JP2003144168A - Cell cycle-relating protein having nuclear export function or nucleus-cytoplasm transport function - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a protein having a cell cycle controlling function, especially a physiological function participating in the checkpoint function of the G2/M period of the cell cycle such as the extranuclear transport function and the nucleus-cytoplasm transfer function, the DNA of the protein and a method for the screening of a substance promoting or suppressing the nuclear export function, cytotoxic function, intranuclear transport function, nucleus-cytoplasm transport function and/or cell cycle controlling function by using the protein or DNA. SOLUTION: An unknown human Nek kinase family member is isolated by cloning a gene expressed in an extremely small amount by using a method comprising the combination of RCE method and Nested-PCR method and its base sequence and the amino acid sequence are determined. An apoptosic cell form is induced by the excessive expression of the Nek kinase in a cell. It has been found that a sequence having physiological functions such as extranuclear transport function and nucleus-cytoplasm transport function is present in the non-catalytic C-terminal site of the Nek kinase.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a cell cycle-related protein having a nuclear export function or a nucleus-cytoplasm transfer function, and a DNA encoding them. In addition, the present invention uses such proteins and DNAs, etc. to use nuclear export function, cytotoxic function, nuclear import function, nucleus-cytoplasm transfer function, and / or cell cycle control function (including nuclear division control function). The present invention relates to a screening method for a substance that promotes or suppresses, a substance that promotes or suppresses the expression of such proteins and DNAs, and the like.

[0002]

2. Description of the Related Art The signal transduction mechanism of cells is one of the basic mechanisms for controlling various cell functions. A known major molecular mechanism of signal transduction is an activity control mechanism by phosphorylation of protein serine, threonine, and tyrosine residues. A complex regulatory mode by multiple protein phosphatases is known.For example, protein phosphatases are intracellular mechanisms such as signal transduction mechanism from receptor on cell membrane, gene expression control, cell cycle control mechanism, etc. It is involved in normal function control in. It is known that abnormalities in these signal transduction pathways are involved in various pathological conditions such as immune diseases, neurological diseases, cancers, etc., and therapeutic agents targeting protein kinases have been developed. Therefore, it is expected that the elucidation of protein kinase will give a great benefit to the diagnostic method of pathological conditions and the development of therapeutic agents.

At present, a large number of protein kinases have been identified, and their characteristics are 250 to 300 due to their structure.
It is divided into a catalytic site (kinase domain) consisting of amino acids and its enzyme activity control site. The kinase domain, which is the catalytic site, has a highly conserved three-dimensional conformation in various protein kinases and can be further classified into 12 subdomains. In addition, the primary structure, which is the amino acid sequence of the kinase domain, is also conserved in each of the 12 subdomains. Due to the similarity between the kinase domains, protein kinases are broadly classified into the tyrosine kinase family, the serine threoin kinase family, and the dear specific kinase. Each is subdivided into subfamilies for more detailed similarity. At present, gene identification of a wide variety of protein phosphatases is progressing, utilizing the structural similarity of kinase domains as a clue and utilizing an explosively increasing gene database led by genomic analysis.

The eukaryotic cell cycle is highly organized and regulated by various protein kinases and phosphatases that have been evolutionarily functionally conserved (Curr. Biol. 5,
1122-1125, 1995, Biochem. J. 317, 633-641, 199.
6). From the filamentous fungus Aspergillus nidulans, NIMA (Never-In M
itosis, gene A) kinase has been isolated (Cell 53,
237-244, 1988). On the other hand, in A. nidulans, NIMA kinase is required for transition to mitosis (Cell 67,
283-291, 1991), and overexpression of NIMA induces nuclear assembly in human HeLa cells (EMBO J. 13, 4926-4937, 1994, Cell 81, 413-424, 1).
995), suggesting the presence of NIMA-related transduction pathways in higher eukaryotic cells.

The above-mentioned NIMA kinase belongs to one of the protein kinase group considered to function in the cell cycle,
Protein kinases structurally related to fungal NIMA kinase have been isolated from various organisms. First discovered NIMA kinase from A. nidulans, PIN1 from fission yeast, Nim- from Neurospora crassa
1 (J. Biol. Chem. 270, 18110-18116, 1995), Schiz
Fin1 from osaccharomyces pombe (J. Cell Sci. 1
11, 967-976, 1998), Kin3 / NPK1 derived from Saccharomyces cerevisiae (Gene 117, 137-140, 1992, Mo).
Genet. 234, 164-167, 1992), and Nek (NIM).
A-related kinase) (Curr.Biol. 5, 1122-1125, 199
5) at least 6 kinds of proteins derived from mammals designated as: Nek1 (EMBO J. 11, 3521-3231, 199)
2), Nek2 (Cell Growth Differ. 5, 625-635, 199)
4), Nek3 (J. Biol. Chem. 274, 13491-13497, 19).
99), Nek4 / STK2 (J. Cancer 54, 571-577, 1
993, Gene 234, 127-137, 1999), Nek6 (Cytogene
Cell Genet. 87, 271-272), and Nek7 (Genomi
cs68, 187-196, 2000), respectively. All these NIMA-related kinases share a primary structure remarkably similar to NIMA at the N-terminal catalytic site. Although it has been speculated that the C-terminal non-catalytic site of NIMA has an important function in cell cycle regulation, such C
Little such sequence similarity is found in the terminal non-catalytic sites (Biochem. J. 317, 633-641, 1996). Thus, at the present knowledge, evolutionarily, in unicellular organisms,
Only one NIMA kinase-like gene has been confirmed in each species, but in mammals such as mice and humans, similar genes that are thought to have different functions have been generated, and physiology is similar if only the gene structure is similar. The physical function could not be inferred.

[0006] Conventionally, NIM-1 (J. Biol. Chem. 270, 18110-1811 has been used as a substance having a functional similarity to NIMA.
6, 1995) and Fin1 (J. Cell Sci. 111, 967-976,
1998), only two types of NIMA-related kinases have been reported, and the functional relationship between fungal-derived NIMA and mammalian-derived Neks is unknown. Meanwhile, Aspergillus NIMA
Kinase and fission yeast PIN1 have similar functions in G2 / M
It is thought to be involved in the control of the period. Among the NIMA family of mammalian origin, the most advanced research is N
It is ek2. Nek2 functions within the centrosome outer periphery by phosphorylation of the physiological substrate c-Nap1 during the cell cycle (EMBO J. 17, 470-481, 1998, J. Cell Biol. 141, 15).
63-1574, 1998, EMBO J. 19, 1816-1826, 2000, J. Cel
l Sci. 113, 1973-1984, 2000), human Nek2 kinase is known to be involved in the regulation of centrosome partitioning in the G2 phase, but is considered to have only a partial function of NIMA kinase. The presence of an unknown Nek kinase family molecule is expected. Nek3 is a cytoplasmic protein that is relatively abundantly expressed in G0 phase.
Even when ek3 is overexpressed, intracellular chromatin mimicry has not been observed (J. Biol. Chem. 274, 13491-13497,
1999). Although little is known about the function of Nek1 and Nek4, mutations in mouse nek1 have been implicated in Kat phenotypes, including polycystic kidney disease, atrophy, male infertility, and Harrahman-Strife syndrome ( Proc. Natl. Acad. Sci. USA 97, 217-221, 2000),
Like nekl and nek2, nek4 is known to be highly expressed in germ cells (Gene 23
4, 127-137, 1999, Development 124, 2167-2177, 199
7, Exp. Cell Res. 237, 264-274, 1997, Oncogenene 1
6, 1813-1827, 1998, Dev. Biol. 208, 456-464, 199
9, Biochem. Biophys. Res Commun. 264, 449-456, 199
9). Nek6 and Nek7 are cytoplasmic proteins lacking the C-terminal regulatory site (Genomics 68, 187-1).
96, 2000), which has recently been reported to be a putative regulator of p70 ribosomal S6 kinase (Curr. Bi).
ol. 11, 1155-1167, 2001). It is suggested from the above report that mammalian-derived NIMA-related kinases that have been reported so far have a different function from fungal-derived NIMA involved in cell cycle processes. The existence of a true functional NIMA homolog has not been revealed in mammals, and it is possible that an unknown NIMA-related kinase functions in the process of mitosis in mammals.

On the other hand, the G2 / M phase in the cell cycle is a period in which chromosome partition and cell division are carried out, and it is considered that many checkpoint mechanisms function to maintain accuracy. NIMA functioning in G2 / M phase
Abnormalities in kinases and Nek2 kinases induce cell cycle dysregulation. Abnormality at this stage induces polyploidy or multinucleation of chromosomal DNA due to abnormal number of chromosomes due to uneven distribution of chromosomes, abnormal genomic structure, and the like. Such a phenomenon is frequently observed in malignant cancer cells in which the molecular mechanism associated with Polo-like kinase and the like is abnormal, and the G2 phase arrest in the cell cycle of infected cells by the accessory protein Vpr of HIV, multinucleation and It is largely involved in the subsequent genomic instability leading to the development of malignant tumors, and is considered to be an important target molecular mechanism in the development of new therapeutic agents in these diseases.

[0008]

The object of the present invention is to provide a physiological function such as a nuclear export function or a nuclear-cytoplasm transfer function related to a cell cycle control function, particularly a checkpoint function in the G2 / M phase of the cell cycle. In addition to functional cell cycle-related proteins and DNA encoding such proteins,
Promote or suppress nuclear export function, cytotoxic function, nuclear import function, nucleus-cytoplasm transfer function, and / or cell cycle control function (including nuclear division control function) using those proteins, DNAs, etc. Another object of the present invention is to provide a method for screening a substance to be used, a method for screening a substance that promotes or suppresses the expression of the above-mentioned proteins, DNA and the like.

[0009]

[Means for Solving the Problems] The present inventors have diligently studied in order to solve the above problems, and clone a gene that is expressed only in a trace amount by a method combining the RACE method and the Nested-PCR method. Protein kinases that regulate the cell cycle in mammals,
In particular, an unknown human Nek kinase family member was discovered, and the nucleotide sequence and amino acid sequence of the Nek kinase were determined using a HeLaS3 cell cDNA library. Further, in order to investigate the function of the Nek kinase, when the Nek kinase was overexpressed in a cell, the cell induced an apoptotic cell morphology. Therefore, it was found that the protein induces an apoptotic cell morphology. . Furthermore, when other functions were examined, at the C-terminal non-catalytic active site of the above-mentioned Nek kinase, nuclear export function, cytotoxic function, nuclear import function, nuclear-cytoplasmic transfer function, cell cycle control function (nuclear division) It was found that a sequence having a physiological function (including a regulatory function) exists, and the present invention has been completed.

That is, the present invention is a DNA or RNA encoding a cell cycle-related protein having a nuclear export function.
(Claim 1) or DNA or RNA encoding the following protein (a) or (b) (a) Cell cycle-related protein (b) consisting of the amino acid sequence shown in SEQ ID NO: 2. A cell cycle-related protein (claim 2) comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence, and having a nuclear export function, or the nucleotide sequence shown in SEQ ID NO: 1. Alternatively, a DNA or RNA consisting of a complementary sequence thereof or a sequence containing a part or all of these sequences (claim 3), or a DNA or RNA hybridizing with the DNA or RNA according to claim 3 under stringent conditions and having a nucleus DNA or RNA encoding a cell cycle-related protein having an externalizing function (claim 4), and a cell cycle-related protein having a nuclear-cytoplasmic transferring function. DNA or RN encoding click protein
A (claim 5) or a DNA or RNA encoding the following protein (a) or (b) (a) a cell cycle-related protein (b) represented by SEQ ID NO: 4 shown in SEQ ID NO: 4 Shown in SEQ ID NO: 3 or a cell cycle-related protein having a function of translocating between nucleus and cytoplasm, which consists of an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence DNA or RN consisting of a nucleotide sequence or a complementary sequence thereof or a sequence containing a part or all of these sequences
A (claim 7) or DNA or RNA which hybridizes with the DNA or RNA according to claim 7 under stringent conditions and which encodes a cell cycle-related protein having a nucleus-cytoplasm transfer function (claim 8). ), DNA or RNA encoding a cell cycle-related protein having a nuclear export function, a cytotoxic function and a nuclear import function (claim 9), or the following (a) or (b) protein: DNA or RNA (a) Cell cycle-related protein consisting of the amino acid sequence shown in SEQ ID NO: 6 (b) The amino acid sequence shown in SEQ ID NO: 6 with one or several amino acids deleted, substituted or added And a cell cycle-related protein having a nuclear export function, a cytotoxic function, and a nuclear import function (claim 10).
Or DNA or RNA consisting of the nucleotide sequence shown in SEQ ID NO: 5 or its complementary sequence, or a sequence containing part or all of these sequences (claim 11), or claim 11
DNA or RNA that hybridizes with the described DNA or RNA under stringent conditions and that encodes a cell cycle-related protein having a nuclear export function, a cytotoxic function and a nuclear import function (claim 12), DNA or RNA encoding a cell cycle-related protein having a nuclear export function, a cytotoxic function, a nuclear import function, and a nuclear-cytoplasmic import function (claim 13), or (a) or
DNA or RNA encoding the protein of (b) (a) Cell cycle-related protein consisting of the amino acid sequence shown in SEQ ID NO: 8 (b) In the amino acid sequence shown in SEQ ID NO: 8, one or several amino acids are deleted , Consisting of a substituted or added amino acid sequence and having a nuclear export function,
A cell cycle-related protein having a cytotoxic function, a nuclear translocation function, and a nuclear-cytoplasm translocation function (claim 14).
Or DNA or RNA consisting of the nucleotide sequence shown in SEQ ID NO: 7 or its complementary sequence, or a sequence containing a part or all of these sequences (claim 15), or claim 15
A DNA that hybridizes with the described DNA or RNA under stringent conditions and that encodes a cell cycle-related protein having a nuclear export function, a cytotoxic function, a nuclear import function, and a nuclear-cytoplasmic import function, or RNA
(Claim 16)

Further, the present invention is characterized by being capable of specifically hybridizing with the DNA or RNA according to claim 3 and inhibiting the expression of a cell cycle-related protein having a nuclear export function. An oligonucleotide characterized by being capable of specifically hybridizing with a nucleic acid (claim 17) or the DNA or RNA according to claim 7 and inhibiting the expression of a cell cycle-related protein having a nucleus-cytoplasm transfer function. Or a peptide nucleic acid (claim 18), or the DNA or RNA according to claim 11.
An oligonucleotide or peptide nucleic acid, which is capable of specifically hybridizing with and inhibiting the expression of a cell cycle-related protein having a nuclear export function, a cytotoxic function and a nuclear import function (claim 19) Or claim 1
The ability to specifically hybridize with the DNA or RNA according to 5, and to inhibit the expression of a cell cycle-related protein having a nuclear export function, a cytotoxic function, a nuclear import function, and a nuclear-cytoplasmic import function. An oligonucleotide or a peptide nucleic acid characterized by (claim 20).

The present invention also relates to a cell cycle-related protein having a nuclear export function (claim 21), a cell cycle-related protein comprising the amino acid sequence shown in SEQ ID NO: 2 (claim 22) and SEQ ID NO: 2. A cell cycle-related protein (claim 23) comprising an amino acid sequence in which one or several amino acids have been deleted, substituted or added in the amino acid sequence shown, and having a nuclear export function (claim 23),
In the cell cycle-related protein having a function of translocating between nucleus and cytoplasm (claim 24), the cell cycle-related protein consisting of the amino acid sequence shown in SEQ ID NO: 4 (claim 25), or the amino acid sequence shown in SEQ ID NO: 4, A cell cycle-related protein consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted or added and having a nuclear-cytoplasmic translocation function (claim 26), a nuclear translocation function, a cytotoxic function and 1 or 2 in the cell cycle-related protein having the function of importing into the nucleus (claim 27), the cell cycle-related protein consisting of the amino acid sequence shown in SEQ ID NO: 6 (claim 28) or the amino acid sequence shown in SEQ ID NO: 6 Consists of an amino acid sequence in which several amino acids have been deleted, substituted or added, and has a nuclear export function, cytotoxic function and nuclear function. A cell cycle-related protein having a translocation function (claim 29), a cell cycle-related protein having a nuclear translocation function, a cytotoxic function, a nuclear translocation function, and a nuclear-cytoplasm translocation function (claim 30), A cell cycle-related protein consisting of the amino acid sequence shown in SEQ ID NO: 8 (claim 31), or the amino acid sequence shown in SEQ ID NO: 8 in which one or several amino acids are deleted, substituted or added. And a cell cycle-related protein (claim 32) having a nuclear export function, a cytotoxic function, a nuclear import function, and a nucleus-cytoplasm transfer function, and one of the proteins according to any one of claims 21 to 23. And a part of the protein according to any one of claims 24 to 26, which is characterized in that it has a nuclear export function. , A peptide having a nucleus-cytoplasmic translocation closing function (claim 34) and a part of the protein according to any one of claims 27 to 29, and the nuclear translocation function, cytotoxic function and / or Or a peptide having a nuclear import function (claim 35) or a part of the protein according to any one of claims 30 to 32, and the nuclear export function, cytotoxic function, and nuclear import function. It relates to a peptide having a function and / or a nuclear-cytoplasmic translocation function (claim 36).

The present invention also relates to a fusion protein or fusion in which the protein according to any one of claims 21 to 32 or the peptide according to any one of claims 33 to 36 is bound to a marker protein and / or a peptide tag. An antibody (claim 38) that specifically binds to the peptide (claim 37), the protein according to any one of claims 21 to 32, or the peptide according to any one of claims 33 to 36, or the antibody is a monoclonal antibody. 39. The antibody according to claim 38, which is a polyclonal antibody (claim 39)
Or a recombinant protein or peptide to which the antibody of claim 38 or 39 specifically binds (claim 40), a recombinant vector containing the DNA of any one of claims 1 to 16 (claim 41), 35. The protein according to any one of claims 21 to 32, the peptide according to any one of claims 33 to 36, the fusion protein or the fusion peptide according to claim 37, the antibody according to claim 38 or 39, or claim 4.
A host cell (claim 42) comprising an expression system capable of expressing the recombinant protein according to claim 0, or claim 21.
33. The protein according to any one of claims 32 to 36,
33. A non-human animal (claim 43) in which the gene function encoding the peptide of any one of claims 1 to 3 is deleted on the chromosome, or the protein of any one of claims 21 to 32, and claim 33 to
The non-human animal that overexpresses the peptide according to any of 36 (claim 44), or the non-human animal is a mouse or rat.
Regarding 5).

The present invention also provides the protein according to any one of claims 21 to 32, the peptide according to any one of claims 33 to 36, or the protein according to any one of claims 21 to 32 or any one of claims 33 to 36. A cell membrane expressing any one of the peptide and a test substance, which is used for nuclear export function, cytotoxic function, nuclear import function, nuclear-cytoplasmic transfer function, and / or A method for screening a substance for promoting or suppressing a cell cycle control function (claim 46), or a cell expressing the protein according to any one of claims 21 to 32 or the peptide according to any one of claims 33 to 36. , Promoting a nuclear export function, cytotoxicity function, nuclear import function, nucleus-cytoplasm transfer function, and / or cell cycle control function characterized by using a test substance Inhibit substance or claim 2,
The protein according to any one of claims 1 to 32 or claim 33 to.
36. A method for screening a substance for promoting or suppressing the expression of the peptide according to any of 36, (claim 47), and
The non-human animal according to any one of 3 to 45 and a test substance are used, the nuclear export function, the cytotoxic function, the nuclear import function, the nuclear-cytoplasmic transfer function, and / or the cell. A method for screening a substance that promotes or suppresses a cycle control function, or a substance that promotes or suppresses expression of the protein according to any one of claims 21 to 32 or the peptide according to any one of claims 33 to 36 (claim 48).

