NaCl force, extraction of 500 ⁇ l of cells The cells were solubilized with a liquid preparation buffer to prepare a cell extract. Next, an immunoprecipitate was prepared from the cell extract using an anti-FLAG antibody (Kodak, USA) and Protein Gagarose beads (Pharmacia) according to a standard method.
  • Chelerythrin Chloride (manufactured by CALBIOCHEM, USA), which is commercially available as a phosphatase inhibitor, was used as an evaluation compound and dissolved in DMSO (GIBO BRL) to 10 mM. This was used as a stock solution and diluted with distilled water to prepare a solution of 400 ⁇ ⁇ . For comparison, a solution prepared by diluting DM SQ with distilled water so as to have the same concentration as the evaluation concentration was used. (3) Method for measuring inhibitory activity
  • an immunoprecipitate was prepared.
  • the non-specifically adsorbed protein was removed twice using 100 ⁇ l of cell extract and 50 ⁇ l of Protein G agarose beads (Pharmacia, Sweden) according to the standard method from the cell extract. went.
  • the cell extract (50 ⁇ l) subjected to the non-specifically adsorbed protein removal treatment was reacted with anti-c-Myc (9E10) -Agarose (Santa Cruz, USA) 101 at 4 ° C. overnight. Agarose after the reaction was washed three times with 100 ⁇ l of cell extract preparation buffer to obtain an immunoprecipitate.
  • the protein after reaction is developed by SDS polyacrylamide gel electrophoresis using 4 to 20% gradient polyacrylamide gel (manufactured by TEFC0, Japan), and the gel is dried to dryness. -Attached to radiography. Then, Myc — DRAK 1, Myc-DRAKl 1 — 345, Myc — DRAK1 1 — 321, which are about 54 kDa, about 45 kDa, about 42
  • the degree of blackening of the kDa protein or the degree of blackening of the MLC protein of about 21 kDa was measured to detect autophosphorylation activity or phosphorylation activity for each MLC. The results are shown in the upper part of Fig. 7 (a). As shown in the upper part of FIG.
  • an immunoprecipitate was prepared.
  • the non-specifically adsorbed protein was removed twice using 100 ⁇ l of cell extract and 50 ⁇ l of Protein G agarose beads (Pharmacia, Sweden) according to the standard method from the cell extract. went. 50 ⁇ l of the cell extract subjected to the non-specifically adsorbed protein removal treatment and 10 ⁇ l of anti-c-Myc (9E10) -Agarose (manufactured by Santa Cruz, USA) were reacted overnight at 4 ° C. The Agarose after the reaction was washed three times with a cell extract preparation buffer 1003 to obtain an immunoprecipitate.
  • Laemml i samp 1 e buiie r manufactured by TEFCO, Japan
  • the phosphorylation reaction was stopped by treating at 94 ° C for 5 minutes.
  • the protein after reaction is developed by SDS polyacrylamide gel electrophoresis using 4-20% gradient polyacrylamide gel (manufactured by TEFC0, Japan), and the gel is evaporated to dryness. The radiography was attached.
  • PT7B 1 ue T-vector, manufactured by Novagen, USA
  • the vector and the DNA were mixed so that the molar ratio thereof was 1: 3, and Ligat was used.
  • DNA was incorporated into the vector using an ion kit (TaKaRa, Japan).
  • the vector T-vector incorporating the DNA was transfected into Escherichia coli DH5a (manufactured by T0Y0B0, Japan), and ampicillin (manufactured by Sigma, USA) was added to 50 g Zml and X-gal ( L-Broth (manufactured by Nakarai Co., Ltd., Japan) containing 200 ⁇ g / m1 Seed on a semi-solid medium plate (manufactured by TaKaRa, Japan) and left at 37 ° C for about 12 hours did.
  • the white colonies that appeared were randomly selected, inoculated in 2 ml of L-Br0th liquid medium containing the same concentration of ampicillin, and cultured with shaking at 37 ° C for about 8 hours. Thereafter, the cells were collected, the plasmid was isolated using Withomid prep (Promega, USA) according to the attached instructions, and the plasmid was separated with the restriction enzyme Ec0RI (Japan, Japan). (T0Y0B0) and restriction enzyme Sa1I (T0Y0B0, Japan). Approximately 300 bp of DNA was excised, confirming that the above PCR product was incorporated into the vector. The nucleotide sequence of the incorporated cDNA was determined for the clone in which the retention of the PCR product was confirmed.
  • the nucleotide sequence of the imported cDNA fragment is determined by the Applied Bi The measurement was performed using a fluorescent sequencer manufactured by O. Systems. Sequence samples are prepared using PRISM, Ready Reaction Dye Term
  • the probe labeled with the radioactive isotope 32 P was prepared as follows.
