Apoptosis-inducing protein and gene encoding the same

An object of the present invention is to provide a protein which induces apoptosis, a base sequence encoding the protein, and an agent for use in the treatment of malignant tumors. The present invention is a protein (ASK1) which has protein kinase activity and enhances SEK1 kinase activity and/or MKK3 kinase activity, or a derivative thereof. Malignant tumors can be treated using the protein according to the present invention or the base sequence encoding the protein.

BACKGROUND OF THE INVENTION
 1. Field of the Invention
 The present invention relates to a protein which induces apoptosis (cell
 death), a gene encoding the same, and a therapeutic agent for malignant
 tumors.
 2. Background Art
 The mitogen-activated protein (MAP) kinase signaling cascade, a signal
 transduction pathway well conserved in cells from yeasts to vertebrates,
 consists of three distinct members of the protein kinase family, including
 MAP kinase (MAPK), MAPK kinase (MAPKK), and MAPKK kinase (MAPKKK) (T.
 Sturgill & J. Wu, Biochim. Biophys. Acta. 1092, 350, 1991; E. Nishida & Y.
 Gotoh, Trends Biochem. Sci., 18, 128, 1993; B. Errede & D. Levin, Curr.
 Opin. Cell Biol., 5, 254, 1993; C. Marshall, Curr. Opin. Genet. Dev., 4,
 82, 1994). MAPKKK phosphorylates and thereby activates MAPKK, and the
 activated form of MAPKK in turn phosphorylates and activates MAPK.
 Activated MAPK translocates to the cell nucleus and regulates the
 activities of transcription factors and thereby controls expression of
 various genes (T. Sturgill & J. Wu, Biochim. Biophys. Acta, 1092, 350,
 1991; E. Nishida & Y. Gotoh, Trends Biochem. Sci., 18, 128, 1993; B.
 Errede & D. Levin, Curr. Opin. Cell Biol., 5, 254, 1993; C. Marshall,
 Curr. Opin. Genet. Dev., 4, 82, 1994).
 Recent studies on MAPK signal transduction pathways have shown that at
 least two distinct MAPKKK-MAPKK-MAPK signal transduction pathways function
 in mammalian cells (R. Davis, Trends Biochem. Sci., 19, 470, 1994; A.
 Waskiewicz & J. Cooper, Curr. Opin. Cell Biol., 7, 798, 1995; J. Kyriakis
 & J. Avruch, J. Biol. Chem., 265, 17355, 1990; B. Derijard et al., Cell,
 76, 1025, 1994; M. Yan et al., Nature, 372, 798, 1994; K. Yamaguchi et
 al., Science, 270, 2008, 1995; J. Kyriakis et al., Nature, 369, 156, 1994;
 I. Sanchez et al., Nature, 372, 794, 1994; B.
 Derijard et al. Science, 267, 682, 1995; S. Matsuda et al., J.
 Biol. Chem., 270, 12781, 1995). These two pathways each consist of the
 Raf-MAPKK-MAPK pathway and the MEKK-SEK1 (or MKK4)-SAPK (or JNK) pathway.
 MKK3/MAPKK6 (or MKK6, a close relative of MKK3) and p38 protein kinase are
 protein kinases corresponding to MAPKK and MAPK, respectively, and are
 known to form another MAPK signal transduction pathway (R. Davis, Trends
 Biochem. Sci., 19, 470, 1994; A. Waskiewicz & J. Cooper, Curr. Opin. Cell
 Biol., 7, 798, 1995; J. Han et al., J. Biol. Chem., 271, 2886, 1996; J.
 Raingeaud et al., Mol. Cell. Biol., 16, 1247, 1996; T. Moriguchi et al.,
 J. Biol. Chem., 271, 13675, 1996).
 Recent studies suggest that the SAPK and/or p38 MAP kinase signaling
 cascades are involved in at least a part of the signal transduction
 pathways which induce apoptosis (Z. Xia et al., Science, 270, 1326, 1995;
 Y. -R. Chen et al., J. Biol. Chem., 271, 631, 1996; N. Johnson et al., J.
 Biol. Chem., 271, 3229, 1996; M. Verheij et al., Nature, 380, 75, 1996).
 Apoptosis herein means cell death different from necrosis, namely program
 cell death. In apoptosis, DNA in each nucleosome is fragmented and the
 fragmented DNAs can be observed like a ladder by electrophoresis.
 Furthermore, apoptosis is considered to be involved in autoimmune
 diseases, HIV infection, neurotic diseases, hepatitis, leukemia, renal
 diseases, skin diseases, eye diseases and aging as well as cancer
 degeneration ("Forefront of Research on Apoptosis." Ed. Masayuki Miura,
 Shigenobu Toya and Sadatoshi Kizaki, Experimental Medicine, Vol. 13,
 1995).
 Tumor necrosis factor-.alpha. (TNF-.alpha.) is known to be a strong
 cellular apoptosis initiation substance. A recent study has shown that
 such cellular apoptosis initiation substances activate the SAPK signal
 transduction system (J. Kyriakis et al., Nature, 372, 794, 1994; J.
 Raingeaud et al., J. Biol. Chem., 270, 7420, 1995).
 However, as far as the inventors know, proteins corresponding to MAPKKK
 present in upstream of the MKK3-p38 pathway and the SEK1-SAPK pathway,
 mechanisms of activation of these pathways, and mechanisms of apoptosis
 through these pathways have not been reprted.
 SUMMARY OF THE INVENTION
 The inventors have now identified a novel mammalian protein (ASK1)
 corresponding to MAPKKK, which activates the MKK3-p38 signal transduction
 pathway as well as the SEK1-SAPK signal transduction pathway. The
 inventors have also found that proinflammatory cytokines activate ASK1 and
 the activated ASK1 is involved in cellular induction of apoptosis through
 the SEK1-SAPK and MKK3-p38 signaling cascades. Furthermore, the inventors
 found that a dominant-negative mutant of ASK1 inhibits apoptosis induced
 by TNF-.alpha.. The present invention is based on these findings.
 Accordingly, an object of the present invention is to provide a protein
 which induces apoptosis, a base sequence encoding the protein, a vector
 comprising the base sequence, a host comprising the vector and a method
 for producing the protein.
 Another object of the present invention is to provide an agent for use in
 the treatment of malignant tumors or a gene therapy agent for use in the
 treatment of malignant tumors.
 A further object of the present invention is to provide a partial peptide
 of the apoptosis-inducing protein and an antibody against the
 apoptosis-inducing protein.