The present invention also provides the nuclear export function, cytotoxicity function, nuclear import function, nucleus-cytoplasm transfer function, and / or cell cycle obtained by the screening method according to any one of claims 46 to 48. 21. A substance promoting or suppressing a control function (claim 49) or a screening method according to any one of claims 46 to 48.
33. The protein according to claim 2 or the peptide according to any one of claims 33 to 36, which is an expression promoting substance or an expression suppressing substance (claim 50); and the protein according to any one of claims 21 to 32, 33. 36. The peptide according to any of 36, the recombinant protein or peptide according to claim 40, the antibody according to claim 38 or 39, and the 49.
Nuclear export function described, cytotoxic function, nuclear import function,
51. Treatment of an immune disease, a neurological disease, or a cancer, which contains as an active ingredient a promoter or suppressor of the nucleus-cytoplasm transfer function and / or cell cycle control function, or the expression promoter or expression suppressor of claim 50. 33. A drug (claim 51), the protein according to any one of claims 21 to 32, and claim 33.
To 36, the recombinant protein or peptide according to claim 40, the antibody according to claim 38 or 39, the nuclear export function, the cytotoxic function, the nuclear import function, and the nucleus according to claim 49. -A diagnostic agent for an immune disease, a neurological disease, or a cancer, which contains a substance that promotes or suppresses the function of intercytoplasmic transition and / or cell cycle control, or the substance that promotes expression or suppresses the expression according to claim 50 as an active ingredient. (Claim 52)

[0016]

BEST MODE FOR CARRYING OUT THE INVENTION The cell cycle-related protein of the present invention is a protein consisting of the amino acid sequence shown in SEQ ID NO: 2, wherein one or several amino acids are deleted or substituted in the amino acid sequence shown in SEQ ID NO: 2. Alternatively, a cell cycle-related protein having an nuclear export function, such as a protein having an added amino acid sequence and having the nuclear export function, a protein consisting of the amino acid sequence shown in SEQ ID NO: 4, the amino acid shown in SEQ ID NO: 4 A cell cycle-related protein having a nuclear-cytoplasmic translocation function, such as a protein having a nuclear-cytoplasmic translocation function, which consists of an amino acid sequence in which one or several amino acids are deleted, substituted or added in the sequence, and a sequence A protein consisting of the amino acid sequence shown in SEQ ID NO: 6, the amino acid sequence shown in SEQ ID NO: 6 In the above, a nuclear export function such as a protein comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added and having a nuclear export function, a cytotoxic function and a nuclear import function, a cytotoxicity 1 in the amino acid sequence shown in SEQ ID NO: 8 or the protein consisting of the amino acid sequence shown in SEQ ID NO: 8
Alternatively, a nuclear export of a protein or the like, which consists of an amino acid sequence in which several amino acids are deleted, substituted or added, and has a nuclear export function, a cytotoxic function, a nuclear import function, and a nuclear-cytoplasmic import function Examples thereof include a cell cycle-related protein having a function, a cytotoxic function, a nuclear import function, and a nucleus-cytoplasm import function. The above-
The intercytoplasmic transfer function refers to the function of serving as a substrate in the nucleus-cytoplasmic transport system. A cell cycle consisting of an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 6 and having a nuclear export function, a cytotoxic function and a nuclear import function. Specific examples of the related protein include Nek9S consisting of the amino acid sequence shown in SEQ ID NO: 10, and in the amino acid sequence shown in SEQ ID NO: 8, one or several amino acids have been deleted, substituted or added. A cell cycle-related protein consisting of an amino acid sequence and having a nuclear export function, a cytotoxic function, a nuclear import function, and a nuclear-cytoplasmic transfer function is a Ne consisting of the amino acid sequence shown in SEQ ID NO: 12.
A specific example is k9L. The protein of the present invention can be prepared by a known method based on its DNA sequence information and the like, and its origin is not particularly limited. Further, the peptide to be the subject of the present invention is composed of a part of the cell cycle-related protein of the present invention, and has an nuclear export function, a cytotoxic function, a nuclear import function, and / or a nucleus-cytoplasm transfer function. Any material may be used as long as it has it. The proteins and peptides that are the subject of the present invention, as well as the recombinant proteins and peptides to which the antibodies that specifically bind to these proteins and peptides specifically bind, may be collectively referred to as "the subject protein / peptide" below. . The protein / peptide of the present invention can be prepared by a known method based on its DNA sequence information and the like, and the origin of such protein is not limited to human.

The polynucleotide of the present invention such as DNA and RNA may be any polynucleotide as long as it encodes the above-mentioned protein / peptide of the present invention.
For example, DNA or RNA encoding a cell cycle-related protein consisting of the amino acid sequence shown in SEQ ID NO: 2.
Or a DNA or RNA coding for a cell cycle-related protein having the nuclear export function, which consists of an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 2, ,
From DNA or RNA encoding a cell cycle-related protein consisting of the amino acid sequence shown in SEQ ID NO: 4, or from the amino acid sequence shown in SEQ ID NO: 4 in which one or several amino acids are deleted, substituted or added And a DNA or RNA encoding a cell cycle-related protein having a nuclear-cytoplasmic transition function, a DNA or RNA encoding a cell cycle-related protein consisting of the amino acid sequence shown in SEQ ID NO: 6, or shown in SEQ ID NO: 6. DNA consisting of an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence described above, and which encodes a cell cycle-related protein having a nuclear export function, a cytotoxic function and a nuclear import function Alternatively, RNA or a cell perimeter composed of the amino acid sequence shown in SEQ ID NO: 8 DNA encoding the relevant protein
Or RNA, or the amino acid sequence shown in SEQ ID NO: 8, which is composed of an amino acid sequence in which one or several amino acids are deleted, substituted or added, and has a nuclear export function,
DN encoding a cell cycle-related protein having a cytotoxic function, a nuclear import function, and a nuclear-cytoplasm import function
A or RNA, DNA or RNA encoding Nek9S consisting of the amino acid sequence shown in SEQ ID NO: 10,
Nek9 comprising the amino acid sequence shown in SEQ ID NO: 12
DNA or RNA encoding L, SEQ ID NOS: 1, 3,
Specific examples thereof include polynucleotides such as DNA and RNA having the nucleotide sequences shown in 5, 7, 9 or 11 or their complementary sequences or sequences containing a part or all of these sequences. Also included in the present invention is an oligonucleotide sequence that can be used for amplification, or a DNA or RNA containing a nucleotide sequence that can hybridize under conditions that can be used as a probe or a marker. These DNAs or RNAs are used for the diagnosis of various diseases related to the expression or function of the protein / peptide of the present invention such as immune diseases, neurological diseases, cancers and the like, or susceptibility to such diseases, and the like. It can be used to examine changes in expression level. These can be prepared by known methods from, for example, human, mouse, rat, rabbit gene libraries or cDNA libraries based on the DNA sequence information and the like.

A DNA or RNA consisting of the nucleotide sequence shown in SEQ ID NO: 1, 3, 5, 7, 9 or 11 or its complementary sequence and a sequence containing a part or all of these sequences is used as a probe. By hybridizing various DNA libraries or various RNA libraries under stringent conditions and isolating DNA or RNA that hybridizes to the probe, the nuclear export function, cytotoxic function, nuclear function It is also possible to obtain DNA or RNA encoding a protein having an internalizing function and / or a nuclear-cytoplasmic importing function. The DNA or RNA thus obtained is also within the scope of the present invention. The hybridization conditions for obtaining the polynucleotide of the present invention such as DNA and RNA include:
For example, hybridization at 42 ° C. and 1 ×
Examples include washing treatment with SSC and a buffer containing 0.1% SDS at 42 ° C., hybridization at 65 ° C., and 0.1 × SSC, 0.1% SDS.
More preferably, the washing treatment at 65 ° C. with a buffer solution containing There are various factors other than the above temperature conditions as factors that affect the stringency of hybridization, and those skilled in the art can appropriately combine various factors and are equivalent to the stringency of hybridization described above. It is possible to achieve the stringency of.

The oligonucleotide or peptide nucleic acid of the present invention includes SEQ ID NO: 1, 3, 5, 7, 9, or 11
Which specifically hybridizes with a DNA or RNA consisting of the nucleotide sequence shown in or a complementary sequence thereof or a sequence containing a part or all of these sequences, and has a nuclear export function,
Those capable of inhibiting the expression of cell cycle-related proteins having a cytotoxic function, a nuclear import function, and / or a nuclear-cytoplasm import function (for example, Bioconjugate Chem. Vol. 5, N
o.1, 1994), there is no particular limitation. The above-mentioned "oligonucleotide" refers to an oligonucleotide or an analogue thereof produced from a naturally occurring base, or a sugar moiety bound by an original phosphodiester bond, or the like, and the naturally occurring base or the original phosphonate. The oligonucleotide produced from a sugar moiety or the like linked by a diester bond refers to a naturally occurring species or a naturally occurring subunit, or a synthetic species produced from a homologue thereof. In addition, the above-mentioned subunit refers to a base-sugar combination in which adjacent subunits are linked by a phosphodiester bond or other bond.

On the other hand, the above-mentioned naturally-occurring base or an analogue of an oligonucleotide produced from a sugar moiety or the like which is bound by an original phosphodiester bond means the same function as the above-mentioned oligonucleotide, but it exists in nature. Not a base such as dihydrouridine, inosine, 4-
Oligonucleotides having modified bases such as acetylcytidine and 1-methyladenosine, phosphate groups, sugar moieties,
It refers to an oligonucleotide or the like in which stability is increased by chemically modifying the 3'and 5'ends. The method for chemically modifying the above-mentioned phosphate group, sugar moiety, and 3 ′, 5 ′ end includes:
For example, a method of substituting one of the oxygen atoms of a phosphodiester group between nucleotides with sulfur to obtain an oligophosphorothioate, or a method of substituting one of the oxygen atoms of a phosphodiester group between nucleotides with -CH ₃ to obtain an oligomethylphosphonate And a method of substituting the phosphodiester bond site with another nonionic and nonchiral structure. However, the present invention is not limited thereto.

The above-mentioned oligonucleotide can be produced by those skilled in the art by a known synthesis method, for example, a solid-phase synthesis method using a synthesizer such as Applied Biosystems. In addition, other oligonucleotide analogues such as phosphorothioate and alkylated derivatives can also be produced by using the same method (Murakami et al., “Chemical synthesis of functional antisense DNA”, Organic synthetic chemistry, 48).
(3): 180-193, 1990). The oligonucleotide or peptide nucleic acid of the present invention obtained by these methods,
It is possible to suppress the production of the subject protein / peptide in animals.

The fusion protein or fusion peptide of the present invention may be any one as long as the protein / peptide of the present invention is bound to a marker protein and / or a peptide tag. It is not particularly limited as long as it is a known marker protein, and specific examples thereof include alkaline phosphatase, Fc region of antibody, HRP, GFP, and the like, and the peptide tag in the present invention includes HA tag. , Myc tag, His tag, FLA
Specific examples of conventionally known peptide tags such as G tag, GST tag, and Xpess tag can be given, but the present invention is not limited thereto, and a leader sequence,
A pro sequence or an amino acid sequence for stable recombinant production can also be mentioned. Such a fusion protein can be produced by a conventional method and contains Ni-NTA and H
Purification of the Protein / Peptide using the affinity of the is tag, detection of the Protein / Peptide, production of antibodies against the Protein / Peptide, quantification of antibodies against the Protein / Peptide, immune diseases, neurological diseases, It is also useful as a therapeutic agent for various diseases with abnormal cell cycle such as cancer, a diagnostic agent for various diseases with immune cycle, nerve disease, cancer and other abnormal cell cycle, and as a reagent for research in the field.

As the antibody that specifically binds to the protein or peptide of the present invention, a monoclonal antibody having a substantially stronger affinity for the protein / peptide of the present invention than for other related polypeptides in the prior art. Specific examples thereof include immunospecific antibodies such as polyclonal antibodies, chimeric antibodies, single chain antibodies, and humanized antibodies. These can be prepared by a conventional method using all or part of the above-mentioned protein / peptide, fusion protein, fusion peptide and the like as an antigen, and among them, a monoclonal antibody is more preferable in terms of its specificity. Antibodies such as such monoclonal antibodies include, for example, quantification of the protein / peptide of the present invention, therapeutic agents for various diseases with abnormal cell cycle such as immune disease, neurological disease, and cancer, and abnormal cell cycle such as immune disease, neurological disease, and cancer. Not only diagnostic agents for various diseases, but also cell cycle mechanism, nuclear export mechanism, cytotoxic function mechanism, nuclear import mechanism, mitotic control system mechanism, nuclear-cytoplasmic transport system It is useful for clarifying the mechanism and the mechanism of cell cycle control.

The above-mentioned antibody of the present invention is administered to an animal (preferably other than human) with a protein / peptide or a fragment thereof containing the protein of the present invention, or a cell expressing the protein on the membrane surface, using a conventional protocol. For example, for the preparation of monoclonal antibodies produced by E. coli, the hybridoma method (Nature 256, 495-497, 197
5), trioma method, human B cell hybridoma method (Imm
unology Today 4, 72, 1983) and EBV-hybridoma method (MONOCLONAL ANTIBODIES AND CANCER THERAPY,
pp.77-96, Alan R. Liss, Inc., 1985).

In order to prepare a single chain antibody against the above-mentioned protein / peptide of the present invention, a method for preparing a single chain antibody (US Pat. No. 4,946,778) can be applied.
In addition, in order to express a humanized antibody, a transgenic mouse or another mammal is used, or by using the above antibody, a clone expressing the protein / peptide of the present invention is isolated / identified, or affinity chromatography is performed. The polypeptide can also be purified with. Antibodies against the protein / peptide of the subject are as described above.
Not only quantification of the protein / peptide, therapeutic agents for various diseases with abnormal cell cycle such as immune diseases, neurological diseases and cancers, diagnostic agents for various diseases with abnormal cell cycle abnormalities such as immune diseases, neurological diseases and cancers , Mechanism of cell cycle, mechanism of nuclear export, mechanism of cytotoxic function, mechanism of nuclear import, mechanism of mitotic control system, mechanism of transport system between nucleus and cytoplasm, mechanism of cell cycle control, etc. It is useful for Recombinant proteins or peptides to which these antibodies specifically bind are also included in the present proteins / peptides of the present invention as described above.

In addition, the above-mentioned monoclonal antibody and the like,
For example, FITC (fluorescein isocyanate) or
Is a fluorescent substance such as tetramethylrhodamine isocyanate
Or ¹²⁵I,³²P,¹⁴C,³⁵S or³Radio iso such as H
Taupe, alkaline phosphatase, peroxidase
Enzyme, β-galactosidase, phycoerythrin, etc.
Labeled with a green fluorescent protein or green fluorescent protein (GF
P) and other fluorescent proteins, etc.
By using the protein, the protein / peptide
Function analysis can be performed. Further, the antibody of the present invention
Examples of immunological measurement methods using RIA include RIA and ELI.
SA method, fluorescent antibody method, plaque method, spot method, blood cell coagulation
Examples include the collection reaction method and the Ouchterlony method.
It

The recombinant vector of the present invention is not particularly limited as long as it is a vector containing the above-mentioned DNA of the present invention, but preferably contains an expression system capable of expressing the protein / peptide of the present invention in a host cell, For example,
Expression systems derived from chromosomes, episomes and viruses, more specifically vectors derived from bacterial plasmids, yeast plasmids, papovaviruses such as SV40, vaccinia virus, adenovirus, fowlpox virus, pseudorabies virus, retrovirus , Vectors derived from bacteriophages, transposons and combinations thereof, such as those derived from genetic elements of plasmids and bacteriophages such as cosmids and phagemids. The expression system may include control sequences that regulate as well as direct expression.

The present invention also relates to a host cell comprising an expression system capable of expressing the protein / peptide of the present invention. Such a gene encoding the protein / peptide of the present invention can be introduced into a host cell by Davis et al. (BASIC MET
HODS IN MOLECULAR BIOLOGY, 1986) and Sambrook et al. (MO
LECULAR CLONING: A LABORATORY MANUAL, 2nd Ed., Cold
Spring Harbor Laboratory Press, Cold Spring Harbo
r, NY, 1989), and many standard laboratory manual methods such as calcium phosphate transfection, DEAE-dextran mediated transfection, transvection, microinjection, cationic lipid mediated transfection. , Electroporation, transduction, scrape loading, ballistic introduction, infection and the like. Then, as the host cell, Escherichia coli, Streptomyces, Bacillus subtilis, Streptococcus, bacterial prokaryotic cells such as Staphylococcus, yeast, fungal cells such as Aspergillus, and insect cells such as Drosophila S2, Spodoptera Sf9, L cells, CHO cells, COS cells, NI
H3T3 cells, HeLa cells, C127 cells, BALB
/ C3T3 cells (including mutant strains deficient in dihydrofolate reductase, thymidine kinase, etc.), BHK21 cells, HEK293 cells, Bowes malignant melanoma cells, and other animal cells, and plant cells.

Any expression system may be used as long as it can express the protein / peptide of the present invention in a host cell. Expression systems derived from chromosomes, episomes, mammals and viruses , For example, bacterial plasmid derived, yeast plasmid derived, SV4
0, such as papovavirus, vaccinia virus, adenovirus, fowlpox virus, pseudorabies virus, retrovirus-derived vector, bacteriophage-derived,
Vectors derived from transposons and combinations thereof, such as those derived from genetic elements of plasmids and bacteriophage, such as cosmids and phagemids can be mentioned. The expression system may include control sequences that regulate as well as direct expression.

The host cell containing the above expression system, the cell membrane of such cell, and the protein / peptide of the present invention obtained by culturing such cell can be used in the screening method of the present invention as described later. For example, as a method for obtaining a cell membrane, F. Pietri-Rouxel (Eur. J. Bioc
hem., 247, 1174-1179, 1997) and the like can be used. In order to recover and purify the protein / peptide of the present invention from cell culture, ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, Known methods including hydroxyapatite chromatography and lectin chromatography, preferably high performance liquid chromatography are used. Particularly, as a column used for affinity chromatography, for example, a column to which an antibody against the protein / peptide of the present invention is bound, or an ordinary peptide tag added to the protein / peptide of the present invention has an affinity for this peptide tag. The protein / peptide of the present invention can be obtained by using a column to which a substance is bound. The above-mentioned method for purifying the protein / peptide of the present invention can also be applied to peptide synthesis.

In the present invention, the non-human animal in which the gene function encoding the protein / peptide of the present invention is deficient on the chromosome means that a part or all of the gene encoding the protein / peptide of the present invention on the chromosome is destroyed / defective. A non-human animal that has been inactivated by gene mutation such as substitution and has lost the function of expressing the protein / peptide of the present invention. It is possible to specifically present a non-human animal that produces a large amount of the protein / peptide of the present invention as compared with the above. The non-human animal in the present invention can specifically include, but is not limited to, non-human animals such as rodents such as mice and rats.

By the way, homozygous non-human animals born according to Mendelian's law include the present protein / peptide deficient type or overexpressed type and its littermate wild type. Since it is possible to carry out an accurate comparative experiment at the individual level by simultaneously using the deficient or overexpressed type and the wild type of the same litter in, the wild type non-human animal, that is, the present protein
It is preferable to use an animal of the same species as the non-human animal in which the gene function encoding the peptide is defective or overexpressed on the chromosome, and further an animal of the same litter, for example, in the screening of the present invention described below. A method for producing a non-human animal in which the gene function encoding the protein / peptide of the present invention is deficient or overexpressed on the chromosome is referred to as Nek9L.
A knockout mouse and a Nek9L transgenic mouse will be described below as examples.