  • the 6th to 304th base sequence of R19772 is restricted to EcoRI (Toyobo, Japan) by the vector ⁇ - ⁇ ecr in which this sequence is incorporated.
  • the cells were cut out with the enzyme Sa1I (manufactured by TOYOBO, Japan) and electrophoresed in a 1.0% agarose 'gel. After staining with Etchizumbu Mide (Made in Japan, Nippon Gene Co., Ltd.), the cells were observed under ultraviolet light to obtain about 300 The bp band was cut out from the gel and purified using Wizard (produced by Promega, USA).
  • the obtained DNA fragment was labeled using a DNA labeling kit (Megaprime DNA labeling system: manufactured by Amersham Co., Ltd.). To 10 to 50 ng / jul of DNA, add 5 ⁇ l of primer solution and deionized water to bring the total volume to 33 ⁇ l, perform a boiling water bath for 5 minutes, and then add 5 ⁇ l of reaction solution. , [a - 32 P] dCTP ( UK, Amersham Corp.) 5/1, and K 1 Enow enzyme solution (Japan, T0Y0B0 companies, Ltd.) was added to 2 mu 1, and a water bath for 10 minutes at 3 7 ° C Then, a radiolabeled R19772 DNA fragment was synthesized.
  • a DNA labeling kit Megaprime DNA labeling system: manufactured by Amersham Co., Ltd.
  • the DNA fragment was purified using a Sephadex column (ProbeQuant G-50 Microcolumns: Pharmacia), and after boiling in a boiling water bath for 5 minutes, ice-cooling for 2 minutes to obtain a probe.
  • the filter prepared by the above method was applied to a SSC solution with a final concentration of 6 times for each component, Denhardt's solution with 5 times the concentration (Wako Pure Chemical Industries, Japan), 1% SDS (Japan, Japan).
  • the product is immersed in a hybridization solution containing denatured salmon sperm DNA (manufactured by Sigma, USA) in a boiling water bath of 100 g / ml and a boiling water bath of 100 g / ml, and is immersed at 0.5 to 1 at 65 ° C. Shake time. Then added 32 P-labeled probe hybridized Daizeshi tio down liquid, to try O by shaking 1 6 h at 6 5 ° C, the hive re Dizesho down line ivy.
  • the purified DNA was digested with the restriction enzyme EcoRI (manufactured by TOYOBO, Japan), and similarly, the plasmid pBluescript II KS (+) digested with the restriction enzyme EcoRI (manufactured by TOYOBO, Japan) ) (Manufactured by Stratagene, Japan).
  • the DNA sequence of these clones was analyzed by a DNA sequencer to determine the full-length sequence of Trad, and was described in SEQ ID NO: 28.
  • the Tradc DNA integrated into Notlsite of plasmid pBluescr ipt 11 KS (+) (Stratagene, USA) was used as type II, and FLAG epitope was added to the N-terminal side.
  • the rad was amplified by PCR. Amplification was performed using Amplitaq (manufactured by Perkin Elmer, Inc., USA), and a synthetic oligonucleotide, a sense primer described in SEQ ID NO: 33 and an antisense primer described in SEQ ID NO: 34, were used. .
  • PCR was performed at 94 ° C for 1 minute, followed by 94 ° C for 30 seconds, 60 ° C for 30 seconds, and 72 ° C for 1 minute for 30 cycles.
  • a phosphorylase-deficient mutant in which the 106th lysine was substituted with alanine was synthesized with CL0NTECH, USA, using the synthetic oligonucleotide of SEQ ID NO: 35.
  • Transformer Site—Di rected Mutagenes is Kit was prepared according to the protocol described in the attached, and pEFBOS-FLAG-hTradK1016A was obtained.
  • the prepared expression vector (PEFBOS-FLAG-hTrad, pEFBOS-FLAG-hTradK1016A) was introduced into Escherichia coli DH5a (manufactured by TOYOBO, Japan) to obtain a transformant.
  • ⁇ - ⁇ EFBOS-FLAG- hT rad was submitted to the Ministry of International Trade and Industry of Japan at the Institute of Biotechnology and Industrial Technology, on March 19, 1999 under the accession number: FE RM BP—6301. International deposit.
  • Wild-type T rad (pEFBOS-FLAG-hTrad), phosphatase deficient 3 (11 (1016 £? 803-? 1 ⁇ 0-11 ⁇ 3 (111016)), and control (pEFB OSmock) were used as COS _ Transiently transfected into 7 cells (ATCC No.
  • NP — 40,1 Cells were solubilized in a 500 ⁇ l cell extract preparation buffer consisting of 0 mM Tris-HC1 (pH 7.5) and 150 mM NaCl, To this cell extract (20 ⁇ l) was added 20 ⁇ l of Laemm1 i sample buffer (manufactured by TEFCO, Japan), and the mixture was treated at 94 ° C. for 5 minutes.
  • the rad phosphorylase domain (SEQ ID NO: 29, 987 to 1241) is highly associated with the calmodulin-dependent phosphorylase family (Cam kinase family). Compared to the amino acid sequences of DAP kinase and T rad, which have homology, it was found that the phosphatase domain was conserved in T rad (see Figure 10). ). Therefore, the phosphatase activity of Trad was directly confirmed. Wild-type FLAG- T rad (p EFBOS-FLAG -hT rad), ⁇ I ⁇ Motokatsu ten students deletion type 1? 1 ⁇ 6-1 ⁇ 3 (111,016 eight £?