 The protein according to the present invention is a protein which has a
 protein kinase activity and enhances the SEK1 kinase activity and/or MKK3
 kinase activity, or derivatives thereof.

DETAILED DESCRIPTION OF THE INVENTION
 Definition
 The term "amino acid" in the present invention includes both optical
 isomers, i.e., the L-isomer and the D-isomer. Thus, the term "protein"
 herein means not only proteins constituted solely by L-amino acids but
 also proteins comprising D-amino acids in part or in total.
 Furthermore, the term "amino acid" herein includes not only the twenty
 .alpha.-amino acids which constitute natural proteins but also other
 .alpha.-amino acids as well as .beta.-, .gamma., and .delta.-amino acids,
 non-natural amino acids, and the like. Thus, amino acids with which
 proteins are substituted or amino acids inserted into proteins as shown
 below are not restricted to the twenty .alpha.-amino acids which
 constitute natural proteins but may be other .alpha.-amino acids as well
 as .beta.-, .gamma.- and .delta.-amino acids, non-natural amino acids, and
 the like. Such .beta.-, .gamma. and .delta.-amino acids include
 .beta.-alanine, .gamma.-aminobutyric acid or ornithine. The amino acids
 other than those constituting natural proteins, or the non-natural amino
 acids include 3,4-dihydroxyphenylalanine, phenylglycine,
 cyclohexylglycine, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid or
 nipecotinic acid.
 In this specification, a specific mutation is notated by the original amino
 acid residue (one letter) first, the position number second and the amino
 acid residue after substitution (one letter) third. For example, "K709R"
 means K (Lys: lysine), the amino residue at position 709, is substituted
 by R (Arg: arginine).
 The term "protein" as used herein includes peptides. Furthermore, the
 expression "protein according to the present invention" includes
 derivatives of the protein.
 Apoptosis-inducing Protein
 The apoptosis-inducing protein according to the present invention is a
 protein which has a protein kinase catalytic region and enhances SEK1
 kinase activity and/or M3 kinase activity, or derivatives thereof.
 The apoptosis-inducing protein is not specifically restricted to any source
 but it may be derived from a mammal including a human, or any other
 sources.
 The apoptosis-inducing protein has protein kinase activity. The term
 "protein having protein kinase activity" in the present invention means a
 protein which is evaluated by those skilled in the art to have protein
 kinase activity, for example, a protein which is evaluated to have protein
 kinase activity when examined under the same conditions as in Example 1.
 The term "protein kinase activity" includes serine/threonine protein
 kinase activity.
 The apoptosis-inducing protein enhances the SEK1 kinase activity and/or
 MKK3 kinase activity. The term "protein enhancing the SEK1 kinase activity
 and/or M3 kinase activity" in the present invention means a protein which
 is evaluated by those skilled in the art to enhance these activities, for
 example, a protein which is evaluated to enhance the SEK1 kinase activity
 and/or MKK3 kinase activity when examined under the same conditions as in
 Examples 3, 4 and 6.
 The protein according to the present invention is characterized by the
 induction of apoptosis. This apoptosis is mediated through the enhancement
 of the SAPK or JNK and/or p38 activity.
 The enhancement of the SEK1 kinase activity and/or MKK3 kinase activity by
 the protein according to the present invention is accelerated by tumor
 necrosis factors (TNFs). An example of the tumor necrosis factor herein is
 TNF-.alpha..
 The term "derivatives of proteins" as used herein includes proteins in
 which the amino groups at the amino terminals (N-terminals) or all or a
 part of the amino groups of the side chains of the amino acids, and/or the
 carboxyl groups at the carboxyl terminals (C-terminals) or all or a part
 of the carboxyl groups of the side chains of the amino acids, and/or the
 functional groups other than the amino groups and carboxyl groups of the
 side chains of the amino acids (e.g., hydrogen, thiol group and amido
 group) have been modified by other appropriate substituents. The
 modification by other appropriate substituents is carried out, for
 example, to protect functional groups in the protein, to improve safety
 and tissue-translocation of the protein or to enhance protein activity.
 The derivatives of the proteins include:
 (1) proteins in which a part or all of the hydrogen atoms of the amino
 groups at the amino terminals (N-terminals) or the amino groups of the
 side chains of the amino acids are replaced by substituted or
 unsubstituted alkyl groups (which may be straight chain, branched chain,
 or cyclic) (e.g., methyl group, ethyl group, propyl group, isopropyl
 group, isobutyl group, butyl group, t-butyl group, cyclopropyl group,
 cyclohexyl, and benzyl group), substituted or unsubstituted acyl groups
 (e.g., formyl group, acetyl group, caproyl group, cyclohexylcarbonyl
 group, benzoyl group, phthaloyl group, tosyl group, nicotinoyl group, and
 piperidinecarbonyl group), urethane-type protective groups (e.g.,
 p-nitrobenzyloxycarbonyl group, p-methoxybenzyloxycarbonyl group,
 p-biphenylisopropyl-oxycarbonyl group, and t-butoxycarbonyl group), or
 urea-type substituents (e.g., methylaminocarbonyl group, phenylcarbonyl
 group, and cyclohexylaminocarbonyl group).
 (2) proteins in which a part or all of the carboxyl groups at the carboxyl
 terminals (C-terminals) or the carboxyl group of the side chains of the
 amino acids are esterified (for example, the hydrogen atom(s) are replaced
 by a methyl group, ethyl group, isopropyl group, cyclohexyl group, phenyl
 group, benzyl group, t-butyl group, or 4-picolyl group), or amidated (for
 example, unsubstituted amides or C1-C6 alkyl amides (e.g., methylamides,
 ethylamides and isopropylamides) are formed); and
 (3) proteins in which a part or all of the functional groups other than the
 amino groups and carboxyl groups of the side chains of the amino acids
 (e.g., hydrogen, thiol group and amino group) are modified by substituents
 similar to those for the abovementioned amino groups or a trityl group.
 Examples of the protein according to the present invention include proteins
 comprising the amino acid sequence of SEQ ID NO: 1 having one or more
 additions, insertions, substitutions and/or deletions, which have protein
 kinase activity and which enhance SEK1 kinase activity and/or MKK3 kinase
 activity. The terms "addition," "insertion," "substitution" and "deletion"
 as used herein refer to those that do not damage the capacity to enhance
 the protein kinase activity and SEK1 kinase activity and/or MKK3 kinase
 activity of the protein comprising the amino acid sequence of SEQ ID NO:
 1. One or more additions, insertions, substitutions and deletions can be
 introduced.