For example, a mouse in which the gene function encoding the Nek9L protein is deficient on the chromosome, that is, N
The ek9L knockout mouse uses a gene fragment obtained by a method such as PCR from a mouse gene library to obtain mouse Nek9 having homology with human Nek9L.
The gene encoding L is screened, the screened gene encoding mouse Nek9L is subcloned using a viral vector or the like, and specified by DNA sequencing. Mouse Nek of this clone
All or part of the gene encoding the C-terminal portion of 9L (the portion corresponding to the protein consisting of the amino acids 289 to 645 of human Nek9) was replaced with pMC1 neo gene cassette or the like, and diphtheria toxin was added to the 3'-terminal side. A targeting vector is prepared by introducing a gene such as the A fragment (DT-A) gene and the herpes simplex virus thymidine kinase (HSV-tk) gene.

The thus prepared targeting vector is linearized, introduced into ES cells by electroporation (electroporation) method or the like, and homologous recombination is performed. From among the homologous recombinants, G418 and ganciclovir are selected. (G
E homologous recombination caused by antibiotics such as ANC)
Select S cells. In addition, it is preferable to confirm whether the selected ES cell is the target recombinant by Southern blotting or the like. The confirmed ES cell clone is microinjected into a mouse blastocyst, and the blastocyst is returned to a foster mouse to prepare a chimeric mouse. By intercrossing this chimeric mouse with a wild-type mouse, a heterozygous mouse can be obtained, and by intercrossing this heterozygous mouse, the Nek9L knockout mouse of the present invention can be produced. . Further, as a method for confirming whether or not the expression ability of Nek9L is deleted in the Nek9L knockout mouse, for example, RNA is isolated from the mouse obtained by the above method and examined by Northern blotting or the like. There is a method of directly examining the expression of Nek9L in mice by Western blotting or the like.

The Nek9L transgenic mouse is
Nek9L derived from human, mouse, rat, rabbit, etc.
CDNA encoding the above gene is fused with a promoter such as chicken β-actin, mouse neurofilament, SV40, and poly A or intron such as rabbit β-globin, SV40 to construct a transgene, and the transgene is a mouse fertilized egg. Microinjected into the pronucleus of
To create a transgenic mouse by culturing the obtained egg cells, transplanting it into the oviduct of a foster mother, and then raising the recipient animal and selecting the offspring mouse having the cDNA from the born offspring mice. You can In addition, the selection of the mouse pups having the cDNA is performed by extracting the crude DNA from the tail or the like of the mouse and using the dot hybridization method using the introduced gene encoding Nek9L as a probe or the PCR method using a specific primer. be able to.

DNA or RNA encoding the protein / peptide of the present invention, the protein / peptide of the present invention,
This protein / peptide and marker protein and /
Or a fusion protein bound to a peptide tag, an antibody against the subject protein / peptide, the subject protein / peptide
A recombinant vector containing a DNA encoding a peptide, a host cell containing an expression system capable of expressing the protein / peptide of the present invention, and the like have a nuclear export function, cytotoxicity, as will be specifically described below. Function, nuclear migration function,
A substance that promotes or suppresses the function of translocation between nucleus and cytoplasm and / or cell cycle control, a substance that promotes or suppresses expression of the protein / peptide of the present invention, and various diseases of abnormal cell cycle such as immune disease, neurological disease, and cancer It is useful as a therapeutic drug and as a diagnostic drug for various diseases of cell cycle abnormalities such as immune diseases, neurological diseases, and cancers. It is also useful for elucidating the mechanism, mechanism of mitotic control system, mechanism of nuclear-cytoplasmic transport system, mechanism of cell cycle control, and the like.

The nuclear export function, cytotoxic function of the present invention,
Examples of the screening method for a substance that promotes or suppresses the nuclear import function, the nuclear-cytoplasmic import function, and / or the cell cycle control function (including the nuclear division control function) include:
A method of using the protein / peptide of the present invention or a cell membrane expressing the protein / peptide of the present invention and a test substance, or a method of using a cell expressing the protein / peptide of the present invention and a test substance Alternatively, a method using a non-human animal such as a knockout mouse or transgenic mouse of the protein / peptide of the present invention and a test substance can be mentioned. Further, as a screening method for a substance that promotes or suppresses expression of the protein / peptide of the present invention, a method of using cells expressing the protein / peptide of the present invention and a test substance, or a knockout mouse or transgenic of the protein / peptide of the present invention A method using a non-human animal such as a mouse and a test substance can be specifically exemplified.

As a screening method using the subject protein / peptide or the cell membrane expressing the subject protein / peptide and the test substance, the subject protein / peptide or the subject protein / peptide expressed on the surface of the cell membrane Specifically, a method of contacting with a test substance to measure and evaluate the biological activity of the protein / peptide of the present invention such as nuclear export function, cytotoxic function, nuclear import function, nucleus-cytoplasm transfer function, etc. Can be mentioned. In addition, cells expressing the subject protein / peptide,
As a screening method using a test substance, cells expressing the protein / peptide of the present invention are brought into contact with the test substance, and the nuclear transfer function, cytotoxic function, and nuclear transfer function of the protein / peptide of the present invention are brought into contact. Specific examples include methods for measuring and evaluating biological activities such as nuclear-cytoplasmic translocation function and changes in the expression level of the subject protein / peptide.

As a screening method using a non-human animal in which the gene function encoding the protein / peptide of the present invention is deficient on the chromosome or a non-human animal overexpressing the protein / peptide of the present invention, and a test substance, A cell, tissue, or organ obtained from a human animal is brought into contact with a test substance in vitro, and the protein
Methods for measuring / evaluating changes in the expression level of the protein / peptide of the subject, as well as the biological activity of the peptide's nuclear translocation function, cytotoxicity function, nuclear translocation function, nuclear-cytoplasmic translocation function, etc. After pre-administering a test substance to a non-human animal whose gene function encoding a protein / peptide is deficient on the chromosome or a non-human animal overexpressing the subject protein / peptide, cells, tissues, or obtained from the non-human animal, or Measurement / evaluation of changes in the expression level of the protein / peptide and the biological activity of the protein / peptide in the organ such as nuclear export function, cytotoxicity function, nuclear import function, and nuclear-cytoplasmic transfer function How to do
The non-human animal in which the gene function encoding the protein / peptide is defective on the chromosome or the protein /
After pre-administering a test substance to a non-human animal that overexpresses the peptide, the nuclear translocation function, cytotoxicity function, nuclear translocation function, nuclear-cytoplasmic translocation function of the protein / peptide of the present invention in the non-human animal Specific examples thereof include a method for measuring / evaluating the biological activity of, and changes in the expression level of the protein / peptide of the present invention. In the above screening, a non-human animal in which the gene function encoding the protein / peptide of the present invention is deficient on the chromosome, or a non-human animal overexpressing the protein / peptide of the present invention and a wild-type non-human animal of the same litter are compared and evaluated. Is more preferable.

The promotion of the nuclear export function, cytotoxicity function, nuclear import function, nucleus-cytoplasm transfer function, cell cycle control function, etc. of the present invention obtained by the above-mentioned screening method, and It can be used for treatment of patients in need of promotion of expression. Further, patients in need of suppression of the nuclear export function, cytotoxicity function, nuclear import function, nuclear-cytoplasmic transfer function, etc. of the present invention obtained by the above screening method, and the suppression of expression of the subject protein / peptide Can be used for the treatment of The protein / peptide of the present invention or an antibody thereto, the above-mentioned nuclear export function, cytotoxic function, nuclear import function, nucleus-cytoplasm transfer function, cell cycle control function or other promoter or inhibitor, or the subject protein -Peptide expression-promoting substances or expression-suppressing substances are therapeutic agents for various diseases having cell cycle abnormalities such as immune diseases, neurological diseases, and cancers, or lead compounds thereof, and various cell cycle abnormalities such as immune diseases, neurological diseases, and cancers. It can be used as an active ingredient such as a diagnostic agent for the above diseases, and can also be used for missile therapy. These therapeutic agents can be administered orally or parenterally. As the orally-administered agent, solid preparations such as powder, granules, capsules and tablets, or liquid preparations such as syrups and elixirs can be used. In addition, parenteral administration agents such as injections, transdermal preparations and suppositories can be used. These preparations can be manufactured according to a conventional method by adding a pharmacologically or pharmaceutically acceptable auxiliary agent to the active ingredient. Examples of the auxiliaries include, for oral agents and mucous membrane administration agents, light anhydrous silicic acid, starch, lactose, crystalline cellulose, excipients such as calcium lactose, disintegrating agents such as carboxymethylcellulose, stearic acid. Ingredients such as lubricants such as magnesium, and in the case of injections, physiological saline, mannitol, propylene glycol and other solubilizers or solubilizers, surfactants and other suspending agents, etc. In the case of the external preparation of the pharmaceutical ingredient (1), a pharmaceutical ingredient such as an aqueous or oily solubilizer or solubilizer and an adhesive is used. In addition, the dose can be appropriately determined depending on the type of target disease, the age, sex, weight of the patient, symptoms, and administration form.

[0041]

The present invention will be described in more detail with reference to the following examples, but the technical scope of the present invention is not limited to these examples. Example 1 (Novel NIMA-related protein kinase Nek
Identification of 9) The EST database was searched to identify putative NIMA-related protein kinase genes. One deduced amino acid sequence in the EST clone (GenBan
k ID: BE388672) was remarkably similar to the sequence of STK2 / Nek4, but 5'and 3'of such cDNA sequence.
A frame shift was observed at the ends. Since the expression of the target gene was at a low level in the process of molecular cloning, it was cloned by 5'- and 3'-end nested RACE-PCR method to identify the full-length cDNA sequence. As a result, two types of full-length cDNAs encoding proteins consisting of 645 and 470 amino acids were found (molecular weights were 74162 and 54006, respectively, and theoretical pI values were 5.0 and 5.66, respectively) (Fig. 1A).
These proteins had a matching N-terminal catalytic site and two coiled coils and two PEST-like motifs could be identified in the C-terminal non-catalytic site. In addition, by genomic BLAST analysis, cDNAs encoding these proteins were all mapped to chromosome 3q21-q22, and it is considered that the difference in exons at the 3'ends of the two cDNAs is due to alternative splicing ( FIG. 1A). Sequencing of multiple sequences of catalytic sites suggests that Nek9 has a unique subdomain of serine / threonine that is closely related to NIMA, and phylogenetic analysis of the catalytic sites of the NIMA kinase family shows that It was suggested that Nek9 identified in 1. was a novel member of the above family (FIG. 1).
B). These proteins are designated as Nek9L (molecular weight 74162) and Nek9S (molecular weight 54006), respectively.
(NIMA-related kinase 9 Long and Short isoform). In addition, it was found from the secondary structure prediction that only Nek9 protein has a coiled coil and a PEST-like motif in the C-terminal half, like NIMA derived from fungi in the mammalian NIMA-related kinase family (Fig. 1B). ). The catalytic site of the human Nek9 protein is most closely related to the Nek3 and Nek4 / STK2 proteins (43% identity to each other) and 33% identity to NIMA from Aspergillus nidulans. Found to have. C-terminal 1
Substructures differ within the NIMA family and such primary structure of Nek9 did not show significant similarity to other NIMA family members.

Example 2 (Expression of Nek9 in various cell lines) By the molecular cloning experiment of Nek9 cDNA,
Although the expression of ek9 mRNA was expected to be at a low level, a signal was not confirmed at all when Northern blot analysis was actually performed using a total RNA sample. However, poly (A) RNA blot analysis using various cell lines confirmed that nek9 mRNA was expressed at a very low level. Lung malignant tumor NCl-H23
In cancer cells such as cells and epidermoid malignant tumor A431 cells, the expression of nek9 mRNA could be confirmed relatively easily. As a result of expression analysis using a multi-tissue array, the expression level of nek9 mRNA was low, and weak signals were observed in tissues such as the left side of the cerebellum, trachea, lung, appendix, and uterus. Germ cell tissue (Gene 234,1
27-137, 1999, Development 124, 2167-2177, 1997, Ex
p. Cell Res. 237, 264-274, 1997, Oncogenene 16, 18
13-1827, 1998, Dev. Biol. 208, 456-464, 1999, Bioc
hem. Biophys. Res Commun. 264, 449-456, 1999), unlike nek1, nek2, and nek4, nek9 mRNA was not highly expressed in testis and proliferative blood cells. Although nek9 mRNA expressed in various cell lines was confirmed in a portion of about 3 kb corresponding to Nek9L (FIG. 2A), nek9S
mRNA was difficult to detect by RNA blot analysis.

To examine the expression of Nek9 in cells, the C-terminal half of Nek9S (polypeptide consisting of amino acid sequence 327 to 470) containing a second coiled coil and two PEST-like motifs (upper panel of FIG. 2B). Bacteria expressing the recombinant protein were recovered by anti-polyclonal antibody. Polyclonal antibody purified by affinity chromatography (α-Ne
k9 polyclonal antibody) or antiserum (α-Nek9 serum) by Western blot analysis, flag-tagged Nek9, GFP-tagged Nek9L, and GFP-t containing either the second coiled coil or two PEST-like motifs.
Although tagged Nek9L (polypeptide consisting of the 379th to 645th amino acid sequence: GLC-ter) was confirmed (lanes 1, 2, 3 and 5 in the lower diagram of FIG. 2B), Nek
GFP-tagged Nek9L (1 to 3 lacking the C-terminal half of 9)
A polypeptide consisting of the 29th amino acid sequence) (GFP-
Catalytic) could not be detected (lane 4 in the lower diagram of FIG. 2B). From these results, it was found that the purified anti-Nek9 polyclonal antibody specifically recognizes the C-terminal portion of Nek9 protein.

Next, it was examined whether or not the exogenous Nek9 protein is expressed in various human cell lines (FIG. 2).
C). As a result, when the whole cell solution was subjected to Western blot analysis, it was found that the α-Nek9 polyclonal antibody recognized an approximately 75 kDa protein (two bands could be confirmed in some samples) (Fig. 2c, upper part). Figure). The molecular weight of the confirmed band was completely in agreement with that of Nek9L (molecular weight 74
162). These results revealed that nek9L is a Nek9 protein that is mainly expressed in somatic cells. The expression level of Nek9 protein is mR.
It was also confirmed that it did not match the expression level of NA (Fig. 2).
Expression of A and 2C, nek9 mRNA was found in HEK29
A431 cells were relatively higher than 3T cells,
Expression of Nek9 protein was the reverse. ). Based on the above, it was considered that the amount of Nek9 might affect some post-translational mechanisms in specific cells.

Example 3 (Cell Cycle Controlled Expression of Nek9) Fungal-derived NIMA, mammalian-derived Nek2 and Nek3
Shows a cell cycle-dependent expression pattern (Cell
Growth Differ. 5, 625-635, 1994, J. Biol. Chem. 27
4, 13491-13497, 1999, J. Cell Biol. 104, 1495-150.
4, 1987), and examined the expression pattern of Nek9 throughout the cell cycle. HeLa cells were synchronized with a dual inhibitor of thymidine / affididecolin, and the expression of nek9 mRNA was examined after the cells passed through the G1 / S transition period (upper panel of FIG. 3A). Using the ethidium bromide (EtBr) staining pattern of poly (A) RNA as a control (middle panel of FIG. 3A), the DNA content at each time was examined by flow cytometry (lower panel of FIG. 3A). As a result, it was confirmed that the expression level of nek9 mRNA was low in the G1 / S transition period (0 hour) but increased in the S to G2 period (6 to 10 hours). The expression level of nek9 mRNA is G2 / M
Reached the peak at the end of the period (9 to 12 hours), and again the G1 period (1
It became low after 4 hours. Ne during the cell cycle
In order to examine the expression level of k9 protein, HeLaS3 cells were pre-synchronized with a thymidine inhibitor, and an Affidecolin inhibitor was added to G2 at the G1 / S transition period.
Synchronized with a nocodazole inhibitor during the / M transition period. After that, after passing through the G1 / S period (left view of FIG. 3B)
And Nek9 in the G2 / M transition period (right panel of FIG. 3B)
The expression level of the protein was examined by Western blotting. Previously, it was reported that the expression level of Nek2 protein increased from S to G2 phase and decreased in mitosis phase (J. Biol. Chem 270, 12899-1290.
5, 1995), and it was reported that the expression level of cyclin B1 (CycB1) reached a peak in the G2 / M phase and rapidly decreased after mitosis (Curr. Opin. Cell).
Biol. 11, 267-273, 1999). From these facts, the expression of Nek2 and cyclin B1 (CycB1) proteins that control the cell cycle in the progression of the cell cycle was also examined (FIG. 3B), and the expression level of tubulin was normalized as a control (FIG. 3B). These results indicate that after reaching the maximum level of Nek2 protein,
There was an increase in the level of Nek9 protein, followed by an increase in cyclin B1 (FIG. 3B, left panel). In addition to this, in parallel with the pattern of cyclin B1 down-regulation, 4 hours after the G2 / M transition phase, N
The expression level of the ek9 protein was decreased (FIG. 3B, right panel). Therefore, it was found that the expression level of Nek9 protein reached the maximum in G2 / M phase in agreement with the expression peak of mRNA. From these, it was revealed that Nek9 is a novel cell cycle regulatory kinase protein that is mainly expressed in the G2 / M phase.

Example 4 (Protein Kinase Activity of Nek9) To examine the protein kinase activity of Nek9, A
Kinase inactive mutant Nek9 (K61R) in which the 61st lysine residue interacting with TP was replaced with arginine
Was produced. Flag-tagged in HEK293T cells
After immunoprecipitation of Nek9, when the autophosphorylation of wild-type Nek9 and kinase inactive Nek9 (K61R) was examined, autophosphorylation of wild-type Nek9 was detected, but
No autophosphorylation of the kinase inactive Nek9 (K61R) could be detected. In the in vitro kinase assay, Nek9 kinase activity was clearly confirmed when Mn ²⁺ ion was used, but Nek9 kinase activity was also confirmed when Mg ²⁺ ion was used. Therefore, the following in vitro kinase analysis of Nek9 was performed using Mn ²⁺ ions.

Myelin basic protein (MBP) and β-casein are derived from fungal-derived NIMA (J. Biol. Chem. 268,
8769-8776, 1993), Nek2 (J. Bio) derived from mammals.
Chem. 270, 12899-12905, 1995), and Nek3 (J. B.
iol. Chem. 274, 13491-13497, 1999). Since the structure of the catalytic site of Nek9 is more related to Nek3 / 4 than to Nek2, we examined whether Nek9 phosphorylates these substrates in vitro. As a result, the Nek9 (K61R) mutant showed inactivity in the in vitro kinase assay,
Mixtures of MBP or histones were phosphorylated by immunoprecipitation with Flag-conjugated wild-type Nek9 (FIG. 4A, top panel). Nek9 was able to slightly phosphorylate the dephosphorylated casein mixture (FIG. 4A, top panel), but α and β casein were found to be poor substrates for Nek9. On the other hand, when a histone protein is used as a substrate, NIMA derived from bacteria phosphorylates histones H1 and H3 (J. Biol. Chem. 268, 8769-8776, 1
993, Cell 102, 293-302, 2000), H1, H2A, H2
N using B, H3, and H4 histone proteins
The substrate specificity of ek9 was examined (Fig. 4B). Nek9
Showed H1, H2A, H in in vitro kinase assay
Phosphorylates histone 3 at similar levels,
4 was found to be phosphorylated at a low level (upper panel of FIG. 4B). It was revealed that Nek9 has many similarities with NIMA and Nek2, but they are not the same in terms of substrate specificity. Nek through cell cycle progression
When assessing the kinase activity of 9, Nek9 was immunoprecipitated from HeLaS3 cells at each time shown in the figure after the G1 / S transition period, and an in vitro kinase assay was performed using histone H2A as a substrate (FIG. 4C). As a negative control, normal rabbit IgG was used for immunoprecipitation, and analysis was performed in parallel with the above. From FIG. 4C, it was revealed that the kinase activity of Nek9 was relatively low in the G1 / S transition period and was promoted during the progression of the cell cycle. The change in the level of Nek9 kinase activity 10 hours after the G1 / S phase showed a correlation with the expression level of the cyclin B1 protein in the G2 / M phase (FIG. 4C). The changes in Nek9 kinase activity paralleled protein expression levels, suggesting that protein levels regulate Nek9 kinase activity throughout the cell cycle.