  • NP — 40 10 mM Tris — HC 1 (pH 7.5)
  • Preparation of 50,000 ⁇ 1 cell extract solution of 150 mM NaC 1 A cell extract was prepared. Next, 100 ⁇ l of the cell extract was treated twice with 50 ⁇ l of Protein G agarose beads (manufactured by FANORE Masia, Sweden) according to a standard method from the cell extract to remove nonspecifically adsorbed proteins twice. went.
  • Phosphorylated buffer 2 consisting of 50 mM Tris_HCl (pH 7.0), 10 mM MgCl 2, and 3 mM MMnCl2 in 20 ⁇ l of immunoprecipitate 0 mu 1 and the 1 0 ⁇ C i [ ⁇ - 32 P] ATP ( UK, Amersham Corp.) was added and promote phosphorylation reaction was incubated for 15 minutes at 3 0 ° C. Thereafter, 20 ⁇ l of Laemml sample le buf fer (manufactured by TEFC 0, Japan) was added, and the mixture was treated at 94 ° C. for 5 minutes to stop the phosphorylation reaction.
  • Laemml sample le buf fer manufactured by TEFC 0, Japan
  • the cells were washed three times with PBS (-), and then blocked with PBS (-) containing 3% BSA (SIGMA, USA) at room temperature for 60 minutes.
  • PBS (-) containing 3% BSA SIGMA, USA
  • primary staining was carried out at room temperature for 60 minutes using lO iUg Zml anti-FLAG antibody (manufactured by Kodak Company, USA) and PBS (-) containing 3% BSA.
  • AX80 (Olympus, Japan) was used for microscopic observation, and the excitation wavelength for detecting FITC was 470-490 nm and the detection wavelength was 515- A 550 nm finalizer was used.
  • a filter having an excitation wavelength of 52 to 550 nm for detecting rhodamin and a detection wavelength of 580 nm or more was used.
  • the peptide synthesis described above was performed by the Fmoc solid-phase synthesis method [Shinsei Kagaku Lecture Course 1 ⁇ Protein VI synthesis and expression, published by Tokyo Chemical Dojin (Japan)], and 2 mg of the obtained synthetic peptide was used.
  • An equivalent amount of the carrier protein KLH (keyhole_l impet hemocyani) (manufactured by PIERCE, USA) was conjugated by the maleimide method and used as an antigen.
  • One 2.5 kg heron NZW, Japan, SLE, Japan
  • Wild-type Trad (pEFBOS-FLAG-hTrad) was transfected into COS-7 cells (AT CC No. CR-1651) by the lipofection method (Mirus, USA). After 36 hours, 0.5% NP — 40,10 mM MTris — HC 1 (pH 7.5), 150 mM M NaC 1 500 ⁇ l cell extract The cells were solubilized using a preparation buffer to prepare a cell extract. Next, immunoprecipitates were prepared from the cell extract using an anti-FLAG antibody (manufactured by Kodak Company, USA) and Protein G agarose beads (manufactured by Pharmacia, Sweden) according to a standard method.
  • an anti-FLAG antibody manufactured by Kodak Company, USA
  • Protein G agarose beads manufactured by Pharmacia, Sweden
  • Chelerythine Chloride (manufactured by CALBIOCHEM, USA), which is commercially available as a phosphatase inhibitor, was used as an evaluation compound and dissolved in DMSO (manufactured by GIBCO BRL, USA) to 10 mM. This solution was used as a stock solution, diluted with distilled water to prepare a solution of 400 ⁇ , and used. For comparison, DMS ⁇ was diluted with distilled water to the same concentration as the evaluation concentration.
  • La emm 1 isample b uiier (manufactured by TEFCO, Japan) was given 201 calories and treated at 94 ° C for 5 minutes to stop the phosphorylation reaction.
  • a 4 to 20% gradient polyacrylamide gel (manufactured by TEFC0, Japan)
  • the Trad after the reaction was developed by SDS polyacrylamide gel electrophoresis.
  • the gel was dried and the degree of autophosphorylation of about 150 kDa protein, which was T rad, was determined by autoradiography by measuring the degree of blackening of the film. .
  • the radioactivity was measured using a BAS-20000 bio-imaging analyzer (manufactured by Fuji Photo Film Co., Ltd., Japan).
  • the novel oxidative enzyme of the present invention By using the novel oxidative enzyme of the present invention, a drug useful for the prevention or treatment of an apoptosis-related disease can be prepared. Furthermore, the oxidative enzyme of the present invention is useful for establishing a method for screening apoptosis-regulating substances and a method for diagnosing apoptosis-related diseases. In addition, the gene encoding the phosphorylase of the present invention is also useful as a gene source used for gene therapy.