 The protein according to the present invention is characterized by the
 enhancement of the SEK1 kinase activity and/or MKK3 kinase activity. SEK1
 and MKK3 are known to be involved in apoptosis. Therefore, the protein
 according to the present invention is useful in elucidating the mechanisms
 of cell functions such as apoptosis.
 The present invention provides a protein comprising the amino acid sequence
 of SEQ ID NO: 1 having one or more additions, insertions, substitutions
 and/or deletions and lacking protein kinase activity, or a derivative
 thereof, (dominant-negative mutants). One or more additions, insertions,
 substitutions and deletions can be introduced.
 This protein can be obtained by modifying a protein which has protein
 kinase activity and enhances SEK1 kinase activity and/or MKK3 kinase
 activity in such a manner so as to destroy the protein kinase activity. An
 example of such modification is a substitution: K709R.
 This modified protein inhibits apoptosis caused by TNF-.alpha.as described
 in Examples thereinafter. Therefore, this modified protein is useful in
 elucidating the living phenomena in which ASK1 is involved in.
 Base Sequence
 The present invention provides a base sequence encoding the protein
 according to the present invention. An example of the base sequence
 encoding the protein according to the present invention is a sequence
 having a part or all of the DNA sequence of SEQ ID NO: 2. Base sequences
 in this specification mean both DNA sequences and RNA sequences.
 When the modified amino acid sequence is given, the base sequence encoding
 such amino acid sequence is easily determined, and a variety of base
 sequences encoding the amino acid sequence described in SEQ ID NO: 1 can
 be selected. The base sequence encoding the protein according to the
 present invention thus means, in addition to a part or all of the DNA
 sequence described in SEQ ID NO: 2, another sequence encoding the same
 amino acid sequence and having degenerate codon(s) in the DNA sequence.
 Furthermore, this base sequence includes RNA sequences corresponding to
 such DNA sequences.
 The base sequence according to the present invention may be derived
 naturally or obtained entirely by synthesis. It may also be synthesized
 using a part of a naturally occurring sequence. DNAs may typically be
 obtained by screening a chromosome library or a cDNA library in accordance
 with a conventional manner in the field of genetic engineering, for
 example, by screening with an appropriate DNA probe obtained based on
 information of the partial amino acid sequence.
 Examples of the base sequence encoding the protein according to the present
 invention include the DNA sequence 268-4392 of SEQ ID NO: 2 (corresponding
 to the open reading frame).
 Vector and Transformed Host Cell
 The present invention provides a vector comprising the abovementioned base
 sequence in such a manner that the vector can be replicable and express
 the protein encoded by the base sequence in a host cell. In addition, the
 present invention provides a host cell transformed by this vector. This
 host-vector system is not particularly restricted and fusion protein
 expression systems with other proteins can also be used. Examples of the
 fusion protein expression system include those using MBP (maltose binding
 protein), GST (glutathione-S-transferase), HA (hemagglutinin), His
 (hexahistidine), myc, Fas, and the like.
 Examples of the vector include plasmid vectors (e.g., expression vectors
 for prokaryotic cells, yeasts, insect cells, and animal cells), virus
 vectors (e.g., retrovirus vectors, adenovirus vectors, adeno-associated
 virus vectors, herpesvirus vectors, Sendai virus vectors, HIV vectors, and
 vaccinia virus vectors), and liposome vectors (e.g., cationic liposome
 vectors).
 In order to express a desired amino acid sequence practically by
 introducing the vector according to the present invention into a host
 cell, the vector may contain, in addition to the base sequence according
 to the present invention, other sequences for controlling the expression
 (e.g., promoter sequences, terminator sequences and enhancer sequences)
 and gene markers for selecting microorganisms, insect cells, animal
 culture cells, or the like (e.g., neomycin resistance genes and kanamycin
 resistance genes). Furthermore, the vector may contain the base sequence
 according to the present invention in a repeated form (e.g., in tandem).
 These base sequences may also be introduced in a vector according to the
 conventional manner, and microorganisms, insect cells, animal cultured
 cells, or the like may be transformed by the vector based on the method
 conventionally used in this field.
 The vector according to the present invention may be constructed based on
 the procedure and manner which have been conventionally used in the field
 of genetic engineering.
 Furthermore, examples of the host cell include Escherichia coli, yeasts,
 insect cells and animal cells such as COS cells (e.g., COS7 cells), mink
 lung epithelial cells (e.g., Mv1Lu), lymphocytes, fibroblasts, CHO cells,
 blood cells, tumor cells, and the like.
 The transformed host cells are cultured in an appropriate medium, and the
 protein according to the present invention may be obtained from the
 culture product. Thus, another aspect of the present invention provides a
 process for preparing the protein according to the present invention. The
 culture of the transformed host cell and culture conditions may be
 essentially the same as those for the cell to be used. In addition, the
 protein according to the present invention may be recovered from the
 culture medium and purified in the conventional manner.
 The present invention can be applied in the gene therapy of malignant
 tumors (e.g., leukemia cells, digestive tract carcinoma cell, lung
 carcinoma cells, pancreas carcinoma cells, ovary carcinoma cells, uterus
 carcinoma cells, melanoma cells, brain tumor cells, etc.) by introducing a
 vector having the base sequence according to the present invention into
 cancer cells of an organism including humans using an appropriate method
 to express the protein according to the present invention, i.e., by
 transforming the cancer cells of cancer patients in situ. For example,
 when the protein according to the present invention is expressed in an
 organism including humans, in particular, in malignant tumor cells,
 apoptosis is induced causing the malignant tumor to shrink, thereby
 enabling treatment of the tumor (see Example 5).
 As for the vectors for gene therapy, see Fumimaro Takahisa, Experimental
 Medicine (extra edition), Vol. 12, No. 15, "Forefront of Gene Therapy"
 (1994).
 Use and Pharmaceutical Composition
 The protein according to the present invention has protein kinase activity
 and enhances SEK1 kinase activity and/or MKK3 kinase activity (Examples 3
 and 4). Furthermore, the protein according to the present invention
 induces apoptosis of immortalized cells (Example 5). Thus, the protein
 according to the present invention is useful in suppressing tumorigenesis
 and/or metastasis in malignant tumors.
 Therefore, according to the present invention, an agent for use in the
 treatment of malignant tumors comprising the protein according to the
 present invention and a pharmaceutically acceptable carrier is provided.