Example 5 (Endogenous Nek in the cell cycle
9. Subcellular localization of endogenous Nek9 protein by laser sc
It measured by the indirect immunofluorescence method using anning confocal microscope (made by Carl Zeiss). Proliferated HeLaS
Three cells were fixed and probed with an anti-Nek9 polyclonal antibody, and such cell nuclei were counterstained with DAPI (see FIG. 5; reference photograph 1). A weak immunostaining signal was confirmed, consistent with the low Nek9 in the cells, but these characteristic staining patterns were only seen in interphase and prophase cells. In interphase cells, small spots such as distinct nuclear foci were stained with the anti-Nek9 antibody (left figure in FIG. 5; see reference photograph 1). In prophase cells, the anti-Nek9 antibody was surrounded by condensed chromosomes (the right figure in FIG. 5; see reference photograph 1). Diffusion staining patterns in other mitotic, metaphase or late cells suggest that the spindle poles and chromosomes have Nek.
It was suggested that 9 was not localized. On the other hand, in the cells at the terminal stage and the cell division stage, they were only slightly stained with the anti-Nek9 antibody. FIG. 3 and FIG. 5 (see reference photograph 1) show that Nk9 in G2 / M phase is cell cycle-regulated and localized in subcells, which shows that Nek9 is from G2 phase to early M phase. Was found to work.

Example 6 (He by overexpression of Nek9)
Apoptotic changes in La cells) Since the characteristic activity of fungal NIMA induces apoptotic nuclear aggregation in HeLa cells, it has been reported that there is a NIMA-related transduction pathway in the human mitotic mechanism ( EMBO J. 13, 4926-4937, 199
4, Cell 81, 413-424, 1995). However, apoptotic cell death has not been reported when mammalian NIMA family kinases were overexpressed in HaLa cells. He9 by overexpression of Nek9L and Nek9S
It has been shown to induce a high frequency of apoptotic cell morphology (cell atrophy and blistering of cells) in La cells, but such function is independent of kinase activity. In addition, by confocal microscopy analysis, flag-tagged N
The nuclear shape of HeLa cells overexpressing ek9L was examined (FIG. 6A; see Reference Photo 2). Flag-tagged Nek9L was probed with an anti-flag antibody and nuclei were visualized by DAPI staining. As a result, about 42% of flags
HeLa cells expressing -tagged Nek9L showed cell atrophy accompanied by nuclear aggregation (middle figure in FIG. 6A; see reference photograph 2) or cell morphology accompanied by apoptotic nuclear fragmentation (right figure in FIG. 6A; reference). (See Photo 2). More specifically, most exogenous Nek9 was detected in the cytoplasmic region, but some Nek9L proteins were localized in nuclear aggregation in atrophied cells (middle panel of FIG. 6A; reference picture 2).
reference). In apoptotic cells, Nek9L protein was detected like a star-like population and accumulated in the vicinity of the cytosolic region, but it was not localized to nuclear fragmentation (Fig. 6A, left panel; Reference photograph 2). reference). From these results, it was found that Nek9 induced the apoptotic change in HeLa cells as well as the apoptotic change induced by the fungal-derived NIMA.

Example 7 (C-terminal non-catalytic site of Nek9 that induces abnormal nuclear morphology in HeLa cells) From the above results, it was revealed that the non-catalytic region functions in the cytotoxicity of Nek9. Furthermore, it was found that the C-terminal non-catalytic site of fungal-derived NIMA functions in the course of the cell cycle, particularly in the G2 / M phase of human HeLa cells. Therefore, it was investigated whether the C-terminal non-catalytic site of Nek9 has another function. The full-length Nek9L, Nek9S, or C-terminal non-catalytic site fused with GFP was expressed in HeLa cells, and the cell morphology and nuclear morphology were analyzed by fluorescence microscopy (Fig. 6B; see Reference Photo 2). As a result, G expressing full-length Nek9L
FP-Nek9L, GFP-Coil1 expressing the first coiled-coil domain, and GFP-L-C-ter expressing the C-terminal side of Nek9L are HeL, respectively.
It was mainly detected in the cytoplasm of a cells. However, neither exogenous GFP-Nek9 chimeric protein was localized in the nucleus. The expression level of GFP-fused Nek9 in the cytoplasm was different from the expression level of exogenous Nek9. However, GFP-Nek9L, GFP-Ne expressing two coiled coil domains
When k9S and GFP-Coil1 / 2 are overexpressed,
It was found to show toxicity to HeLa cells (arrow head in FIG. 6B; see reference photograph 2). Most G
The cells expressing FP-Coil1 / 2 were atrophied (FIG. 6B).
2nd tier from the top right column; see Reference Photo 2.) On the other hand, GFP-Coil1 lacking the second coiled coil motif
Did not induce cell atrophy (FIG. 6B, third column from the left column, see reference photograph 2). Therefore, 330-4
The second coiled-coil motif consisting of the 00th amino acid sequence was revealed to be important in the cytotoxic effect of Nek9.

Some GFP-full length Nek9 expressing cells (approximately 22%), and GFP-Coil1 expressing cells and GFP-LC-ter expressing cells (approximately 43%).
˜44%), an abnormal morphonuclear morphology (arrow in FIG. 6C; see reference photograph 2) was observed. GFP-Coil
In the case of 1 and GFP-LC-ter expressing cells, the induced abnormal nuclear morphology was dependent on the expression level of GFP chimeric protein. GFP-Co with strong green fluorescence
In the cells highly expressing il1, no significant change due to the tubulin staining pattern was observed, but a deformed nucleus was seen (left column of FIG. 6C; see reference photo 2). Deformation of the nucleus in the cells expressing the intermediate level was observed, and the spindle-shaped pole-like GFP signal was frequently detected in the nucleus (right column of FIG. 6C; see reference photo 2). GFP alone did not induce such abnormalities. These abnormal nuclear forms have not been reported by other NIMA-related kinases, and Nek
It was revealed that the non-catalytic region of 9 has a functionally unique activity.

Example 8 (Nucle9-to-cytoplasm translocation function of Nek9 mediated by CRM1-dependent transduction pathway) Exogenous Nek9 protein was detected in the nucleus of interphase cells (see FIG. 5; reference photograph 1). ), The overexpressed full-length Nek9 protein or the GFP-fused chimeric Nek9 protein was mainly detected in the cytoplasmic region (FIG. 6A; see reference photo 2). In order to examine this difference, two types of cell extracts were prepared by dissolving the cytoplasmic fraction or the insoluble fraction containing the nucleus in 0.1% NP40, and the endogenous or extrinsic Nek9 subcells were analyzed by Western blotting. Localization was analyzed (Fig. 7A). Endogenous Nek9 protein was detected in the insoluble nuclear fraction in HeLaS3 and HEK293T cells (Fig. 7A, left panel), and these results were consistent with the results by confocal microscopy analysis (Fig. 5; see Reference Photo 1). On the other hand, overexpressed Nek9
Most of them were detected in cytosolic fraction lysates,
Only a small amount of exogenous Nek9 protein was detected in the insoluble fraction containing the nucleus (right panel of FIG. 7A). The reason for this is considered to be that since the expression level of the endogenous Nek9 protein is low in cells, only a small amount of Nek9 remains in the nucleus and an excessive amount of Nek9 flows into the cytoplasm.

It has been reported that various proteins shuttle between the nucleus and the cytoplasm by a specific traffic mechanism mediated by CRM1 (Cell 90, 105).
1-1060, 1997). Leptomycin B inhibits CRM1
It is known to inhibit the translocation of target proteins between the nucleus and the cytoplasm. Therefore, leptomycin B causes N
It was investigated whether the ek9 protein was inhibited between the nucleus and the cytoplasm. The result is shown in FIG. 7B. This confirmed the exogenous Nek9 protein that should be detected in the cytoplasm in the nucleus after treatment with leptomycin B for 4 hours. In addition to this, flag-tagged N
ek9L was co-immunoprecipitated with CRM1 (FIG. 7C). From this result, it was found that Nek9 is a substrate in the nuclear export mechanism mediated by CRM1. However, typical nuclear localization and translocation signals could not be predicted in the Nek9 protein by computer analysis. To clarify which region of Nek9 corresponds to the nuclear-cytoplasmic transport system, various GFP fusion N
We investigated the effect of Leptomycin B on the cellular localization of the ek9 construct. The result is shown in FIG. From this,
Various elements in Nek9, particularly the C-terminal non-catalytic site, have been found to be involved in the nuclear-cytoplasmic transport mechanism. The first coiled-coil motif located in the 289th to 329th amino acid sequence is involved in the nuclear export mechanism, and the second coiled coil motif located in the 330th to 400th amino acid sequence is involved in the nuclear targeting of Nek9. Was. Furthermore, it was found that the amino acid sequence at the 512th to 645th positions in the C-terminal region of Nek9L contains factors involved in nuclear targeting and nuclear translocation. From these things, N
It was revealed that overexpression of ek9 is continuously transported between the nucleus and the cytoplasm through the C-terminal non-catalytic site.

Filamentous fungus Aspergillus nidarans (A. n
NIDA is an important regulator in the G2 / M transitional phase (Curr. Biol. 5, 1122-1125, 1).
995, Biochem. J. 317, 633-641, 1996). However, it was not clear if there was a NIMA homologue with its true function in mammals. In this study, Nek
We identified 9 new members of the mammalian NIMA family and characterized them.

Aspergillus nidarans (A. nidulan
s) requires NIMA kinase for mitotic separation (Cell 67, 283-291, 1991), and the coiled coil motif containing the C-terminal non-catalytic site of NIMA is associated with nuclear aggregation, nuclear localization, and G2. It has been reported to have a functionally important function of inducing a dominant negative phenotype in the stagnation / lag phase (EMBO J. 13, 4926-4937, 199).
4, Cell 81, 413-424, 1995, EMBO J. 13, 2103-2113,
1994). FIG. 9 shows the functions of Nek9, but C of Nek9
The terminal non-catalytic sites have a variety of activities. Surprisingly, Nek9 is the only NIMA-related mammalian kinase
Has an activity of inducing apoptotic nuclear aggregation. As shown in FIG. 9, the second coiled coil of Nek9 is
Corresponding to cytotoxicity and nuclear targeting, it can be seen that it has NIMA-related activity.

The new properties of Nek9 were also investigated in parallel. First, the C-terminal non-catalytic site of Nek9, which corresponds to the amino acid sequence at positions 289-329 and 379-645, frequently induces an abnormal nuclear morphology such as a round overhang,
After that, cells containing many divided nuclei were observed. Next, we found that the C-terminal non-catalytic site of Nek9 is a substrate for CRM1-mediated nuclear-cytoplasmic transport. Also, from FIGS. 8 and 9, 289th to 329th and 3rd
Two coiled coils consisting of 30th to 400th are N
It was revealed that ek9 is independently involved in translocation and translocation into the nucleus. In addition, 512-64
C of Nek9L corresponding to the 5th amino acid sequence
It was found that there is another nuclear-cytoplasmic transport factor in the terminal extension. Since the expression of Nek9 reaches the peak in the G2 / M phase, it is considered that the C-terminal non-catalytic site inhibits a part of the nuclear division control system and / or the nuclear-cytoplasmic transport mechanism. Furthermore, since the expression level of endogenous Nek9 protein is low in cells, Nek
It is presumed that some functional factor is required to transfer the excessive expression level of 9 into the nucleus.

NIMA expression peaked during the G2 / M transition (J. Cell Biol. 104, 1495-1504, 1987), and the degradation of NIMA by cyclosomes paralleled the degradation of cyclin B during mitosis. It is known to be carried out (Mol. Biol. Cell 9, 3019-3030, 1998). The expression of Nek9, which is considered to be a cell cycle regulator, peaks at G2 / M phase, and down-regulation of Nek9 protein occurs in parallel with cyclin B1 in mitosis. Therefore, the expression profile of Nek9 is similar to that of fungal NIMA. Regarding localization in cells, fungal NIMA has a putative nuclear localization signal (Biochem. J. 317, 633-641, 1996) and is also increased in the nucleus during mitosis. (Cell 102, 293-30
2, 2000). Similarly, endogenous Nek9 could be detected in cell nuclei in interphase, but the typical nuclear localization signal by Nek9 could not be deduced. Interestingly, it was possible to find a unique localization of Nek9 in premetaphase cells, surrounded by aggregated chromosomes. Aspergillus nidalans (A. nid
ulans) did not match the mitotic process of mammals, and the intracellular localization of human Nek9 was different from that of fungal NIMA in the early M phase. The intracellular localization pattern of such Nek9 is
It is entirely independent in the mammalian NIMA family, and Nek9 is thought to have a unique role in prophase.

Nek9 as a protein kinase
It has similar but different properties to NIMA, mammalian Nek2, and Nek3. Several physiological substrates for the NIMA family have already been identified, eg N
Histone H3 for IMA (Cell 102, 293-302, 200
0) and c-Nap1 (J. Cell Biol. 14) against Nek2.
1, 1563-1574, 1998), and p70 against Nek6 / 7
S6 kinase (Curr. Biol. 11, 1155-1167, 2001) and the like are known. Histone H3 autophosphorylation and phosphorylation was examined by in vitro kinase analysis of Nek9 and histones were not phosphorylated by Nek9 in vitro. The identification of physiological substrates is important for understanding the physiological functions of Nek9 and needs to be studied in the future.

From the above, Nek9 could be found as a NIMA-related factor, and a new property of Nek9 in the C-terminal non-catalytic site, particularly in the coiled coil motif was found. Because coiled-coil motifs are often involved in protein-protein interactions,
Identification of target molecules for the coiled-coil motif in Nek9 will bring new insights into the physiological function / regulation of such Nek9 kinase. However, from the above results, it should be considered that the NIMA family member including Nek9 is required in the mammalian NIMA-related transduction pathway. Although it is still early to determine that Nek9 is a true mammalian homologue of fungal NIMA, this study provides a new clue for Nek9's understanding of mammalian NIMA-related transduction pathways in mitosis. Needless to say.

Method 1 (Molecular Cloning of Nek9 cDNA) From the EST database at the National Center for Biotechnology Information (NCBI), novel protein kinases and ATP interaction motifs of known protein kinases were searched using the TBLASTN program (FASEB J. .9
5756-596, 1995). One deduced amino acid sequence of the EST clone (Genbank ID: BE388672) is STK2 /
It turned out to be quite similar to Nek4. Also,
For full-length cDNA molecular cloning, refer to the SMART ^TM RACE cDNA amplification kit (Cl
ontech Laboratories, Palo Alto, California). cDNA was synthesized from poly (A) RNA of HeLaS3 cells and synthesized with a synthetic primer [5'-AGGGAGA
GAAGGTGTGTCACCTTCAACGAT-3 '; Primer used for 5'-end RACE method: SEQ ID NO: 13 (P1), 5'-TTGGAAGCCCAAC
TCCTCTCCAAGCTGGAC-3 ′; primer used for 3′-end RACE method: SEQ ID NO: 14 (P2)] was used for first PCR amplification. The Nested second PCR method uses the nested primer [5'-CACAG
GATCCCATTAGAAGTCGAG-3 '; Primer used for 5'-end nested RACE method: SEQ ID NO: 15 (P3), 5'-GCTGCATGAAT
CATGCATTCGCTGGC-3 ';3'-end nested RACE primer used in the method: SEQ ID NO: 16 (P4)].
The product obtained by the RACE method was subcloned into pCR ^R 2.1-TOPO plasmid (Invitrogen, CH Groningen, The Netherlands), and the sequences of both chains were determined using ABI model 373 automated sequencer. To determine the nucleotide sequence of the target gene, the consensus sequence of 10 individually obtained RACE clones was determined, and the intron-exon structure of the cDNA sequence was predicted by BLAST analysis using the human gene database. Pyrobest ^TM DNA Polymerase (TaKaRa
(Japan) and synthetic primer [5'-CGAATTCCATGCTGAAA
TTCCAAGAGGCAGC-3 '; Primer for the 5'end of nek9L: SEQ ID NO: 17 (P5), 5'-CTAGAGATGGTTCTG
GAGAGTAGG-3 ′; primer for 3 ′ end of nek9L: SEQ ID NO: 18 (P6), 5′-CTGAAAGTCAGCTGTGAGTTG
CATG-3 '; primer for the 3'end of nek9S:
By the PCR method using SEQ ID NO: 19 (P7)].
After obtaining cDNAs containing the complete open reading frame (ORF) from the cDNA of eLaS3 cells, these cDNAs were cloned into pCR ^R -BluntII-TOPO plasmid (Invitro
gen). All the two strands of the PCR product were confirmed, and these plasmids were used for pCR / n
ek9L and pCR / nek9S.

Method 2 (Computer analysis of Nek9) The intron-exon nucleotide structures of nek9L and nek9S were determined by human genome Blast analysis. Profilescan program (http: //www.isrec.isb-sib.
ch / software / PFSCAN # form.html), PSORT program (htt
p: //psort.ims.u-tokyo.ac.jp/), PESTfind program (h
ttp: //www.at.embnet.org/embnet/tools/bio/PESTfin
d) and Coils program (http://www.ch.embnet.org/s
(oftware / COILS # form.html) was used to predict the secondary structure of NIMA family kinases. Clustal W (v. 1.8)
(http://www.ddbj.nig.ac.jp/E-mail/clustalw-j.html)
By using the amino acid sequence containing the N-terminal catalytic site (A. nid
ulans-derived NIMA, N. crassa Nim-1, S. pombe Fin
1, S. cerevisiae KIN3, C. elegans F19H6.1, D. mela
A phylogenetic tree was created by arranging nogaster CG17256, murine Neks, and human Nek9) and comparing them. The calculation method for the tree diagram is performed by the proximity coupling method of Kimura's correction using Clustral W (v1.8),
Tree view software (http: //tazonomy.zoology.
gla.ac.uk/rod/treeview.html).