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Abstract

L'invention concerne: une nouvelle kinase ayant une activité induisant l'apoptose, ainsi qu'un ADN codant pour cette kinase, cette dernière permettant d'obtenir des médicaments utiles dans la prévention et le traitement de maladies associées à l'apoptose, et pouvant également être utilisée pour l'établissement d'un procédé de criblage de substances régulant l'apoptose et d'un procédé de diagnostic de maladies associées à l'apoptose; un ADN constitué d'au moins douze bases de la séquence des bases de l'ADN susmentionné, ou du brin complémentaire ou bien d'un dérivé de cet ADN; un ADN de recombinaison pouvant être répliqué, construit par intégration de l'ADN susmentionné ou de son brin complémentaire dans un vecteur d'expression pouvant être répliqué; un micro-organisme ou une cellule transformés par cet ADN de recombinaison pouvant être répliqué; un procédé de criblage de substances diminuant ou potentialisant au moins une des activités de la kinase susmentionnée; et un anticorps pouvant se lier à ladite kinase.
PCT/JP1998/005974 1997-12-26 1998-12-25 Nouvelle kinase Ceased WO1999033961A1 (fr)

Priority Applications (1)

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AU16914/99A AU1691499A (en) 1997-12-26 1998-12-25 Novel kinase

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JP36764097 1997-12-26
JP9/367640 1997-12-26
JP9/367641 1997-12-26
JP36764197 1997-12-26
JP10/108150 1998-04-17
JP10/108149 1998-04-17
JP10814998 1998-04-17
JP10815098 1998-04-17

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000073469A3 (fr) * 1999-05-28 2001-11-29 Sugen Inc Proteines kinases
WO2008148216A1 (fr) * 2007-06-06 2008-12-11 Centre Hospitalier De L'universite De Montreal Expression de drak2 associée au diabète

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5683910A (en) * 1996-09-13 1997-11-04 Incyte Pharmaceuticals, Inc. Human phosphorylase kinase gamma subunit

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5683910A (en) * 1996-09-13 1997-11-04 Incyte Pharmaceuticals, Inc. Human phosphorylase kinase gamma subunit
US5833981A (en) * 1996-09-13 1998-11-10 Incyte Pharmaceuticals, Inc. Human phosphorylase kinase gamma subunit

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000073469A3 (fr) * 1999-05-28 2001-11-29 Sugen Inc Proteines kinases
WO2008148216A1 (fr) * 2007-06-06 2008-12-11 Centre Hospitalier De L'universite De Montreal Expression de drak2 associée au diabète

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