 The term "treatment" in the present invention also refers to "prevention."
 Examples of malignant tumors include leukemia (for example, myelocytic
 leukemia, lymphocytic leukemia such as Burkitt lymphoma), digestive tract
 carcinoma, lung carcinoma, pancreas carcinoma, ovary carcinoma, uterus
 carcinoma, brain tumor, malignant melanoma, other carcinomas, and
 sarcomas.
 The agent for use in the treatment of malignant tumors according to the
 present invention may be administered orally or parenterally (e.g.,
 intramuscular injection, intravenous injection, subcutaneous
 administration, rectal administration, transdermal administration, nasal
 administration, and the like), preferably orally. The pharmaceutical agent
 may be administered to a human and other animals in a variety of dosage
 forms suited for oral or parenteral administration.
 The agent for use in the treatment of malignant tumors can be formulated in
 a variety of form including oral agents such as tablets, capsules,
 granules, dispersible powders, pills, fine particles and troches,
 injections such as intravenous injections and intramuscular injections,
 rectal agents, fatty suppositories and aqueous suppositories, for example,
 depending on their intended uses. These preparations may be prepared
 according to methods well known in the art with conventional excipients,
 fillers, binding agents, wetting agents, disintegrating agents,
 surfactants, lubricants, dispersing agents, buffering agents,
 preservatives, dissolution aids, antiseptics, flavorings, analgesic agents
 and stabilizing agents. Examples of the abovementioned possible nontoxic
 additives to be used include lactose, fructose, glucose, starch, gelatin,
 magnesium carbonate, synthetic magnesium silicate, talc, magnesium
 stearate, methylcellulose, carboxymethylcellulose or a salt thereof, gum
 arabic, polyethylene glycol, syrup, Vaseline, glycerine, ethanol,
 propylene glycol, citric acid, sodium chloride, sodium sulfite, and sodium
 phosphate.
 The content of the protein according to the present invention in a
 pharmaceutical agent varies depending on its dosage forms. The composition
 may contain about 1-50% by weight, preferably about 1-20% by weight, of
 the protein.
 The dose of the protein for the treatment of malignant tumors may
 appropriately be determined in consideration of its uses and the age, sex
 and condition of a patient, and is desirably in the range of about 0.1-500
 mg, preferably about 0.5-50 mg, per day for an adult, which may be
 administered at once or divided into several portions a day.
 According to the present invention, a base sequence encoding the protein
 according to the present invention or a vector comprising the base
 sequence may be used to suppress tumorigenesis and/or metastasis of
 malignant tumors by transforming a target cell. In other words, the base
 sequence and vector can be used as a gene therapeutic agent for use in the
 treatment of malignant tumors (the gene therapy agent). The method of
 administration, effective dosage, possible carriers to be included, and
 other parameters of a gene therapeutic agent can conform to those for an
 antitumor agent.
 The gene therapeutic agent according to the present invention can be
 administered to a mammal, including a human, and other animals by the HVJ
 liposome method (Kaneda, Experimental Medicine, Vol. 12, No. 2, 78(184),
 1994; Morishita, et al., Experimental Medicine, Vol. 12, No. 15,
 158(1928), 1994), a method in which the base sequence according to the
 present invention is administered as is by injection or the like, the
 calcium phosphate method, the DEAE-dextran method, the electroporation
 method, the gene gun method (T. M. Klein et al., Bio/Technology 10,
 286-291, 1992), the lipofection method (Nabel et al., Science 244, 1285,
 1990), a method using an appropriate vector (e.g., adenovirus vector,
 adeno-associated virus vector, herpes virus vector, vaccinia virus vector,
 and retrovirus vector), or the like.
 In considering the local or temporary induction of apoptosis, the gene
 therapy agent according to the present invention is preferably
 administered in such a manner that ASK1 is transiently present in the
 body. Examples of such a manner include parenteral administration methods
 such as administering the base sequence according to the present invention
 as is by injection or the like, the lipofection method, the HVJ liposome
 method, a method using an adenovirus vector, and a method using a vaccinia
 virus vector.
 Another aspect of the present invention provides a use of the protein, base
 sequence or vector, in particular, a use for the manufacture of a
 medicament.
 Still another aspect of the present invention provides a method for
 treating malignant tumors in mammals comprising administrating the
 protein, base sequence or vector according to the present invention. The
 effective dosages, methods of administration and dosage forms can apply
 for this method.
 Peptide and Antibody
 The present invention provides a peptide consisting of the amino acid
 sequence 654-669 of SEQ ID NO: 1 and a peptide comprising the amino acid
 sequence 654-669 of SEQ ID NO: 1.
 Examples of the peptide comprising the amino acid sequence 654-669 of SEQ
 ID NO: 1 include a peptide in which an optional amino acid sequence is
 added to the N-terminal and/or C-terminal of said amino acid sequence,
 including the protein according to the present invention.
 The peptide can be used as an antigen to obtain an antibody against the
 protein according to the present invention. Furthermore, the protein
 according to the present invention is closely involved in mechanisms of
 apoptosis as mentioned above. Therefore, the protein according to the
 present invention is useful in elucidating these mechanisms of apoptosis.
 The present invention provides the antibody against the peptide. The
 antibody in the present invention includes a polyclonal antibody and a
 monoclonal antibody.
 The antibody according to the present invention can be produced by a method
 generally known by those skilled in the art. For example, the polyclonal
 antibody can be obtained by injecting the abovementioned peptide into an
 animal (e.g., a rabbit, goat, rat, mouse, and sheep) with an optional
 carrier (e.g., bovine serum albumin) and purifying the serum of the animal
 after a certain period. The monoclonal antibody can be prepared by the
 hybridoma fusion technique. For example, see the following literature for
 reference: Kohler and Milstein, Nature, 256: 495-97, 1975; Brown et al.,
 J. Immunol., 127(2), 539-46, 1981; Brown et al., J. Biol. Chem., 255,
 4980-83, 1980; Yeh et al., Proc. Natl. Acad. Sci. (USA), 76(6), 2927-31,
 1976; and Yeh et al., Int. J. Cancer, 29, 269-75, 1982; Zola et al., in
 Monoclonal Hybridoma Antibodies: Techniques and Applications, Hurell
 (ed.), pp. 51-52 (CRC Press, 1982).
 The peptide according to the present invention is a part of the amino acid
 sequence of the protein according to the present invention. Therefore, a
 specific reaction (i.e., immuno cross reaction) of the antibody according
 to the present invention can be an index for the presence of the protein
 according to the present invention.