Method 3 (plasmid formation) PCR-based QuickChange ^™ si according to the manufacturer's manual
te-Directed Mutagenesis kit (Stratagene, La Jo
A negative kinase Nek9 in which the 61st lysine residue of Nek9 was replaced with arginine was prepared by the site-directed mutagenesis method using Ila, CA). Composite D
NA primer [5'-GGAGAGGAATTAAAGGTACTTAGGGAAATAT
CTGTTGGAGAACTA-3 '(SEQ ID NO: 20: P8) and 5'-TAGT
TCTCCAACAGATATTTCCCTAAGTACCTTTAATTCCTCTCC-3 '(SEQ ID NO: 21: P9)] as template pCR / nek9
Used for PCR reaction of plasmid. All mutated N
The ek9L or Nek9S cDNA clones were sequenced and designated as Nek9 (K61R). flag
-tagged Nek9 and EGFP (enhanced green fluorescen
t protein) -ORF in order to produce tagged Nek9
CDNA of Nek9L or Nek9S containing
PCR / nek9L or pCR / nekpS by digestion
PFlag CMV2 plasmid (Eastman Kodak, Rochester, NY) or pEGFP-C1 plasmid (Clonte
ch company), the resulting plasmid vector pFlag-Nek9L, pFlag-Nek9L (K61R), pFlag-
Nek9S, pFlag-Nek9S (K61R), pGFP-Nek9L, pGFP-Nek9L (K
61R), pGFP-Nek9S, pGFP-Nek9S (K61R). Ne
The cDNA fraction corresponding to the amino acid sequence consisting of the 289th to 400th amino acids of k9L was used as a synthetic primer [5'-GGAATTCCG
AGCAGCTACAGAACC-3 '(SEQ ID NO: 22: P10) and 5'-G
GAATTCCATTCCTTTTAAATGTG-3 '(SEQ ID NO: 23: P1
1)], template plasmid pCR / nek9L,
And the PCR method using pyrobest ^™ DNA polymerase. The PCR product was cloned into pCR ^R -Blunt II-TOPO plasmid (Invitrogen) and sequenced. Further, in the case of producing a GFP-Nek9L (amino acid sequence consisting of the 289th to 400th amino acids) expression plasmid, it was subcloned into the EcoRI site of the pEGFP-C1 plasmid, and this was named pGFP-Coil1 / 2. Nek9
The deletion mutation of was prepared as follows. 33 of Nek9L
To remove the polypeptide consisting of the 0-645th amino acid sequence and the 330-400th amino acid sequence, the pGFP-Nek9L or pGFP-Coil1 / 2 plasmid was used as Ps.
Digest it with t I, join the resulting partial sequences, and
A GFP-Nek9 (polypeptide consisting of the amino acid sequence of positions 1 to 329 or 289 to 329) expression vector plasmid was prepared. Also, EG
In order to obtain a C-terminal domain expression plasmid of FP-Nek9, Ne was isolated from pGFP-Nek9L or pGFP-Nek9S plasmid.
It was digested with Bgl III to remove the polypeptide consisting of the amino acid sequence 1 to 378 in k9. After that, it connects itself, and EGFP-Nek9L
(Polypeptide consisting of 379-645th amino acid sequence) Expression vector plasmid, and EGFP-Nek
A 9S (polypeptide consisting of the 379 to 470th amino acid sequence) expression vector plasmid was prepared, and
It was named pGFP-LC-ter and pGFP-SC-ter. pGFP-LCt
er was further digested with Bam HI and Nek9L (3
(Polypeptide consisting of the 79-645th amino acid sequence), after removing the polypeptide consisting of the 512-645th amino acid sequence, it is joined together to form EGFP-Nek9 (polypeptide consisting of the 379-511th amino acid sequence). ) Expression plasmid pGFP-PES
T1 / 2/3 was made.

Method 4 (Preparation of Nek9-Specific Polyclonal Antibody) To prepare a recombinant antigen protein, Nek9S c was prepared.
Eco-DNA of pTrcHisC plasmid (Invitrogen)
Subcloned into RI site, and polyhistidine tag (Hi
s-tag) Nek9 expression plasmid pTrcHis-Nek9S was prepared. However, since it was difficult to express the full-length histidine-tagged Nek9S protein in Escherichia coli, the cDNA fragment encoding the amino acid sequence of 1 to 326th was removed by Pst I digestion, and the histagged Nek9S (327 to 470th The plasmid pTrcHis-SC-ter expressing the polypeptide consisting of the amino acid sequence of p. Such Histag Nek
9S was subjected to isopropyl-β-thiogalactopyranoside (IPTG) induction for 2 hours to produce E. coli JM1.
09 and expressed in denaturing buffer (8 M urea and 50 mM
A cell lysate was prepared with Na-phosphate buffer (pH 8). Histag Nek9S expressed by E. coli
The (327-470th amino acid) protein was adsorbed to a Ni-NTA agarose gel (Qiagen GmbH, Germany) in a denatured state and eluted with a denaturing buffer (pH 4), followed by 10 mM Na-phosphate buffer (pH 7). ) Was used for dialysis. On the other hand, the antigen [recombinant histag Nek9S (327th to 470th amino acid) protein] obtained by immunization from the precipitate not dissolved in the denaturing buffer (pH 8) was repurified and obtained after dialysis. The lysed fraction was purified using an affinity column prepared with antiserum. Furthermore, the above-mentioned Histag Nek9S (327
~ 470th amino acid) protein is redissolved in a denaturing buffer and purified using a Ni-NTA agarose gel, and a buffer containing urea (2 M urea and 0.1 M Na).
-Phosphate; pH7) was dialyzed. Purified histag N
Rabbits were immunized with ek9S protein (100 μg × 6) in a denatured state. Affinity purification of antiserum was performed using HiTrap NHS-activated column (Amercham Pharmaci
a Biotech, Buckinghamshire, UK), recombinant Histag Nek9S (327-) in the lysed fraction after dialysis.
(Amino acid at position 470) A protein was bound thereto. In addition, as a pretreatment for purifying anti-Nek9 polyclonal antibody from antiserum, E. coli
The Ni-NTA agarose gel affinity column pre-adsorbed with the lysate was used to absorb the non-specifically binding antibody to the column, after which the Histag Nek9S (32
(Amino acids 7 to 470) Protein-bound Hi
Anti-Nek9 polyclonal antibody was purified using Trap NHS-activated column. The purified polyclonal antibody was diluted with 50% glycerol containing 0.05% bovine serum albumin and stored at -20 ° C.

Method 5 (Immunoprecipitation and Immune Complex Kinase Assay) In order to examine immunoprecipitation in transient expression, FuGENE ^™ 6 reagent (Roche Molecular) was used according to the manufacturer's manual.
HEK293T cells were transfected with the expression plasmid (10 ⁶ cells / 2 μg plasmid / 60 mm dish; 2 × 10 ⁵ cells / 0.5 μg plasmid / 12 well plate) using Biochemicals, Indianapolis, Indiana). . 38-44 hours after transfection, cells were placed in Nek lysis buffer (50
mM Hepes-NaOH; pH 7.5, 100 mM NaC
1, 5 mM KCl, 10 mM MgCl ₂ , 5 mM MnCl ₂ , 0.1% NP-40, 1 mM PMS
F, 2 μg / ml leupeptin, 2 μg / ml pepstatin A, 2 μg / ml heparin, 1 μg M okad
aic acid) and suspended on ice for 10 minutes. Endogenous Ne
In the k9 protein, HeLaS3 cells were
k9 lysis buffer (20 mM Hepes-NaOH; pH 7.5,
420 mM NaCl, 2 mM MgCl ₂ , 0.1%
NP-40, 1 mM PMSF, 2 μg / ml leupeptin, 2 μg / ml pepstatin A, 2 μg / m
1 heparin, 1 μM okadaic acid) on ice
Dissolved for 0 minutes. The obtained cell lysate was once frozen with liquid nitrogen and then centrifuged at 4 ° C. for 10 minutes (× 1
2000 g) to obtain a soluble cell extract. HeLaS3 extract was diluted with an equal volume of dilution buffer (20 mM
Hepes-NaOH; pH 7.5, ₂ mM MgCl ₂ , 0.
1% NP-40, 0.5% deoxycholate, 1 mM PMSF, 2 μg / ml leuptipene, 2 μg / ml
Of Pepstatin A, 1 μM okadaic acid). Dilute the cell extract with a stirrer at 4 ° C for 10
Minute, proteinG 4FF sepharose (Amersham Pharacia Bi
Otech). Pre-treated cell extracts were treated with antibody (4 μg anti-Flag M2 antibody obtained from 2 × 10 ⁶ HEK293T cells, or 1 mg He.
After incubation with 3 μg of anti-Nek9 polyclonal antibody or rabbit IgG) obtained from LaS3 cell extract on ice for 60 minutes, protein G 4
FF sepharose was added and the mixture was stirred at 4 ° C for 30 minutes. on the other hand,
The immune complex used for in vitro kinase assay was washed once with lysis buffer and then washed with HS buffer (0.1% N 2
P-40, 50 mM Hepes-NaOH; pH 7.5, 1 M NaCl) (1 ml x 5) and 50 mM Hepes-NaOH
It was washed with (pH 7.5) (1 ml × 1). The kinase reaction was performed using 30 μl of kinase buffer (50 mM Hepe
s-NaOH; pH 7.5, 10 μg / ml heparin, 5 m
M MnCl ₂ , 10 μM ATP, 100 μCi / m
1 [γ ³² -P] -ATP (Amersham Pharmacia Biotech
20) at 30 ° C. in 0.2 mg / ml substrate)
I went for a minute. After stopping the reaction by adding 4 × Laemmli SDS sample buffer (10 μl), 95 ° C.
Heat treated in. The denatured sample was separated by SDS-PAGE and analyzed using BAS 1800 bio-image analyzer (Fuji Film Co., Japan). The immune complex used to detect CRM1 was Nek lysis buffer (1 ml x
It was washed in 6) and analyzed by Western blotting.

Method 6 (Western Blot Analysis) To prepare whole cell lysates, cells were lysed with urea buffer (8 M urea, 0.1 M sodium phosphate; pH 7.0) and sonicated. Processed. For subcellular fraction analysis, the soluble fraction was prepared using Nek buffer and insoluble molecules were washed once with Nek buffer and the same amount of 1
X Laemmli SDS sample buffer was dissolved and then sonicated. Lysed cell extracts other than the above,
Preparation was performed using the same Nek or Nek9 lysis buffer as above, and the protein concentration was measured by BCA protein assay kit (Pierce, IL). A sample containing the same amount of protein was mixed with 4 × Laemmli SDS sample buffer, heat denatured, and then separated by SDS-PAGE, Immobilon ^™ PVDF membranes (Millipor).
e Corp., Bedford, MA). Such a membrane was treated with 5% low-fat milk powder / TBS-T (20 mM Tris-C
l; pH 7.5, 137 mM NaCl, 0.1% Twe
It was treated with en-20) and hybridized with an antibody diluent diluted with the same buffer at room temperature. Then, a secondary antibody conjugated with horseradish peroxidase was hybridized, and the signal increased by chemiluminescence was detected by ECL dete.
ction reagent (manufactured by Amersham Pharmacia Biotech).

Method 7 (RNA blot analysis) TRIZOT reagent (Life Tehchnologies, Grand Islan
(New York, New York), total RNA was isolated, and the poly (A) RNA was isolated from Oligotex ^™ -dT30 <Super> (Daiichi Pur).
e Chemicals, Japan). Poly (A) RNAs obtained from various cell lines were electrophoresed on a 1% agarose gel containing 2.2M formaldehyde (1
μg / lane), nylon membrane (Hybond-N +, Amersham Ph)
armacia Biotech). Many human-derived tissue expression arrays were purchased from Clontech. Such a tissue expression array and full length nek9Lc labeled with ³² P
BAS 1800 bio-image a by hybridizing with DNA
It was analyzed by a nalyzer.

Method 8 (Cell Cycle Synchronization and Nek9 Expression Analysis) For cell cycle synchronization, HeLa or HeLaS3 cells were seeded and cultured at 3 × 10 ⁶ cells per 100 mm dish.
The next day, the cells were pre-synchronized by treatment with 2.5 mM thymidine for 14 hours and then washed 3 times with phosphate buffered saline (PBS). Then, the cells were cultured for 11 hours, and in order to prevent cell growth in the G1 / S phase, aphidicolin (1 μg /
ml) was added and treated for 14 hours. In the G2 / M arrest phase, after thymidine-treated cells were cultured for 5 hours,
Nocodazole (0.5 μM) was added and treated for 19 hours,
The cells were washed 3 times with PBS, and the medium was further exchanged for culturing. The cells were collected at each time point, with the exchange time point being zero. To examine the expression of nek9 mRNA,
(A) RNA (1 μg / lane) was isolated and subjected to Northern blot analysis. Western blotting analysis was performed using each protein (25 μg / lane) obtained from the whole cell lysate to examine the expression of Nek9 protein. FACS
Calibur (Becton Dickinson) and CellQuest softwar
Flow cytometric analysis was performed using e.

Method 9 (Immunofluorescence Microscopy Analysis) In order to carry out an immunofluorescence microscopy experiment of exogenous Flag-tagged or GFP-tagged Nek9 protein, Effectene ^™ reagents (manufactured by Qiagen) were used according to the manufacturer's manual. , Expression plasmid (0.15 μg) was added to HEK29
3T or HeLa cells (4-well multi-chamber slides
Inject 5 × 10 ⁴ cells per well into Asahi Techno
Glass, Japan). To analyze the endogenous Nek9 protein, the above multi-cham
HeLaS3 cells were cultured on ber slides. 38-42 hours after transfection, the cells were washed once with PBS and for 10 minutes, 4% formaldehyde / PB
After fixing with S buffer, it was treated with 99.5% ethanol at room temperature for 2 minutes. Furthermore, the treated cells are treated with PBS.
After washing once with, the primary antibody (anti-Nek9 antibody (1 μg
/ Ml), anti-β tubulin antibody (× 200), anti-vimentin antibody (× 50), or anti-flag M2 antibody (0.3 μg / ml) diluted with PBS, and inject into mild chamber at room temperature. Incubated for 2 hours. Then, wash the slides 3 times with PBS,
PBS solution containing secondary antibody (TRITC-conjugated goat anti-rabbit IgG antibody (× 40
0), FITC-conjugated sheep anti-mouse IgG antibody (× 20
0), Cy3-conjugated goat anti-mouse IgG antibody (× 40
0)). Again, such slides were washed 3 times with PBS and treated with DAPI (Vectashi containing antifade).
Mounted by Eld, Vector Laboratories Inc). Immunostained cells were analyzed using the Carl Zeiss LSM 510 system.
Was examined with a confocal laser scanning microscope. In some embodiments, flag binding Nek9 or G.
Surviving cells from HEK293T cells expressing FP-linked chimeric Nek9 were analyzed with a fluorescence microscope (Olympus, IX70, Japan) and photographed with Sensia II film (Fuji Film).

(Cells and Materials Used in Examples and the Above Methods) Fibroblast HEK293T cells transformed with human embryonic kidney, uterine epithelial malignant tumors HeLa and HelaS3 cells, epidermal malignant tumor A431 cells, osteosarcoma U2OS cells,
Glioblastoma U251 and SF268 cells, lung small cell malignant tumor NCI-H23 cells, gastric malignant tumor St-4 and MKN2
8 cells, colorectal adenocarcinoma WiDr and KM12 cells, thymic malignant tumor MCF-7 and MDA-MB-231 cells, pancreatic malignant tumor MIA PaCa-2 cells, gallbladder malignant tumor HAG-
1 cell, ovarian adenocarcinoma OVCAR-3 and SKOV-3 cells were 10% heat-treated fetal bovine serum (FBS), respectively.
(Sigma) and 5 μg / ml gentamicin (Lif
The cells were cultured in Dulbecco's modified Eagle medium (DMEM) (Sigma, ST. Louis, MO) supplemented with e Technologies). In addition, human monoblastic leukemia U937 cells and erythroblastic leukemia K562 cells were treated with 10% FB treated with heat.
The cells were cultured in RPMI 1640 medium (Sigma) supplemented with S (Sigma) and 5 μg / ml gentamicin.
Anti-β tubulin antibody, anti-Flag M2 antibody, anti-vimentin antibody, FITC-conjugated anti-mouse IgG
Antibodies and TRICT-conjugated anti-rabbit IgG antibodies are
I bought it from gma. In addition, anti-CRM1 antibody, anti-GFP
Antibodies and anti-cyclin B1 antibody are from Santa Cruz Biotech
Purchased from nology (Santa Cruz, CA).
Anti-Nek2 antibody is available from Transduction Laboratories (Lex
Cyton, KY) and Cy3-conjugated anti-mouse IgG antibody was purchased from Amersham Pharmacia Biotech. Nocodazole, aphidicolin, thymidine, and leptomycin B were purchased from Sigma and okadaic acid was purchased from Calbiochem (San Diego, CA). Dephosphorylated casein and myelin basic protein (MBP) from Sigma
Histone proteins were purchased from Roche Molecular Biochemicals.

[0070]

INDUSTRIAL APPLICABILITY According to the present invention, a cell cycle-related protein having a nuclear export function, a cytotoxic function, a nuclear import function, and / or a nucleus-cytoplasm transfer function, and DNA or RNA encoding them are provided. can do. In addition, by using these proteins, DNA or RNA, not only screening of substances useful for the prevention and treatment of various diseases of cell cycle abnormalities such as immune diseases, neurological diseases and cancers, but also screening of substances useful for diagnosis, and also the mechanism of cell cycle ,
Mechanism of nuclear export, mechanism of cytotoxic function,
It is possible to elucidate the mechanism of nuclear import, the mechanism of mitotic control system, the mechanism of nuclear-cytoplasmic transport system, the mechanism of cell cycle control, and the like.

[0071]

[Sequence list]                                SEQUENCE LISTING <110> JAPAN SCIENCE AND TECHNOLOGY CORPORATION       NATIONAL INSTITUTE OF INFECTIOUS DISEASES PRESIDENT <120> Cell cycle-related proteins with nuclear export       function or nucleo-cytoplasmic shuttling function <130> 13-263 <140> <141> <160> 23 <170> PatentIn Ver. 2.1 <210> 1 <211> 123 <212> DNA <213> Homo sapiens <400> 1 gagcagctac agaacctaat gtgtagatat tcagaaatga ctctggaaga caaaaatttg 60 gattgtcaga aggaggctgc tcatataatt aatgccatgc aaaaaaggat ccacctgcag 120 act 123 <210> 2 <211> 41 <212> PRT <213> Homo sapiens <400> 2 Glu Gln Leu Gln Asn Leu Met Cys Arg Tyr Ser Glu Met Thr Leu Glu   1 5 10 15 Asp Lys Asn Leu Asp Cys Gln Lys Glu Ala Ala His Ile Ile Asn Ala              20 25 30 Met Gln Lys Arg Ile His Leu Gln Thr          35 40 <210> 3 <211> 402 <212> DNA <213> Homo sapiens <400> 3 cagcctgctt acagaacaaa ccaacaggac agtgatatcg aagcgttggc caggtgtttg 60 gaaaatgtcc tgggttgcac ttctctagac acaaagacca tcaccaccat ggctgaagac 120 atgtccccag gaccaccaat tttcaacagt gtgatggcca ggaccaagat gaaacgcatg 180 agggaatcag ccatgcagaa gctggggaca gaagtatttg aagaggtcta taattacctc 240 aagagagcaa ggcatcagaa tgctagcgaa gcagagatcc gcgagtgttt ggaaaaagtg 300 gtgcctcaag ccagcgactg ttttgaagtg gaccagctcc tgtactttga agagcagttg 360 ctgatcacga tgggaaaaga acctactctc cagaaccatc tc 402 <210> 4 <211> 134 <212> PRT <213> Homo sapiens <400> 4 Gln Pro Ala Tyr Arg Thr Asn Gln Gln Asp Ser Asp Ile Glu Ala Leu   1 5 10 15 Ala Arg Cys Leu Glu Asn Val Leu Gly Cys Thr Ser Leu Asp Thr Lys              20 25 30 Thr Ile Thr Thr Met Ala Glu Asp Met Ser Pro Gly Pro Pro Ile Phe          35 40 45 Asn Ser Val Met Ala Arg Thr Lys Met Lys Arg Met Arg Glu Ser Ala      50 55 60 Met Gln Lys Leu Gly Thr Glu Val Phe Glu Glu Val Tyr Asn Tyr Leu  65 70 75 80 Lys Arg Ala Arg His Gln Asn Ala Ser Glu Ala Glu Ile Arg Glu Cys                  85 90 95 Leu Glu Lys Val Val Pro Gln Ala Ser Asp Cys Phe Glu Val Asp Gln             100 105 110 Leu Leu Tyr Phe Glu Glu Gln Leu Leu Ile Thr Met Gly Lys Glu Pro         115 120 125 Thr Leu Gln Asn His Leu     130 <210> 5 <211> 336 <212> DNA <213> Homo sapiens <400> 5 gagcagctac agaacctaat gtgtagatat tcagaaatga ctctggaaga caaaaatttg 60 gattgtcaga aggaggctgc tcatataatt aatgccatgc aaaaaaggat ccacctgcag 120 actctgaggg cactgtcaga agtacagaaa atgacgccaa gagaaaggat gcggctgagg 180 aagctccagg cggctgatga gaaagccagg aagctgaaaa agattgtgga agaaaaatat 240 gaagaaaata gcaaacgaat gcaagaattg agatctcgga actttcagca gctgagtgtt 300 gatgtactcc atgaaaaaac acatttaaaa ggaatg 336 <210> 6 <211> 112 <212> PRT <213> Homo sapiens <400> 6 Glu Gln Leu Gln Asn Leu Met Cys Arg Tyr Ser Glu Met Thr Leu Glu   1 5 10 15 Asp Lys Asn Leu Asp Cys Gln Lys Glu Ala Ala His Ile Ile Asn Ala              20 25 30 Met Gln Lys Arg Ile His Leu Gln Thr Leu Arg Ala Leu Ser Glu Val          35 40 45 Gln Lys Met Thr Pro Arg Glu Arg Met Arg Leu Arg Lys Leu Gln Ala      50 55 60 Ala Asp Glu Lys Ala Arg Lys Leu Lys Lys Ile Val Glu Glu Lys Tyr  65 70 75 80 Glu Glu Asn Ser Lys Arg Met Gln Glu Leu Arg Ser Arg Asn Phe Gln                  85 90 95 Gln Leu Ser Val Asp Val Leu His Glu Lys Thr His Leu Lys Gly Met             100 105 110 <210> 7 <211> 1071 <212> DNA <213> Homo sapiens <400> 7 gagcagctac agaacctaat gtgtagatat tcagaaatga ctctggaaga caaaaatttg 60 gattgtcaga aggaggctgc tcatataatt aatgccatgc aaaaaaggat ccacctgcag 120 actctgaggg cactgtcaga agtacagaaa atgacgccaa gagaaaggat gcggctgagg 180 aagctccagg cggctgatga gaaagccagg aagctgaaaa agattgtgga agaaaaatat 240 gaagaaaata gcaaacgaat gcaagaattg agatctcgga actttcagca gctgagtgtt 300 gatgtactcc atgaaaaaac acatttaaaa ggaatggaag aaaaggagga gcaacctgag 360 ggaagacttt cttgttcacc ccaggacgag gatgaagaga ggtggcaagg cagggaagag 420 gaatctgatg aaccaacttt agagaacctg cctgagtctc agcctattcc ttccatggac 480 ctccacgaac ttgaatcaat tgtagaggat gccacatctg accttggata ccatgagatc 540 ccagaagacc cacttgtggc tgaagagtac tacgctgatg catttgattc ctattgtgta 600 gagagtgatg aggaggaaga agaaatagcg ttagaaagac cagagaaaga aatcaggaat 660 gagggatccc agcctgctta cagaacaaac caacaggaca gtgatatcga agcgttggcc 720 aggtgtttgg aaaatgtcct gggttgcact tctctagaca caaagaccat caccaccatg 780 gctgaagaca tgtccccagg accaccaatt ttcaacagtg tgatggccag gaccaagatg 840 aaacgcatga gggaatcagc catgcagaag ctggggacag aagtatttga agaggtctat 900 aattacctca agagagcaag gcatcagaat gctagcgaag cagagatccg cgagtgtttg 960 gaaaaagtgg tgcctcaagc cagcgactgt tttgaagtgg accagctcct gtactttgaa 1020 gagcagttgc tgatcacgat gggaaaagaa cctactctcc agaaccatct c 1071 <210> 8 <211> 357 <212> PRT <213> Homo sapiens <400> 8 Glu Gln Leu Gln Asn Leu Met Cys Arg Tyr Ser Glu Met Thr Leu Glu   1 5 10 15 Asp Lys Asn Leu Asp Cys Gln Lys Glu Ala Ala His Ile Ile Asn Ala              20 25 30 Met Gln Lys Arg Ile His Leu Gln Thr Leu Arg Ala Leu Ser Glu Val          35 40 45 Gln Lys Met Thr Pro Arg Glu Arg Met Arg Leu Arg Lys Leu Gln Ala      50 55 60 Ala Asp Glu Lys Ala Arg Lys Leu Lys Lys Ile Val Glu Glu Lys Tyr  65 70 75 80 Glu Glu Asn Ser Lys Arg Met Gln Glu Leu Arg Ser Arg Asn Phe Gln                  85 90 95 Gln Leu Ser Val Asp Val Leu His Glu Lys Thr His Leu Lys Gly Met             100 105 110 Glu Glu Lys Glu Glu Gln Pro Glu Gly Arg Leu Ser Cys Ser Pro Gln         115 120 125 Asp Glu Asp Glu Glu Arg Trp Gln Gly Arg Glu Glu Glu Ser Asp Glu     130 135 140 Pro Thr Leu Glu Asn Leu Pro Glu Ser Gln Pro Ile Pro Ser Met Asp 145 150 155 160 Leu His Glu Leu Glu Ser Ile Val Glu Asp Ala Thr Ser Asp Leu Gly                 165 170 175 Tyr His Glu Ile Pro Glu Asp Pro Leu Val Ala Glu Glu Tyr Tyr Ala             180 185 190 Asp Ala Phe Asp Ser Tyr Cys Val Glu Ser Asp Glu Glu Glu Glu Glu         195 200 205 Ile Ala Leu Glu Arg Pro Glu Lys Glu Ile Arg Asn Glu Gly Ser Gln     210 215 220 Pro Ala Tyr Arg Thr Asn Gln Gln Asp Ser Asp Ile Glu Ala Leu Ala 225 230 235 240 Arg Cys Leu Glu Asn Val Leu Gly Cys Thr Ser Leu Asp Thr Lys Thr                 245 250 255 Ile Thr Thr Met Ala Glu Asp Met Ser Pro Gly Pro Pro Ile Phe Asn             260 265 270 Ser Val Met Ala Arg Thr Lys Met Lys Arg Met Arg Glu Ser Ala Met         275 280 285 Gln Lys Leu Gly Thr Glu Val Phe Glu Glu Val Tyr Asn Tyr Leu Lys     290 295 300 Arg Ala Arg His Gln Asn Ala Ser Glu Ala Glu Ile Arg Glu Cys Leu 305 310 315 320 Glu Lys Val Val Pro Gln Ala Ser Asp Cys Phe Glu Val Asp Gln Leu                 325 330 335 Leu Tyr Phe Glu Glu Gln Leu Leu Ile Thr Met Gly Lys Glu Pro Thr             340 345 350 Leu Gln Asn His Leu         355 <210> 9 <211> 2080 <212> DNA <213> Homo sapiens <220> <221> CDS <222> (294) .. (1706) <400> 9 ccaaacagcc gtggcccgcg gtgtctggcg ctcggtgggt gtggttgccc ctagtttgag 60 gcctgcccga ttacccgcaa gacttgggca gccccgggcg ccgctccgac cacgacaggg 120 aaaggaacct taatctcatc tttaaaataa ggagaattac tgagtgacct gaaggaccct 180 tttcagctgg aaagtctgaa ctgaccaaca ctggatgaat ttgaccattt cttaggagac 240 tggaatgtta agtttctata aatgaatgaa ccagttctct cttgtttttgga gca atg 296                                                            Met                                                              1 ctg aaa ttc caa gag gca gct aag tgt gtg agt gga tca aca gcc att 344 Leu Lys Phe Gln Glu Ala Ala Lys Cys Val Ser Gly Ser Thr Ala Ile               5 10 15 tcc act tat cca aag acc ttg att gca aga aga tac gtg ctt caa caa 392 Ser Thr Tyr Pro Lys Thr Leu Ile Ala Arg Arg Tyr Val Leu Gln Gln          20 25 30 aaa ctt ggc agt gga agt ttt gga act gtc tat ctg gtt tca gac aag 440 Lys Leu Gly Ser Gly Ser Phe Gly Thr Val Tyr Leu Val Ser Asp Lys      35 40 45 aaa gcc aaa cga gga gag gaa tta aag gta ctt aag gaa ata tct gtt 488 Lys Ala Lys Arg Gly Glu Glu Leu Lys Val Leu Lys Glu Ile Ser Val  50 55 60 65 gga gaa cta aat cca aat gaa act gta cag gcc aat ttg gaa gcc caa 536 Gly Glu Leu Asn Pro Asn Glu Thr Val Gln Ala Asn Leu Glu Ala Gln                  70 75 80 ctc ctc tcc aag ctg gac cac cca gcc att gtc aag ttc cat gca agt 584 Leu Leu Ser Lys Leu Asp His Pro Ala Ile Val Lys Phe His Ala Ser              85 90 95 ttt gtg gag caa gat aat ttc tgc att atc acg gag tac tgt gag ggc 632 Phe Val Glu Gln Asp Asn Phe Cys Ile Ile Thr Glu Tyr Cys Glu Gly         100 105 110 cga gat ctg gac gat aaa att cag gaa tat aaa caa gct gga aaa atc 680 Arg Asp Leu Asp Asp Lys Ile Gln Glu Tyr Lys Gln Ala Gly Lys Ile     115 120 125 ttt cca gaa aat caa ata ata gaa tgg ttt atc cag ctg ctg ctg gga 728 Phe Pro Glu Asn Gln Ile Ile Glu Trp Phe Ile Gln Leu Leu Leu Gly 130 135 140 145 gtt gac tac atg cat gag agg agg ata ctt cat cga gac tta aag tca 776 Val Asp Tyr Met His Glu Arg Arg Ile Leu His Arg Asp Leu Lys Ser                 150 155 160 aag aat gta ttt ctg aaa aat aat ctc ctt aaa att gga gat ttt gga 824 Lys Asn Val Phe Leu Lys Asn Asn Leu Leu Lys Ile Gly Asp Phe Gly             165 170 175 gtt tct cga ctt cta atg gga tcc tgt gac ctg gcc aca act tta act 872 Val Ser Arg Leu Leu Met Gly Ser Cys Asp Leu Ala Thr Thr Leu Thr         180 185 190 gga act ccc cat tat atg agt cct gag gct ctg aaa cac caa ggc tat 920 Gly Thr Pro His Tyr Met Ser Pro Glu Ala Leu Lys His Gln Gly Tyr     195 200 205 gac aca aag tcg gac atc tgg tca ctg gca tgc att ttg tat gag atg 968 Asp Thr Lys Ser Asp Ile Trp Ser Leu Ala Cys Ile Leu Tyr Glu Met 210 215 220 225 tgc tgc atg aat cat gca ttc gct ggc tcc aat ttc tta tcc att gtt 1016 Cys Cys Met Asn His Ala Phe Ala Gly Ser Asn Phe Leu Ser Ile Val                 230 235 240 tta aaa att gtt gaa ggt gac aca cct tct ctc cct gag aga tat cca 1064 Leu Lys Ile Val Glu Gly Asp Thr Pro Ser Leu Pro Glu Arg Tyr Pro             245 250 255 aaa gaa cta aat gcc atc atg gaa agc atg ttg aac aag aat cct tca 1112 Lys Glu Leu Asn Ala Ile Met Glu Ser Met Leu Asn Lys Asn Pro Ser         260 265 270 tta aga cca tct gct atc gaa att tta aaa atc cct tac ctt gat gag 1160 Leu Arg Pro Ser Ala Ile Glu Ile Leu Lys Ile Pro Tyr Leu Asp Glu     275 280 285 cag cta cag aac cta atg tgt aga tat tca gaa atg act ctg gaa gac 1208 Gln Leu Gln Asn Leu Met Cys Arg Tyr Ser Glu Met Thr Leu Glu Asp 290 295 300 305 aaa aat ttg gat tgt cag aag gag gct gct cat ata att aat gcc atg 1256 Lys Asn Leu Asp Cys Gln Lys Glu Ala Ala His Ile Ile Asn Ala Met                 310 315 320 caa aaa agg atc cac ctg cag act ctg agg gca ctg tca gaa gta cag 1304 Gln Lys Arg Ile His Leu Gln Thr Leu Arg Ala Leu Ser Glu Val Gln             325 330 335 aaa atg acg cca aga gaa agg atg cgg ctg agg aag ctc cag gcg gct 1352 Lys Met Thr Pro Arg Glu Arg Met Arg Leu Arg Lys Leu Gln Ala Ala         340 345 350 gat gag aaa gcc agg aag ctg aaa aag att gtg gaa gaa aaa tat gaa 1400 Asp Glu Lys Ala Arg Lys Leu Lys Lys Ile Val Glu Glu Lys Tyr Glu     355 360 365 gaa aat agc aaa cga atg caa gaa ttg aga tct cgg aac ttt cag cag 1448 Glu Asn Ser Lys Arg Met Gln Glu Leu Arg Ser Arg Asn Phe Gln Gln 370 375 380 385 ctg agt gtt gat gta ctc cat gaa aaa aca cat tta aaa gga atg gaa 1496 Leu Ser Val Asp Val Leu His Glu Lys Thr His Leu Lys Gly Met Glu                 390 395 400 gaa aag gag gag caa cct gag gga aga ctt tct tgt tca ccc cag gac 1544 Glu Lys Glu Glu Gln Pro Glu Gly Arg Leu Ser Cys Ser Pro Gln Asp             405 410 415 gag gat gaa gag agg tgg caa ggc agg gaa gag gaa tct gat gaa cca 1592 Glu Asp Glu Glu Arg Trp Gln Gly Arg Glu Glu Glu Ser Asp Glu Pro         420 425 430 act tta gag aac ctg cct gag tct cag cct att cct tcc atg gac ctc 1640 Thr Leu Glu Asn Leu Pro Glu Ser Gln Pro Ile Pro Ser Met Asp Leu     435 440 445 cac gaa ctt gaa tca att gta gag gat gcc aca tct gac ctt gga tac 1688 His Glu Leu Glu Ser Ile Val Glu Asp Ala Thr Ser Asp Leu Gly Tyr 450 455 460 465 cat gca act cac agc tga ctttcagagc agtgtttttt ccaaagctgg 1736 His Ala Thr His Ser                 470 aatagcctga gactctaggg cctgggtcta caaggagcat gactccctac cccaccattc 1796 agatgtggag aatgcagtga agtaggcctt ggctgcttac ccacatcctg accagctgtc 1856 acctcttcat ttcctgttca gccactcttt aagcaatcac tgagcccaat gctcagagat 1916 agctcagacc tgtaggtggt tgtgctttgc ttgcctgtag gagccccaca tgccctttac 1976 agttctgggc tgtctatgac ttacgatatc acctcttctt tttaaatgtc caagtggaaa 2036 taaagtgatt tgaactttgc caaaaaaaaa aaaaaaaaaa aaaa 2080 <210> 10 <211> 470 <212> PRT <213> Homo sapiens <400> 10 Met Leu Lys Phe Gln Glu Ala Ala Lys Cys Val Ser Gly Ser Thr Ala   1 5 10 15 Ile Ser Thr Tyr Pro Lys Thr Leu Ile Ala Arg Arg Tyr Val Leu Gln              20 25 30 Gln Lys Leu Gly Ser Gly Ser Phe Gly Thr Val Tyr Leu Val Ser Asp          35 40 45 Lys Lys Ala Lys Arg Gly Glu Glu Leu Lys Val Leu Lys Glu Ile Ser      50 55 60 Val Gly Glu Leu Asn Pro Asn Glu Thr Val Gln Ala Asn Leu Glu Ala  65 70 75 80 Gln Leu Leu Ser Lys Leu Asp His Pro Ala Ile Val Lys Phe His Ala                  85 90 95 Ser Phe Val Glu Gln Asp Asn Phe Cys Ile Ile Thr Glu Tyr Cys Glu             100 105 110 Gly Arg Asp Leu Asp Asp Lys Ile Gln Glu Tyr Lys Gln Ala Gly Lys         115 120 125 Ile Phe Pro Glu Asn Gln Ile Ile Glu Trp Phe Ile Gln Leu Leu Leu     130 135 140 Gly Val Asp Tyr Met His Glu Arg Arg Ile Leu His Arg Asp Leu Lys 145 150 155 160 Ser Lys Asn Val Phe Leu Lys Asn Asn Leu Leu Lys Ile Gly Asp Phe                 165 170 175 Gly Val Ser Arg Leu Leu Met Gly Ser Cys Asp Leu Ala Thr Thr Leu             180 185 190 Thr Gly Thr Pro His Tyr Met Ser Pro Glu Ala Leu Lys His Gln Gly         195 200 205 Tyr Asp Thr Lys Ser Asp Ile Trp Ser Leu Ala Cys Ile Leu Tyr Glu     210 215 220 Met Cys Cys Met Asn His Ala Phe Ala Gly Ser Asn Phe Leu Ser Ile 225 230 235 240 Val Leu Lys Ile Val Glu Gly Asp Thr Pro Ser Leu Pro Glu Arg Tyr                 245 250 255 Pro Lys Glu Leu Asn Ala Ile Met Glu Ser Met Leu Asn Lys Asn Pro             260 265 270 Ser Leu Arg Pro Ser Ala Ile Glu Ile Leu Lys Ile Pro Tyr Leu Asp         275 280 285 Glu Gln Leu Gln Asn Leu Met Cys Arg Tyr Ser Glu Met Thr Leu Glu     290 295 300 Asp Lys Asn Leu Asp Cys Gln Lys Glu Ala Ala His Ile Ile Asn Ala 305 310 315 320 Met Gln Lys Arg Ile His Leu Gln Thr Leu Arg Ala Leu Ser Glu Val                 325 330 335 Gln Lys Met Thr Pro Arg Glu Arg Met Arg Leu Arg Lys Leu Gln Ala             340 345 350 Ala Asp Glu Lys Ala Arg Lys Leu Lys Lys Ile Val Glu Glu Lys Tyr         355 360 365 Glu Glu Asn Ser Lys Arg Met Gln Glu Leu Arg Ser Arg Asn Phe Gln     370 375 380 Gln Leu Ser Val Asp Val Leu His Glu Lys Thr His Leu Lys Gly Met 385 390 395 400 Glu Glu Lys Glu Glu Gln Pro Glu Gly Arg Leu Ser Cys Ser Pro Gln                 405 410 415 Asp Glu Asp Glu Glu Arg Trp Gln Gly Arg Glu Glu Glu Ser Asp Glu             420 425 430 Pro Thr Leu Glu Asn Leu Pro Glu Ser Gln Pro Ile Pro Ser Met Asp         435 440 445 Leu His Glu Leu Glu Ser Ile Val Glu Asp Ala Thr Ser Asp Leu Gly     450 455 460 Tyr His Ala Thr His Ser 465 470 <210> 11 <211> 2939 <212> DNA <213> Homo sapiens <220> <221> CDS <222> (294) .. (2231) <400> 11 ccaaacagcc gtggcccgcg gtgtctggcg ctcggtgggt gtggttgccc ctagtttgag 60 gcctgcccga ttacccgcaa gacttgggca gccccgggcg ccgctccgac cacgacaggg 120 aaaggaacct taatctcatc tttaaaataa ggagaattac tgagtgacct gaaggaccct 180 tttcagctgg aaagtctgaa ctgaccaaca ctggatgaat ttgaccattt cttaggagac 240 tggaatgtta agtttctata aatgaatgaa ccagttctct cttgtttttgga gca atg 296                                                            Met                                                              1 ctg aaa ttc caa gag gca gct aag tgt gtg agt gga tca aca gcc att 344 Leu Lys Phe Gln Glu Ala Ala Lys Cys Val Ser Gly Ser Thr Ala Ile               5 10 15 tcc act tat cca aag acc ttg att gca aga aga tac gtg ctt caa caa 392 Ser Thr Tyr Pro Lys Thr Leu Ile Ala Arg Arg Tyr Val Leu Gln Gln          20 25 30 aaa ctt ggc agt gga agt ttt gga act gtc tat ctg gtt tca gac aag 440 Lys Leu Gly Ser Gly Ser Phe Gly Thr Val Tyr Leu Val Ser Asp Lys      35 40 45 aaa gcc aaa cga gga gag gaa tta aag gta ctt aag gaa ata tct gtt 488 Lys Ala Lys Arg Gly Glu Glu Leu Lys Val Leu Lys Glu Ile Ser Val  50 55 60 65 gga gaa cta aat cca aat gaa act gta cag gcc aat ttg gaa gcc caa 536 Gly Glu Leu Asn Pro Asn Glu Thr Val Gln Ala Asn Leu Glu Ala Gln                  70 75 80 ctc ctc tcc aag ctg gac cac cca gcc att gtc aag ttc cat gca agt 584 Leu Leu Ser Lys Leu Asp His Pro Ala Ile Val Lys Phe His Ala Ser              85 90 95 ttt gtg gag caa gat aat ttc tgc att atc acg gag tac tgt gag ggc 632 Phe Val Glu Gln Asp Asn Phe Cys Ile Ile Thr Glu Tyr Cys Glu Gly         100 105 110 cga gat ctg gac gat aaa att cag gaa tat aaa caa gct gga aaa atc 680 Arg Asp Leu Asp Asp Lys Ile Gln Glu Tyr Lys Gln Ala Gly Lys Ile     115 120 125 ttt cca gaa aat caa ata ata gaa tgg ttt atc cag ctg ctg ctg gga 728 Phe Pro Glu Asn Gln Ile Ile Glu Trp Phe Ile Gln Leu Leu Leu Gly 130 135 140 145 gtt gac tac atg cat gag agg agg ata ctt cat cga gac tta aag tca 776 Val Asp Tyr Met His Glu Arg Arg Ile Leu His Arg Asp Leu Lys Ser                 150 155 160 aag aat gta ttt ctg aaa aat aat ctc ctt aaa att gga gat ttt gga 824 Lys Asn Val Phe Leu Lys Asn Asn Leu Leu Lys Ile Gly Asp Phe Gly             165 170 175 gtt tct cga ctt cta atg gga tcc tgt gac ctg gcc aca act tta act 872 Val Ser Arg Leu Leu Met Gly Ser Cys Asp Leu Ala Thr Thr Leu Thr         180 185 190 gga act ccc cat tat atg agt cct gag gct ctg aaa cac caa ggc tat 920 Gly Thr Pro His Tyr Met Ser Pro Glu Ala Leu Lys His Gln Gly Tyr     195 200 205 gac aca aag tcg gac atc tgg tca ctg gca tgc att ttg tat gag atg 968 Asp Thr Lys Ser Asp Ile Trp Ser Leu Ala Cys Ile Leu Tyr Glu Met 210 215 220 225 tgc tgc atg aat cat gca ttc gct ggc tcc aat ttc tta tcc att gtt 1016 Cys Cys Met Asn His Ala Phe Ala Gly Ser Asn Phe Leu Ser Ile Val                 230 235 240 tta aaa att gtt gaa ggt gac aca cct tct ctc cct gag aga tat cca 1064 Leu Lys Ile Val Glu Gly Asp Thr Pro Ser Leu Pro Glu Arg Tyr Pro             245 250 255 aaa gaa cta aat gcc atc atg gaa agc atg ttg aac aag aat cct tca 1112 Lys Glu Leu Asn Ala Ile Met Glu Ser Met Leu Asn Lys Asn Pro Ser         260 265 270 tta aga cca tct gct atc gaa att tta aaa atc cct tac ctt gat gag 1160 Leu Arg Pro Ser Ala Ile Glu Ile Leu Lys Ile Pro Tyr Leu Asp Glu     275 280 285 cag cta cag aac cta atg tgt aga tat tca gaa atg act ctg gaa gac 1208 Gln Leu Gln Asn Leu Met Cys Arg Tyr Ser Glu Met Thr Leu Glu Asp 290 295 300 305 aaa aat ttg gat tgt cag aag gag gct gct cat ata att aat gcc atg 1256 Lys Asn Leu Asp Cys Gln Lys Glu Ala Ala His Ile Ile Asn Ala Met                 310 315 320 caa aaa agg atc cac ctg cag act ctg agg gca ctg tca gaa gta cag 1304 Gln Lys Arg Ile His Leu Gln Thr Leu Arg Ala Leu Ser Glu Val Gln             325 330 335 aaa atg acg cca aga gaa agg atg cgg ctg agg aag ctc cag gcg gct 1352 Lys Met Thr Pro Arg Glu Arg Met Arg Leu Arg Lys Leu Gln Ala Ala         340 345 350 gat gag aaa gcc agg aag ctg aaa aag att gtg gaa gaa aaa tat gaa 1400 Asp Glu Lys Ala Arg Lys Leu Lys Lys Ile Val Glu Glu Lys Tyr Glu     355 360 365 gaa aat agc aaa cga atg caa gaa ttg aga tct cgg aac ttt cag cag 1448 Glu Asn Ser Lys Arg Met Gln Glu Leu Arg Ser Arg Asn Phe Gln Gln 370 375 380 385 ctg agt gtt gat gta ctc cat gaa aaa aca cat tta aaa gga atg gaa 1496 Leu Ser Val Asp Val Leu His Glu Lys Thr His Leu Lys Gly Met Glu                 390 395 400 gaa aag gag gag caa cct gag gga aga ctt tct tgt tca ccc cag gac 1544 Glu Lys Glu Glu Gln Pro Glu Gly Arg Leu Ser Cys Ser Pro Gln Asp             405 410 415 gag gat gaa gag agg tgg caa ggc agg gaa gag gaa tct gat gaa cca 1592 Glu Asp Glu Glu Arg Trp Gln Gly Arg Glu Glu Glu Ser Asp Glu Pro         420 425 430 act tta gag aac ctg cct gag tct cag cct att cct tcc atg gac ctc 1640 Thr Leu Glu Asn Leu Pro Glu Ser Gln Pro Ile Pro Ser Met Asp Leu     435 440 445 cac gaa ctt gaa tca att gta gag gat gcc aca tct gac ctt gga tac 1688 His Glu Leu Glu Ser Ile Val Glu Asp Ala Thr Ser Asp Leu Gly Tyr 450 455 460 465 cat gag atc cca gaa gac cca ctt gtg gct gaa gag tac tac gct gat 1736 His Glu Ile Pro Glu Asp Pro Leu Val Ala Glu Glu Tyr Tyr Ala Asp                 470 475 480 gca ttt gat tcc tat tgt gta gag agt gat gag gag gaa gaa gaa ata 1784 Ala Phe Asp Ser Tyr Cys Val Glu Ser Asp Glu Glu Glu Glu Glu Ile             485 490 495 gcg tta gaa aga cca gag aaa gaa atc agg aat gag gga tcc cag cct 1832 Ala Leu Glu Arg Pro Glu Lys Glu Ile Arg Asn Glu Gly Ser Gln Pro         500 505 510 gct tac aga aca aac caa cag gac agt gat atc gaa gcg ttg gcc agg 1880 Ala Tyr Arg Thr Asn Gln Gln Asp Ser Asp Ile Glu Ala Leu Ala Arg     515 520 525 tgt ttg gaa aat gtc ctg ggt tgc act tct cta gac aca aag acc atc 1928 Cys Leu Glu Asn Val Leu Gly Cys Thr Ser Leu Asp Thr Lys Thr Ile 530 535 540 545 acc acc atg gct gaa gac atg tcc cca gga cca cca att ttc aac agt 1976 Thr Thr Met Ala Glu Asp Met Ser Pro Gly Pro Pro Ile Phe Asn Ser                 550 555 560 gtg atg gcc agg acc aag atg aaa cgc atg agg gaa tca gcc atg cag 2024 Val Met Ala Arg Thr Lys Met Lys Arg Met Arg Glu Ser Ala Met Gln             565 570 575 aag ctg ggg aca gaa gta ttt gaa gag gtc tat aat tac ctc aag aga 2072 Lys Leu Gly Thr Glu Val Phe Glu Glu Val Tyr Asn Tyr Leu Lys Arg         580 585 590 gca agg cat cag aat gct agc gaa gca gag atc cgc gag tgt ttg gaa 2120 Ala Arg His Gln Asn Ala Ser Glu Ala Glu Ile Arg Glu Cys Leu Glu     595 600 605 aaa gtg gtg cct caa gcc agc gac tgt ttt gaa gtg gac cag ctc ctg 2168 Lys Val Val Pro Gln Ala Ser Asp Cys Phe Glu Val Asp Gln Leu Leu 610 615 620 625 tac ttt gaa gag cag ttg ctg atc acg atg gga aaa gaa cct act ctc 2216 Tyr Phe Glu Glu Gln Leu Leu Ile Thr Met Gly Lys Glu Pro Thr Leu                 630 635 640 cag aac cat ctc tag gcaactatca aaaagaagca gaagttcaag tggacaaatt 2271 Gln Asn His Leu             645 tatgtgaaaa ttcatttaac atataagctg aactctatta tggggaatgg atacaaaagc 2331 agagctccca tcttgacttt caattcctca tcagaagtac tggcttcttt agagagtagt 2391 aagcatggct gcctatgctt ggagtcataa gtgttatttg gactataccc tgagataagc 2451 ttatagatca agtttggctc ccttgaaaag catttctctc atgtgcgccc tcagggcttc 2511 cagcaggatt gagtcaccct gacgatgacc ggggagaagc cgtgtgctct tcattatttt 2571 cagctggagg acagagctca gtgcctgact gcctagggtc tcatggactg taggcagcct 2631 gccagtgaag gtcactggac tctagcctac aacatgctga gctacagccc agaagccaga 2691 catgcctgtc ttagctgacc tgtttttggt ccacttttgc ccttccatga ctaataagga 2751 agatatgtgt gtatttcata cacacacaag gacctggatt aaaaatccaa aaagtgattc 2811 tcttctatga tttatttcaa actcatccat agataattca agatttgtat tcaaaataaa 2871 catagttttc acagttacaa aataaatcac ctattttatc tttagaaaaa aaaaaaaaaa 2931 aaaaaaaa 2939 <210> 12 <211> 645 <212> PRT <213> Homo sapiens <400> 12 Met Leu Lys Phe Gln Glu Ala Ala Lys Cys Val Ser Gly Ser Thr Ala   1 5 10 15 Ile Ser Thr Tyr Pro Lys Thr Leu Ile Ala Arg Arg Tyr Val Leu Gln              20 25 30 Gln Lys Leu Gly Ser Gly Ser Phe Gly Thr Val Tyr Leu Val Ser Asp          35 40 45 Lys Lys Ala Lys Arg Gly Glu Glu Leu Lys Val Leu Lys Glu Ile Ser      50 55 60 Val Gly Glu Leu Asn Pro Asn Glu Thr Val Gln Ala Asn Leu Glu Ala  65 70 75 80 Gln Leu Leu Ser Lys Leu Asp His Pro Ala Ile Val Lys Phe His Ala                  85 90 95 Ser Phe Val Glu Gln Asp Asn Phe Cys Ile Ile Thr Glu Tyr Cys Glu             100 105 110 Gly Arg Asp Leu Asp Asp Lys Ile Gln Glu Tyr Lys Gln Ala Gly Lys         115 120 125 Ile Phe Pro Glu Asn Gln Ile Ile Glu Trp Phe Ile Gln Leu Leu Leu     130 135 140 Gly Val Asp Tyr Met His Glu Arg Arg Ile Leu His Arg Asp Leu Lys 145 150 155 160 Ser Lys Asn Val Phe Leu Lys Asn Asn Leu Leu Lys Ile Gly Asp Phe                 165 170 175 Gly Val Ser Arg Leu Leu Met Gly Ser Cys Asp Leu Ala Thr Thr Leu             180 185 190 Thr Gly Thr Pro His Tyr Met Ser Pro Glu Ala Leu Lys His Gln Gly         195 200 205 Tyr Asp Thr Lys Ser Asp Ile Trp Ser Leu Ala Cys Ile Leu Tyr Glu     210 215 220 Met Cys Cys Met Asn His Ala Phe Ala Gly Ser Asn Phe Leu Ser Ile 225 230 235 240 Val Leu Lys Ile Val Glu Gly Asp Thr Pro Ser Leu Pro Glu Arg Tyr                 245 250 255 Pro Lys Glu Leu Asn Ala Ile Met Glu Ser Met Leu Asn Lys Asn Pro             260 265 270 Ser Leu Arg Pro Ser Ala Ile Glu Ile Leu Lys Ile Pro Tyr Leu Asp         275 280 285 Glu Gln Leu Gln Asn Leu Met Cys Arg Tyr Ser Glu Met Thr Leu Glu     290 295 300 Asp Lys Asn Leu Asp Cys Gln Lys Glu Ala Ala His Ile Ile Asn Ala 305 310 315 320 Met Gln Lys Arg Ile His Leu Gln Thr Leu Arg Ala Leu Ser Glu Val                 325 330 335 Gln Lys Met Thr Pro Arg Glu Arg Met Arg Leu Arg Lys Leu Gln Ala             340 345 350 Ala Asp Glu Lys Ala Arg Lys Leu Lys Lys Ile Val Glu Glu Lys Tyr         355 360 365 Glu Glu Asn Ser Lys Arg Met Gln Glu Leu Arg Ser Arg Asn Phe Gln     370 375 380 Gln Leu Ser Val Asp Val Leu His Glu Lys Thr His Leu Lys Gly Met 385 390 395 400 Glu Glu Lys Glu Glu Gln Pro Glu Gly Arg Leu Ser Cys Ser Pro Gln                 405 410 415 Asp Glu Asp Glu Glu Arg Trp Gln Gly Arg Glu Glu Glu Ser Asp Glu             420 425 430 Pro Thr Leu Glu Asn Leu Pro Glu Ser Gln Pro Ile Pro Ser Met Asp         435 440 445 Leu His Glu Leu Glu Ser Ile Val Glu Asp Ala Thr Ser Asp Leu Gly     450 455 460 Tyr His Glu Ile Pro Glu Asp Pro Leu Val Ala Glu Glu Tyr Tyr Ala 465 470 475 480 Asp Ala Phe Asp Ser Tyr Cys Val Glu Ser Asp Glu Glu Glu Glu Glu                 485 490 495 Ile Ala Leu Glu Arg Pro Glu Lys Glu Ile Arg Asn Glu Gly Ser Gln             500 505 510 Pro Ala Tyr Arg Thr Asn Gln Gln Asp Ser Asp Ile Glu Ala Leu Ala         515 520 525 Arg Cys Leu Glu Asn Val Leu Gly Cys Thr Ser Leu Asp Thr Lys Thr     530 535 540 Ile Thr Thr Met Ala Glu Asp Met Ser Pro Gly Pro Pro Ile Phe Asn 545 550 555 560 Ser Val Met Ala Arg Thr Lys Met Lys Arg Met Arg Glu Ser Ala Met                 565 570 575 Gln Lys Leu Gly Thr Glu Val Phe Glu Glu Val Tyr Asn Tyr Leu Lys             580 585 590 Arg Ala Arg His Gln Asn Ala Ser Glu Ala Glu Ile Arg Glu Cys Leu         595 600 605 Glu Lys Val Val Pro Gln Ala Ser Asp Cys Phe Glu Val Asp Gln Leu     610 615 620 Leu Tyr Phe Glu Glu Gln Leu Leu Ile Thr Met Gly Lys Glu Pro Thr 625 630 635 640 Leu Gln Asn His Leu                 645 <210> 13 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: P1 <400> 13 agggagagaa ggtgtgtcac cttcaacgat 30 <210> 14 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: P2 <400> 14 ttggaagccc aactcctctc caagctggac 30 <210> 15 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: P3 <400> 15 cacaggatcc cattagaagt cgag 24 <210> 16 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: P4 <400> 16 gctgcatgaa tcatgcattc gctggc 26 <210> 17 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: P5 <400> 17 cgaattccat gctgaaattc caagaggcag c 31 <210> 18 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: P6 <400> 18 ctagagatgg ttctggagag tagg 24 <210> 19 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: P7 <400> 19 ctgaaagtca gctgtgagtt gcatg 25 <210> 20 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: P8 <400> 20 ggagaggaat taaaggtact tagggaaata tctgttggag aacta 45 <210> 21 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: P9 <400> 21 tagttctcca acagatattt ccctaagtac ctttaattcc tctcc 45 <210> 22 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: P10 <400> 22 ggaattccga gcagctacag aacc 24 <210> 23 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: P11 <400> 23 ggaattccat tccttttaaa tgtg 24