 Thus, another aspect of the present invention provides a protein which can
 be recognized by the antibody according to the present invention, and the
 protein according to the present invention which can be recognized by the
 antibody.
 EXAMPLE
 Example 1
 Cloning of ASK1 cDNA by Polymerase Chain Reaction (PCR) Method and
 Determination of Amino Acid Sequence of ASK1
 (1) Isolation of cDNA
 A degenerate PCR-based strategy was used in an attempt to obtain a novel
 serine/threonine kinase cDNA according to the method described in P. ten
 Dijke et al., Oncogene, 8, 2879, 1993; P. Franzen et al., Cell, 75, 681,
 1993, and P. ten Dijke et al., Science, 264, 101, 1994.
 As a result, several human cDNA fragments encoding more distantly related
 protein kinases whose functions are unknown along with the recipient-type
 serine/threonine kinase family, were obtained.
 First, a PCR fragment was obtained using a set of PCR primers derived from
 the conserved subdomains VII and VIII of the serine/threonine kinase
 family (S. Hanks et al., Science, 241, 42, 1988). Using this fragment, a
 corresponding nearly full-length cDNA clone was isolated (the
 serine/threonine kinase encoded by this cDNA is hereinafter referred to as
 activator of SEK1 and MKK3 (ASK1) because of its characteristics).
 More specifically, an amplified oligo(dT)-primed .lambda.gt 11 cDNA library
 from human erythroleukemia (HEL) cells (M. Poncz et al., Blood, 69, 219,
 1987) was screened with a .sup.32 P-labeled PCR fragment. Hybridization
 and purification of positive bacteriophage were performed as described in
 H. Ichijo et al., J. Biol. Chem., 268, 14505, 1993. Base sequencing was
 done on both strands with a Sequenase DNA sequencing kit (U.S. Biochemical
 Corp.). Among 18 clones obtained, the three longest clones (clones 20, 27
 and 72) were entirely sequenced. The sequence of clone 72 started from the
 middle of the open reading frame and ended by a stretch of poly A. The
 sequences of clones 20 and 27 covered the 5' part of ASK1 cDNA, and the
 overlapping parts with clone 72 were identical in sequence. The ASK1 cDNA
 sequence, combining the clone 20 and clone 72, yielded a 4533-base pair
 sequence with an ATG codon starting at position 268 followed by a 4125-bp
 open reading frame encoding a protein consisting of 1375 amino acids (FIG.
 1). This protein (ASK1 protein) has an estimated molecular weight of
 154,715 Da.
 On the other hand, another clone (clone 27) was obtained in which an open
 reading frame starts from the site corresponding to an amino acid at
 position 375 of clone 20. Because clone 27 contained a 4-bp deletion at
 position 805 to 808 of ASK1 cDNA, in-frame upstream stop codons were
 formed.
 The serine/threonine kinase domain of ASK1 was found in the middle part of
 the ASK1 protein and had long N-terminal and C-terminal flanking sequences
 (FIG. 1). Furthermore, RNA blot analysis revealed a single 5-kb transcript
 that was expressed in various human tissues (FIG. 3). Blots with mRNAs
 from various human tissues (Clontech) were probed with ASK1 cDNA labeled
 by random priming.
 (2) Homology Search by Database
 A database search of ASK1 sequence outside its kinase domain showed that a
 short amino acid sequence in the N-terminal part contains a motif for an
 FK506-binding protein (FKBP)-type peptidyl-prolyl cis-trans isomerase
 (FIG. 1, underlined). In contrast, the kinase domain of ASK1 has evident
 sequence homology with members of the MAPKKK family. The degree of
 homology was 30.0% with MEKK1 in mammal and 32.3% and 30.4% with SSK2 and
 STE11 in Saccharomyces cerevisiae, respectively.
 Phylogenetic comparison suggested that ASK1 is distantly related to
 mammalian MAPKKKs (RAF-1, KSR-1, TAK-1, and TPL-2) but most closely
 related to the SSK2/SSK22 family of yeast MAPKKK protein, which are
 upstream regulator proteins of yeast HOG1 MAPK (T. Maeda et al., Science,
 269, 554, 1995). (FIG. 2).
 Comparison of amino acid sequences between the kinase domain of ASK1 and
 kinase domains of other MAPKKKs was carried out using the clustal computer
 alignment program of laser gene program (DNASTAR) (D. Higgins & P. Sharp,
 Comput. Appl. Biosci., 5, 151, 1989).
 Example 2
 Molecular Genetic Analysis of ASK1 Kinase Activity Using Yeast
 Overall structures of ASK1 and yeast MAPKKKs, SSK2/SSK22, are different
 (namely, the kinase domain of SSK2 or SSK22 is located in the C-terminal
 part of these proteins (T. Maeda et al., Science, 269, 554, 1995)).
 However, it was of interest to examine whether ASK1 might act as a
 functional kinase in yeast and thereby complement the loss of yeast
 MAPKKK.
 First, ASK1 cDNA was introduced into a yeast expression vector pNV11 (H.
 Shibuya et al., Nature, 357, 700, 1992), and whether ASK1 can restore SSK2
 or SSK22 MAPKK signal deletion in a yeast mutant strain TM257-H1
 (ssk2.DELTA., ssk22.DELTA., sho1.DELTA.) ( ), which grows in a normal YPD
 medium but not in a hyperosmotic medium was investigated. In this
 connection, SHO1 is an SH3 domain-containing trans-membrane osmosensor
 that relates to another signaling pathway leading to various
 hyperosmolarity responses by way of HOG1 activation independently of
 SSK2/SSK22. Single or double mutant strains of SHO1, SSK2 or SSK22 are
 resistant to hyperosmotic medium. However, it is known that if SHO1, SSK2
 and SSK22 are simultaneously destroyed, the yeast cells are unable to grow
 in hyperosmotic medium.
 Accordingly, transformants were tested for growth in the presence of 1.5 M
 sorbitol (FIG. 4). Specifically, five independent transformants were
 selected and grown on YPD plates in the presence or absence of 1.5 M
 sorbitol. Photographs in FIG. 4 were taken after the growth for 6 days at
 30.degree. C.
 Transformants with PNV11 vector alone or ASK1(K709R) (a mutant strain in
 which kinase catalytic activity was inactivated by substituting Lys 709
 with Arg) vector were also tested.