[Brief description of drawings]

FIG. 1 is a diagram showing the secondary structures of cDNA and amino acids of nek9L and nek9S of the present invention (A), and the phylogenetic tree of the NIMA family (B). A: The upper panel of each nek9 shows the intron-exon nucleotide structure of the nek9L and nek9S genes.
The middle figure shows the structure of the full-length cDNA. The straight line indicates the untranslated region, and the black box and the shaded region are n
The code region between ek9L and nek9S is the same or different. The predicted secondary structure of each Nek9 is shown in the figure below. In the figure, "○" indicates the catalytic site, gray squares indicate the estimated coiled-coil, and "●" indicates PE.
Each ST-like motif is shown. B: Sequence (NIMA from A. nidulans, Nim-1 from N. crassa, Fin1 from S. pombe, S. cerevi
KIN3 from siae, F19H6. from C. elegans.
1, N-terminal catalytic sites of CG17256 derived from D. melanogaster, Neks derived from mouse, and Nek9 derived from human were arranged by Clustal W (v1.8). Such a phylogenetic tree was created by comparing these catalytic sites. The corresponding secondary structure is shown on the right. In addition, "○" in the figure
Indicates a catalytic site, gray indicates a putative coiled-coil motif, “●” indicates a PEST-like motif, black arrowheads indicate leucine zipper motif (L-Zip), and “■” in NIMA and Nek4 indicate a typical nucleus. The localization signal (NLS) and “2” of Nek2 indicate the KEN-box.

FIG. 2 shows the results of Northern blot analysis (A) and Western blot analysis (B and C) of endogenous Nek9. A: Shows the results of Northern blot analysis of nek9 mRNA. Poly (A) RNA samples (1 μg / lane) were blotted and hybridized with the ³² P-labeled full length nek9 cDNA probe. The figure above shows the membrane hybridized with ³² P as a BAS 1800 imaging ana
The results obtained by lysis are shown below. 28S rRNA is shown below.
The result of investigating the amount of extraction is shown with the band of as a control. B: The result of having investigated the specific reaction with an anti-Nek9 polyclonal antibody is shown. C-terminal non-catalytic site of Nek9S (3
A polypeptide consisting of the 27th to 470th amino acid sequences) was used as an antigen (upper figure). Flag tagged Nek9
L (lane 1), Flag tagged Nek9S (lane 2), GFP-
Nek9L (lane 3), GFP-Catalytic (polypeptide consisting of 1 to 329th amino acid sequence) (lane 4), or GFP-LC-ter (polypeptide consisting of 379 to 645th amino acid sequence) (lane 5) HEK2 respectively
It was expressed in 93T cells. Lane 6 shows the negative control sample. Western blot analysis was performed using anti-Nek9 polyclonal antibody (α-Nek9 PoAb), anti-Nek9 serum (α-Nek9 serum), anti-Flag monoclonal antibody (α-Flag), or anti-GFP polyclonal antibody (α-GFP). . C: Shows the detection results of endogenous Nek9 protein in various cells. SD of whole cell lysate (14 μg / lane)
Endogenous Nek9 was separated by S-PAGE and by Western blot using anti-Nek9 polyclonal antibody.
Protein was detected. The arrow indicates the position of the endogenous Nek9 protein, and the lower panel shows the results using tubulin as a control.