 Results showed that expression of ASK1, but neither vector alone nor ASK1
 (K709R), complemented TM257-H1 growth in the hyperosmotic environment
 (FIG. 4). ASK1 could not restore the osmotic response in a PBS2
 (downstream target protein of SHO1, SSK2, and SSK2 (Maeda, T. et al.,
 Science, 269, 554, 1995))-defective yeast strain (data not shown). This
 observation strongly suggests that ASK1 activity observed in TM257-H1 was
 mediated by the PBS-HOG1 signaling pathway but not by any pathway other
 than the HOG1 activation.
 These results, together with the fact that the mammalian counterpart of
 yeast HOG1 is p38 MAP kinase (J. Rouse et al., Cell, 78, 1027, 1994; J.
 Han et al., Science, 265, 808, 1994; J. Lee et al., Nature, 372, 739,
 1994), suggested that ASK1 may be a novel mammalian MAPKKK and involved in
 activation of MKK3-p38 signal transduction pathway by phosphorylating
 MKK3.
 Example 3
 Cell Biological Analysis of ASK1 Kinase Activity Using Mammalian Cells
 To investigate whether ASK1 may function as an MAPKKK in mammalian cells,
 an ASK1 plasmid was transfected into COS7 cells together with known MAPK
 and MAPKK expression plasmids (FIG. 5). All the MAPK and MAPKK constructs
 were hemagglutinin (HA) epitope-tagged, expressed with or without ASK1,
 and immunoprecipitated with antibody to HA. Specifically, the following
 procedure was used.
 The cDNAs encoding Xenopus MAPK (Y. Gotoh et al., EMBO J., 10, 2661, 1991)
 and Xenopus MAPKK (H. Kosako et al., EMBO J., 12, 787, 1993) were cloned
 as previously described. Coding regions for rat SAPK.alpha. (J. Kyriakis
 et al., Nature, 369, 156, 1994), human p38 (J. Han et al., Biochim.
 Biophys. Acta, 1265, 224, 1995), mouse SEK1 (I. Sanchez et al., Nature,
 372, 794, 1994), and human MKK3 (B. Derijard et al., Science, 267, 682,
 1995) were amplified by PCR method. An HA tag was introduced into the
 BglII-EcoRI sites of a mammalian expression vector pSR.alpha.456 (Y.
 Takebe et al., Mol. Cell. Biol., 8, 466, 1988), yielding pSR.alpha.-HA1.
 The cDNAs encoding MAPK, SAPK.alpha., p38, MAPKK, SEK1, and MKK3 were
 subcloned into the BglII site of pSR.alpha.-HA1. ASK1 cDNA was introduced
 into another mammalian expression vector, pcDNA3 (Invitrogen). For
 transient expression, COS7 cells were transfected with lipofectamine (Life
 Technologies) according to the manufacturer's instructions. For preparing
 extracts, cell were lysed in a buffer solution (20 mM tris-HCl (pH 7.5),
 12 mM .beta.-glycerophosphate, 150 mM NaCl, 5 mM EGTA, 10 mM NaF, 1%
 Triton X-100, 0.5% deoxycholate, 3 mM dithiothreitol (DTT), 1 mM sodium
 vanadate, 1 mM phenylmethylsufonyl fluoride (PMSF), and aprotinin (20
 .mu.g/ml)). Cell extracts were clarified by centrifugation at 15,000 g for
 10 min.
 For immunoprecipitation, the supernatants were incubated with monoclonal
 antibody to HA (12CA5) for 1 hour at 4.degree. C. After the addition of
 protein A-Sepharose (Pharmacia Biotech), the lysates were incubated for an
 additional 1 hour. The beads were washed twice with a solution (500 mM
 NaCl, 20 mM tris-HCl (pH 7.5), 5 mM EGTA, 1% Triton X-100, 2 mM DTT, and 1
 mM PMSF), then twice with a solution (150 mM NaCl, 20 mM tris-HCl (pH
 7.5), 5 mM EGTA, 2 mM DTT, and 1 mM PMSF), and subjected to kinase assays.
 The precipitated immune complexes were subjected to a phosphorylation
 assay. In the assay, exogenous proteins were added as substrates. The
 substrate proteins used were myelin basic protein (MBP) (Sigma) for MAPK,
 c-Jun for SAPK, ATF-2 for p38, kinase-negative MAPK for MAPKK, and
 kinase-negative p38 (MPK2) for SEK1 and MKK3. ATF2 used herein was
 prepared according to the method previously described (S. Gupta et al.,
 Science, 267, 389-393, 1995). Hexahistidine (His)-tagged c-Jun (S. Matsuda
 et al., J. Biol. Chem., 270, 12781, 1995) and glutathione-S-transferase
 (GST)-kinase-negative Xenopus MAPK (K57D) were prepared as described in H.
 Kosako et al., EMBO J., 12, 787, 1993. MPK2 (J. Rouse et al., Cell, 78,
 1027, 1994), a Xenopus counterpart of mammalian p38, was used as a
 substrate protein for SEK1 and MKK3 in the assay.
 His-tagged kinase-negative MPK2 (K54R) was prepared according to the method
 described in T. Moriguchi et al., J. Biol. Chem., 270, 12969, 1995. To
 measure the activity to phosphorylate MBP, c-Jun, ATF2, kinase-negative
 MAPK, and kinase-negative MPK2, the immune complex was incubated for 30
 minutes at 30.degree. C. with 3 .mu.g of each substrate protein in a final
 volume of 25 .mu.l of a solution (20 mM Tris-HCl (pH 7.5), 10 mM
 MgCl.sub.2, 100 .mu.M [.gamma.-.sup.32 P]ATP (0.3 .mu.Ci)). The reaction
 was stopped by addition of Laemmli's sample buffer and boiling. After
 SDS-polyacrylamide gel electrophoresis (PAGE), phosphorylation of these
 proteins was quantified with an image analyzer (FujiX BAS2000).
 Results showed that ASK1 expression induced 7.6- and 5.0-fold activation of
 SAPK and p38 MAP kinase, respectively, but only weakly activated MAPK
 (FIG. 5).
 Furthermore, ASK1 activated MKK3 and SEK1 up to 11.8- and 7.0-fold,
 respectively. In contrast, no detectable activation of MAPKK was observed
 (FIG. 5).
 Example 4
 In Vitro-coupled Kinase Assay with Recombinant Proteins
 To investigate whether the MKK3 activation observed in FIG. 5 was a direct
 effect by ASK1, an in vitro-coupled kinase assay with recombinant SEK1,
 MKK3, MAPKK6, and recombinant kinase-negative p38 (MPK2) proteins was
 used. In this Example, ASK1 expressed in COS7 cells was immunoprecipitated
 with polyclonal antibody as described in Example 3, and the resulting
 immune complex was used as an ASK1 enzyme standard. The anti-ASK1
 polyclonal serum used in the immunoprecipitation was raised against the
 peptide sequence (SEQ ID NO:3)(TEEKGRSTEEGDCESD), corresponding to amino
 acids 654 to 669 of ASK1, that was coupled to keyhole limpet hemocyanin by
 a glutaraldehyde method, mixed with Freund's adjuvant, and used to
 immunize rabbits according to the method described in H. Ichijo et al., J.
 Biol. Chem., 270, 7420, 1995. The coupled kinase assay was carried out
 using recombinant SEK1, MKK3, MAPKK6, and recombinant kinase-negative p38
 proteins together with this immune complex according to the following
 procedure.
 His-tagged Xenopus MAPKK and human MKK3 were expressed in Escherichia coli
 and purified as described in Y. Gotoh et al., Oncogene, 9, 1891, 1994. To
 measure the activity of an immune complex to activate MAPKK or MKK3, 0.2
 .mu.g of His-MAPKK or His-MKK3 was first incubated with the immune complex
 for 15 minutes at 30.degree. C. in a final volume of 25 .mu.l of a
 solution (20 mM Tris-HCl (pH 7.5), 10 mM MgCl.sub.2, and 100 .mu.M ATP).
 Subsequently, 0.3 .mu.Ci of [.gamma.32P] ATP and 3 .mu.g of
 GST-kinase-negative MAPK (to MAPKK) or His-kinase-negative MPK2 (to MKK3)
 were incubated in the same buffer solution (final volume, 35 .mu.l) for 7
 minutes at 25.degree. C. Samples were then analyzed by SDS-PAGE and image
 analyzer. Results are shown in FIG. 6.
 ASK1 immunoprecipitates from COS7 cells strongly activated SEK1, MKK3, and
 MAPKK6 activity (greater than 40-fold for each), and phosphorylation of
 p38 was observed only when ASK1 was present in the kinase assay.
 ASK1-dependent phosphorylation of p38 was further confirmed to result in
 the activation of p38 using wild-type p38 and ATF2. In contrast, ASK1
 weakly activated MAPKK (2.2-fold) even in the presence of MAPKK.
 When Raf was used as MAPKKK for a positive control, a 27.8-fold activation
 of MAPKK was observed (date not shown). These results (Example 4) and the
 results in Example 3 indicated that ASK1 is a novel MAPKKK, which
 selectively activates the SEK1-SAPK and MKK3/MAPKK6-p38 pathways.
 Example 5
 Induction of Apoptosis by ASK1 Expression
 (1) Confirmation of ASK Expression
 The biological activity of ASK1 was investigated using mink lung epithelial
 (Mv1Lu) cell lines that were stably transfected with metallothionein
 promoter-based expression plasmids. To avoid the possibility that
 constitutively expressed ASK1 might induce cell death, resulting in a
 failure to obtain stable transformants, a metallothionein-inducible
 promoter system was used.
 ASK1 and ASK1(K709R) cDNA were subcloned into pMEP4 vector (Invitrogen) at
 convenient enzyme cleavage sites. Transfection of cDNAs was done with
 Transfectam (Promega) according to the manufacturer's instructions.
 Selection by hygromycin B was done by the method described in M. Saitoh et
 al., J. Biol. Chem., 271, 2769, 1996. Several independent clones were
 ring-cloned, and the expression of ASK1 protein was determined by
 immunoprecipitation (H. Ichijo et al., J. Biol. Chem., 268, 14505, 1993)
 with antiserum to ASK1. Two independent positive clones were used for the
 assays with essentially the same results.
 Cells were metabolically labeled with a mixture of [.sup.35 S]methionine
 and [.sup.35 S]cysteine in the presence or absence of 100 .mu.M ZnCl.sub.2
 for 5 hours. The cellular lysates were then subjected to
 immunoprecipitation with antiserum to ASK1, SDS-PAGE, and fluorography.
 Results are shown in FIG. 7. It was revealed that ASK was highly expressed
 only when induced by ZnCl2. ASK1(K709R)-transfected cells expressed the
 recombinant protein in similar amounts.
 (2) Effects on Thymidine Incorporation
 To investigate the effects of ASK1 on cellular growth, Mv1Lu cells stably
 transfected with vector alone (FIG. 8, black squires), ASK1 (FIG. 8, black
 circles), and ASK1(K709R) (FIG. 8, white circles) were incubated in MEM
 containing 1% fetal bovine serum (FBS) and the indicated concentration of
 ZnCl.sub.2 for 16 hours. The cells were then pulse-labeled with [.sup.3
 H]thymidine for 1 hour, and [.sup.3 H] radioactivity incorporation into
 the DNA was determined using a liquid scintillation counter. Results are
 shown in FIG. 8.
 Drastic inhibition of [.sup.3 H]thymidine incorporation was observed in the
 cells transfected with ASK1. In contrast, no inhibition was observed in
 the cells transfected with the vector alone or ASK1(K709R) vector (FIG.
 8). Correlation between dose-dependent inhibition of [.sup.3 H]thymidine
 incorporation by ZnCl.sub.2 and the dose-dependent expression and
 activation of ASK1 was investigated. The ASK1-transfected Mv1Lu cells were
 treated with the indicated amount of ZnCl.sub.2 for 5 hours, and then the
 level of ASK1 was determined by immunoprecipitation (FIG. 9, top).
 Furthermore, the cells were treated with the indicated amount of
 ZnCl.sub.2 for 5 hours, ASK1 was recovered from the cells by
 immunoprecipitation and then subjected to the MKK3-MPK2 coupled kinase
 assay (FIG. 9, bottom). Results showed that the dose-dependent inhibition
 of [.sup.3 H]thymidine incorporation by ZnCl.sub.2 correlated well with
 the dose-dependent expression and activation of ASK1 (FIG. 9).
 (3) Effects on Enhancement of SAPK and p38 Kinase Activity
 The following experiment was carried out to investigate correlation between
 ASK1 activity and endogenous SAPK and p38 activation.