FIG. 3 shows the results of cell cycle regulated expression of Nek9 of the present invention. A: shows the expression of nek9 mRNA. HeLa cells were synchronized with the G1 / S transition phase, and after a lapse of a G1 / S arrest period, poly (A) RNA was isolated for Northern blot analysis at a certain time point, and further for flow cytometry analysis. Cells were prepared as. Above is nek
The result of the Northern blot analysis of 9 is shown. The figure below shows the results of staining the gel with ethidium bromide (EtBr), and the DNA content was examined by flow cytometric analysis compared to the loaded control. B: Shows the results of the expression pattern of endogenous Nek9 protein in the cell cycle. Thymidine / in the G1 / S transition period
Doubly synchronized by aphidicolin (left) or G2 /
During the M transition phase, thymidine / nocodazole were synchronized in duplicate (right panel) to synchronize HeLaS3 cells. Whole cell lysates were prepared after a certain period of time after each arrest period. Such whole cell lysates were analyzed by Western blot analysis. In addition, Nek9
Was analyzed using an anti-Nek9 polyclonal antibody as a probe. In addition, cell cycle-regulated expression of Nek2 protein and cyclin B1 protein was also examined, and expression of tubulin was also analyzed as a control.

FIG. 4 is a diagram showing the results on the kinase activity of Nek9 protein of the present invention. A: The result of having investigated the selectivity of the substrate of Nek9 is shown. Control (N), wild type Nek9L with attached flag
HEK293T cells expressing W.K., and HEK293 expressing kinase-inactive Nek9L (K61R)
T cells (KR) were subjected to immunoprecipitation using an anti-flag antibody, and the substrate, dephosphorylated caseins (Caseins), myelin basic protein (MBP), and histone mixture (Hi
In vitro kinase analysis was performed using stones). [
^In vitro kinase analysis was performed in the presence of ³² P-γ] -ATP, after which each sample was separated by SDS-PAGE. The upper and lower panels show the ³² P autoradiogram and Coomassie blue staining pattern of the sample gel, respectively. The arrowheads in the figure indicate the position of each protein, and the arrows indicate the heavy and light chains of the immunoglobulin used for immunoprecipitation. B: The result of having investigated the phosphorylation of the histone protein in vitro by Nek9 is shown. Immunoprecipitated wild type Nek9L protein (W) or kinase inactive Nek
In vitro kinase analysis was performed on 9L protein (KR) using histone protein as a substrate.
The upper and lower panels show the ³² P autoradiogram and Coomassie blue staining pattern of the sample gel, respectively.
The arrowheads in the figure indicate the position of each protein, and the arrows indicate the heavy and light chains of the immunoglobulin used for immunoprecipitation. C: Shows the results of examining the kinase activity of the endogenous Nek9 protein in the cell cycle. HeLaS3 cells to G1
The whole cell lysate was prepared at a certain time point after the G1 / S transition period after synchronization with the / S transition period. Anti-Nek
Immunoprecipitation (IP) was performed with each protein (1 mg) using 9 polyclonal antibodies (lane N) and rabbit IgG (lane C) as a negative control, respectively. In vitro kinase analysis was performed using histone H2A as a substrate and analyzed by BAS 1800 imaging analyzer (upper figure). The amount of Nek9 protein and cyclin B1 protein in the cell lysate was confirmed by Western blotting (IB) (lower panel).

FIG. 5 shows the results of intracellular localization of endogenous Nek9 in HeLaS3 cells. Endogenous Ne
The intracellular localization of k9 protein was examined in asynchronously grown HeLaS3 cells. The characteristic localization of Nek9 could be confirmed in metaphase and pre-metaphase cells. The endogenous Nek9 protein was probed with an anti-Nek9 polyclonal antibody (α-Nek9), and rabbit IgG was used as a primary antibody as a negative control (control IgG). TRITC-conjugated anti-rabbit IgG
Was used as a secondary antibody and DNA was counterstained with DAPI. Then, indirect immunofluorescence analysis was performed using a confocal laser scanning microscope.

FIG. 6 shows the results of morphological changes due to overexpression of Nek9 protein of the present invention. A: Shows the result of inducing the apoptotic morphology by Flag-tagged Nek9L. Flag-tagged Nek9L was expressed in HeLa cells and these cells were fixed 32 hours after transfection. Flag-tagged Nek9L was probed with anti-flag monoclonal antibody and Cy3-conjugated secondary antibody, and DNA was counterstained with DAPI. Indirect immunofluorescence analysis was performed using a confocal laser scanning microscope. The number of apoptotic cells in EGFP-positive cells was counted by a fluorescence microscope, and 300 or more cells were analyzed. B: Morphological changes due to the C-terminal non-catalytic site of Nek9. GFP-Nek9L (G-Nek9L), GFP-Nek
9S (G-Nek9S), GFP-catalytic site (G-Catalytic),
GFP-LC-ter (GLC-ter), GFP-PE
ST1 / 2/3 (G-PEST1 / 2/3) and GFP (GFP) as a control were transiently expressed in HeLa cells for 44 hours. The nuclear morphology of EGFP-positive viable cells was examined by fluorescence microscopy and over 300 cells were analyzed. The arrowheads in the figure indicate cell atrophy, and the arrows indicate cells with abnormal nuclear morphology. C: Shows the nuclear morphology of GFP-Coil1-expressing HeLa cells. GFP-Coil1-expressing cells were fixed in the same manner as in FIG. A and stained with an anti-β tubulin antibody. DNA to D
Counterstained with API and examined by confocal microscopy. The left figure is
The GFP-Coil1 high level expressing cells showing strong green fluorescence are shown, and the right figure shows the GFP-Coil1 medium level expressing cells, respectively.

FIG. 7 is a diagram showing the result of Nek9 translocation between nucleus and cytoplasm of the present invention. A: Expression of each intracellular fraction of Nek9 is shown. 0.
A cytoplasmic fraction (S) soluble in 1% NP-40 or an insoluble fraction (P) containing nuclei was prepared by the previous method. Endogenous Nek9 protein was probed with anti-Nek9 polyclonal antibody (α-Nek9) to produce endogenous Nek9
The results of examining the expression of 9 proteins are shown in the left figure, and the over-expressed Flag-tagged Nek9L or Flag-tagged Nek9S was treated with anti-f.
Endogenous Ne by probing with a lag antibody (α-Flag)
The right figure shows the results of examining the expression of k9 protein. The expression results of Nek2 and tubulin used as controls are shown in the middle and lower figures, respectively. B: Results of examination on nuclear transfer of exogenous Nek9 by leptomycin B are shown. Flag-tagged in HEK293T cells (lower panel) treated with control (upper panel) and leptomycin B (LMB) (4 hours at 20 nM).
Nek9L was probed with anti-flag antibody and FITC conjugated secondary antibody. The localization of Nek9 was confirmed with a fluorescence microscope and photographed. C: Shows the relationship between Nek9 and CRM1. Nek9
Flag-tagged Ne
Immunoprecipitation with an anti-flag antibody was performed on k9L-expressing HEK293T cells or HEK293T cells as a control. Co-precipitated CRM1 was detected by Western blot with anti-CRM1 antibody (upper panel). Endogenous CRM1 and Flag-tagged Nek9 in lysates
The expression levels of L are shown below.

FIG. 8 shows the results of the C-terminal non-catalytic site involved in the nuclear-cytoplasmic transition of Nek9 of the present invention. G
FP fusion Nek9 protein in HeLa cells or HEK
It was expressed in 293T cells, and the subcellular amount was examined by a fluorescence microscope. Cell atrophy was frequently observed in HeLa cells (Toxic), but less so in HEK293T cells. Observe major localization in cytoplasm (Cyto), nucleus (nuc), and diffusion pattern (Cyto / Nuc)
Using EK293T cells, leptomycin B (LM
After treatment with B) (4 hours at 20 nM), Nek9
I checked the move.

FIG. 9 shows a comparison of functional features between Nek9 and NIMA of the present invention. NIMA and Nek9
The structural motif of Nek9 and NIMA (Cur
r. Biol. 5, 1122-1125, 1995, Biochem. J. 317, 633-6.
41, 1996) and compared the characteristic functions at the C-terminus. It is noteworthy that the second coiled-coil motif of Nek9 showed a similar effect to the coiled-coil motif of fungal NIMA. In the figure, "○" indicates a catalytic site, gray indicates a putative coiled-coil motif, "●" indicates a PEST-like motif, and "■" of NIMA indicates a predicted typical nuclear localization signal. Shown respectively.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) A61K 45/00 A61P 35/00 4B065 A61P 25/00 37/00 4C084 35/00 43/00 105 4C085 37 / 00 111 4H045 43/00 105 C07K 16/40 111 19/00 C07K 16/40 C12N 1/15 19/00 1/19 C12N 1/15 1/21 1/19 9/12 1/21 C12Q 1/02 5 / 10 G01N 33/15 Z 9/12 33/50 Z C12Q 1/02 C12P 21/08 G01N 33/15 C12N 15/00 ZNAA 33/50 5/00 A // C12P 21/08 A61K 37/48 (72 ) Inventor Shusuke Fukasawa 7-18-26-806 Tanashi-cho, Nishi-Tokyo, Tokyo (72) Inventor Yuko Murakami 6-19-6 F Term, Kugahara, Ota-ku, Tokyo (reference) 2G045 AA34 AA35 BB10 BB14 BB24 BB51 CB01 DA13 DA36 FA16 FB01 FB02 FB03 4B024 AA01 AA11 BA10 CA04 CA11 DA03 HA14 HA17 4B050 CC03 DD07 LL01 LL03 4B063 QA18 QQ08 QQ27 QR07 QR77 4B064 AG27 CA20 CC24 DA13 4B065 AA93X AA93Y AB01 BA01 CA29 CA44 4C084 AA02 AA07 AA17 BA01 BA02 BA08 BA19 BA21 BA23 CA18 DC01 NA14 ZA011 ZB011 ZB261 ZC012 ZC412 4C085 AA13 AA14 AA16 BB11 CC03 CC21 DD62 EE01 4H045 AA10 AA11 AA20 AA30 BA10 BA41 CA40 DA76 EA20 EA50 FA74

Claims (52)

[Claims] 1. A DNA or RNA encoding a cell cycle-related protein having a nuclear export function. 2. A DNA or RNA encoding the following protein (a) or (b). (a) a cell cycle-related protein consisting of the amino acid sequence shown in SEQ ID NO: 2 (b) an amino acid sequence shown in SEQ ID NO: 2 in which one or several amino acids are deleted, substituted or added, Cell cycle-related protein with nuclear export function 3. A DNA or RNA consisting of the nucleotide sequence shown in SEQ ID NO: 1 or its complementary sequence or a sequence containing a part or all of these sequences. 4. D which hybridizes with the DNA or RNA of claim 3 under stringent conditions and which encodes a cell cycle-related protein having a nuclear export function.
NA or RNA. 5. A DNA or RNA encoding a cell cycle-related protein having a nuclear-cytoplasmic transfer function. 6. A DNA or RNA encoding the following protein (a) or (b). (a) a cell cycle-related protein consisting of the amino acid sequence shown in SEQ ID NO: 4 (b) an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 4, And cell cycle-related protein having nuclear-cytoplasmic transition function 7. A DNA or RNA comprising the nucleotide sequence shown in SEQ ID NO: 3 or its complementary sequence or a sequence containing a part or all of these sequences. 8. A DNA or RNA which hybridizes with the DNA or RNA according to claim 7 under stringent conditions and which encodes a cell cycle-related protein having a nucleus-cytoplasm transfer function. 9. A DNA or RNA encoding a cell cycle-related protein having a nuclear export function, a cytotoxic function, and a nuclear import function. 10. A DNA or RNA encoding the following protein (a) or (b). (a) a cell cycle-related protein consisting of the amino acid sequence shown in SEQ ID NO: 6 (b) consisting of an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 6, And cell cycle related protein having nuclear export function, cytotoxicity function and nuclear import function 11. A DNA or RNA comprising the nucleotide sequence shown in SEQ ID NO: 5, its complementary sequence, or a sequence containing a part or all of these sequences. 12. A DNA which hybridizes with the DNA or RNA according to claim 11 under stringent conditions and which encodes a cell cycle-related protein having a nuclear export function, a cytotoxic function and a nuclear import function, or RN
A. 13. A DNA or RNA encoding a cell cycle-related protein having a nuclear export function, a cytotoxic function, a nuclear import function, and a nuclear-cytoplasmic import function. 14. A DNA or RNA encoding the following protein (a) or (b). (a) a cell cycle-related protein consisting of the amino acid sequence represented by SEQ ID NO: 8 (b) an amino acid sequence represented by SEQ ID NO: 8 in which one or several amino acids are deleted, substituted or added, And cell cycle-related protein having nuclear export function, cytotoxic function, nuclear import function, and nucleus-cytoplasm transfer function 15. A DNA or RNA comprising the nucleotide sequence shown in SEQ ID NO: 7 or its complementary sequence, or a sequence containing a part or all of these sequences. 16. A hybridizing with the DNA or RNA according to claim 15 under stringent conditions and having a nuclear export function, a cytotoxic function, a nuclear import function, and a nucleus-.
DNA or RNA that encodes a cell cycle-related protein having a cytoplasmic transition function. 17. An oligonucleotide or peptide nucleic acid, which is capable of specifically hybridizing with the DNA or RNA of claim 3 and inhibiting the expression of a cell cycle-related protein having a nuclear export function. 18. An oligonucleotide or peptide nucleic acid, which is capable of specifically hybridizing with the DNA or RNA according to claim 7 and inhibiting the expression of a cell cycle-related protein having a nucleus-cytoplasm translocation function. . 19. It is possible to inhibit the expression of a cell cycle-related protein that hybridizes specifically with the DNA or RNA of claim 11 and has a nuclear export function, a cytotoxic function and a nuclear import function. Characterized oligonucleotide or peptide nucleic acid. 20. A cell cycle-related protein that hybridizes specifically with the DNA or RNA according to claim 15 and has a nuclear export function, a cytotoxic function, a nuclear import function, and a nucleus-cytoplasm transfer function. An oligonucleotide or peptide nucleic acid, which is capable of inhibiting the expression of 21. A cell cycle-related protein having a nuclear export function. 22. A cell cycle-related protein consisting of the amino acid sequence shown in SEQ ID NO: 2. 23. A cell cycle-related protein which comprises the amino acid sequence shown in SEQ ID NO: 2 in which one or several amino acids have been deleted, substituted or added, and which has a nuclear export function. 24. A cell cycle-related protein having a nuclear-cytoplasmic transfer function. 25. A cell cycle-related protein comprising the amino acid sequence shown in SEQ ID NO: 4. 26. A cell cycle-related protein having an amino acid sequence represented by SEQ ID NO: 4 in which one or several amino acids are deleted, substituted or added, and having a nucleus-cytoplasm transfer function. 27. A cell cycle-related protein having a nuclear export function, a cytotoxic function, and a nuclear import function. 28. A cell cycle-related protein comprising the amino acid sequence shown in SEQ ID NO: 6. 29. An amino acid sequence represented by SEQ ID NO: 6 comprising an amino acid sequence in which one or several amino acids have been deleted, substituted or added, and has a nuclear export function, a cytotoxic function and a nuclear import function. A cell cycle-related protein having: 30. A cell cycle-related protein having a nuclear export function, a cytotoxic function, a nuclear import function, and a nuclear-cytoplasmic import function. 31. A cell cycle-related protein consisting of the amino acid sequence shown in SEQ ID NO: 8. 32. An amino acid sequence represented by SEQ ID NO: 8, which comprises an amino acid sequence in which one or several amino acids have been deleted, substituted or added, and has a nuclear export function, a cytotoxic function, and a nuclear import function. , And a cell cycle-related protein having a nuclear-cytoplasmic transition function. 33. A peptide comprising a part of the protein according to any one of claims 21 to 23 and having a nuclear export function. 34. A peptide comprising a part of the protein according to any one of claims 24 to 26 and having a nuclear-cytoplasmic translocation closing function. 35. A peptide comprising a part of the protein according to any one of claims 27 to 29 and having a nuclear export function, a cytotoxic function and / or a nuclear import function. 36. It comprises a part of the protein according to any one of claims 30 to 32 and has a nuclear export function, a cytotoxic function, a nuclear import function, and / or a nucleus-cytoplasm transfer function. Characterized peptide. 37. A fusion protein or fusion peptide in which the protein according to any one of claims 21 to 32 or the peptide according to any one of claims 33 to 36 is bound to a marker protein and / or a peptide tag. 38. An antibody that specifically binds to the protein according to any one of claims 21 to 32 or the peptide according to any one of claims 33 to 36. 39. The antibody according to claim 38, wherein the antibody is a monoclonal antibody or a polyclonal antibody. 40. A recombinant protein or peptide to which the antibody according to claim 38 or 39 specifically binds. 41. The DN according to any one of claims 1 to 16.
A recombinant vector containing A. 42. The protein according to any one of claims 21 to 32, the peptide according to any one of claims 33 to 36, the fusion protein or the fusion peptide according to claim 37, the antibody according to claim 38 or 39, or Claim 4
A host cell comprising an expression system capable of expressing the recombinant protein according to 0. 43. A non-human animal in which the gene function encoding the protein according to any one of claims 21 to 32 or the peptide according to any one of claims 33 to 36 is deleted on the chromosome. 44. A non-human animal that overexpresses the protein according to any one of claims 21 to 32 or the peptide according to any one of claims 33 to 36. 45. The non-human animal according to claim 43 or 44, wherein the non-human animal is a mouse or a rat. 46. The protein according to any one of claims 21 to 32, the peptide according to any one of claims 33 to 36, or the protein according to any one of claims 21 to 32, or any one of claims 33 to 36. Cell membrane expressing the peptide described, and a nuclear export function characterized by using a test substance, cytotoxic function, nuclear import function,
A method for screening a substance that promotes or suppresses a nucleus-cytoplasm transfer function and / or a cell cycle control function. 47. Nuclear translocation, wherein a cell expressing the protein according to any one of claims 21 to 32 or the peptide according to any one of claims 33 to 36 and a test substance are used. 21. A substance that promotes or suppresses a function, a cytotoxic function, a nuclear translocation function, a nuclear-cytoplasm translocation function, and / or a cell cycle control function, or 21.
32. The protein according to any of 32 or 37 to 36.
A method for screening a substance for promoting or suppressing the expression of the peptide according to any one of 1. 48. A nuclear export function, a cytotoxic function, a nuclear import function, a nucleus-cytoplasm characterized by using the non-human animal according to any one of claims 43 to 45 and a test substance. A method for screening a substance that promotes or suppresses a translocation function and / or a cell cycle control function, or a substance that promotes or suppresses expression of the protein according to any one of claims 21 to 32 or the peptide according to any one of claims 33 to 36. . 49. A nuclear export function, a cytotoxic function, a nuclear import function, a nucleus-cytoplasm transfer function, and / or a cell cycle control function obtained by the screening method according to claim 46. Promoter or suppressor. 50. An expression promoting substance or expression suppression of the protein according to any one of claims 21 to 32 or the peptide according to any one of claims 33 to 36 obtained by the screening method according to any one of claims 46 to 48. material. 51. The protein according to any one of claims 21 to 32, the peptide according to any one of claims 33 to 36, the recombinant protein or peptide according to claim 40, the antibody according to claim 38 or 39, Item 49. An enhancer or suppressor of the nuclear export function, cytotoxic function, nuclear import function, nucleus-cytoplasm transfer function, and / or cell cycle control function of Item 49, or the expression promoter of Claim 50, or A therapeutic agent for an immune disease, a neurological disease, or cancer containing an expression inhibitor as an active ingredient. 52. The protein according to any of claims 21 to 32, the peptide according to any of claims 33 to 36, the recombinant protein or peptide according to claim 40, the antibody according to claim 38 or 39, Item 49. An enhancer or suppressor of the nuclear export function, cytotoxic function, nuclear import function, nucleus-cytoplasm transfer function, and / or cell cycle control function of Item 49, or the expression promoter of Claim 50, or A diagnostic agent for an immune disease, a neurological disease, or cancer, which contains an expression inhibitor as an active ingredient.