 Mv1Lu cells stably transfected with ASK1 were incubated with the indicated
 concentration of ZnCl.sub.2 for 5 hours. To measure the activity of SAPK,
 each cell extract was subjected to a kinase detection assay (in-gel kinase
 assay) within a polyacrylamide gel containing c-Jun as a substrate protein
 according to the method described in S. Matsuda et al., J. Biol. Chem.,
 270, 12781, 1995. To examine the activity of p38, p38 was
 immunoprecipitated with polyclonal antibody to p38 (C-20, Santa Cruz)
 according to the method described in Example 3 except for the presence of
 0.1% SDS during the immunoprecipitation. The kinase activity was then
 detected using ATF2 as a substrate protein. Results are shown in FIG. 10.
 It was revealed that endogenous SAPK and p38 were also activated in
 parallel with the ASK1 activities. (4) Effects on cell morphology and DNA
 fragmentation
 It was revealed that morphological changes (namely, cytoplasmic shrinkage
 and cellular condensation) were induced within 6 hours after addition of
 ZnCl.sub.2 when cells were treated with 100 .mu.M ZnCl.sub.2 and ASK1 was
 continuously expressed (data not shown). These morphological changes were
 not observed in the cells in which ASK1(K709R) was expressed. Cells were
 incubated with MEM containing 1% FBS in the presence or absence of 100
 .mu.M ZnCl.sub.2 for 26 hours. The typical morphological properties of
 apoptotic cells, i.e., cytoplasmic shrinkage and cellular condensation,
 became most evident after induction for long hours (26 hours) (FIG. 11,
 top).
 Whether ASK1 induces the apoptotic cell death was investigated by an in
 situ staining of cells with the terminal deoxynucleotidyl
 transferase-mediated dUTP nick end labeling (TUNEL) method (FIG. 11,
 bottom) as well as by genomic DNA fragmentation. More specifically, Mv1Lu
 cells transfected with ASK1 were incubated with MEM without FBS in the
 presence or absence of 100 .mu.M ZnCl.sub.2 for 25 hours and then stained
 by TUNEL method with a situ cell death detection kit (Boehringer Mannheim)
 (FIG. 11, bottom), or the total DNA was isolated and subjected to 2%
 agarose gel electrophoresis (FIG. 12). As a result, apoptosis and DNA
 fragmentation were observed after induction of ASK1 expression by
 ZnCl.sub.2 (FIG. 11, bottom, and FIG. 12).
 Example 6
 ASK1 Activation by TNF-.alpha.
 In this Example, whether the treatment of cells with TNF-.alpha. resulted
 in the activation of ASK1 was examined. Mv1Lu cells transfected with ASK1
 were first treated with 50 .mu.M ZnCl.sub.2 for 5 hours to induce ASK1
 expression. The cells were then stimulated with TNF-.alpha. (100 ng/ml)
 for the indicated time. ASK1 immunoprecipitates derived from
 TNF-.alpha.-treated cells were subjected to a coupled kinase assay with
 MKK3 and kinase-negative p38 (FIG. 13, top and bottom, and FIG. 14).
 The results showed that the treatment of cells with TNF-.alpha. activated
 the ASK1 in ASK1-transfected Mv1Lu cells within 5 minutes (FIG. 13, top
 and bottom). The ASK1 was activated by TNF-.alpha. in a dose-dependent
 manner (FIG. 14).
 ASK1-nontransfected 293 cells and A673 cells were treated with TNF-.alpha.
 (100 ng/ml). The results showed that endogenous ASK1 was also activated by
 TNF-.alpha. in other various cell types in which apoptosis is induced by
 TNF-.alpha. (data not shown), including human 293 embryonal kidney cells,
 A673 rhabdomyosarcoma cells (FIG. 13, bottom), Jurkat T cells, and KB
 epidermal carcinoma cells (data not shown).
 Furthermore, ASK1(K709R) was transiently transfected into 293 cells (FIG.
 15) or Jurkat T cells (FIG. 16). Specifically, the experiment was carried
 out according to the following method. 293 cells (2.times.10.sup.6) were
 transiently transfected with 2 .mu.g of pcDNA3 control vector or
 pcDNA3-ASK1(K709R) by the use of Tfx-50 (Promega) according to the
 manufacturer's protocol. Eight hours after transfection, cells were
 treated with TNF-.alpha. (100 ng/ml) with or without 300 nM actinomycin D
 (ActD) for 16 hours. Apoptotic cells detached from culture plate were
 collected, and total DNA was isolated and analyzed by 2% agarose gel
 electrophoresis (FIG. 15).
 Furthermore, the pcDNA3-ASK1 (K709R) was transfected into Jurkat cells by
 DMRIE-C reagent (Life Technologies) together with pHook-1 plasmid
 (Invitrogen). Further, the pHook-1 plasmid encodes a single-chain antibody
 fusion protein directed to the hapten phox
 (4-ethoxymethylene-2-phenyl-2-oxazolin-5-one). Therefore, it is possible
 to selectively isolate transfected cells with magnetic beads coated with
 phOx.
 ASK1(K709R)-transfected populations of cells (cotransfection efficiency was
 nearly 100% as determined by .beta.-galactosidase staining) were isolated
 on phOx-coated magnetic beads with the Capture-Tec kit (Invitrogen), after
 which the cells were incubated with various concentrations of TNF-.alpha.
 for 5.5 hours. Cytoplasmic small fragmented DNA was isolated as described
 (Selins, K. & Cohen, J., J. Immunol., 139, 3199, 1987) with minor
 modifications. Cells (3.times.10.sup.6) were lysed with 200 .mu.l of a
 buffer solution (20 mM Tris-HCl (pH 7.5), 10 mM EDTA, 0.5% Triton X-100).
 The resulting lysate was incubated with proteinase K (0.2 mg/ml) and RNase
 N (0.1 mg/ml) at 42.degree. C. for 1 hour. DNA was then purified by
 phenol-chloroform extraction after ethanol extraction. The extracted
 cytoplasmic DNA was analyzed by 2% agarose gel electrophoresis (FIG. 16).
 The results showed that DNA fragmentation induced by TNF-.alpha. was
 effectively reduced. Further, nontransfected Jurkat cells and isolated
 Jurkat cells (that were transfected with pHook-1 and control pcDNA3
 plasmid) were similarly sensitive to TNF-.alpha. in the DNA fragmentation
 assay (date not shown). This observation suggested that ASK1(K709R) acts
 as a dominant-negative mutant, and more importantly, that ASK1 is
 essential for the TNF-.alpha.-induced apoptotic response.